Tissue Engineering and Regenerative Medicine
Volume 7 - 2019 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fbioe.2019.00109">https://doi.org/10.3389/fbioe.2019.00109</a>
Foam sclerotherapy is clinically employed to treat varicose veins
It involves intravenous injection of foamed surfactant agents causing endothelial wall damage and vessel shrinkage
Foam production methods used clinically include manual techniques
such as the Double Syringe System (DSS) and Tessari (TSS) methods
Pre-clinical in-vitro studies are conducted to characterize the performance of sclerosing agents; however
the experimental models used often do not replicate physiologically relevant physical and biological conditions
physical vein models (PVMs) were developed and employed for the first time to characterize the flow behavior of sclerosing foams
PVMs were fabricated in polydimethylsiloxane (PDMS) by replica molding
and were designed to mimic qualitative geometrical characteristics of veins
Foam behavior was investigated as a function of different physical variables
namely (i) geometry of the vein model (i.e.
The experimental set-up consisted of a PVM positioned on an inclined platform
a syringe pump to control the flow rate of a blood substitute
The static pressure of the blood surrogate at the PVM inlet was measured upon foam administration
The recorded pressure-time curves were analyzed to quantify metrics of foam behavior
with a particular focus on foam expansion and degradation dynamics
Results showed that DSS and TSS foams had similar expansion rate in the physiological PVM
whilst DSS foam had lower expansion rate in the varicose PVM compared to TSS foam
The degradation rate of DSS foam was lower than TSS foam
the background flow rate had a significant effect on foam behavior
enhancing foam displacement rate in both types of PVM
It was however designed for usage under static fluidic conditions
and it did not replicate the varicose vein architecture
In order to address these limitations of previous test methods
the work in this study aims to develop physical models replicating qualitative architectural characteristics of varicose veins and to employ them as a screening platform for comparing the flow behavior of different foam formulation methods
The developed biomimetic-inspired vein model (referred to as physical vein model
or PVM) allows recapitulating features of physiological and varicose veins
and physiologically relevant flow conditions
PVMs were employed to compare the flow performance of polidocanol-based PCFs
as a function of vessel geometry (straight vs
it was demonstrated that models can be coated with endothelial cells
enabling future investigations of both mechanical and biological performance of sclerosing agents
Assembled PVMs representing simplified physiological (A) and varicose (B) vein models
and demonstration of perfusion using a red dye
These models were employed to test the flow behavior of foams
The main channel was punctured with a needle (16G)
in order to mimic the clinical process of injection more closely
Inlet/outlet ports were connected with silicone tubing (4 mm outer diameter
Schematic of PVMs production via replica molding
(A) a CAD design of positive molds was generated and 3D printed
(B) two PDMS layers were produced from the molds
and permanently bonded via oxygen plasma treatment to obtain a fully circular channel cross-section (C)
the main channel was punctured with a 16G needle (D)
the foam was produced by passing the polidocanol solution (liquid phase) from one syringe
10 times into and out of the other syringe initially containing room air
The pressure transducer was connected to a National Instruments I/O module (NI-DAQ
The NI-DAQ system supports analog and digital inputs
and communicates with the NI-DAQ software (National Instrument
USA) script was employed to store pressure data in an automated fashion
Schematic illustrating the set-up for evaluating the flow behavior of foams in the PVM model
A steady flow is imposed using a syringe pump (A)
and a pressure transducer (B) is positioned in line with the inlet tubing and prior to the PVM (C)
The foam is injected through the 16G needle into the main channel (D)
The pressure transducer is connected to a National Instruments I/O module
A Matlab code is employed to store pressure data in an automated fashion (E)
The corresponding inlet Reynolds number was calculated using Equation 1
where &#x003C1; is the density of the blood surrogate (kg/m3)
v is the mean velocity of the blood surrogate (m/s)
D is the hydraulic diameter of the vein model (m)
and &#x003BC; is the dynamic viscosity of the blood surrogate (Pa&#x000B7;s)
The Reynolds number in these experiments ranged from 1.65 to 3.34, which is &#x0007E;100&#x02013;200 times lower than physiological values (<a href="#B30">Raju et al., 2004</a>)
in order to replicate quasi-static or impaired flow conditions occurring in diseased veins
(ii) an almost linear increase in pressure due to the expansion of the foam within the PVM
and (iii) an almost linear decrease in pressure caused by foam degradation and &#x0201C;washing out.&#x0201D; The CfAS calculated the slope of phases (ii) and (iii)
which were referred to as expansion rate (ER) and degradation rate (DR)
expansion time (ET) and degradation time (DT) were also quantified
The plots were divided into three phases: (i) an increase of the pressure due to the expansion of the foam inside the channel
(ii) a decrease of the pressure caused by the degradation of the foam
and (iii) an initial peak due to the injection
A representative pressure profile is illustrated in <a href="#F4">Figure 4</a>
from which four different phases can be identified:
This phase was associated with a rapid pressure spike
likely due to the insertion of the needle within the PVM or other mechanical perturbations associated with the injection procedure
Peak pressure values in this phase ranged from 10 to 50 mmHg
The CfAS allowed quantifying the slope of the plug expansion phase
which was herein referred to as expansion rate (ER)
It is hypothesized that more cohesive foams would fractionate more slowly and dilute less rapidly with the blood substitute
thus resulting in lower ER and higher peak pressures
The expansion time (ET) was also calculated from the pressure-time curve; higher ET corresponds to a longer contact time between the foam and the inner surface of the PVM
The CfAS allowed quantifying the slope of the plug degradation phase
which was referred to as degradation rate (DR)
It is hypothesized that more cohesive foams would destabilize more slowly
The degradation time (DT) was also calculated from the pressure-time curve; higher DT corresponds to a longer contact time between the foam and the inner surface of the PVM
The pressure level at the end of the degradation phase could be equal or greater than the initial pressure (i.e.
bubbles accumulated at the top surface of the vein model
they would remain in place for the duration of the pressure recording (up to 100 s)
causing the residual pressure to be greater than the initial pressure
Comparisons between foam production methods were performed using an unpaired Student&#x00027;s t-test for two groups analysis
or one-way ANOVA in the case of more than two groups
Statistical significance was assumed for p &#x0003C; 0.05
All statistical tests were performed with Prism software (GraphPad Software Inc.
All data were reported as the mean &#x000B1; SD of at least six independent repeats of the same experiment
HUVECs (human umbilical venous endothelial cells) were seeded in the PVM models
HUVECs were extracted from umbilical cords
and this was carried out in accordance with the Human Tissue Act (2004) and the recommendations of Southampton &#x00026; South West Hampshire Research Ethics Committee B with Governance provided by the University of Southampton Research Governance Office
Umbilical cords were collected from the Princess Anne Hospital (Southampton
UK) from non-complicated natural vaginal births following agreed ethical collection protocols [Local Research Ethical Committee (LREC); Ref: 07/H0502/83]
the PDMS devices were washed four times with 70% ethanol for sterilization
HBSS (Hanks&#x00027; Balanced Salt Solution
USA) was conveyed through the channels to remove ethanol traces
the inner surfaces of the device were coated with 3 mL of different proteins: 50 &#x003BC;l/mL of rat type I collagen (100 &#x003BC;g/mL; Gibco&#x02122;
UK) or (ii) 100 &#x003BC;l/mL of fibronectin (Sigma
The device was subsequently placed at 37&#x000B0;C in a 5 % CO2 incubator for 2 h
The HUVECs suspension was injected into the proteins-coated channels (at a concentration of 4&#x02013;5 &#x000D7; 106 cells/mL)
The device was finally incubated overnight to promote cell attachment
Bright field images of HUVECs within the PVM models were acquired with an optical microscope (Olympus
Images were taken of live samples every 24&#x02013;48 h
The PVM models developed in this study have been employed to characterize the flow behavior of sclerosing foams
by measuring static pressure of a blood surrogate at the PVM inlet
only the pressure spike corresponding to foam injection was present
due to the absence of a background pressure-driven flow
Pressure plots obtained from the CfAS while injecting DSS (A,C) and TSS (B,D) foams
The upper panels show the recordings obtained using the straight channel geometry
whereas the lower panels show the recording obtained using the serpentine-like channel geometry
the pressure was measured without injecting foam
at a background constant flow rate (72 mL/h)
The pressure inside the vein models was also measured while injecting the foam in the absence of a blood-surrogate flow (F)
Expansion rate values at the different flow rates investigated
and different foam production methods (TSS blue bars
Measurements were obtained from the straight (A) and curved (B) channel geometry
Data represent the average of 6 measurements &#x000B1; SD
An asterisk (*) indicates that differences between mean values are statistically significant (p &#x0003C; 0.05)
and results are reported as mean value &#x000B1; standard deviation
A one-way ANOVA was conducted to evaluate whether ER of both types of foam depended on the inlet flow rate
a significant difference was observed between all flow rates investigated; indeed
the average ER value increased with increasing the inlet flow rate
increasing the background flow caused a reduction of ET
likely due to a &#x0201C;washing out&#x0201D; effect of the blood surrogate
DSS foam demonstrated greater ability to oppose this effect
resulting in higher contact time with the vessel wall
Expansion time values at the different flow rates investigated
Two asterisks (**) indicate that differences between mean values are very statistically significant (p &#x0003C; 0.01)
Degradation rate values at the different flow rates investigated
<a href="#F8">Figure 8</a> shows DR values determined for both TSS and DSS foams
DSS foam had a lower DR (0.26 &#x000B1; 0.01 mmHg/s) compared to TSS foam (0.32 &#x000B1; 0.03 mmHg/s)
Significant difference in DR between TSS (0.45 &#x000B1; 0.06 mmHg/s) and DSS (0.37 &#x000B1; 0.09 mmHg/s) foams was found at 72.0 mL/h
At the highest flow rate (125.0 mL/h) both types of foam had similar DR (0.48 &#x000B1; 0.03 mmHg/s for TSS and 0.47 &#x000B1; 0.07 mmHg/s for DSS)
suggesting that foam degradation performance is dominated by the background flow at these higher flow rates
A one-way ANOVA was performed to evaluate the effect of background flow rate on DR
a significant difference was observed with increasing the inlet flow rate; indeed
the average DR value increased with increasing the flow rate
no significant difference was found by varying the flow rate
Both foams had comparable degradation performance across the two PVM geometries
suggesting that once a foam plug has been established into the vein
its degradation dynamics is not significantly affected by the vessel architecture
<a href="#F9">Figure 9</a> shows DT values determined for both TSS and DSS foams at all flow rates investigated
DSS foam had a statistically higher DT (20.73 &#x000B1; 2.60 s) compared to TSS foam (15.46 &#x000B1; 2.30 s)
the average DT value was still higher compared to TSS (16.38 &#x000B1; 3.70 s and 10.90 &#x000B1; 3.40 s
At the highest flow rate (125.0 mL/h) both PCFs had comparable DT (9.97 &#x000B1; 5 s for TSS and 7.8 &#x000B1; 5.6 s for DSS)
foams with lower degradation rate had a longer degradation time
Similar observations were made using the varicose PVM model
where at the lowest flow rate (62.5 mL/h) DSS foam had statistically higher DT (24.8 &#x000B1; 6.20 s) compared to TSS
Differences between foam types reduced with increasing the inlet flow rate
both types of foam presented similar DT (9.1 &#x000B1; 5.3 s for TSS and 11.07 &#x000B1; 4.1 s for DSS)
A one-way ANOVA was conducted to evaluate the effect of flow rate on DT for both types of foam
results show that there is a significant difference between DTs measured at increasing flow rates
DT was not significantly influenced by the PVM geometry
no significant difference was found by varying the background flow rate
Degradation time values at the different flow rates investigated
An asterisk (*) indicates that differences between mean values are statistically significant (p &#x0003C; 0.05) and two asterisks (**) indicate that differences between mean values are very statistically significant (p &#x0003C; 0.01)
Bright field microscope images of HUVECs cultured within the fully circular PVM channels
Images on the left show the lower channel wall coated with collagen (A) and fibronectin (C)
Images on the right show the upper channel wall coated with collagen (B) and fibronectin (D)
Images (4x magnification) were taken after 48 h from cell seeding
a novel experimental method to quantify and compare the flow behavior of sclerosing foams was developed
The method provided a quantitative determination of fluid pressure upon foam administration
within models of either physiological or varicose veins (referred to as physical vein model
When a cohesive foam is injected into the PVM, it forms a plug that displaces the blood substitute. The foam plug however degrades over time, due to its intrinsic instability and the &#x0201C;washing out&#x0201D; action of the background flow. Using our model system, we were able to characterize these phenomenological behaviors for the first time, by measuring the static pressure of a blood surrogate at the PVM inlet (<a href="#F5">Figure 5</a>)
It is well-known that sclerosing foams produced using different techniques differ in their &#x0201C;static&#x0201D; physical properties
we evaluated for the first time the dynamic flow behavior of sclerosing foams
by analyzing their expansion and degradation within qualitative models of both physiological and varicose veins
the behavior of different PCFs was compared
at varying volumetric flow rates (in the range 62.5&#x02212;125.0 mL/h)
Results also demonstrated that the flow field within the target vein can significantly influence the expansion dynamics of sclerosing foams
DSS foam was slightly less sensitive to changes in the background flow rate
suggesting that more cohesive foams may offer higher resistance to the &#x0201C;washing out&#x0201D; effect of the blood flow during expansion
Reducing blood flow rate during administration (i.e.
via vein compression) may thus be preferable to enhance therapeutic efficacy
With respect to the degradation dynamics of PCFs (<a href="#F8">Figures 8</a>, <a href="#F9">9</a>), at the lowest flow rate investigated DSS foam had lower degradation rate compared to TSS foam. This was likely due to the slower coarsening and drainage rate of DSS foams, coherently with previous studies (<a href="#B4">Carugo et al., 2015</a>)
Increasing the inlet flow rate resulted in PCFs having comparable degradation rate
suggesting that foam degradation performance is dominated by the background flow in these conditions
there was no significant difference in the degradation dynamics between the two PVM geometries investigated; suggesting that once a foam plug has been established into the vein
It is important to highlight that expansion and degradation dynamics taken at the lowest flow rate are likely to be more representative of the flow conditions in a diseased (i.e.
DSS presented a slightly superior performance compared to TSS
Finally, it was demonstrated that PVM models can be lined with endothelial cells in order to recreate the endothelial layer (<a href="#F10">Figure 10</a>)
The degree of endothelial damage upon treatment with foam can be employed as an indicator of therapeutic efficacy
we will employ these cell-coated PVM models to investigate the biological effects of sclerosing foams and correlate them with foam mechanical behavior
we described the development of physical vein models replicating the qualitative architecture of physiological and varicose veins
and their utility as model platforms to screen the flow behavior of sclerosing foams
upon different formulation and administration conditions
A simple method to manufacture vein models was developed
which aimed at generating channels with circular section and with a geometry that recapitulates some characteristics of the varicose vein
An experimental protocol was also established to investigate the flow performance of foams at conditions relevant to their clinical administration
the experimental set-up replicated some aspects of the clinical process of foam injection
and the presence of a background blood flow
Fluid pressure at the PVM inlet was measured during foam administration
which revealed different phases of the foam expansion and degradation dynamics
Particular emphasis was given to expansion and degradation of the foam plug
As reported in previous studies (<a href="#B4">Carugo et al., 2015</a>), the cohesiveness of foams is highly dependent on their rheological properties, which in turn are influenced by the bubble size distribution and foam drainage kinetics. Previous results showed that foam produced using the DSS method were more stable and presented longer dwell time compared to TSS foams (<a href="#B4">Carugo et al., 2015</a>)
Consistently with these previous observations
in our dynamic study DSS foam had longer degradation time and slower degradation rate than TSS foam
no significant difference between the two foam formulations was found in the physiological vein model
whereas DSS had slower expansion in the varicose model
Differences in foam behavior across different model geometries could be attributed to the broader bubble size distribution of TSS foam compared to DSS foam; although these aspects merit further investigation
the physical vein models and experimental methods developed in this study provide a novel technology platform to measure the behavior of different formulations of sclerosing foams
at physical conditions that resemble their clinical administration
They could therefore be employed as an additional test method in the pre-clinical pipeline
to innovate foam formulation and administration procedures
we demonstrated that PVM models are suitable for coating with endothelial cells
which enables future investigations to correlate flow performance of sclerosing agents with their biological effects
It should be noted that the PVM models reported in this study do not replicate the presence of venous valves
or the mechanical properties of the vein wall
which may affect the flow behavior of foams
Ongoing research is focusing on the incorporation of these additional architectural and functional characteristics
a more faithful replication of the physiological boundary conditions (including changes due to clinical practices; i.e.
vein compression) will be considered in the future
EB conducted all experiments and data analyses
DC co-designed all experimental procedures
co-wrote the manuscript and supervised the project
and VP contributed to the design and implementation of the research and to the analysis of the results
All the other authors helped supervise the project
This research was supported by Doctoral Training Partnership funded from EPSRC and Biocompatibles UK Ltd
The funding was awarded by the Faculty of Engineering and Environment (University of Southampton)
EB is in receipt of a Doctoral Training Partnership funded from EPSRC and Biocompatibles UK Ltd
and VP are paid employees of Biocompatible UK Ltd
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
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Millar TM and Carugo D (2019) Physical Vein Models to Quantify the Flow Performance of Sclerosing Foams
Received: 31 October 2018; Accepted: 01 May 2019; Published: 21 May 2019
Copyright &#x000A9; 2019 Bottaro, Paterson, Zhang, Hill, Patel, Jones, Lewis, Millar and Carugo. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY)</a>
distribution or reproduction in other forums is permitted
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This paper provides an analysis of microfluidic techniques for the production of nanoscale lipid-based vesicular systems
In particular we focus on the key issues associated with the microfluidic production of liposomes
production method (including microchannel architecture)
and drug loading in determining liposome characteristics
we propose microfluidic architectures for the mass production of liposomes with a view to potential industrial translation of this technology
They are composed of one or more closed shells
consisting of a phospholipid bilayer and enclosing a small volume of aqueous liquid
Their dimensions can vary from tens of nanometres up to hundreds of micrometres depending on the preparation protocol and the final use
liposomes used in medical applications are unilamellar with an average size of ~100 nm
although they are less frequently employed in association with commercial formulations
Over the past few decades different techniques have been proposed for the preparation of liposomes and liposome size, dispersity (Đ)<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 9" title="Stepto, R. F. T. Dispersity in polymer science (IUPAC Recommendations 2009). Pure Appl. Chem. 81, 351–353 (2009)." href="/articles/srep25876#ref-CR9" id="ref-link-section-d444357001e602">9</a>
lamellarity and entrapped liquid volume may all vary depending on the method used
The majority of the existing methods can be categorised into one of two groups of bulk methods
where the term “bulk” is used throughout this manuscript to describe macroscale or batch techniques
More recently, improvements have been made with the development of microfluidic production methods<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 10" title="van Swaay, D. &amp; DeMello, A. Microfluidic methods for liposomes. Lab Chip 13, 752–767 (2013)." href="/articles/srep25876#ref-CR10" id="ref-link-section-d444357001e623">10</a>
in which liposome formation occurs within a confined microenvironment
Microfluidic methods have demonstrated potential for achieving higher control over the physical properties of the end product
There are however concerns about the viability of this technology on an industrial scale
These concerns arise mostly from the lack of experimental evidence demonstrating: (i) a systematic superiority compared to macroscale bulk methods
(ii) suitability for producing clinically relevant liposome formulations
(iii) efficiency in loading bioactive compounds
it should be noted that efforts have been made in the industrial environment to develop controlled precipitation processes for producing liposomes of clinically relevant standards
we attempt to address these issues by critically analysing state-of-the art microfluidic methods reported in the literature and by performing additional experiments
Many of the aforementioned techniques however result in the production of relatively large vesicular systems (i.e.
Giant or Large Unilamellar Vesicles) or microtubules
limiting their potential for the production of nanoscale drug delivery vehicles
being simply a miniaturised version of their analogous bulk counterparts
many of the aforementioned methods cannot be regarded as strictly microfluidic
the microfluidic hydrodynamic focusing (MHF) approach presents the typical physical characteristics of microfluidic systems (i.e.
including low Reynolds number and diffusion dominated mass transfer) and
represents the only viable microfluidic method for producing lipid-based nanoscale vesicular systems with potential for clinical application
Only limited attention has been devoted to this technique in previous publications<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Herzog, C. et al. Eleven years of Inflexal V-a virosomal adjuvanted influenza vaccine. Vaccine 27, 4381–4387 (2009)." href="/articles/srep25876#ref-CR5" id="ref-link-section-d444357001e685">5</a> however
and a critical evaluation of the method has not been carried out
which are routinely employed in the industry
may not represent a scaled up version of microfluidic flow focusing regimes
which may be more suitable for medical or biotechnological applications (e.g
as delivery systems for anticancer drugs or as transfection reagents)
and which are routinely used in the industry
Although this effect could be reduced by significantly diluting the liposome suspension prior to dimensional analysis
this may not be applicable to many MHF techniques which suffer from a relatively low liposome concentration in the end-product
there has been little or no systematic characterisation of drug encapsulation efficiency or the effect of drug encapsulation on liposome size
different device architectures have been considered for liposome production
Devices include microscale chips (cross-sectional dimensional range of 100–320 μm) with mixing channel displaying distinct architectural features (i.e.
and containing micropillar structures) and scaled-up versions of microscale flow focusing architectures
with cross-sectional channel dimension in the millimetre range
Considering the spectrum of different architectures described in the literature
the following general principles have been considered in this study for designing and constructing chips employed for liposome production
The total width and length of the chip should be defined to comply with the dimensional constraints imposed by the typical size of microscope stages that are routinely used to assess the correct functioning of the chips
the microchannel architecture should fit within the maximum width and length of conventional glass slides for microscopy (i.e.
the thickness of the device should comply with the working distance of microscope objectives
It should be noted that whilst in situ microscope observation may not be required in an industrial setting
we consider it as a desirable requirement at a research and development stage
The inlet channels should be long enough to allow the fluid flows to fully develop before they intersect with each other
allowing for a stable and predictable flow focusing regime
Channels should also be designed so as to guarantee sufficient spacing between the inlet ports
allowing for robust and practical connection with tubing and pumping units
A typical inlet channel length for MHF chips is in the range of 5–10 mm; note that longer channels result in higher backpressure
which could potentially compromise device usability
the mixing channel should be designed to allow for complete mixing of the solvents (ethanol or isopropanol
and water) at the selected operating flow rates
and to comply with the dimensional requirements imposed by microscope interfacing
(iii) Relative orientation of the inlet channels
the angle between the side and central inlet channels should be defined so as to minimise fluid dynamic perturbations at the intersection between flows
particularly if devices are designed to operate at high throughput regimes
An angle in the range 30°–60° was deemed suitable for the applications described in the present study
The constitutive materials should satisfy different requirements
compatibility with microfabrication and bonding techniques
PDMS has been identified as the optimal material choice
given its compatibility with pharmaceutical-grade solvents (i.e.
ease of surface treatment by exposure to oxygen plasma
and potential for bonding with commercially available glass slides
Traditional soft lithographic methods based on SU-8 moulding can be employed for producing PDMS microchannel architectures
which can be irreversibly coupled to glass substrates using plasma bonding techniques
glass is chemically inert and its surfaces smoothness is suitable for optical microscopy
With an aim to develop cost-effective and facile microfabrication methods with potential for large-scale chip fabrication
we also employed an in-house developed technique which combines micromilling with epoxy-based replica moulding
Both these methods allow for obtaining well-defined cross-sectional channel features
which may be complex to achieve using wet etching techniques
Microfluidic flow focusing was selected as a microfluidic-based regime for nanoscale liposome production
and its performance compared with other micromixing geometries
MHF operated at laminar flow conditions has been previously demonstrated to allow for precise control over the interfacial boundaries between solvent and co-solvent streams
resulting in diffusion dominated mass transfer and leading to liposomes of relatively uniform physical properties
liposome size and dispersity in MHF chips has the advantage of being potentially controlled on-demand by finely adjusting the hydraulic boundary conditions
MHF devices were compared with other micromixing geometries
including those containing serpentine-like microchannels and micropillar arrays
</a>Panel A shows a schematic representation of a microfluidic device
and the process of liposome (SUV) self-assembly
Panel B shows a schematic representation of the ethanol injection procedure
Panel C illustrates the geometrical characteristics of the chips employed for the microfluidic experiments
#chip1-MHF comprises three inlet microchannels with 30° intersection angle between each other
#chip2-YJ comprises two inlet microchannels with 120° intersection angle between each other
#chip3-MP comprises three inlet microchannels with 90° intersection angle between each other
and a mixing microchannel with three pillar mixing elements
TeflonTM tubes were used to connect the microfluidic platform with syringes
The volumetric flow rate was controlled using syringe pumps
a detailed analysis is provided of the effects of the operating parameters (especially FRR) on liposome dimensions
taking into consideration the effect of residual alcohol on the viscosity of the liposomal samples and thus on the determination of liposome size by light scattering measurements
Liposomes produced using MHF were compared with those obtained by the bulk ethanol injection method
Ethanol injection was selected as a bulk technique since
in our opinion it strongly resembles MHF liposome formation (in terms of simplicity and physical conditions) relative to other bulk techniques
this method results in the formation of small unilamellar vesicles (SUV); therefore it does not require post-processing homogenisation steps
phospholipids are firstly dissolved in ethanol; a small amount of the lipid solution is then injected into water
</a>Effect of the variation of lipid concentration (A)
ethanol content (B) and simultaneous variation of both parameters (C) on the dimension of liposomes produced by ethanol injection
data relative to liposomes prepared by MHF microfluidics are also reported in panel C (dashed line)
Liposomes were constituted of PC/cholesterol 4.0−0.4 mM
and data represent the mean of three independent samples
</a>Effect of the variation of lipid composition on the size (upper part
panel B) of liposomes produced by MHF microfluidics
Liposomes were constituted of PC/DDAB 9.0−1.0 mM (filled circles
dashed line) or PC/cholesterol 9.0−1.0 mM (open circles
and are reported as the mean of three independent samples
cryo-TEM and macroscopic aspect (insets) of empty PC/cholesterol (A) and PC/DDAB (C) are reported
images of the corresponding ivermectin loaded liposomes are also reported (B,D)
It can therefore be concluded that microfluidic methods are potentially suitable for the preparation of liposomal suspensions to be included in commercial
in order to minimise the content of toxic constituents we demonstrate that ethanol is a suitable substitute for the more toxic isopropanol in microfluidic experiments
particularly in view of a potential translation of this technology into the industrial environment
</a>Data refer to Z-average of liposomes produced by #chip1-MHF (circles)
#chip2-YJ (hexagons) or #chip3-PM (triangles)
Liposomes were constituted of PC/DDAB 9.0−1.0 mM
produced at the indicated FRR and TFR = 37.50 μl/min
Data are reported as the mean of three independent samples
the size of empty (water-filled) liposomes is also reported (open circles)
Liposomes were produced by #chip1-MHF at TFR = 37.50 μl/min and FRR = 30
</a>(A) Geometrical characteristics of easy-to-build chips for liposome production
and potential parallelized network for mass production of liposomes
(C) Schematic of #chip5-MHF-LC and related fabrication method developed in house (μMi-REM)
</a>(A,B) Dependence of liposome mean size (A) and dispersity index (B) on the flow rate ratio (FRR
ranging from 5 to 100) at a fixed TFR of 6 ml/min
(C,D) Dependence of liposome mean size (A) and dispersity index (B) on the total flow rate (TFR
ranging from 3 to 18 ml/min) at a fixed FRR of 100
Liposome size was observed to reduce with increasing TFR (at a fixed FRR of 100)
while increasing FRR resulted in increased liposome size
This finding appears to be contrary to previous results obtained using different microscale geometries and therefore merits further investigations (i.e.
particularly on the fluid dynamic and mass transport phenomena occurring within these scale-up architectures)
these components also present low inter-sample variability which is of particular importance for parallelisation purposes
Despite the potential advantages offered by the proposed microfluidic systems
a more pervasive understanding of the effect of the microfluidic architecture on liposome characteristics may be beneficial to optimise device performance and their scaling-up for mass production of liposomes
This will represent the subject of future investigations
The potential for microfluidic-based technology to produce nanoscale lipid vesicles for medical applications has already been demonstrated
the translation of this method to the industrial environment has been hindered by several limiting factors
we critically review the state-of-the-art microfluidic methods and perform additional experiments in an attempt to address the associated research questions
The following concluding remarks can be drawn from our studies:
(i) A suitable bulk counterpart to MHF microfluidic methods has been identified (referred to as “controlled ethanol injection”)
which makes it possible to carry out a systematic comparison between the two methods
we have demonstrated that liposomes produced using microfluidics were smaller and more uniform in size than the ones produced by controlled injection
whilst no appreciable differences in liposome dimensional stability were found over time (data not shown)
MHF thus has the potential advantage of being able to generate a wider range of mean liposome sizes
whilst maintaining lower size dispersity compared to bulk counterparts
application-specific tuning of liposome size through changes of the hydrodynamic boundary conditions
(ii) Both lipid and ethanol concentration have a significant effect on liposome properties (in both bulk and microfluidic methods)
This is of particular importance when results taken at different FRRs are compared and interpreted
changing FRR not only causes changes in the fluid dynamic field (i.e.
width of the focused stream) but also in the physico-chemical properties of the fluidic environment
(iii) The lipid formulation plays an important role in determining liposome properties
charged lipids generated smaller vesicles compared to uncharged lipids
MHF microfluidic methods were also found to be suitable for producing different liposome formulations
(iv) Microfluidic methods are suitable for producing small and uniform liposomes at relatively high concentrations of lipids and using solvents with relatively low toxicity (i.e.
This has important implications for the potential utility of this technology in the pharmaceutical industry
freezing and thawing) is required with MHF methods to obtain liposomes of desirable characteristics
(v) It is possible to efficiently encapsulate biologically active compounds in liposomes produced using microfluidics
on-chip liposome loading with bioactive compounds (both hydrophilic and lipophilic) has seen a limited number of advancements in recent years
and may represent an exciting avenue of research in the near future
(vi) High-throughput and easy-to-build microfluidic architectures can be constructed for mass production of liposomes
without significantly influencing the quality of the end-product compared to conventional MHF devices
we have demonstrated liposome production at volumetric flow rates of up to 18 ml/min
Highly pure phosphatidylcholine (PC) 90% from soybean was purchased from Phospolipon 90G Lipoid
Germany; and dimethyldioactdecyl-ammoniumbromide (DDAB) from Sigma-Aldrich
1,2-dimyristoyl-sn-slycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (14:0 PEG2000 PE) were purchased from Avanti Polar Lipids; polydimethylsiloxane (PDMS) monomer Sylgard® 184 and curing agent were purchased from Dow Corning (USA); SU-8 photoresist was obtained from Chestech (UK); trichloro (1 H,1 H,2 H,2 H)-perfluorooctylsilane
and suramin sodium salt were purchased from Sigma-Aldrich (UK)
All the other regents and solvents not included in this section were also purchased from Sigma-Aldrich (UK)
The “Cross-flow” #chip1-MHF consists of polydimethylsiloxane (PDMS) and glass layers
and was produced with conventional soft lithography techniques
an SU-8 mould with the designed microchannel pattern
consisting of three inlets and one reaction channel
was prepared following the standard lithography protocols
the mould was covered with a layer of a 10: 1 (w/w) PDMS precursor and curing agent mixture and heated for 1 h at 80 °C for the polymer to cure
The PDMS sheet with the microchannel architecture fabricated on the surface was then removed from the mould and permanently bonded to a glass slide after oxidizing its surface via plasma treatment
The “Snake mixer slide” #chip2-YJ was obtained from Thinxxs
The mixer was integrated on a microscope slide made of cyclic olefin copolymer (COC)
For the fabrication of the “three-inlets pillars” #chip3-PM
a photolithography/wet etching procedure was used
the channel network was designed using AutoCAD drawing software first
A film negative of the desired final size was then prepared by a commercial photo mask producer to form the optical mask
Crown white glass plates (thickness of 1.5 mm) coated with a thin layer of chromium metal mask plus an upper layer of positive photoresist were used for channel network fabrication
the pattern of interconnecting channels was transferred from the negative film to the photoresist layer on the glass which was then developed and removed
to reveal the channel areas of the glass to be etched
The glass plate was baked in an oven at 80 °C overnight to dry and harden the mask on glass
The channels were then etched using 1% hydrofluoric acid buffered with 5% ammonium fluoride solution at 65 °C
under ultrasonic agitation (Ultrasonic Cleaner
the etched glass was thermally bonded (595 °C for 3 h) with a top plate of the same material into which outlet and inlet holes had been previously drilled
the cross-sectional profile of the etched microchannel was measured using a surface profilometer
The ethanol injection method was applied to prepare liposomes
The required quantities of lipids were dissolved in ethanol: PC 90G (40–90 mM)
The resulting organic solution (0.5 ml) was then injected by a syringe pump (KD scientific
New Era pump System) at flow rate of 500 μl/min under magnetic stirring (300 rpm) in an appropriate volume of water (4.5 ml)
Spontaneous liposome formation occurred as soon as the ethanolic solution came into contact with the aqueous phase
The liposome suspension was then kept under stirring by vortexing for 5 minutes at room temperature
The obtained liposomal suspension was stored at 20 °C
Liposomes were prepared by injecting a lipid mixture (PC 90G 90 mM DDAB 10 mM) dissolved in ethanol into the central channel of the microfluidic network of #chip1-MHF; water was injected into two oblique side channels intersecting with the central channel
The flow rate ratio (FRR) is defined here as the ratio between the water volumetric flow rate and the ethanol volumetric flow rate
Liposome formation at different shear forces was investigated by changing the total flow rate (TFR) from 18.75 to 75.00 μl/min
The flow focusing was observed and monitored with a Dino-Eye Eyepiece camera (Dino-lite Digital Microscope)
Liposomes were prepared via a similar procedure to that applied for the #chip1-MHF apart from the fact that the lipid mixture (PC 90 mM and DDAB 10 mM) dissolved in ethanol was injected into one channel of the Y geometry
while water was injected into the other channel
TFR was set between 18.75 and 75.00 μl/min
The preparation of liposomes by the “Three-inlets pillars” chip (#chip3-PM) was analogous to the procedure described for “cross-flow” (#chip1-MHF)
The TFR was varied from 18.75 to 75.00 μl/min and the FRR was varied from 10 to 50
Microchip constituents were obtained from IDEX Health &amp; Science
#chip4-OFF3 components were made from PEEK and specifically designed with a 0.006” thru-hole that delivers a low swept volume and includes F-112 and P-416 fittings
To connect the “off-the-shelf” devices to the syringes
TeflonTM tubes with an inner diameter of 750 μm were employed
USA) were used to control the flow rate of liquids pumped through the devices
Liposomes were again prepared using a similar procedure to that above for #chip1-MHF apart from the fact that the central ethanol stream contained DMPC
Cholesterol and 14:0 PEG2000 PE at a concentration of 10
The TFR was varied from 3 to 18 ml/min and the FRR was varied from 5 to 100
All dimensional analysis of the liposome dispersions were performed using a DLS Zetasizer Nano-ZS (Malvern Instruments
UK) with a backscattering detection angle of 173°
and a 4.0 mW power source was used to report the intensity mean diameter (Z-average) and the dispersity of the liposome formulations
The mean particle size was obtained from the results of three experiments
The size distribution was evaluated in terms of the dispersity index (Đ)
The measurements of vesicle size and dispersity were carried out at 21 °C in water
A 3 mL aliquot of sample solution was pipetted on to plasma-treated (Gatan Solarus Model 950 Advanced Plasma System
time 30 s) carbon copper grids (Quantifoil R 3.5/1) in the environmental chamber of a fully automated vitrification device for plunge freezing (FEI Vibrot)
having relative air humidity of 100% and a temperature of 22 °C
The excess solution was removed by blotting with filter paper for 2 s followed by 1 s draining and plunging of the samples into 1:1 mixture of liquid ethane and liquid propane
Vitrified samples were cryo-transferred into a Jeol JEM3200FSC cryo-TEM operating at −194 °C
The temperature of the samples was −187 °C during image acquisition
The microscope was operated in the bright field mode
using a 300 kV acceleration voltage; the in-column energy filter was set to 0–20 eV energy-loss range (zero-loss imaging)
Micrographs were recorded with a Gatan Ultrascan 4000 CCD camera
The determination of drug entrapment efficiency in liposomes was performed by loading 250 μl of a liposome dispersion into a Sheparose® 4B column (1.0 cm in diameter and 10 cm long; GE Healthcare
The void volume peak fractions containing drug loaded liposomes were collected and quantitated for drug content
UV-VIS measurement (at 253 nm) was conducted by diluting 300 μl of each fraction with 700 μl of ethanol
UV spectra were recorded with a Hewlett-Packard 8452 diode array spectrophotometer
Drug encapsulation efficiency (EE) was calculated as follows:
where C0 and C1 correspond to the total and liposome-associated amount of drug
Liposome production by microfluidics: potential and limiting factors
Preparation of vesicles (liposomes) In Encyclopedia of Nanoscience and Nanotechnology (ed
Development of liposomal formulations: From concept to clinical investigations
Liposomal drug delivery systems: From concept to clinical applications
Effectiveness and Safety of Short Course Liposomal Amphotericin B (AmBisome) as First Line Treatment for Visceral Leishmaniasis in Bangladesh
Eleven years of Inflexal V-a virosomal adjuvanted influenza vaccine
Porcine surfactant (Curosurf) for acute respiratory failure after near-drowning in 12 year old
Clinical development of liposome based drugs: formulation
Liposome Drug Products-Guidance for Industry
Dispersity in polymer science (IUPAC Recommendations 2009)
Functional reconstitution of a voltage-gated potassium channel in giant unilamellar vesicles
e25529; doi: 10.1371/journal.pone.0025529 (2011)
Vesicles of variable sizes produced by a rapid extrusion procedure
Unilamellar vesicle formation and encapsulation by microfluidic jetting
Controllable monodisperse multiple emulsions
Novel method for obtaining homogeneous giant vesicles from a monodisperse water-in-oil emulsion prepared with a microfluidic device
Preparation of cell-encapsulation devices in confined microenvironment
Octanol-assisted liposome assembly on chip
A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array
Microfluidic directed formation of liposomes of controlled size
Microfluidic mixing and the formation of nanoscale lipid vesicles
Microfluidic directed self-assembly of liposome-hydrogel hybrid nanoparticles
Formation of liposome by microfluidic flow focusing and its application in gene delivery
Microfluidic hydrodynamic focusing based synthesis of POPC liposomes for model biological systems
Continuous flow production of cationic liposomes at high lipid concentration in microfluidic devices for gene delivery applications
Microfluidic synthesis of PEG- and folate-conjugated liposomes for one-step formation of targeted stealth nanocarriers
Microfluidic preparation of liposomes to determine particle size influence on cellular uptake mechanisms
High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization
Microfluidic-controlled manufacture of liposomes for the solubilisation of a poorly water soluble drug
Microfluidic and lab-on-a-chip preparation routes for organic nanoparticles and vesicular systems for nanomedicine applications
High-throughput continuous flow production of nanoscale liposomes by microfluidic vertical flow focusing
Controlled self-assembly of monodisperse niosomes by microfluidic hydrodynamic focusing
characterization and applications of liposomes: State of the Art
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Authors would like to thank Dr Xunli Zhang (University of Southampton
UK) for providing microfluidic devices fabricated using photolithography and wet etching
and James Fisk and David Salisbury (Institute of Biomedical Engineering
UK) for fabricating PMMA moulds used in the μMI-REM microfabrication method
Mechatronics and Bioengineering Science research groups
Faculty of Engineering and the Environment
Department of Life Science and Biotechnology
Elisabetta Bottaro &amp; Claudio Nastruzzi
All authors contributed to the writing process of the paper and all reviewed the final manuscript
performed the experiments and prepared some of the figures
designed the experiments and made the remaining figures
The authors declare no competing financial interests
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1: Structure of the guanidinase enzyme of the comammox species Nitrospira inopinata
The presumed entrance to a tunnel leading to the active site is highlighted in the left image
the tunnel is shown as a green line and guanidine as a stick model
The structure was elucidated by the team led by Kristina Djinović-Carugo
C: Kristina Djinović-Carugo/University of Vienna
Research team identifies unconventional energy source for recently discovered &quot;green&quot; nitrifying bacteria
An international research team led by the Centre for Microbiology and Environmental Systems Science (CeMESS) at the University of Vienna has discovered that comammox bacteria
This unique ability opens new avenues for targeted cultivation of these enigmatic microbes and could also provide a key to reducing agricultural nitrous oxide emissions
The research findings were recently published as an article in the prestigious journal Nature
the conversion of ammonia via nitrite to nitrate
is carried out by specialized microorganisms called nitrifiers
This process is extremely important for the global biogeochemical nitrogen cycle in virtually all ecosystems
but it plays an ambivalent role in global change
nitrification contributes to the emission of the potent greenhouse gas and ozone-depleting substance nitrous oxide and leads to massive fertilizer losses in agriculture
resulting in the eutrophication of water bodies
nitrification is indispensable as a biological purification step for nutrient removal in wastewater treatment plants
thus protecting water bodies from excessive nitrogen input from wastewater
The study authors have now found a way that may promote nitrifiers in the environment that emit less nitrous oxide
Comammox bacteria are considered &quot;green&quot; nitrifiers because
they produce only small amounts of nitrous oxide as a byproduct of their metabolism and efficiently remove nitrogen compounds from wastewater in treatment plants
Since the discovery of nitrifiers in the 19th century
it was assumed that these microorganisms could only respire ammonia and urea
the research groups led by Michael Wagner and Holger Daims demonstrated that some nitrifiers could also use the chemically unstable cyanate for their energy metabolism
our team has now shown that comammox bacteria can also grow with the unconventional substrate guanidine,&quot; explains Marton Palatinszky
&quot;The comammox bacteria use a transporter and an enzyme
structurally and functionally characterized in detail by us
which allows them to produce ammonium from guanidine in a highly energy-efficient manner within the cell.&quot;
Guanidine is a metabolic product of microorganisms and plants
Little is known about its role in human and animal metabolism
It is formed in soils during the degradation of synthetic fertilizer additives and in wastewater during the breakdown of the commonly used drug metformin
little is known about the distribution and further processing of guanidine in the environment
including microbiologists from the Helmholtz Centre for Environmental Research in Leipzig; Germany and Aalborg University in Denmark
demonstrated that guanidine is present not only in human urine but also in livestock excreta and that comammox bacteria utilize guanidine in wastewater treatment plants
They also showed that guanidine is metabolized by nitrifiers in agricultural soils
New Opportunities for Cultivation and Nitrous Oxide Reduction
The Vienna microbiologists are now attempting to enrich and isolate the widespread comammox bacteria from environmental samples using guanidine
as only one strain is currently available in pure culture worldwide
&quot;This seems particularly promising as none of the other nitrifier strains we tested could grow with guanidine as the sole energy and nitrogen source,&quot; explains Katharina Kitzinger
The team also wants to investigate whether adding guanidine to agricultural fertilizers could increase the abundance of comammox bacteria in arable soils
thereby reducing agricultural nitrous oxide emissions
&quot;This work would not have been possible without the close collaboration of many researchers involved in the 'Microbiomes Drive Planetary Health' Cluster of Excellence
We extend our sincere thanks to the Austrian Science Fund (FWF) for this special support,&quot; says study leader Michael Wagner
Palatinszky M , Herbold CW, Sedlacek CJ, Pühringer D, Kitzinger K, Giguere AT, Wasmund K, Nielsen PH, Dueholm MKD, Jehmlich N, Gruseck R, Legin A, Kostan J, Krasnici N, Schreiner C, Palmetzhofer J, Hofmann T, Zumstein M, Djinovic-Carugo K, Daims H, Wagner M. Growth of complete ammonia oxidizers on guanidine. Nature.DOI: <a href="https://www.nature.com/articles/s41586-024-07832-z" target="_blank" class="external-link-new-window">10.1038/s41586-024-07832-z</a>
Volume 10 - 2023 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fmolb.2023.1155629">https://doi.org/10.3389/fmolb.2023.1155629</a>
This article is part of the Research TopicWhen Predictions Meet Experiments: The Future of Structure Determination<a class="Link Link--linkType Link--maincolor Link--medium Link--icon Link--chevronRight Link--right" aria-label="View all 6 articles" href="https://www.frontiersin.org/research-topics/46164/when-predictions-meet-experiments-the-future-of-structure-determination/magazine" target="_self" data-event="customLink-linkType-a_viewAll6Articles">View all 6 articles</a>
Protein structure prediction and structural biology have entered a new era with an artificial intelligence-based approach encoded in the AlphaFold2 and the analogous RoseTTAfold methods
More than 200 million structures have been predicted by AlphaFold2 from their primary sequences and the models as well as the approach itself have naturally been examined from different points of view by experimentalists and bioinformaticians
we assessed the degree to which these computational models can provide information on subtle structural details with potential implications for diverse applications in protein engineering and chemical biology and focused the attention on chalcogen bonds formed by disulphide bridges
We found that only 43% of the chalcogen bonds observed in the experimental structures are present in the computational models
suggesting that the accuracy of the computational models is
insufficient to allow the detection of chalcogen bonds
according to the usual stereochemical criteria
High-resolution experimentally derived structures are therefore still necessary when the structure must be investigated in depth based on fine structural aspects
In 2021, sensational progress was made in protein structure prediction with AlphaFold2, the artificial intelligence system developed by DeepMind (<a href="#B17">Jumper et al., 2021</a>). These predictions became freely available in the AlphaFold Protein Structure Database (AlphaFold DB), created by EMBL-EBI, which presently includes more than 200 million predictions (<a href="#B30">Tunyasuvunakool et al., 2021</a>) (<a href="#B31">Varadi et al., 2022</a>)
Here, we seek to assess the degree to which these computational models can provide information on subtle details that may be important in various applications in protein engineering, chemical biology, and biotechnology. As an example, we focus on chalcogen bonds (referred to as ChB according to (<a href="#B1">Aekeroy et al., 2019</a>)) formed by disulphide bridges (<a href="#B1">Aekeroy et al., 2019</a>) (<a href="#B32">Vogel et al., 2019</a>)
This is an interesting test case because chalcogen bonds are not yet parameterized in any molecular mechanics/dynamics force field
their presence cannot be affected by energy minimization protocols
these moderate clashes can be tolerated if compensated by a good and native-like packing around them
involving attractive interactions like hydrogen bonds
(A) Schematic representation of a chalcogen bond; the position of the nucleophilic atom
relative to the sulfur atom is monitored with two variables
and &#x003b1; &#x0003d; 180 &#x02013; &#x003b8;
where &#x003b8; is the angle defined by the nucleophile
and the atom covalently bound to the sulfur
the differences between the distances d observed in the predicted structures (Alpha Fold DB) and in the experimental structures (PDB)
the differences between the angles &#x003b1; observed in the predicted structures (Alpha Fold DB) and in the experimental structures (PDB)
(D) Scatter plot of the Delta-&#x003b1; versus the Delta-d values
Delta-d and Delta-&#x003b1; values are given in &#x000c5; and degrees
ChBs are an interesting test case because they are not yet parameterized in any molecular mechanics/dynamics force field
their presence cannot be favoured by energy minimization protocols
they would be considered inter-atomic clashes and consequently disfavoured
A ChB can be formed by a nucleophilic atom and a chalcogen atom that is covalently bound to another atom (<a href="#F1">Figure 1A</a>). The nucleophilic atom must be positioned along the prolongation of the covalent bond, or along the prolongation of one of the two covalent bonds if the chalcogen is divalent. As a consequence, there are two parameters that must be monitored, the distance d between the nucleophilic and the chalcogen atom and the angle &#x003b1; (<a href="#F1">Figure 1A</a>)
According to the IUPAC recommendations, the distance d must be shorter than the sum S of the van der Waals radii of the nucleophilic and chalcogen atoms (<a href="#B1">Aekeroy et al., 2019</a>), despite the fact that the use of van der Waals radii in determining non-bonding interactions may need to be revised (<a href="#B25">Politzer et al., 2007</a>)
to account for the lower accuracy of macromolecular structures relative to small molecule structures
a in a ChB d must not be larger than S &#x0002b; 0.1&#x000a0;&#x000c5;
which is supplementary to the angle &#x003b8;
In chemical crystallography and material science
a ChB is usually characterized by a value smaller than 20&#x000b0;
to account for the lower accuracy of macromolecular structures
we increased this threshold value to 25&#x000b0;
Similar settings were previously used (<a href="#B10">Carugo, 2023</a>) (<a href="#B8">Carugo, 2023</a>) and can be compared with estimated average positional standard errors of 0.046 &#x000c5;
which imply estimate errors of about 0.06 &#x000c5; on bond distances and of about 2&#x000b0; on bond angles
About one-half (43%) of the ChBs observed in the experimental structures are present in the computational models if the same stereochemical criteria are used
This means that about one-half of the ChBs observed experimentally in high resolution crystal structures are not observed in the models deposited in AlphaFold DB
Does this indicate that these models are wrong
models available in AlphaFold DB are extremely similar to the experimental crystal structures
which are very small (0.046 &#x000b1; 0.002&#x000a0;&#x000c5;) as expected for high-resolution structures
the average absolute value of Delta-d is about three times greater than that calculated on the contacts (shorter than 3.5&#x000a0;&#x000c5;) between main-chain oxygen and nitrogen atoms involving the residues that form ChBs (0.106 &#x000b1; 0.008&#x000a0;&#x000c5;)
ChB predictions are much less accurate than main-chain hydrogen bond predictions
<a href="#F2">Figure 2A</a> shows an example in which the ChB is detected in both the experimental structures and in the computational model. The sulfur-oxygen distance d is even shorter in the AlphaFold DB model and the angle &#x003b1; is even closer to 0&#x000b0; in the AlphaFold model. <a href="#F2">Figure 2B</a> shown an example in which the ChB is detected in the experimental structure and not in the AlphaFold DB model
The local stereochemistry is quite well predicted but the sulfur-oxygen distance is slightly too long (the threshold is sum of the van der Waals radii
1.52&#x000a0;&#x000c5; for oxygen and 1.80&#x000a0;&#x000c5; for sulfur
with and small positive tolerance of 0.1&#x000a0;&#x000c5;
but in terms of chemical interactions is crucial
Comparison between the experimental and computation ChBs in three selected examples
The experimental structure is represented with ball and sticks and the computational model only with sticks
In (A) the ChB is detectable in both structures; in (B)
the ChB is detectable only in the experimental structures since the distance d is slightly too long in the computational model; in (C)
the ChB is detected in the experimental structures and it cannot be detected in the computational model where the disulphide bond is absent&#x02013;this is a region partially unstructured of the protein
carbon light grey in the experimental structure and azure in the AlphaFold DB model
it would be possible to increase the threshold values of d and &#x003b1; that allow to automatically detect ChBs in such a way to increase the number of ChBs in the models of AlphaFold DB
the values of these thresholds strictly depend on the laws of chemistry and physics and nothing indicates here that this is justified
AlphaFold2 is a powerful tool for predicting protein three-dimensional structures
There are only a few cases where the predicted structure is very different from the experimental one. For example, in ten cases with Delta-&#x003b1; &#x0003e; 30&#x000b0;, most of them have large Delta-ds (<a href="#F1">Figure 1D</a>)
the average pLDDT values of the residues bridged by the ChB are &#x0003c;90
indicating that side-chains might not be predicted reliably
suggesting that predictions should be treated with caution
Only three predicted structures (PDB: 3soj
despite Delta-&#x003b1; &#x0003e;30&#x000b0;
<a href="#F2">Figure 2C</a> shows one of the rare examples where AlphaFold DB models seem to be completely inadequate
The local stereochemistry&#x02013;this a partially disordered part of the protein&#x02013;is wrong
the cysteine 4 is misplaced and the disulphide bonds is broken
We conclude that computational models produced with AlphaFold2 and stored in AlphaFold DB are accurate&#x02013;we note that for these proteins a high-resolution crystal structure is available in the PDB
they show contacts between sulphur atoms of disulphide bridges and protein nucleophilic atoms that are comparable to the experimental ones
the accuracy of the computational models is
insufficient to allow the detection of ChBs
The original contributions presented in the study are included in the article/<a href="#SM1">Supplementary Material</a>
further inquiries can be directed to the corresponding authors
OC and KD-C contributed to the conception and design of the study
OC performed the statistical analysis and wrote the first draft of the manuscript
All authors contributed to the article and approved the submitted version
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations
Any product that may be evaluated in this article
or claim that may be made by its manufacturer
is not guaranteed or endorsed by the publisher
The Supplementary Material for this article can be found online at: <a href="https://www.frontiersin.org/articles/10.3389/fmolb.2023.1155629/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fmolb.2023.1155629/full&#x00023;supplementary-material</a>
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Citation: Carugo O and Djinovi&#x00107;-Carugo K (2023) Automated identification of chalcogen bonds in AlphaFold protein structure database files: is it possible?
Received: 31 January 2023; Accepted: 26 June 2023;Published: 06 July 2023
Copyright &#x000a9; 2023 Carugo and Djinovi&#x00107;-Carugo. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY).</a> The use
&#x0002a;Correspondence: Oliviero Carugo, <a id="encmail">b2xpdmllcm8uY2FydWdvQHVuaXZpZS5hYy5hdA==</a> Kristina Djinovi&#x00107;-Carugo, <a id="encmail">a3Jpc3RpbmEuZGppbm92aWNAdW5pdmllLmFjLmF0</a>
Nearly a third of renal cell carcinoma patients develop metastatic disease
oncologists have struggled to understand why.Credit: Rasi Bhadramani/ Getty Images 
Most patients with renal cell carcinoma (RCC)
with slow-growing tumours that typically respond to treatment
But nearly a third will develop aggressive metastatic disease
from which the likelihood of surviving five years past diagnosis can be as low as 15%
Clinicians have struggled to understand why
New research from a team led by oncologist Giannicola Genovese and postdoctoral fellow Luigi Perelli at The University of Texas MD Anderson Cancer Center
in collaboration with Alessandro Carugo at IRBM in Italy
now reveals the series of genetic changes that make relatively indolent tumours malignant (Perelli
The Genovese lab develops experimental systems for reconstructing the natural history of cancer
“We genetically engineer the key drivers of the disease in experimental model systems — mostly the mouse — and then follow tumour evolution,” says Genovese
“We’re trying to understand how tumours become aggressive
the team engineered mouse models in which a subset of kidney cells were genetically manipulated to include various mutations with an established link to aggressive RCC
One change proved particularly problematic
the deletion of a section of mouse chromosome 4 that contains two genes that regulate cell division gave rise to extremely aggressive cancers
Genovese and colleagues determined that this disruption creates conditions that lead to the acquisition of further genomic damage
cells found in metastatic growths routinely lost a sizable segment of a second mouse chromosome containing genes that allow cells to respond to immune signaling molecules known as interferons
Genovese and Perelli believe this deletion may make tumour cells resistant to the immune response
allowing the cancer to progress more rapidly
the researchers were able to map all the changes they observed in mice back to equivalent chromosomal disruptions in patient-derived RCC tumours
“It parallels with the human data,” says Perelli
This highlights the relevance of a well-designed model for studying human cancers
and Genovese’s team is now looking into opportunities to apply their findings to improve care for RCC patients
checkpoint inhibitor drugs that dramatically boost the anti-tumour immune response can be highly effective against RCC
but less than half of patients respond meaningfully to this treatment
The group is looking into how the disruption of the interferon response observed in this study might influence the success or durability of checkpoint inhibitor treatment
They also see opportunities to use insights from this mouse model to guide how clinicians approach early-stage tumours based on the presence or absence of genomic features associated with metastasis
“This will be very important for teasing out patients who should be treated less aggressively right now
and to identify those who may need additional treatment.”
This same approach to modelling tumour evolution could also illuminate distinctive roads to progression followed by other tumour types
and Genovese says his team is now looking at the extent to which these routes overlap or diverge in pancreatic cancer as well as rarer forms of kidney cancer
“We're working with clinicians to model those tumour types and understand if there are vulnerabilities that can be exploited.”
To read the full paper in Nature Cancer, <a href="https://www.nature.com/articles/s43018-023-00584-1" data-track="click" data-label="https://www.nature.com/articles/s43018-023-00584-1" data-track-category="body text link">click here</a>. 
Volume 3 - 2023 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fruro.2023.1335414">https://doi.org/10.3389/fruro.2023.1335414</a>
This article is part of the Research TopicModelling the Impact of Medical Devices in the Urinary Tract: From Bench to Bedside<a class="Link Link--linkType Link--maincolor Link--medium Link--icon Link--chevronRight Link--right" aria-label="View all 4 articles" href="https://www.frontiersin.org/research-topics/50255/modelling-the-impact-of-medical-devices-in-the-urinary-tract-from-bench-to-bedside/magazine" target="_self" data-event="customLink-linkType-a_viewAll4Articles">View all 4 articles</a>
Ureteral stents are hollow tubes that are inserted into the ureter to maintain the flow of urine from the kidney to the bladder
the use of these indwelling stents is associated with potential complications
an organized consortium of bacterial species embedded within a self-producing extracellular matrix
can attach to the outer and inner surfaces of ureteral stents
encrustation - defined as the buildup of mineral deposits on the stent surface - can occur independently or in parallel with biofilm formation
Both phenomena can cause stent obstruction
which can lead to obstructive pyelonephritis and make stent removal difficult
Understanding the influence of flow on the development of biofilm and encrustation and the impact of small mechanical environmental changes (e.g.
wall shear stress distribution) is key to improve the long-term performance of stents
Identifying the optimal stent properties to prevent early bacterial attachment and/or crystal deposition and their growth
would represent a breakthrough in reducing biofilm-/encrustation-associated complications
This review identifies the most prevalent bacterial strains and crystal types associated with ureteral stents
and the process of their association with the stent surface
which often depends on patient comorbidities
we focus on the often-overlooked role of fluid dynamics on biofilm and encrustation development in ureteral stents
from micro- to macro-scale) with the aim of providing a knowledge base to inform the development of safer and more effective ureteral stents
We refer to encrustation as the build-up of mineral deposits/crystals
on the surface of ureteral stents or within the matrix of a biofilm
translating computational and experimental findings into actual stent improvements is a challenging endeavor that is often ignored or underestimated
This comprehensive review aims to introduce and discuss the methods used to identify
and manage biofilm and encrustation in ureteral stents
It first describes the bacterial species involved in biofilm formation
and the interplay between biofilm and encrustation
It subsequently discusses ways to prevent or minimize the growth of biofilms on ureteral stents that are specifically optimized based on fluid dynamics
These include methods based on computer simulations and experimental tests to spatially resolve relevant flow metrics
particularly WSS at different dimensional scales
it identifies a range of WSS values acting on the stent surface and elucidates the impact of WSS on biofilm formation and crystal deposition
This finding underlies the necessity to explore new techniques to prevent the conditioning film from forming in the first place
in addition to preventing bacterial attachment to the stent material
While numerous factors contribute to biofilm development, bacteria remain the primary cause of its formation (a summary on biofilm formation in ureteral stents is provided in <a href="#box1">Box 1</a>). Biofilms often comprise a combination of bacterial species, which compete and/or cooperate to form multi-species biofilms (<a href="#B51">51</a>)
To effectively combat biofilm formation in ureteral stents
it is crucial to identify and understand the specific bacterial species that thrive in this environment
Summary of biofilm formation in ureteral stents
&#x2022; Ureteral stents are susceptible to biofilm development
&#x2022; Conditioning film forms from proteins and polysaccharides on stent surfaces
&#x2022; Bacteria adhere to stents and the conditioning film
&#x2022; Bacteria use flagella for strong catch bonds to resist shear forces
&#x2022; EPS matrix provides protection and nutrients to bacteria
&#x2022; Biofilms are usually flat under high-shear stress and thick under low-shear stress
Influence of bacterial presence on crystal types in ureteral stents
&#x2022; Crystal type on ureteral stents can vary based on the bacterial presence
calcium oxalate monohydrate crystals are the main crystals in ureteral stents
struvite and hydroxyapatite crystals predominate
&#x2022; Certain bacteria accelerate stent encrustation
these studies highlight the significance of the interplay between bacteria and crystal deposition in the development of encrustation
Bacteria-induced encrustation forms more rapidly and&#xa0;is often thicker
Since bacteria survive within the encrusting material
effective removal of encrusting fragments is key to preventing recurrence
Furthermore, while some antimicrobial coatings can reduce biofilm formation on polymeric stents, thus enhancing their durability, the effectiveness of these coatings can be compromised by the conditioning film and subsequent crystal deposition (<a href="#B57">57</a>). Since crystals have a higher affinity to various components of the conditioning film than to the stent material itself (<a href="#B39">39</a>)
it is crucial to address the problem of crystal deposition in parallel with biofilm reduction
Detection and analysis of bacterial colonization and encrustation in ureteral stents
&#x2022; Sterile urine cultures may underestimate the bacterial colonization on stents
&#x2022; Stent culture after vortexing and sonication is more effective than standard urine culture
&#x2022; Techniques like 16S rRNA sequencing
and SEM improve understanding of stent microbiomes
&#x2022; SEM with EDX is commonly used to identify crystal types in stents
and &#xb5;CT are used to quantify stent encrustation
Besides identifying the crystal type, some studies also used SEM as a qualitative method to estimate the extent of encrustation on different sections of ureteral stents. An in vitro study performed by Cauda et&#xa0;al. (<a href="#B76">76</a>) used this technique to compare different stent materials
qualitatively identifying those developing less encrustation
and the percentage of the analyzed ureteral stents associated with the presence of bacteria (colonization rate)
and research challenges in ureteral stent studies
&#x2022; Escherichia coli and Enterococcus faecalis
and Pseudomonas aeruginosa are also frequently found on ureteral stents
&#x2022; Longer indwelling time increases microbial colonization of stents
&#x2022; Bacterial load and encrustation are normally low under six weeks
&#x2022; The high variability of the research methods used in the field
complicates the Interpretation of the results and the drawing of clear conclusions
Table&#xa0;1 Characteristics of ureteral stents and associated bacterial colonization
it emerges that Escherichia coli is consistently identified as the predominant bacterial species found in ureteral stents retrieved from patients
are also identified in most of these studies
Staphylococcus epidermis is the most prevalent one
more than five bacterial species were found in most studies
the identification of a &#x2018;safe indwelling time&#x2019; is challenging
due to the independent nature of most studies
and variations in patients&#x2019; conditions and comorbidities
the integration of obstruction monitoring methods with quantification techniques holds the potential to determine the appropriate time for stent removal and potentially identify regions of the stent that are most problematic
Ureteral stents can exhibit different encrustation and biofilm patterns depending on the specific segment of the stent (a summary of the distribution patterns is provided in <a href="#box5">Box 5</a>)
A stent can be divided into its two pigtails (located in the renal pelvis and the bladder
respectively) and a straight central segment
This straight part can be subdivided into three regions: i) proximal section (near the ureteropelvic junction or UPJ)
ii) middle section (within the middle ureter)
and iii) distal section (near the ureterovesical junction or UVJ)
Location matters in ureteral stent bacterial colonization and encrustation
&#x2022; Microbial colonization is more pronounced in the distal and proximal sections of ureteral stents
&#x2022; Encrustations are typically more visible in the proximal section
&#x2022; Colonization and encrustation patterns can vary based on the patient type
&#x2022; Future consideration should be given to developing patient-specific stents
which is in disagreement with most other studies in this area
the possibility of developing patient-specific stents should be considered in the future
future scientific efforts should focus on combining information on stent sections that are more prone to develop encrustation (also based on patient-specific conditions) with the analysis of local fluid dynamics in these regions
and WSS ranges observed on both the internal and external walls of the stent and side holes
Table&#xa0;2 Summary of study parameters and wall shear stress value ranges in ureteral stents
Firstly, microfluidic Stent-on-Chip (SOC) models (<a href="#B32">32</a>) were employed to quantify the WSS distribution in a ureteral stent
and the effect of side hole shape and stent wall thickness on the WSS field
these values should be interpreted cautiously as the intrinsic simplifications of microfluidic models prevent them from reproducing the full complexity of the stented ureter (e.g.
In an attempt to provide more physiological WSS values, Mosayyebi et&#xa0;al. (<a href="#B33">33</a>) used CFD to compute WSS in a tapered stented ureter that mimicked the ureter of a pig, and identified variations in WSS levels depending on the longitudinal position along the ureter. They also evaluated WSS acting over the inner wall of side holes, as shown in <a href="#T2">Table&#xa0;2</a>
the ureteropelvic junction (UPJ) had a larger diameter than the ureterovesical junction (UVJ)
This configuration resulted in elevated WSS levels within the mid and distal sections
A comprehensive CFD and particle image velocimetry study by Zheng et&#xa0;al. (<a href="#B2">2</a>) employed a full-scale model to assess WSS distribution over the inner walls of side holes
the ureter was modeled as an unobstructed tube with a constant diameter
The mean WSS was higher in the first and last side holes of the straight part of the stent
while the side holes in the middle section exhibited lower WSS
These differences may be attributed to the passive nature of side holes in the middle section
since there is no significant flow exchange through these holes (&#x2018;passive&#x2019; side holes)
whereas the first and last side holes promote such flow exchange (&#x2018;active&#x2019; side holes)
Their findings also indicated that smaller side holes experienced significantly greater WSS levels
providing a potential means to mitigate encrustation and biofilm formation in these regions of the stent
due to the correlation between these phenomena and WSS
Vogt et&#xa0;al. (<a href="#B31">31</a>) conducted a small-scale CFD simulations on DJ ureteral stents
emphasizing the importance of utilizing smooth stent designs
since grooves and imperfections on the stent surface lead to stagnation zones (with low WSS levels) which can act as traps for crystals and promote encrustation
corresponding to a wide range of fluid shear forces
Wall shear stress values across various ureteral stent locations
&#x2022; Stent design affects shear stress and encrustation patterns
&#x2003;o Proximal side holes show very low wall shear stress (WSS) below 0.001 Pa
&#x2003;o Distal side holes have higher WSS
&#x2003;o WSS varies from 10-6 Pa to 0.19 Pa on the stent&#x2019;s internal wall
accurate WSS measurements in stented ureters depend on various factors and the study design
none of the studies reviewed have considered the impact of reflux
which substantially influence WSS values and add complexities to a model
To better understand the true impact of WSS on ureteral stents
future research must diligently consider and incorporate these factors into their models (whether in vitro and/or in silico)
At the micro-scale, the relationship between WSS and bacterial interactions with surfaces has been extensively explored in various domains (<a href="#B115">115</a>) (see Section 4). However, this critical association remains largely unexplored in the context of ureteral stents. De Grazia et&#xa0;al. (<a href="#B34">34</a>) conducted experimental investigations using a microfluidic SOC approach
were the primary sites of bacterial attachment
followed by side holes and the intra-luminal surface
This study marked the first attempt to examine bacterial attachment on ureteral stent architectures
The study revealed a correlation between bacteria coverage area
Pseudomonas fluorescens was used as a bacterial model
which is not a highly prevalent bacterium in the urinary tract and may limit the clinical relevance of the study and its generalization
streamlined) side holes could reduce particle deposits in stents as a result of increased WSS levels
Besides the SOC study, Mosayyebi et&#xa0;al. (<a href="#B33">33</a>) also investigated the relationship between WSS and crystal deposition at the macroscale level
Their study stands out as the only macroscale model investigating this relationship
It was observed that the accumulation of particles was more pronounced in the side holes situated in the proximal region of the stent compared to those in the distal region
as expected by the higher shear stress levels in the former
their full-scale CFD study showed that during the voiding stage
the average WSS at side holes located in the distal section is larger than in the middle and proximal sections
could be perfused with both artificial urine and urinary bacteria
Significance of wall shear stress in bacterial attachment and encrustation in ureteral stents
&#x2022; Experimental studies showed that areas with low WSS (below 0.04 Pa) are primary sites for bacterial attachment
&#x2022; Experimental studies showed that particle deposition is more common in areas with lower WSS
thinner walls and streamlined side holes) influences shear stress and encrustation patterns
&#x2022; Particle accumulation is higher in proximal side holes (WSS &lt; 0.001 Pa) compared to distal ones (WSS &gt; 0.01 Pa)
&#x2022; Experimental studies showed that vesicoureteral reflux (VUR) reduces encrustation
&#x2022; There is a lack of comprehensive studies integrating flow dynamics with biofilm formation and encrustation
Valuable insights into bacterial attachment and wall shear stress from related research
&#x2022; Despite limited experimental studies directly linking bacterial attachment and WSS in the urinary tract
valuable insights are derived from other research areas
&#x2022; Microfluidics is used to explore how shear stress affects biofilm formation
&#x2022; Experimental studies showed that bacterial colonization decreases with WSS &gt; 0.02 Pa
&#x2022; Different bacteria can exhibit different attachment rates depending on the level of WSS
the development of fluid-mechanical-based stent designs
maximizing WSS to reduce bacterial attachment
<a href="#T3">Table&#xa0;3</a> provides an overview of microfluidic studies that have established a correlation between WSS levels (within the range of values observed in ureteral stents) and the behavior of bacteria that are prevalent in the urinary tract
The studies were identified and selected using the Scopus database with specific keywords (&#x201c;*&#x201d; AND (&#x201c;shear stress&#x201d; OR &#x201c;biofilm formation&#x201d; OR &#x201c;biofilm growth&#x201d;) AND (&#x201c;microfluidics&#x201d; OR &#x201c;microfluidic&#x201d; OR &#x201c;micro-fluidic&#x201d; OR &#x201c;flow chamber&#x201d;)
where * represent all the bacteria selected above)
Table&#xa0;3 The impact of wall shear stress on biofilm formation in microfluidic experiments
coli to bladder T24 transitional cells and type IV collagen reaches its highest level under minimal shear stress (0.01 Pa) conditions
even when subjected to higher shear stress (0.59 Pa)
coli cells remain attached after binding to host cells or collagen
though detachment rates escalate with the magnitude of shear stress
the reported WSS in the distal section of the ureteral stent spans from 10-2 to 10-1 Pa
those in the proximal section range from 10-5 to 10-4 Pa
Considering several reports suggesting the complete suppression or substantial reduction of biofilm thickness beyond 0.02 Pa
this becomes a possible reason for the observed phenomenon
a flawless stent design has not yet been achieved
and none of the existing stents can completely prevent the formation of biofilms or encrustation on their surface
Advanced materials and coatings: Continued exploration of novel materials and coatings could provide solutions to reduce encrustation and biofilm formation
Researchers might focus on materials that minimize friction during placement and removal while maximizing long-term performance
Biofilm and encrustation control: Exploring innovative strategies
such as targeted antimicrobial/encrustation therapies or biofilm/encrustation-disrupting techniques
could offer new avenues to prevent stent-related complications
Patient-specific and practice-driven design: For short-term stenting (less than two weeks)
clinicians may prefer ureteral stents with better cost-effectiveness due to the small likelihood of encrustation and biofilm formation
a special focus on the current stent designs is crucial since small changes already proved beneficial in reducing attachment
Special care should be taken in the pigtail sections
Micro- and macro-scale research areas are also presented
followed by a description of the WSS ranges within ureteral stents
Innovative stents: New strategies and technologies are emerging
such as smart stents that incorporate sensors enabling real-time monitoring of urinary tract conditions
This could allow for early detection of complications and timely interventions
Multidisciplinary approach: Encouraging collaboration between diverse disciplines
will be essential to tackle the challenges associated with stent complications
Regulatory Guidelines: Collaboration between researchers and regulatory bodies could lead to the development of standardized guidelines for stent testing
Writing&#xa0;&#x2013;&#xa0;review &amp; editing
The author(s) declare financial support was received for the research
The work is supported by the Swiss National Science Foundation (SNSF
DC is co-inventor on patent application PCT/GB2018/052522 &#x201c;Stent with streamlined side holes&#x201d;
The author(s) declared that they were an editorial board member of Frontiers
This had no impact on the peer review process and the final decision
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Received: 09 November 2023; Accepted: 19 December 2023;Published: 16 January 2024
Copyright &#xa9; 2024 Amado, Zheng, Lange, Carugo, Waters, Obrist, Burkhard and Clavica. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY)</a>
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Takotsubo syndrome (TTS) is a transient heart disease that has been historically related to the occurrence of psychological (emotional) factors (“broken heart” syndrome). We aimed to conduct a systematic review analyzing the role of psychological factors in TTS.
All studies on TTS and psychological factors from January 1991 through April 2019 were scrutinized according to the Cochrane Collaboration and the PRISMA statements. Selected studies were additionally evaluated for the Risk of Bias according to the Newcastle-Ottawa Scale (NOS).
Fifteen case-control studies (by Mayo Clinic criteria) were finally selected. Most studies analyzed stressful life-events or trauma, although with conflicting findings, while a likely role of long-lasting psychological distress seemed to be a homogenous result. Among life-time psychopathology, only anxiety appeared to have a significant role. Some studies outlined a likely role of personality, but findings are conflicting.
Our findings do not lead to any definitive assumption on the specific role of psychological factors in TTS, also for scant strong methodology of the most part of the studies. More studies with stronger research methodology are needed to better characterize psychological elements in TTS.
Volume 10 - 2019 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fpsyg.2019.02742">https://doi.org/10.3389/fpsyg.2019.02742</a>
Objective: Takotsubo syndrome (TTS) is a transient heart disease that has been historically related to the occurrence of psychological (emotional) factors (&#x201C;broken heart&#x201D; syndrome)
We aimed to conduct a systematic review analyzing the role of psychological factors in TTS
Methods: All studies on TTS and psychological factors from January 1991 through April 2019 were scrutinized according to the Cochrane Collaboration and the PRISMA statements
Selected studies were additionally evaluated for the Risk of Bias according to the Newcastle-Ottawa Scale (NOS)
Results: Fifteen case-control studies (by Mayo Clinic criteria) were finally selected
Most studies analyzed stressful life-events or trauma
while a likely role of long-lasting psychological distress seemed to be a homogenous result
only anxiety appeared to have a significant role
Some studies outlined a likely role of personality
Conclusion: Our findings do not lead to any definitive assumption on the specific role of psychological factors in TTS
also for scant strong methodology of the most part of the studies
More studies with stronger research methodology are needed to better characterize psychological elements in TTS
Takotsubo syndrome (TTS) is a form of transient heart failure syndrome often mimicking acute myocardial infarction. It is also known as stress cardiomyopathy, broken heart syndrome, or apical ballooning syndrome, and was firstly described in 1990 (<a href="#B15">Dote et al., 1991</a>), even if some Authors dated it earlier (<a href="#B55">Wittstein, 2008</a>)
no systematic reviews have been realized analyzing the likely causative link between psychological factors and TTS
with the existing reviews detecting other factors (e.g.
the role of drugs or pathophysiologic mechanisms)
The objective of the present study was to systematically review all the studies on psychological factors (as antecedents) (traumatic/stressful events
psychopathology and personality) in patients with TTS diagnosis in order to understand their likely role in this syndrome
We conducted a systematic review of the literature on psychological factors (psychopathologic disorders
stressful life-events/psychological trauma
All observational studies were included in the review by the ascertainment of a case-control study design
The search was limited to English-written publications
and to the period from January 1991 to April 2019
An additional analysis of the reference list in each selected paper was also performed
&#x2022; Studies with an analytical study design as defined by <a href="#B24">Grimes and Schulz (2002)</a> (i.e.
an observational study with a comparison or control group Both retrospective and perspective studies have been included to consider the highest number of studies
&#x2022; Diagnosis of TTS by the Mayo Clinic criteria or by the new TTS criteria (<a href="#B40">Parodi et al., 2014</a>; <a href="#B34">Lyon et al., 2016</a>)
&#x2022; Studies adopting standardized and validated tests
&#x2022; Studies written in English language
&#x2022; Studies with intra-group control (e.g.
&#x2022; Pharmacological and behavioral intervention trials
&#x2022; Number of subjects per group &#x2264; 5
Study selection was performed by two independent reviewers with research expertise in clinical psychology and cardiology (FG and FB) who assessed the relevance of the study for the objectives of this review
This first round of selection was based on the title
If the reviewers did not reach a consensus or the abstract did not contain sufficient information the full text was reviewed
In the second phase (screening), full-text reports were evaluated to detect whether the studies met the inclusion criteria (<a href="#F1">Figure 1</a>)
PRISMA flow diagram of literature search and selection of publications
all full-texts were retrieved and a final check was made to exclude papers not responding to inclusion/exclusion criteria
and reaching the final consensus to decide the final number of studies to be selected
A standardized data extraction form was prepared; data were independently extracted by two of the authors (FG and FB) and inserted in a study database. A process of discussion/consensus moderated by a third reviewer (SC) (<a href="#B20">Furlan et al., 2009</a>) resolved discrepancies between reviewers
For variables expressed as median and 25th&#x2013;75th percentile it was not possible to calculate Cohen&#x2019;s d
Quality assessment of each of the included studies was evaluated following the Newcastle-Ottawa Scale (NOS) for case-control studies on a 9-star model (<a href="#B53">Wells et al., 2014</a>)
Studies scoring above the median NOS value were considered as high quality (low risk of bias) and those scoring below the median value were considered as low quality (high risk of bias)
two reviewers (FB and FG) independently extracted relevant information and data from all eligible reports that met the above inclusion criteria
We found 15 studies meeting inclusion criteria (<a href="#F1">Figure 1</a>), for a total of 2581 subjects (1152 TTS patients; 1069 other heart diseases; 360 healthy controls). The description of the samples, psychological variables, study design, psychometric scales (tests, interview or retrospective medical records analysis), key findings, Cohen&#x2019;s d or Odd ratios and 95% confidence intervals of selected studies are reported in <a href="#T1">Table 1</a>
Of interest, the time elapsing between the cardiac event and the psychological assessment was extremely variable, ranging from hours (<a href="#B13">Delmas et al., 2013</a>) to years (<a href="#B23">Goh et al., 2016</a>)
History of long-lasting psychological distress not temporally related to the cardiac events was evidenced in four studies (<a href="#B13">Delmas et al., 2013</a>; <a href="#B29">Kastaun et al., 2014</a>; <a href="#B31">Lacey et al., 2014</a>; <a href="#B44">Rosman et al., 2017</a>)
Some studies differentiated emotional (27&#x2013;38%) from physical (36&#x2013;50%) triggers (<a href="#B11">Compare et al., 2014</a>; <a href="#B49">Templin et al., 2015</a>; <a href="#B23">Goh et al., 2016</a>; <a href="#B44">Rosman et al., 2017</a>)
with 12&#x2013;28% of patients with indeterminable reasons
acute myocardial infarction with emotion triggers vs
TTS without emotion trigger and found a significant prevalence only in the two groups with emotion triggers
Other studies (<a href="#B11">Compare et al., 2014</a>; <a href="#B29">Kastaun et al., 2014</a>) did not find any role for psychopathology in the history of TTS patients
Half of the studies reflected the median value (&#x03BC; = 5), four were above it and three below (<a href="#T2">Table 2</a>). Four studies were quoted as high quality (low risk of bias) by NOS (see <a href="#T2">Table 2</a>)
Ottawa-Newcastle risk of bias for case-control studies
Although the role of psychological factors has been extensively studied in TTS
only fifteen studies fulfilled the criteria to perform a systematic review
because of the small number of selected studies and the heterogeneous methodology used for the psychological assessment (no studies shared the same psychological assessment tools)
Most studies attempted to understand if stressful events (or trauma) could have a role (trigger) in TTS, but findings are conflicting. As suggested by the recent Expert Consensus Document on TTS (<a href="#B28">Jelena-Rima et al., 2018</a>)
one of the key questions to answer is which role triggering factors have in the stress response of the heart
the etymology of &#x201C;stress&#x201D; cardiomyopathy requires specific attention for the role of psychological stressor as possible etiological factor
our review does not allow any conclusion by this side
The first point that warrants attention is the difference between studies drawing data from standardized psychometric tools or from retrospective assessment of medical records
none allowed any conclusion toward a role for psychological trigger events in comparison to other cardiac events (control group)
all studies that draw data from medical records (usually based on clinical interview at admission) evidenced a role for psychological trigger events compared to controls
this opens both to methodological and clinical considerations
the use of standardized measures of assessment would lead to strongest conclusions
but in a direction making questionable the evidence of a role of psychological factors closely involved in the etiology of TTS
homogenous clinical observations by medical records suggest implementing further case-control studies to support the role of psychological triggers in TTS
The unanswered question is why a person develops TTS and another one other disorders
Not finding any differences between TTS and myocardial infarction as regards to psychological triggers may delineate at some level the involvement of similar mechanisms
Furthermore, homogeneous (albeit still limited) findings are driven from the analysis of studies evidencing a role for long-lasting psychological distress. A role for early traumatic psychological experience has been evidenced as predisposing factor for patients with cardiovascular diseases (<a href="#B51">Thurston et al., 2014</a>, <a href="#B50">2017</a>; <a href="#B4">Bomhof-Roordink et al., 2015</a>; <a href="#B54">Winning et al., 2016</a>)
and deserves more attention in future studies
This finding may help to explain why only some patients experience a stressful event as trigger of TTS
the comorbid occurrence of anxiety and depression should be considered as non-specific of TTS
A final note on the unique study (<a href="#B45">Saffari et al., 2017</a>) evidencing worsening of the quality of life and sexuality in TTS
there is the impossibility to make a meta-analysis
the small number of studies and the differences in the psychometric tools
timing of observation and different design among different studies did not allow to pursuit the initial aim
A or D personality) not totally supported by strong evidence (not included in DSM 5)
so that any sound conclusion on this topic is not allowed and further research need to be addressed on this topic
the rigor of methodology we relied on (Cochrane Collaboration and the PRISMA statements) allowed to get strong results and conclusion
on the basis of our systematic review we cannot evidence a clear-cut role for psychological trauma preceding TTS onset
but a possible role of long-lasting emotional distress
we can suggest a role for life-time anxiety disorders (more than depression)
but studies are needed to clarify if differences exist with other cardiac events
we cannot conclude in the direction of specific patterns differentiating TTS from other cardiac disorders
We need studies with stronger methodology addressing the involvement of emotional events by structured interviews conducted shortly after the onset of TTS
The timing of interviewing patients should be carefully delineated (no more than 6 months) to avoid recall bias
multicentre studies are warranted to recruit a large number of patients and increase sample size for this relatively rare entity
The choice of an adequate control groups needs attention
because one of the main questions is whether TTS actually differs from other cardiac disorders
as regards to personality and comorbid psychopathologic disorders
Finally, we stress the importance of a multidisciplinary approach to TTS; such an approach should involve a collaborative process between cardiologists and clinical psychologists from the diagnosis to treatment. Evidence are accumulating on the efficacy of psychological interventions for cardiac diseases (<a href="#B43">Richards et al., 2018</a>)
and SC contributed to the conception and design of the study
FG organized the database and wrote the first draft of the manuscript
FG and FB made the bibliographic research and selected papers for the systematic review (in case of doubt made confirmation with SC)
FB and SC read and approved the submitted version of the manuscript
All authors contributed to the manuscript revision
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Copyright &#x00A9; 2019 Galli, Bursi and Carugo. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY)</a>
*Correspondence: Federica Galli, <a id="encmail">ZmVkZXJpY2EuZ2FsbGkxQHVuaW1pLml0</a>
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is a cell surface protein involved in homotypic cell–cell adhesion via intercellular oligomerization and proliferative signalling via proteolytic cleavage
Despite its use as a diagnostic marker and being a drug target
structural details of this conserved vertebrate-exclusive protein remain unknown
Here we present the crystal structure of a heart-shaped dimer of the extracellular part of human EpCAM
The structure represents a cis-dimer that would form at cell surfaces and may provide the necessary structural foundation for the proposed EpCAM intercellular trans-tetramerization mediated by a membrane-distal region
we show how proteolytic processing at various sites could influence structural integrity
oligomeric state and associated functionality of the molecule
We also describe the epitopes of this therapeutically important protein and explain the antigenicity of its regions
the outlined aspects of EpCAM biology are far from being well-defined
especially from the structural point of view
since to date no representative structure was available to interpret the experimental observations
we present the structure of a non-glycosylated mutant form of EpEX (EpEXΔ) at 1.86-Å resolution
a first and exemplary structure of the unique GA733 protein family
EpEXΔ forms a cis-dimer corresponding to a half of the proposed trans-tetrameric intercellular unit
We reveal the cis-dimerization interface between the two subunits involving the thyroglobulin type-1A (TY) domain and thereby describe a novel role of the versatile TY protein module
EpCAM cis-dimer could be additionally stabilized by interaction of the TM helices of the two cis-oriented subunits as we explore by molecular dynamics (MD) simulations
We show that the lateral protein surfaces of the wt EpCAM cis-dimer are partially covered with glycan chains as inferred from the position of known N-glycosylation sites
The structure explains the high antigenicity of the surface-exposed small amino-terminal domain that harbours conformational epitopes of the majority of anti-EpCAM antibodies
We provide a model of the trans-tetrameric intercellular unit where the trans-interactions involve the glycan-free membrane-distal platform-like surface of EpEXΔ
we comment on the design of efficient EpCAM-targeted binder molecules and explain the detrimental effect of CTE-causing mutations on EpCAM structure and function
It lacks the TM and intracellular part (EpIC) of full-length EpCAM
it contains three N-glycosylation-abolishing mutations (indicated by black lines)
(b) Cartoon representation of the complete EpEXΔ chain contained in the asymmetric unit shown in two different orientations
Secondary structure elements are labelled as they appear along the polypeptide chain from N to C terminus (for example
The three glutamine residues (N-glycosylation abolishing mutations) are shown as black sticks
and all disulphide bridges are shown as sticks
The N-terminal region tethered to the βA sheet is coloured in pale cyan
Zoomed-in part shows contact between ND (green) and its copy from the adjacent asymmetric unit (grey)
Detailed view (top) of TY-loop residues (dark purple) interacting with residues of βC sheet (grey
side chains within 4 Å of TY loop shown in dark pink)
The Arg80 and Arg81 (dark teal sticks) interact with Asp194
Gln74 and Gln111 (black sticks) introduced by glycosylation-abolishing mutations within TY correspond to glycosylated Asn residues in wt EpEX
The three disulphide bonds of TY are shown as sticks
the 6 and 28 kDa fragments (ND plus α1 and the rest of TY plus CD
respectively) remain connected by the Cys66-Cys99 bond
the C-terminal region of EpEXΔ (from Met261 to Lys265 plus His266) is not shown
The top membrane-distal part of the molecule formed by RCD and ND is free of glycan chains
the same fragment pattern could be generated using cathepsin K
which also cleaves at the Gly79-Arg80 site
such downward orientation would bring the C-terminal regions of the two subunits into close proximity
it is plausible to assume that also the TM part (single helix per chain) forms a dimer with potential to additionally stabilize the EpCAM cis-dimer
To investigate the dimerization propensity of the EpCAM TM helix
we performed coarse-grained (CG) MD simulations of two modelled EpCAM TM helices embedded in a lipid bilayer
The two EpCAM TM helices were allowed to diffuse freely within the bilayer patch from their initial positions separated by 50 Å
</a>(a) Interhelical distance calculated from six CG MD simulations started from the same initial state
(b) Interhelical crossing angle distribution in combined simulation runs 1 and 3–6 (red)
For combined runs only those parts where the two helices formed a dimer were used for calculation
(c) Representative CG dimer model TM helices of EpCAM calculated by MD simulation
Backbones of the two helices are shown as light and dark gray sticks
isoleucine and valine residues are shown as blue
Labels follow residue numbering in full-length EpCAM
which are known modifiers of adhesion and signalling functions of TM molecules
which is formed by the ND and the α2 helix plus RCD of the CD
may represent the environment-sensing part of EpCAM while the lateral parts may be involved in interactions with other (trans)membrane proteins on the surface of the same cell
the RCD and possibly the nearby α2 are proposed to be directly involved in EpCAM trans-tetramerization as described in our paper
The role of all these cleavages and the exact mechanism by which they occur remains to be elucidated
This latter mutation could impair formation of EpCAM cis-dimers or even prevent normal folding of EpCAM and adoption of the compact overall structure
</a>The identified epitope regions and the most antigenic N-domain are depicted as intensively shaded surface regions
Listed below are other anti-EpEX antibodies for which the exact epitope is unknown
Antibody names are color-coded by the domains they recognize (ND
17-1A and MT201 are targeted by Catumaxomab
no adhesive function has been reported to date
It is important that the similarities/differences are taken into account when developing diagnostic/prognostic and therapeutic approaches and great care is needed when analysing the results with regard to both EpCAM and Trop2
We prepared two EpEX variants—wt EpEX (residues 24-265 of EpCAM) and the non-glycosylated mutant variant (EpEXΔ)
both with an uncleavable His6-tag at the C terminus
the region of EpCAM cDNA (clone ID IRAKp961G0321Q; Source BioScience imaGenes) coding for Gln24-Lys265 was amplified by PCR
In case of EpEXΔ three mutations were introduced: Asn74Gln
5′- and 3′-ends of the DNA fragment were extended by PCR to introduce the N-terminal melittin signal peptide (MKFLVNVALVFMVVYISYIYA) and a C-terminal His6-tag
The resulting fragment was cloned into pFastBac1
which was in turn used to prepare recombinant baculoviruses using the Bac-to-Bac system (Invitrogen)
Expression was performed using suspension cultures of Spodoptera frugiperda Sf9 cells at multiplicity of infection of 5
The recombinant protein was collected from the medium 36 h post infection by dialysis and subsequent immobilized metal affinity chromatography using Ni2+-charged HisTrap columns (GE Healthcare)
The peak eluate fractions were pooled and EDTA (pH 8.0) was added to a final concentration of 5 mM
Following dialysis and ion-exchange chromatography on Mono Q column (GE Healthcare)
the final buffer exchange was done by size-exclusion chromatography on a Superdex 200 column (GE Healthcare) equilibrated in 20 mM Na-HEPES pH 8.0
The peak fraction was pooled and concentrated using Amicon centrifugal filter unit with 10 kDa cutoff membrane (Millipore)
EpEX was prepared using the same procedure; however, the ion-exchange chromatography step was omitted. This recombinant EpEX variant is heterogeneously glycosylated as revealed by the presence of multiple bands on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel, all at higher Mr than the EpEXΔ variant (<a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/ncomms5764#MOESM758">Supplementary Fig. 1a</a>)
The interhelical distance was defined as the distance between centres of the two helices
The interhelical crossing angle was defined as the angle between vectors describing the two helices; vector of each helix was drawn between centres of two groups of backbone atoms (Ala270-Val273 and Ala281-Val284)
EpEX and EpEXΔ were incubated with human recombinant cathepsins L
a HEPES buffer with pH 7.2 and an additional low-pH buffer were used (sodium acetate pH 5.5 or Bis-Tris pH 6.0)
All buffers were 0.1 M and contained 2 mM EDTA and 2 mM DTT
Cathepsin/EpEX(Δ) molar ratio was 10−2 or 10−3
Reaction mixtures were incubated at 37 °C for 1 h
Reactions were stopped by adding reducing SDS–PAGE sample buffer followed by boiling
Fragments were visualized on 15% SDS–PAGE by Coomassie staining
Cleavage site was determined by N-terminal amino-acid sequencing (Edman degradation) of electrophoretically separated fragments transferred to a polyvinylidene difluoride membrane
Crosslinking of intact or trypsin-cleaved EpEXΔ was performed in PBS by treating the protein with 1.3 mM BS3 (Pierce) at 21 °C for 30 min
The reaction was quenched by adding Tris buffer (pH 7.4) to a final concentration of 50 mM followed by 30 min incubation at 21 °C
Reaction mixtures were analysed on 15% SDS–PAGE under non-reducing and reducing conditions
Protein bands were visualized by Coomassie staining
The N-terminal pyroGlu residue was changed to Gln because modified residues are not generally accepted in docking simulations
Histidine protonation states and flexible segments were defined automatically and centre of mass restraint was used to enforce contact between the molecules
C2 point symmetry restraint was imposed between the two dimers (AB–CD)
An additional calculation was performed where C2 point symmetry restrains between subunits in each subunit pair (A–C
B–D) were used to limit calculation results to tetrameric units with perpendicular C2 rotation axes
Docking solutions were first ranked according to their HADDOCK and Z-scores
the latter indicating separation of a cluster of solutions from the average in terms of score (better solutions have more negative Z-scores)
and further ranked according to the buried surface area and contacts between ND
In the models from the best docking cluster (Z=−1.2)
the relative angle between the two cis-dimers was 30°
while in the models from second (Z=−0.9) and third best clusters (Z=0.1)
the relative angle was 150° (ND’s already in contact) and 75°
the best model from additional symmetry-restrained docking run was used
Accession codes: Coordinates and structural factors have been deposited in the Protein Data Bank under accession code <a href="http://www.pdb.org/pdb/search/structidSearch.do?structureId=4MZV">4MZV</a>
Crystal structure and its bearing towards an understanding of key biological functions of EpCAM
Efficient selection of human tumor growth-inhibiting monoclonal antibodies
Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon
CD marker expression profiles of human embryonic stem cells and their neural derivatives
reveal a novel CD marker for exclusion of pluripotent stem cells
Characterization of epithelial cell adhesion molecule as a surface marker on undifferentiated human embryonic stem cells
Epithelial cell adhesion molecule-targeted drug delivery for cancer therapy
immunogenicity and bioactivity of the therapeutic antibody catumaxomab intraperitoneally administered to cancer patients
Absence of cell-surface EpCAM in congenital tufting enteropathy
Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule
Evidence for a role of the epithelial glycoprotein 40 (Ep-CAM) in epithelial cell-cell adhesion
The structural analysis of adhesions mediated by Ep-CAM
Oligomeric state of the colon carcinoma-associated glycoprotein GA733-2 (Ep-CAM/EGP40) and its role in GA733-mediated homotypic cell-cell adhesion
Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions
Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule
Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins
Expression of Ep-CAM shifts the state of cadherin-mediated adhesions from strong to weak
The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7
EpCAM contributes to formation of functional tight junction in the intestinal epithelium by recruiting claudin proteins
Epithelial cell adhesion molecule (EpCAM) regulates claudin dynamics and tight junctions
Nuclear signalling by tumour-associated antigen EpCAM
Initial activation of EpCAM cleavage via cell-to-cell contact
Determination of disulphide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A
Solution structure of mouse Cripto CFC domain and its inactive variant Trp107Ala
a new tool for fast protein structure alignment in three dimensions
WW domain-containing proteins: retrospectives and the future
Characterization of the type-1 repeat from thyroglobulin
a cysteine-rich module found in proteins from different families
Structure of the RNA binding domain of a DEAD-box helicase bound to its ribosomal RNA target reveals a novel mode of recognition by an RNA recognition motif
Structure of the mature ectodomain of the human receptor-type protein-tyrosine phosphatase IA-2
crystallization and preliminary X-ray characterization of the human epithelial cell-adhesion molecule ectodomain
Inference of macromolecular assemblies from crystalline state
Glycosylation is crucial for stability of tumour and cancer stem cell antigen EpCAM
Biosynthesis and glycosylation of the carcinoma-associated antigen recognized by monoclonal antibody KS1/4
Biochemical analysis of a human epithelial surface antigen: differential cell expression and processing
Isolation and characterization of a cDNA encoding the KS1/4 epithelial carcinoma marker
Biochemical and immunological characterization of the human carcinoma-associated antigen MH 99/KS 1/4
Helix-helix interactions in membrane proteins: coarse-grained simulations of glycophorin a helix dimerization
Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L
Dual concentration-dependent activity of thyroglobulin type-1 domain of testican: specific inhibitor and substrate of cathepsin L
Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins
Diversity and evolution of the thyroglobulin type-1 domain superfamily
and tetraspanins promotes colorectal cancer progression
In vivo imaging of extracellular pH using 1H MRSI
Cysteine cathepsins and the cutting edge of cancer invasion
Tumor-specific glycosylation of the carcinoma-associated epithelial cell adhesion molecule EpCAM in head and neck carcinomas
Crystal structure of ICAM-2 reveals a distinctive integrin recognition surface
C-cadherin ectodomain structure and implications for cell adhesion mechanisms
Urinary EpCAM in urothelial bladder cancer patients: characterisation and evaluation of biomarker potential
Regulated intramembrane proteolysis and degradation of murine epithelial cell adhesion molecule mEpCAM
Crystal structure of an active form of BACE1
an enzyme responsible for amyloid beta protein production
EpCAM proteolysis: new fragments with distinct functions
Identification of EpCAM as the gene for congenital tufting enteropathy
Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics
Trop2: a possible therapeutic target for late stage epithelial carcinomas
Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling
PHENIX: a comprehensive Python-based system for macromolecular structure solution
Substructure search procedures for macromolecular structures
structure refinement and density modification with the PHENIX AutoBuild wizard
Automated protein model building combined with iterative structure refinement
‘Conditional Restraints’: restraining the free atoms in ARP/wARP
Refinement of macromolecular structures by the maximum-likelihood method
Coot: model-building tools for molecular graphics
MolProbity: all-atom structure validation for macromolecular crystallography
GlyProt: in silico glycosylation of proteins
Knowledge-based protein secondary structure assignment
MultiSeq: unifying sequence and structure data for evolutionary analysis
The MARTINI force field: coarse grained model for biomolecular simulations
GROMACS 4: algorithms for highly efficient
The HADDOCK web server for data-driven biomolecular docking
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supported by the national GRID Initiatives of Belgium
the Netherlands (via the Dutch BiG Grid project)
Taiwan and the Latin America GRID infrastructure via the Gisela project is acknowledged for the use of web portals
This work was supported by the Slovenian Research Agency (grant J1-2017)
Faculty of Chemistry and Chemical Technology
Kristina Djinović-Carugo &amp; Brigita Lenarčič
Department of Structural and Computational Biology
analysed the results and wrote the manuscript
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We compared the topologies of protein and small molecule crystals
which have many common features – both are molecular crystals with intermolecular interactions much weaker than intramolecular interactions
They also have different features – a considerably large fraction of the volume of protein crystals is occupied by liquid water while no room is available to other molecules in small molecule crystals
We analyzed the overall and local topology and performed multilevel topological analyses (with the software package ToposPro) of carefully selected high quality sets of protein and small molecule crystal structures
Given the suboptimal packing of protein crystals
which is due the special shape and size of proteins
it would be reasonable to expect that the topology of protein crystals is different from the topology of small molecule crystals
we discovered that these two types of crystalline compounds have strikingly similar topologies
This might suggest that molecular crystal formations share symmetry rules independent of molecular dimension
This was explained by the model of deformable molecules
in contrast to the model of rigid ones used by Kitaigorodskii
We discarded structures containing nucleic acids and structures with an average B-factor smaller than 10 Å2 or larger than 40 Å2
We considered only structures with 50–500 amino acids in the asymmetric unit
We discarded structures deposited without their experimental diffraction data
Redundancy was reduced to 30% sequence identity
Structures with missing protein atoms or with protein atoms deposited with zero occupancy were discarded as well as the structures where non-protein and non-water atoms are more than 5% of the total number of atoms
resolution and R-factor thresholds ensure that unreliable structures are excluded from the analyzed data sets; moreover
we considered a homogeneous set containing only low temperature crystal structures
since packing might depend on temperature; furthermore
limitations on the average B-factor and on protein dimension ensure that anomalous structures – much larger or smaller or much more or less flexible than customary proteins – are removed from the data sets; and eventually
redundancy reduction to 30% sequence identity
It is also important to observe that the exclusion of structures containing too many (more than 5%) hetero-atoms ensures that crystal contacts due to the presence of co-crystallized small molecules (the so called packing bridges) are minimized
where some of the protein atoms/residues were undetected
ensures that no protein-protein crystal packing contacts are neglected
We prepared three ensembles of protein crystal structures
we collected only monomeric proteins that crystallized with only one molecule per asymmetric unit (monomer set; 394 structures)
we grouped dimeric proteins that crystallized with only one dimer per asymmetric unit (dimer set; 207 structures)
we pulled together structures of monomeric proteins with two molecules in the asymmetric unit (double set; 164 structures)
The identification codes of all these crystal structures are listed in the Supplementary Information (Table <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-017-12699-4#MOESM1">S1</a>)
Crystallographic data for organic molecular crystals consisting of chemically equivalent molecules were taken from the CSD (release 5.38)
We have not analyzed molecular crystals of coordination or organometallic compounds
which have different chemical nature compared to proteins
which contain one independent molecule and 10,240 structures with two independent chemically equivalent molecules in the asymmetric unit
or disordered structures as well as those with Rf &gt; 10% were excluded; no other restrictions were applied
The crystal contacts were identified as described previously<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 13" title="Carugo, O. &amp; Djinović-Carugo, K. How many packing contacts are observed in protein crystals? J. Struct. Biol. &#xA;                           180, 96–100 (2012)." href="/articles/s41598-017-12699-4#ref-CR13" id="ref-link-section-d427961216e652">13</a>
The asymmetric unit was transformed according to all symmetry operations and translated up to three times along the axes a
and c in both negative and positive directions
is symmetry related to the first – were considered to be in contact if at least one atom of one of them is closer than 4.5 Å from an atom of the other one
We refer to this method below as ‘Distance’ method
The ‘Domains’ method is designed to process large samples of crystal structures in an automated mode
this method was never used for proteins; thus
it was important to check if this method gives similar results for proteins as the ‘Distance’ method
combined from the faces of their Voronoi polyhedra
which correspond to the intermolecular contacts
This method was also used to compute coordination numbers of some protein molecules for comparison with the simple geometrical approach described above
its center of mass and centers of mass of four neighboring molecules (pink balls)
which represent a fragment of the underlying net
where N is the series of coordination numbers for all independent nodes
indicating the dimensionality of the net (C
respectively) and n is the number of the net with a given ND sequence in the TTD collection
the 16T4 symbol means a three-periodic net with 16-coordinated nodes (i.e
any molecule has 16 neighbors in the packing) and it is the fourth 16-coordinated net in the TTD collection
Since the strength of intermolecular contacts is quite different
the topology of a molecular packing strongly depends on which contacts are taken into account
Ignoring interactions of a particular level of strength
we generate different underlying nets for the same crystal structure
Each underlying net specifies the packing topology at a given level of interaction and corresponds to a structure representation
hcp topology; (b) 10 coordinated underlying net
bct topology; (c) 8 coordinated underlying net
hex topology; (d) 6 coordinated underlying net
hxl topology; (e) 4 coordinated underlying net
connected at different levels of interactions are in yellow
strengths of crystal packing contacts were estimated based on their dissociation free energies as described below
which were generated by applying all the symmetry operations to the asymmetric unit and by applying three translations along all the axes in the negative and in the positive directions
Intermolecular contacts were defined as pairs of atoms
one in the reference molecule and the other in a satellite molecule
which are determined by the ‘Domains’ method
Distances between the molecular centers of mass are given in Å
Thus protein crystals follow the close packing topologies but with some gaps
which are obviously filled by the solvent molecules and decrease coordination numbers of protein molecules
It is interesting to observe that while few topologies are extremely more frequent than others in small molecule crystals
30% of the small molecule crystals are associated with the most frequent topology (bcu-x) and 65% of them have one of the four most common topologies (bcu-x
only 6% of the protein crystals are associated with the most frequent topology (fcu) and only 19% of them have one of the four most common topologies (fcu
This suggests that the suboptimal packing of the protein molecules in the crystal state allows a wider number of topologies and none of them can be much more frequent than the others
</a>Distribution of the coordination numbers (%) for all the structures of proteins examined (see full data in Table <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-017-12699-4#MOESM1">S2</a>) compared to the CN for small molecules
we see that the most frequent coordination number of small molecules (14) is observed in a large fraction of crystals (52%) while the most frequent coordination number for proteins (7 or 8) is observed only in a smaller fraction of crystals (about 20%)
small molecules can adopt 25 different coordination number values (from 4 to 32) while proteins can have only 14 different coordination number values (ranging from 1 to 16 – coordination numbers 2 and 15 are never observed)
All these observations point out that proteins
which are suboptimally packed in their crystals
can hardly by surrounded by numerous other proteins and that the palette of their coordination numbers is considerably more limited
This means that from 5 to 26% of protein molecules essentially differ from small molecules by their ability to be packed
In order to reach a better understanding of the differences between the topologies of protein and small molecule crystals
we performed the multilevel analysis for the molecular packings in 394 monomeric protein structures and 105,549 structures of small molecules
1,799 and 1,144,539 structure representations were generated
</a>Distribution of the underlying nets observed in the multilevel topological analysis of the proteins and small molecule crystals ranked by CN and scaled to 100 on the most frequent in each set (dia for proteins and layers sql for small molecules)
</a>Most typical underlying motifs in packings of protein and small organic molecules
protein crystals nucleation occurs at very high levels of supersaturation
often two or three orders of magnitude greater than that required to sustain crystal growth
proteins may assume several distinctive solid states that include amorphous precipitates
Further studies are necessary and additional data must be considered to find out the rationale why topology seems to be independent of packing efficiency and crystallization
Die Chemismus in der thierischen Organisation
Criteria to extract high quality Protein Data Bank subsets for structure users
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Krissinel for his advices on the use of PISA and on the interpretation of the results
thanks Kristina Djinović for her kind hospitality in Vienna
are grateful to the Russian Government (Grant 14.B25.31.0005) for support
Samara Center for Theoretical Materials Science (SCTMS)
School of Materials Science and Engineering
The authors declare that they have no competing interests
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DOI: https://doi.org/10.1038/s41598-017-12699-4
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What would a Kim Dolce &amp; Gabbana show be like
prepping for the Kim Dolce &amp; Gabbana show.
We&#x27;re doing fittings and everything&#x27;s kind of crazy
I&#x27;ve been spending a lot of time over the years
I have a really big Dolce &amp; Gabbana archive.
So when they saw a lot of the looks that I had,
And we just started having these conversations
with this like retrospective of the last 25 years
And then when Kourtney got married at their home,
it kind of reignited a whole new love and friendship.
We all have such different style and personalities.
So to see everyone wear Dolce &amp; Gabbana
There&#x27;s something just extra sensual about Italian style
by the &#x27;90s supermodels and early 2000s.
And to get every single one of those looks reimagined
I didn&#x27;t really understand that each look
to express what we&#x27;re trying to express.
that there&#x27;s a science behind it all.
I have, I think every single piece in the show.
Just on a regular day in Milan wearing this?
This whole process has been so eye opening
I wore these bottoms to Kourtney and Travis&#x27;s wedding.
you cannot beat Dolce &amp; Gabbana early &#x27;90s,
Some of my favorites is this little bolero
And we had it remade with all of the crystal,
which I feel like was a little bit more me.
And then of course we have all of the original corsets.
it came in, I think three different colors of crystals.
Who doesn&#x27;t remember Gisele wearing this bra.
and all I wanted to do was save up for this little bra.
I think crystals are definitely making a comeback
Seeing their relationship together is so inspiring.
and that&#x27;s always what I&#x27;ve been about.
I signed your praises earlier so I was like...
So thank you Vogue for coming on this journey with us.
And I can&#x27;t wait for you to see the show.
Kristina Djinovic-Carugo (bottom left) and her team at the Center for Optimized Structural Studies (COSS)
who have successfully extended their funding as a "Laura Bassi Centre of Excellence"
boosting structural research in Vienna with 1 million euro until 2016
colored by its electrostatic potential (red representing negative charges and blue representing positive charges)
Utl.: &quot;Laura Bassi Centre of Excellence&quot; of Kristina Djinovic-Carugo extended for another three years