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Above the downstairs room containing sewing machines
The industrial kite designer lives and works in the medieval city of Utrecht
there is a relentless obsession he seems compelled to follow in many areas of his life
I’m working on something to do with flying objects,” he says
Bryan grew up in a small village where he felt there was nothing to do
The one thing he did find was power kiting
“My father was really into flying trick kites and some power kites,” he explains
Bryan eventually moved from his small home village to Utrecht
which meant kitesurfing became more accessible now he was closer to the sea
“I really loved the hang-time because you got the feeling of flying
I didn’t know we could fly in the Netherlands and I hadn’t seen anyone soaring
so this was the most realistic way to fly in my mind.”  
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as a non-invasive and easily accessible biofluid
has been shown to contain RNA biomarkers for prediction and diagnosis of several diseases
systematic analysis done by our group identified two problematic issues not coherently described before: (1) most of the isolated RNA originates from the oral microbiome and (2) the amount of isolated human RNA is comparatively low
The degree of bacterial contamination showed ratios up to 1:900,000
so that only about one out of 900,000 RNA copies was of human origin
but the RNA quality (average RIN 6.7 + /− 0.8) allowed for qRT-PCR
Using 12 saliva samples from healthy donors
we modified the methodology to (1) select only human RNA during cDNA synthesis by aiming at the poly(A)+-tail and (2) introduced a pre-amplification of human RNA before qRT-PCR
the manufacturer’s criteria for successful pre-amplification (Ct values ≤ 35 for unamplified cDNA) had to be replaced by (3) proofing linear pre-amplification for each gene
increasing the number of evaluable samples up to 70.6%
When considering theses three modifications unbiased gene expression analysis on human salivary RNA can be performed
As a more robust but—due to the high background—less sensitive alternative
we examined whether whole saliva could be used
The great majority of studies dealing with disease biomarkers based on gene expression have been performed using blood or tissue that requires collection by a trained person
the drawbacks of saliva samples (low RNA yield and high levels of non-human RNA) could be mitigated with methodologic improvements in the laboratory approach
We recently became interested in isolating RNA from saliva
Contradictory results indicated two major challenges:
Overwhelming bacterial contamination of the samples
Low abundance of human RNA compared with blood
we qualitatively and quantitatively describe these two challenges and how we sought to overcome them
A consistent method for performing gene expression analysis in saliva and dealing with these limitations has never been coherently described before and this work may improve efforts of others in trying to extract RNA in a consistent and well documented way
we focussed on mRNAs which have shown to be promising biomarkers in other types of body tissues and fluids
In order to process human whole saliva to gene expression analysis, we modified several aspects of manufacturer recommendations in the workflow of RNA isolation, cDNA synthesis and Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) in specific ways as described in Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41598-020-67825-6#Tab1">1</a>
We introduced (1) a cDNA synthesis of only human RNA species
(2) a pre-amplification step and (3) its linearity control
we isolated whole blood total RNA to compare quality and quantity of extracted RNA to that extracted from human saliva
The experimental design was granted by the Ethics committee (Bayerische Landesärztekammer
RNA quantity/quality and TaqMan qRT-PCR) were performed according to the standard operating procedures implemented in our laboratory in 2008 when the Bundeswehr Institute of Radiobiology became DIN-accredited by TÜV Süd München
Whole saliva samples were collected using 4 ml ORAgeneRNA (RE-100) vial collection kits from DNA Genotek according to the manufacturer’s instructions (DNA Genotek Inc.
The kit is an all-in-one system for the collection
stabilization and transportation of RNA from saliva
Unstimulated whole saliva was collected from twelve healthy donors (5 females
The self-collection kits are provided with an RNA stabilizing solution to prevent degradation of RNA by RNases
Two ml of saliva was expectorated into the provided vial and mixed with 2 ml stabilizing solution in the lid
The whole saliva was collected from 9 to 10 am and was preserved in the OrageneRNA Self-Collection Kits after having been shaken vigorously
Samples were stored at room temperature overnight and placed in a freezer (− 20 °C or lower) for storage within 1 day
donors were not allowed to eat or smoke 2 h prior to collection or to drink at least 1 h prior to collection
whole blood samples (2.5 ml) from six of the twelve healthy donors were drawn into PAXgene Blood RNA tubes (Qiagen
Germany) at the Bundeswehr Institute of Radiobiology on the day of saliva collection
kept at room temperature overnight and then stored at − 20 °C
All samples were anonymized and obtained with informed consent from all donors
Sampling methods were carried out in accordance with the institutional guidelines and regulations
Whole saliva samples were processed following a combination of the ORAgene RNA purification protocol and the mirVana kit protocol (Invitrogen
the frozen (− 20 °C) ORAgene RNA collection tubes were thawed at room temperature
The first steps were performed according to the ORAgene RNA purification protocol<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 25" title="Oragene ® • RNA purification protocol using the Qiagen RNeasy Micro Kit for volumes up to 1 , 000 µL. 3–5 (2012)." href="/articles/s41598-020-67825-6#ref-CR25" id="ref-link-section-d456251102e760">25</a>: after vigorous shaking
the samples were heated in a 50 °C water bath for one hour in order to homogenize the solution
After taking aliquots of 500 µl (5–6 aliquots per sample) and incubating them at 90 °C for 15 min
samples were cooled to room temperature and 20 µl ORAgene neutralizer solution (1/25 of total volume) was added to each aliquot as buffer and to precipitate the nucleic acids
After incubation on ice for 10 min and centrifugation at 13,000g (rcf
the cell-free clear supernatant was pipetted to a fresh Eppendorf tube
while the separated pellet containing the turbid impurities was discarded
At this step we diverged from the ORAgene RNA purification protocol by skipping the precipitation step with Ethanol and adopted the mirVana kit protocol<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 26" title="Life Technologies. mirVanaTM miRNA Isolation Kit. 33 (2011)." href="/articles/s41598-020-67825-6#ref-CR26" id="ref-link-section-d456251102e772">26</a>
We added the Lysis/Binding Solution from the mirVana kit
Then total RNA including small RNA species was isolated by combining a Phenol–Chloroform RNA precipitation with further processing and purification using a silica membrane
After several washing procedures to purify RNA from other residual debris
DNA ingredients became digested on the membrane (RNAse free DNAse Set
RNA was eluted with 100 µl RNAse free water in a collection tube and the aliquots were pooled for each sample before freezing at − 20 °C
and the tubes were stored until quantitative and qualitative analysis
The PAXGene tubes containing the whole blood (n = 6) were thawed
washed and centrifuged according to the PAXgene Blood RNA system protocol (BD Diagnostics
Cells in the supernatant were lysed (Proteinase K; BD Diagnostics
then the Lysis/Binding Solution was added and further steps were performed according to mirVana kit protocol described above
Quantity of isolated total RNA from saliva and blood was measured spectrophotometrically using NanoDrop One Microvolume UV–Vis spectrophotometer (NanoDrop
The results were displayed in ng/µl along with ratio absorbance value at 260 and 280 nm
The ratio of absorbance at 260 and 280 nm (260/280) was used to assess the purity of RNA in the sample
RNA integrity/quality was assessed by the 2100 Agilent Bioanalyzer (Life Science Group
Germany) and DNA contamination was controlled by conventional PCR using a β-actin primer
Taking known quantitative and qualitative baselines from RNA specimens isolated from whole blood
a ratio of A260/A280 ≥ 2.0 (Nanodrop) and a RNA integrity number (RIN) ≥ 6 would indicate adequate quality of the saliva samples for qRT-PCR analysis
Every single strand of isolated RNA was converted into cDNA via reverse transcription using random primers
The amount of RNA input was always determined to 1 µg
The calculated sample input volume was diluted in RNase free water until a final volume of 50 µl was obtained
Total RNA was reverse transcribed in 100 µl of total reaction mixture containing 10 µl of 10 × RT buffer
4 µl of 10 mM deoxynucleotide triphosphate (dNTP)
Each RNA sample was reverse transcribed using a two-step PCR protocol: 2 h and 15 min in a cycle of 25 °C for 10 min
37 °C for 2 h and 85 °C for 5 min followed by infinite hold at 8 °C
Because of the presence of specific abundant transcripts
the synthesized cDNA was serially diluted using a buffer II (KCl
Three different cDNA dilutions were prepared out of the stock cDNA solution: 10 ng/10 µl
The concentration with the least amount of cDNA (0.01 ng/10 µl) was used for qRT-PCR amplification of 16S and 18S
The diluted cDNA samples from the High Capacity Kits were stored in − 20 °C until qRT-PCR
The quantity of synthesized cDNA was measured fluorometrically via Qubit 2.0 Fluorometer (Invitrogen
The SuperScript III first-strand synthesis system was newly introduced to synthesize cDNA from the isolated salivary RNA in order to subsequently perform gene expression analysis of human origin
The SuperScript III reverse transcriptase catalyzed reaction utilizes Oligo (dt)20 primers which selectively target and amplify the 3′ poly(A) + tail of the vast majority of eukaryotic messenger RNA (mRNA) and thereby is specific for human RNA only
pan-bacterial RNA will be excluded from further processing
This decreased non-human RNA amplification bias afterwards which is common in the presence of RNAs of non-human origin
According to the kit description, the amount of starting material can vary from 1 pg to 5 μg of total RNA and the maximum input volume of RNA is 8 µl<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 28" title="RevTsc, T. SuperScript III Reverse Transcriptase (200U/µL) #18080–085. Manual 1–4 (2004)." href="/articles/s41598-020-67825-6#ref-CR28" id="ref-link-section-d456251102e819">28</a>
In order to increase the amount of input for downstream gene expression analysis
our samples were steamed at 45 °C for 90 min followed by elution with 30 µl of RNase free water
Using the concentration from repeated NanoDrop measurements
the amount of RNA input was defined as 0.5 µg
conforming to the maximum input volume of 8 µl
Each reverse transcription reaction contained 1 µl of Annealing Buffer
2 µl of SuperScript III/RNaseOUT Enzyme mix and RNase/DNase free water (depending on the existing sample volume to obtain a total of 20 µl)
The ingredients were processed in a 0.2 ml PCR tube according to the manufacturer’s instructions in a two-step PCR
the tubes were cooled on ice and stored at − 20 °C until subsequent pre-amplification
the reactions were performed twice for each sample and later combined into one tube prior to pre-amplification and qRT-PCR
The pre-amplification product was then used for qRT-PCR
using the corresponding TaqMan Gene Expression Assays
To ensure linearity of the pre-amplification step
the thermal cycling was set to 10 and 14 cycles for each sample in order to show linearity and
a Ct value of 32 before pre-amplification would result in expected Ct values of 22 and 18 after 10 and 14 cycles of pre-amplification
two kinds of cDNAs were utilized for qRT-PCR: for human (18S rRNA
Hs99999901_g1) and pan-bacterial (16S rRNA
cDNA with a low concentration such as 0.01 ng/10 µl from High-capacity cDNA reverse transcription kit was used
whereas for the other primer probe designs (CDKN1A
SuperScript III First-Strand Synthesis SuperMix synthesized
human cDNA with 10 × and 14 × as well as without pre-amplification was used for detection of each of these low abundant genes in each sample (10 ng/10 µl)
The qRT-PCR reaction contained TaqMan Universal PCR Master Mix and one of the inventoried TaqMan Gene Expression Assays for separate detection of candidate transcripts
Using a 96-well-forat TaqMan qRT-PCR platform
Germany) and transferred into the ABI PRISM 7900HT sequence detection system
All technical procedures for accurate qRT-PCR were performed in accordance with standard operating procedures implemented in our laboratory in 2008 when the Bundeswehr Institute of Radiobiology became certified according to DIN EN ISO 9001/2008
All chemicals using TaqMan chemistry were provided by Life Technologies
the 18S/16S rRNA ratio was calculated between raw Ct values of 18S and 16S to compare the quantitative relation of human RNA to bacterial RNA
The fold change was determined using the formula: fold change = 2^ (Ct 18S rRNA – Ct 16S rRNA)
the observed and expected fold difference of 18S rRNA between 14 and 10 × pre-amplification as well as 14 ×/10 × and unamplified SuperScript III First-Strand Synthesis SuperMix synthesized cDNA was assessed to evaluate a linear and unbiased pre-amplification among the different samples and genes
The correspondence of the manufacturer’s prediction of a successful pre-amplification (&lt; 35 Ct-values for genes before amplification) and our measurements (proofing the linear amplification using unamplified Ct-values and two amplification rounds) was examined for each gene and comparison using a two-by-two contingency table comprising:
TP (manufacturer predicts success and proof of linear amplification was demonstrated)
TN (manufacturer predicts failure and no linear amplification was examined)
but no linear amplification was examined) and
FP in percent (FPx100/(TP + TN + FP + FN))
FN in percent (FNx100/(TP + TN + FP + FN))
that 100% of reactions worked in our measurements when the manufacturer says it should work (according to a Ct value without pre-amplification &lt; 35)
that 100% of reactions didn’t work in our measurements when the manufacturer says it shouldn’t work (according to a Ct value without pre-amplification &gt; 35) resulting in an agreement
in which the pre-amplification should work according to the manufacturer
but our findings (proof of linear pre-amplification) provide insufficient measurements and indicate disagreement
in which the pre-amplification shouldn’t work according to the manufacturer
but our findings provide reliable measurements
Excel 2010 (Microsoft) was used for descriptive statistics (n, mean, standard error of mean, standard deviation, min, max) and generating the tables. SigmaPlot Version 14 (Systat Software, Inc., San Jose California USA, <a href="https://www.systatsoftware.com">https://www.systatsoftware.com</a>) was used for graphical presentations and comparisons among groups (p-values with parametric t-tests or non-parametric tests
</a>Box plots show the amounts of total RNA (human and bacterial) in µg isolated from whole saliva (2 ml
Quality of isolated RNA is shown using RNA integrity numbers (RIN) for saliva samples and blood samples (B)
Gel-like images created by the Agilent bioanalyzer display bands of 28S and 18S rRNA for eight randomly selected saliva samples and are shown as an inserted picture in (B)
</a>The box plots display the 18S rRNA raw Ct-values for both whole saliva (n = 12) and whole blood (n = 6) samples
the input amount for cDNA synthesis for each sample was 1 µg and the amount of cDNA input for qRT-PCR was 20 ng—regardless of whether saliva or blood was used
amplification plots for 18S rRNA are shown for one saliva sample and the corresponding blood sample as an inserted figure
Droplines (vertical dashed lines) provide the corresponding cycles required for the fluorescent signal to cross the threshold (Ct values)
Ct values for detection of human 18S rRNA show a Ct-difference of about 10 Ct-values corresponding to 2^ 10 ~ 1,000-fold differences in RNA copy numbers
similar quantities of total RNA input for saliva and blood RNA samples resulted in about 549 times lower human 18S rRNA copy numbers in saliva compared to 18S rRNA measurements in whole blood
These findings led us to the following questions:
Was there significant bacterial contamination
which was not statistically different from each other
This observation does not imply inhibition of cDNA synthesis
PCR efficiency was close to 100% and the slopes were not statistically significantly different for 18S Ct values below 35 (mean 3.52
SD + /− 0.015 for saliva compared to mean 3.58
18S rRNA raw Ct-values differed for saliva and blood by about 8 Ct values
But this shift was similar for all dilutions with 18S rRNA Ct values below 35
which means that linearity between blood and saliva persists in a range below Ct values of about 35
curves tend to reach a saturation point and linearity disappears because amounts of input for qRT-PCR are too low
These results clearly suggest that there is no inhibition of qRT-PCR when using human salivary material
These results quantitatively show a high bacterial contamination in saliva samples
</a>The figure depicts three exemplary amplification plots (unamplified
10 × and 14 × pre-amplified) from one saliva sample (sample ID 7) and one gene (CDKN1A) created with the ABI PRISM 7900HT sequence detection system
The amplification plot (A) represents the unamplified sample
the one marked with (B) the 10 × pre-amplified sample and the one with (C) the 14 × pre-amplified sample
Linearity of 10 × pre-amplification is shown as a ΔCt of 10 in horizontal solid line 1 (Ct (no-Amp)—Ct (14X pre-Amp))
and corresponding ΔCt of 4 in horizontal dotted line 2 (Ct (10X pre-Amp)—Ct (14X pre-Amp))
ΔRn (normalized reporter value): the Rn value of an experimental reaction minus the Rn value of the baseline signal
Considering, that according to the manufacturer, a minimum Ct value of 35 is necessary for unbiased pre-amplification, we observed different patterns (Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41598-020-67825-6#Tab3">3</a>, Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-67825-6#Fig4">4</a>):
</a>The figure depicts a two-by-two contingency table for each gene and for all genes together in order to statistically quantify the correspondence of the manufacturer’s prediction of a successful pre-amplification and our measurements
Numbers in bold represent the false negatives (FN
Fields in grey depict the values for FP and FN in percent
For CDKN1A (Ct value without pre-amplification &lt; 35: best results
obviously unbiased) our findings are consistent with the manufacturer (PPV 100%
In FDXR our results were satisfying regarding Ct values after pre-amplification (mean Ct of 23.9 after 14 ×)
with a mean Ct value of 37.4 without pre-amplification and a majority of non-pre-amplified samples being undetermined during qRT-PCR
the results should be biased according to the manufacturer
This shows a high number of false negatives
which indicates that pre-amplification is linear although it shouldn’t work according to the manufacturer
For DDB2 the corresponding mean ΔCt (observed vs expected) was 1.8 after 10 × pre-amplification, respectively 2.33 after 14 × pre-amplification (Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41598-020-67825-6#Tab3">3</a>)
This means that linearity of pre-amplification was suboptimal compared to CDKN1A
With a mean Ct value without pre-amplification of 36.1
pre-amplification shouldn’t work according to the manufacturer
Although this was true for the majority of comparisons
we found false negative results in 16.6% (PPV 100%
FN 16.7%; Ct value without pre-amplification &gt; 35: limitations
We found a different pattern for GAPDH: while the mean raw Ct value without pre-amplification was below the recommended 35 cycles (33.5) compared to DDB2
indicating enough RNA copy numbers for an unbiased pre-amplification and qRT-PCR
the corresponding ΔCt (observed vs expected) was 5.78 after 10 × pre-amplification and 6.69 after 14 × pre-amplification
These outcomes are biased and indicate that the pre-amplification results in an almost 2^6.69 = 103 times difference compared to the expected values
This dysregulation results in a high number of false positives (58.3%)
the recommended Ct values of target genes below 35 before pre-amplification proved to be not sufficient of itself and had to be augmented by the criteria of showing a linear (unbiased) pre-amplification
Taken together, predictions based on the manufacturer’s criteria are biased and in our examinations can result in as much as 58.3% false positive and up to 70.6% false negative results (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-67825-6#Fig4">4</a>)
a considerable number of samples and genes could not be used for analysis
But according to our measurements checking uniformity of pre-amplification
they can be used when following our newly introduced criteria (proofing linearity of the pre-amplification step)
but the method behind has never been explicitly described in detail before
that have to be coped with when working with saliva
Our aim was to provide a robust method to process whole saliva for downstream gene expression studies
Saliva was not a sterile medium compared to blood or other body tissues
Due to the complex contamination of human whole saliva
RNA from this medium is extremely sensitive to rapid degradation of salivary RNA
Endonucleases like RNAses mainly contribute to that
the RNA integrity is sufficiently well in order to process human salivary RNA for downstream gene expression analysis using the modified workflow
Or in other words: the salivary transcriptome is stable enough
two challenges had to be addressed within our effort:
How to deal with the overwhelming bacterial contamination of the samples
How to deal with the low abundance of human RNA
in order to detect gene expression changes of human origin only
which had a high inter-individual variance
actually posed the main problem when working with whole saliva
We tried to overcome these problems by focusing on the poly(A)+-tails of human RNA using only Oligo (dt)20 primers for cDNA synthesis and were able to amplify specific human RNA
But still having to deal with the low yields of material from human eukaryotic origin
downstream pre-amplification became indispensable in the adapted methods we devised to obtain reliable results in downstream gene expression analysis
This further supports augmentation of the recommended Ct value of target genes below 35 before pre-amplification by the criteria of showing a linear (unbiased) pre-amplification for each target gene
the criteria of Ct values &lt; 35 before pre-amplification appeared important but not primarily decisive (see false negatives for FDXR)
the suboptimal pre-amplification (∆Ct observed vs expected) in DDB2 (compared to CDKN1A) is most likely due to the fact that Ct values without pre-amplification were already &gt; 35
whereas CDKN1A was &lt; 35 and showed best results (confirming the manufacturer’s specifications)
The observed and expected Ct-values for CDKN1A are very close and represent a variance as accepted for technical replicate measurements (&lt; 0.5 Ct-values difference)
These findings suggest that a minimum amount of input is required such that uniformity of pre-amplification should always be checked
one solution was to synthesize more cDNA from human RNA with the SuperScript kit in order to increase the input for pre-amplification and qRT-PCR
we demonstrated that in 17–71% of the comparisons
a false prediction is made when considering the Ct-value &gt; 35 in un-amplified samples only
This means that gene expression analysis works in 17–71% of the samples with Ct values above 35
even when the manufacturer maintains that it doesn’t work (false negatives)
pre-amplification doesn’t work in 58.3% of certain gene measurements
an improved outcome was achieved if pre-amplification showed a linear (unbiased) relation and that it is necessary to check the linearity
By taking these exemplary genes and checking the linearity individually
we are able to show different patterns of disagreement described above: false positives and false negatives
Due to the moderate sample size we judge our study more as a “proof of concept” study where we explore the potential of mRNAs in human whole saliva
even increasing the sample size will not alter the three newly introduced modifications which are: (1) Selective cDNA synthesis of mRNA species of human origin
(2) pre-amplification before qRT-PCR and (3) check uniformity of pre-amplification
These are prerequisites for successful future efforts with respect to processing human RNA for biomarker studies
18S rRNA is not synthesized to cDNA when using the SuperScript III First-Strand Synthesis System
because 18S rRNA -transcripts lack a poly(A)+-tail
cannot be used as a normalizer in gene expression analysis in the current application
A new housekeeping gene except for rRNA needs to be detected when working on gene expression analysis in human whole saliva
Further investigations will show if this poses a limitation of its use as a biomarker
We decided to use whole saliva as a source for RNA biomarkers instead of salivary supernatant due to the numerous advantages described above
a higher background due to a high amount of non-human RNA is a drawback but using whole saliva is much easier than harvesting supernatant and isolating exosomes
whether the oral microbiome alters gene expression patterns in the salivary glands and to what extend this affects the robustness of salivary transcriptomics as a diagnostic tool
less elaborate and high through-put approach with high utility for future studies
we report a robust modified methodology to process human whole saliva as a non-invasive and easily accessible biofluid for gene expression analysis
described here will be important to overcome the two challenges of an overwhelming bacterial contamination and low abundance of human RNA in the source material for future applications
We suggest that modifications such as ours will render human saliva as a more attractive material for an expanded number of biomarker studies
The datasets generated and analysed during the present study are available from the corresponding author upon request
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Saliva: An emerging biofluid for early detection of diseases
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The majority of microRNAs detectable in serum and saliva is concentrated in exosomes
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This work was supported by the German Ministry of Defence
Bundeswehr Institute of Radiobiology affiliated to the University of Ulm
Faculty of Military Health Sciences in Hradec Kralove
University Hospital and Faculty of Medicine
Nuclear Physics Institute of the Czech Academy of Sciences
Institute for Hematology and Blood Transfusion
wrote the main manuscript text and prepared the figures
contributed to the conception of the study
The authors declare no competing interests
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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outside at home doing what he loved to do with his dog by his side
to the late Raymond and Maxine (Houser) Heffley
He graduated from high school and entered into the United States Army
He honorably served his country during the Vietnam War
He married Bethany Jo Thomas on November 27
David was a farmer and construction worker
David was preceded in death by his parents and a sister
A celebration of David&#39;s life will take place at a later date
Memorial donations may be sent to Disabled Veterans of America.&nbsp;
The following is a <a href=http://www.courier-journal.com/story/news/local/oldham/2015/09/29/oldham-history-woman-offers-snapshot-life-nazi-germany/72992286/>continuation of an interview</a> I conducted with Waltraud Thomas Vaughan
she gives her perspective about how village life affected her as a child and her family during World War II
"We were lucky living in a small town because all of us had a garden
So we ate a lot of vegetables but didn’t have much meat
My mother and father all had to work to make things that the Germans needed for the war
we were sent to collect things for the war effort
like certain plants they used to make medicine out of
We would bring the plants to school every Friday
You were required to bring something in and we would often go long ways to find the plants
We were given a small amount of money for the plants we turned in and then we were forced to buy pictures of Hitler with the money
One day a couple of us decided we would buy something we could eat instead of a picture of Hitler
So when the time came to turn in our money
We were punished and slapped across the face
They made it clear to us not to do that again
even though my mother didn’t think we should collect those things
she began going with me to look for plants so I wouldn’t get punished
I was also required to work in a factory to make uniforms for the soldiers
"Hitler wanted the kids to have more food so families with kids were rationed more food
A couple of summers I was sent away to live with a family in another part of Germany that had more food
but I was sent to a family that owned a drugstore and I thought they were rich because they had a maid
I was able to eat with the family at their table
They were nice people and the next year I was invited back
"We also were required to take in prisoners of war in our town
We had two French officers that lived upstairs in our house
They (the POW’s) were allowed to live in civilian homes
Sometimes they would give me small pieces of chocolate
The higher ranking soldiers lived in homes while the longer ranked lived as a group in the town’s bowling alley
You could check soldiers out to work and they would help the farmers all day in the field
That was a time when the army was segregated
There was a rumor that when the Americans came they would all kill us
We had very little food and a friend and I ran downtown because we heard a shipment of honey was coming
The soldiers gave us chocolate — and we were so happy
My mother and the girl’s mother came running downtown because they heard the Americans had come in town and were worried for us
We had just eaten the chocolate and my mother said “oh no
it could be poisoned.” But of course it wasn’t and we were OK
After that we didn’t go outside for days because we were afraid the Americans would kill us
I helped to take care of a woman’s children; she was a dressmaker
It was during that time I met my future husband
There was a movie that we (friends and I) could go to once a week and on the way home
 A girl that I knew was engaged to an American soldier and she told me there was somebody that wanted to meet me and that was him
 I spoke no English and he spoke no German
He asked if he could come to my home and meet me
My parents were not crazy about me dating an American soldier but after they met him
I wanted to say something to her about being sorry about what the German’s did
I said I was sorry about a lot of things they did
we didn’t know about it when it was happening
She dropped her basket of clothes and ran over to hug me
She said she knew I didn’t have anything to do with what happened and she loved me and I was a nice person
I thought they would all hate Germans because of what happened
"My husband was in the Army for 30 years (WWII through Vietnam) and the last place we lived was in Fort Knox
we settled in Louisville where he got a job working at the Post Office
four grandkids and seven great-grandchildren."
You can contact Nancy at: nancystheiss@gmail.com
16-year-old Anika Pierce considered herself an unofficial Boy Scout
tagged along with her brother and his Cub Scout troop to events and activities
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WNT3 and POU2AF1 is a promising approach for identification of clinically relevant groups (unexposed
low- and high exposed) after radiological/nuclear events
results from international biodosimetry exercises have shown differences in dose estimates based on radiation-induced GE of the four genes
differences in GE using next-generation-sequening (NGS) and validation with quantitative real-time polymerase chain reaction (qRT-PCR) was reported
These discrepancies could be caused by radiation-responsive differences among exons of the same gene
We performed GE analysis with qRT-PCR using TaqMan-assays covering all exon-regions of FDXR
Peripheral whole blood from three healthy donors was X-irradiated with 0
After 24 and 48 h a dose-dependent up-regulation across almost all exon-regions for FDXR and DDB2 (4–42-fold) was found
A down-regulation for POU2AF1 (two- to threefold) and WNT3 (&lt; sevenfold) at the 3’-end was found at 4 Gy irradiation only
this confirms our hypothesis for radiation-responsive exon-regions for WNT3 and POU2AF1
we identified the most promising TaqMan-assays for FDXR (e.g
Hs01573371_m1) for biodosimetry purposes and acute radiation syndrome prediction
considering several criteria (detection limit
To explain fold-differences in up- or down-regulated genes reported by various teams we hypothesized that while TaqMan assays target the same genes
they assess different exon-pairs within that gene
These exon-pairs might be more or less responsive to radiation exposure
This might be explained considering the conventional NGS method sums the reads across all exons of a gene and are attributed to that gene
qRT-PCR TaqMan assays selectively recognize one specific exon-pair of one gene
The different findings with respect to GE changes might be explained depending on whether the whole gene (NGS) or only an exon-region of the gene is interrogated (qRT-PCR)
We questioned whether the "best coverage" TaqMan assay was the most suitable for investigating radiation-induced gene expression changes
we examined radiation-induced gene expression changes of FDXR
and POU2AF1 at the exon level using qRT-PCR
We evaluated all exon-pairs of these genes for the most radiation-responsive region
We also considered detection limits (sufficient baseline gene copy numbers)
Study design overview including X-irradiation of SH-CPT blood
RNA isolation and gene expression measurements with TaqMan assays covering all exon-regions of FDXR
SH-CPT = Cell Preparation Tube with Sodium Heparin
Due to the minimal-invasive collection and the fully anonymized processing of the samples
the local ethical commission (Ethics committee
Germany) decided that experiments can be performed in agreement with ethical standards and do not require an additional approval
All samples were obtained with informed consent
All experiments were performed in accordance with relevant guidelines and regulations
RNA from mononuclear cells was isolated following the RNeasy Mini Kit (Qiagen)
cells were lysed (RLT-buffer) and RNA precipitated by adding ethanol
Samples were transferred on a silica-membrane which binds RNA
samples washed again and RNA eluted in 60 µl RNase-free water
The isolated RNA was quantified spectrophotometrically (NanoDrop
RNA integrity was assessed by 4200 TapeStation System (Agilent Technologies
Possible contamination by sample genomic DNA was controlled by PCR using primers for the actin gene
RNA specimens with a ratio of A260/A280 nm ≥ 2.0 and RNA integrity number (RIN) ≥ 8 were processed for qRT-PCR analysis
The ratio/fold-change (FC) relative to the unexposed sample at the same time point (reference) was determined by the delta-delta Ct-approach (FC = 2(-∆∆Ct))
A FC of one corresponds to a gene expression similar to unexposed samples
A FC higher or lower than one refers to a several-fold over- or under-expression of the gene of interest after exposure relative to the reference
All experimental work was performed according to the standard operating procedures implemented in our laboratory since 2008 when the Bundeswehr Institute of Radiobiology became DIN-certified by TÜV Süd München
The following inclusion criteria were applied to select the most promising TaqMan assays for biodosimetry purposes:
Baseline (raw Ct-values) that were &lt; 33 (up-regulated genes) and &lt; 30 (down-regulated genes) were chosen to allow for a dose-dependent deregulation of gene expression within the linear dynamic range of qRT-PCR
a difference in fold change ± one unit between time points was determined
if the differential gene expression was 10.5-fold after 24 h (relative to unexposed)
the differential gene expression after 48 h had to be between 9.5 and 11.5
Inter-individual variability of the three donors was determined as a further characteristic of radiation-responsive exon-pairs
the sum of the standard deviations of the dose categories was calculated for each TaqMan assay
Descriptive statistics were performed using Excel
To examine the dose dependency of the inter-individual variability
repeated measurements analysis of variance (ANOVA) followed by Tukey’s test as a post-hoc test were used to compare the coefficient of variation (CV
These analysis and graphical representations were performed using SPW (SigmaPlot
Assay labels starting with the two capital letters “AR” can be found after creating an account at Thermo Fisher web site
entering the “Reorder Custom Assays” section with the implemented search function in the “Custom TaqMan Assay Design Tool”
</a>Box plots of coefficient of variations of fold changes of FDXR
POU2AF1 across all assays per dose category
Horizontal lines within boxplots represent medians
The upper and lower whiskers correspond to 1.5 × of the interquartile range
p-Values were calculated between exposure groups (0 Gy
4 Gy) using repeated measurements ANOVA with Tukey’s Test as a post-hoc test and p-values ≤ 0.01 and ≤ 0.001 are marked with one and two asterisks
These might be explained by employment of different TaqMan assays targeting different exon-pairs
the aim of this study was to improve detection of GE fold changes by identifying the most radiation-responsive exon-regions of these genes
Our results indicate marginal differences between TaqMan assays for FDXR and DDB2
genes often used in biodosimetry and effect prediction
because radiation-induced regulations are comparable over all exon-regions of both genes
which was opposite with what we found for POU2AF1 and WNT3 that showed radiation-induced downregulation only at exon-pairs on the 3’ end
other housekeeping genes or differences in qRT-PCR data analysis using other baselines and thresholds
might have had an impact on differences among laboratories of these international exercises
differences in dose estimates deduced from calibration curves might be caused when applying different regression models
In the context of the RENEB 2021 inter-laboratory comparison exercise
the impact of these other factors is currently under investigation (unpublished results)
We found that the so called “best coverage assays” as recommended by the manufacturer for qRT-PCR analysis did not necessarily perform best in the present radiation exposure study
For FDXR and WNT3 the best coverage assays were among the ones suitable for biodosimetry purposes
Hs00172068_m1 (DDB2) showed no GE persistency over 48 h after 4 Gy irradiation
and a mean baseline of 32 Ct-values was too low for Hs01573369_m1 (POU2AF1) to allow radiation-induced down-regulation of GE (resulting in increased Ct-values) in the linear dynamic range of qRT-PCR
other assays were superior to the best coverage TaqMan assays
the best coverage assay does not automatically fulfill all the criteria needed for a good GE biomarker used for biodosimetry or health effect prediction purposes
The assay sequence length in qRT-PCR contains at least 60 bp and is therefore unambiguous for a specific location in the genome
are affected by single nucleotide polymorphisms (SNPs)
In qRT-PCR SNPs can prohibit the development of an amplification plot and therefore cause undetectable Ct-values
which argues against differences in PCR reaction efficiency inherent to the employed TaqMan assays
raw Ct-values of our genes were well within the linear-dynamic range of the method
so that a slight shift to higher Ct-values would not alter our results
EDTA and lithium-heparin are suitable for examining the radiation induced gene expression changes examined within this study
this study represents the most comprehensive study on often used genes for biodosimetry and ARS prediction on an exon-level of these four genes
a comparable induction of GE after irradiation observed in most FDXR and DDB2 exon-regions does not explain huge differences in dose estimates reported by different teams in large biodosimetry exercises
Exon-related GE differences in POU2AF1 and WNT3 underline difficulties in validation of NGS data using qRT-PCR
magnitude of radiation-induced differential GE
time persistency and inter-individual variability
we identified several TaqMan assays that could be used for biodosimetry purposes and identification of clinically relevant groups
Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans–joint RENEB and EURADOS inter-laboratory comparisons
Examining radiation-induced in vivo and in vitro gene expression changes of the peripheral blood in different laboratories for biodosimetry purposes: First RENEB gene expression study
Comparable dose estimates of blinded whole blood samples are obtained independently of culture conditions and analytical approaches
Realising the European network of biodosimetry (RENEB)
Realising the European network of biodosimetry: RENEB–status quo
Laboratory intercomparison of gene expression assays
Rapid high-throughput diagnostic triage after a mass radiation exposure event using early gene expression changes
Validating baboon ex vivo and in vivo radiation-related gene expression with corresponding human data
Identifying a diagnostic window for the use of gene expression profiling to predict acute radiation syndrome
Acute radiation syndrome-related gene expression in irradiated peripheral blood cell populations
Gene expression-based biodosimetry for radiological incidents: Assessment of dose and time after radiation exposure
FDXR is a biomarker of radiation exposure in vivo
High and low dose responses of transcriptional biomarkers in ex vivo X-irradiated human blood
Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise
Predicting the radiation sensitivity of male and female rhesus macaques using gene expression
Thermo Fisher Scientific. TaqMan Gene Expression Assay solutions. <a href="http://tools.thermofisher.com/content/sfs/brochures/taqman-gex-brochure.pdf" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="http://tools.thermofisher.com/content/sfs/brochures/taqman-gex-brochure.pdf">http://tools.thermofisher.com/content/sfs/brochures/taqman-gex-brochure.pdf</a> (2018)
Correcting false gene expression measurements from degraded RNA using RTQ-PCR
The ultimate qPCR experiment: Producing publication quality
Gene expression changes in irradiated baboons: A summary and interpretation of a decade of findings
Radiation-induced alternative transcription and splicing events and their applicability to practical biodosimetry
Alternative transcript initiation and splicing as a response to DNA damage
DNA Repair Genes: Alternative transcription and gene expression at the exon level in response to the DNA damaging agent ionizing radiation
Lazaruk, K. et al. The design process for a new generation of quantitative gene expression analysis tools. <a href="http://tools.thermofisher.com/content/sfs/manuals/cms_040599.pdf" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="http://tools.thermofisher.com/content/sfs/manuals/cms_040599.pdf">http://tools.thermofisher.com/content/sfs/manuals/cms_040599.pdf</a> (2006)
In vivo validation of alternative FDXR transcripts in human blood in response to ionizing radiation
Impact of inter-individual variance in the expression of a radiation-responsive gene panel used for triage
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We greatly appreciate Razan Muhtadi for her professional and precise work in RNA isolation
RNA quality/quantity measurement and qRT-PCR
Open Access funding enabled and organized by Projekt DEAL
Bundeswehr Institute of Radiobiology Affiliated to the University Ulm
contributed to the design of the experiments
the tables and figures as well as drafted the manuscript
contributed to the data analysis and interpretation and revised the final manuscript
provided critical laboratory resources and revised the final version of the article
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DOI: https://doi.org/10.1038/s41598-022-13577-4
Cross Country&#8217;s editor Ed Ewing talks to paragliding guide and Mastering Paragliding author Kelly Farina
getting back into it after a layoff and more
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a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease
In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming
we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR
this approach was independently validated on 91 samples from 61 healthy donors
we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis
which then ensured comparable analysis using the same base human input material
we examined relative levels of ten known housekeeping genes
and assessed inter- and intra-individual differences in 61 salivary RNA isolates
while considering effects of demographical factors (e.g
epidemiological factors comprising social habits (e.g
previous radiological diagnostic procedures (e.g
number of CT-scans) and saliva collection time (circadian periodic)
Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02)
None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined
ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples
Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance
our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva
The inserted table shows the number of samples used in the different tasks as well as the time points of sampling
and previous radiological diagnostic procedures (e.g
was isolated by combining a Phenol–Chloroform RNA precipitation with further processing using silica membranes
DNA residuals were digested on the membrane (RNAse-free DNAse Set
RNA was eluted with 100 µl RNAse free water in a collection tube and the aliquots were pooled for each sample
In order to increase the input RNA amount for downstream gene expression analysis
samples were steamed at 45 °C for 90 min followed by elution with 30 µl of RNase free water before freezing at − 20 °C
Quality and quantity of isolated total RNA were measured spectrophotometrically using NanoDrop™ One Microvolume UV–Vis spectrophotometer (NanoDrop
RNA integrity was assessed by the 2100 Agilent Bioanalyzer (Life Science Group
Germany) and DNA contamination was controlled by conventional PCR using actin primers
The amount of total RNA input was always determined to 500 ng measured by NanoDrop™ One
cDNA was diluted to a concentration of 0.01 ng/10 µl
which was used for qRT-PCR detection of 16S and 18S rRNA
</a>The flow chart displays the different steps (rows) in gene expression analysis including our modified workflow
the required kits and the detour for adjustment of human RNA input as well as its confirmation (boxes in darker grey)
The boxes in brighter grey depict the advanced methodological workflow for gene expression analysis in whole saliva samples
Using the generated ratio, a RNA input of 4 ng total human RNA was determined individually for each sample for a second cDNA synthesis followed by 2nd qRT-PCR (confirmation for methodological reasons in this context, Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-022-05670-5#Fig2">2</a> boxes in darker grey)
cDNA from High-capacity cDNA reverse transcription kit was used
whereas for the primer probe designs representing the potential house-keeping genes (ACTB
SuperScript™ III First-Strand Synthesis SuperMix synthesized
human cDNA with and without 14× pre-amplification was used for detection of each of these genes in each sample
The qRT-PCR reaction contained TaqMan® Universal PCR Master Mix and one of the inventoried TaqMan® Gene Expression Assays for separate detection of transcripts
using a 96-well-format TaqMan® qRT-PCR platform and the QuantStudio™ 12 K Flex Real-Time PCR System
maximum [max]) were calculated for continuous variables such as gene expression data and age
Frequency tables of categorical data were examined for statistical differences using the chi-square test for equal proportion
Comparisons of categorical variables with gene expression values were performed using the non-parametric Kruskal–Wallis (KW) test
We assessed the assumptions of normality (Kolmogorov–Smirnov) and boxcox transformed the continuous variable “age”
All calculations were performed using SAS (release 9.4
Graphical presentations were performed using Sigma Plot 14 (Jandel Scientific
The merged set of raw data is provided within supplemental Table <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-022-05670-5#MOESM3">2</a>
RNA quantity/quality and TaqMan® qRT-PCR) were performed according to the standard operating procedures implemented in our laboratory in 2008 when the Bundeswehr Institute of Radiobiology became DIN-certified by TÜV Süd München
All samples and data was processed anonymously without exception and only for this specific purpose
All data is handled according the European General Data Protection Regulation
From 91 samples an average of 93.3 µg (SD ± 141.7) total RNA per 2 ml saliva could be isolated
high purity RNA with OD260/280 ratios at a mean of 2.1 was isolated from saliva
A mean RNA integrity number (RIN) of 5.9 (SD ± 1.4) was detected for saliva samples and all saliva samples showed gel-like image bands of human 28S and 18S rRNA that did not provide any indications for severe degradation
No DNA contamination could be detected by beta actin PCR in all samples (data not shown)
</a>The box plots in (A) display the human 18S rRNA and bacterial 16S rRNA raw Ct values (threshold cycles) for all whole saliva samples (n = 91)
solid lines the median and dots the outliers
The input amount for cDNA synthesis for each sample was 0.5 µg
The inserted table shows the calculated ratio between raw Ct values of human 18S rRNA and bacterial 16S rRNA and provides descriptive statistics: mean
standard deviation [stdev] and standard error of the mean [sem]
The box plots in (B) represent the human 18S rRNA raw Ct-values before and after adjustments accounting for input differences from left to right
The left part shows Ct values from 1st qRT-PCR performed using cDNA with an input of 500 ng total RNA (bacterial and human RNA) and the right boxplot the corresponding results when taking 4 ng of human RNA (calculated via the 18S/16S-ratio together with total RNA concentration values measured)
Asterisks (**) refer to a P value &lt; 0.001 using 500 ng total RNA measurements as reference
Using the generated ratio (ratio = 2^(Ct18S rRNA—Ct16S rRNA)), a defined amount of human RNA (4 ng) was reverse transcribed in a second cDNA synthesis, again using High-capacity cDNA reverse transcription kit, followed by qRT-PCR analysis for detection of human 18S rRNA and bacterial 16S rRNA Ct-values (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-022-05670-5#Fig3">3</a>B)
The 50% interquartile range of human 18S rRNA quantification in 51 saliva samples changed significantly (P &lt; 0.001) from 7 Ct values of unadjusted input RNA (comprising an unknown mixture of human and bacterial RNA) to 1.5 Ct values after considering the degree of bacterial contamination and adjusting for it
The 61 healthy donors comprised almost equal proportions of females (44.3%) and males (55.7%, Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41598-022-05670-5#Tab1">1</a>)
Donors were mostly Caucasians (77.1%) and about half of them were aged 19–30 years at time of saliva collection
Only 18% smoked over at least 5 years and another 16% of former smoker smoked at least 2 years and stopped smoking about a year ago
Alcohol consumption and diet was reported by 41% and 28%
Most (83.6%) of the donors brushed their teeth at least twice a day and about half of them used flossing and about one-third mouthwash
Braces (3.3%) and dentures (11.5%) were reported less frequently and oral problems like periodontitis in 23%
Acute and chronic diseases such as rheumatism or disease of the thyroid gland were reported in 6.6% and 16.4%
Radiological examinations during the last six months (mainly X-rays and CT-scans) were reported in 18% and none of our donors received radiotherapy
Total RNA concentration increased almost three-fold with higher alcohol consumption (data not shown)
Only human RNA (18S rRNA) amount as well as the ratio of 18S/16S rRNA appeared significantly associated with age on a categorical (P = 0.07
which was not shown for bacterial RNA (16S rRNA)
We built two groups based on relatively high (18S rRNA Ct value ≤ 30) and relatively low (18S RNA Ct value &gt; 30) amounts of human RNA
Within the younger age group (&lt; 30 years) the mean human RNA amount (18S rRNA as representative) was 676-fold higher between both human 18S rRNA groups
but neither sociodemographic nor epidemiological parameters appeared significantly associated (data not shown)
In the older (&gt; 30 years old) compared to younger donor group (≤ 30 years old)
examinations regarding the high yield human 18S rRNA group showed significantly more alcohol consumption per week (P = 0.012)
higher frequency of former smoker (P = 0.0058)
radiological examinations (P = 0.012) and dentures (P = 0.032)
</a>Box plots in (A) show the human 18S rRNA and bacterial 16S rRNA raw Ct-values for whole saliva samples (total n = 40) per time point (each n = 10: 9 am—0 h; 3 pm—6 h; 9 pm—12 h; 9 am next day—24 h)
The box plots in (B) represent the quality of isolated RNA using RNA integrity numbers (RIN) for saliva samples (total n = 40) per time point (each n = 10)
The asterisk (*) refers to a P value &lt; 0.05 using 9 am measurements as the reference
</a>Raw Ct values (threshold cycles) of three candidate house-keeping genes (ACTB
ATP6 and B2M) as well as a combination of them (arithmetic mean) are depicted over time (four time point each: 9 am—0 h; 3 pm—6 h; 9 pm—12 h; 9 am next day—24 h) for each donor
They were fulfilling the criteria for being an appropriate housekeeping gene in this context
Our study indicated no significant differences in RNA amounts between the collection time points
but intra-individual differences were high (&gt; 1000-fold)
RIN values slightly improved at later (3 pm and 9 pm) relative to early collection time points (9 am)
indicating that saliva samples can be collected during the whole day
widening the applicability of this approach
We did not observe any significant differences concerning sociodemographic and epidemiological characteristics that would explain the observed magnitude of inter-individual variance of human RNA yields
the differences in amounts of human RNA (raw Ct value for 18S rRNA was ≤ 30) between the samples could not be explained by sociodemographic or epidemiological characteristics
the addressed sociodemographic and epidemiological conditions seemed to be of minor relevance for interpretation of saliva gene expression results
ACTB and B2M as housekeeping genes for expression studies using human saliva will increase the robustness and
planned future studies on saliva samples from irradiated donors will finally show whether radiation impacts these three identified genes
which would render them unsuitable as housekeeping genes
The applicability of suggested housekeeping genes has to be proven in future independent studies
some limitations of this manuscript need to be considered
The major limitation lies in the fact that epidemiological data was gathered from the healthy donors by anamnesis
the oral health status was self-reported and not confirmed by medical examination
a main strength of this study is that it represents a comprehensive examination of different facets when working with human whole saliva
The enhancement of the methodology and the proper examination of potential confounders like the influence of sociodemographic and epidemiologic characteristics that could potentially influence salivary isolates are completely novel findings
Considering saliva as an emerging source of body fluid for gene expression examinations underlines the importance of those findings
A key strength of the present study was the sample size: 91 samples from 61 donors in total are remarkable numbers considering molecular biological studies
These numbers and the numerous endpoints in this study are sufficient for creating reliable hypotheses
we (I) improved the comparability of gene expression measurements among different saliva samples
(II) demonstrated that quality and quantity of RNA isolates is highly robust considering potential confounding factors such as demographics/epidemiologic and the saliva sampling time
making the approach of saliva collection even more attractive for further biomarker studies and (III) identified a set of potential housekeeping genes (ATP6
ACTB and B2M) and suggested their combination to increase robustness of saliva-based gene expression studies
Extracellular RNAs: Development as biomarkers of human disease
Genome-wide analysis of microRNA and mRNA expression signatures in cancer
miRNAs as biomarkers in disease: Latest findings regarding their role in diagnosis and prognosis
Neonatal salivary analysis reveals global developmental gene expression changes in the premature infant
Practical evaluation of an RNA-based saliva identification method
Analysis of saliva gene expression during head and neck cancer radiotherapy: A pilot study
The utilization of saliva as an early diagnostic tool for oral cancer: microRNA as a biomarker
Salivary transcriptome diagnostics for oral cancer detection
Saliva diagnostics—Current views and directions
Exosomes from human saliva as a source of microRNA biomarkers
Salivary biomarkers: Toward future clinical and diagnostic utilities
Salivary diagnostics: Enhancing disease detection and making medicine better
Overcoming challenges in human saliva gene expression measurements
Oragene ® • RNA purification protocol using the Qiagen RNeasy Micro Kit for volumes up to 1000 µL
SuperScript III Reverse Transcriptase (200U/µL) #18080-085
TaqMan PreAmp Master Mix User Guide (2018)
Saliva liquid biopsy for point-of-care applications
Usefulness of saliva samples for biomarker studies in radiation research
Validation of housekeeping genes for normalizing RNA expression in real-time PCR
High resolution of microRNA signatures in human whole saliva
and clinical utility for oral cancer detection
RNA sequencing analysis of salivary extracellular RNA
Quantitative real-time reverse transcription polymerase chain reaction: Normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41598-022-05670-5?format=refman&amp;flavour=references">Download references</a>
This study includes work from Selamawit Worku Alemu Master thesis
We highly appreciate the work of Thomas Müller
who carefully and patiently introduced SW Alemu in techniques performed in the course of this project and constantly supported her qRT-PCR experiments
This work was supported by the German Ministry of Defence and the Czech Ministry of Defence (A long-term developmental plan 1011)
University Hospital and Medical Faculty in Hradec Kralove
<a data-test="citation-link" data-track="click" data-track-action="download article citation" data-track-label="link" data-track-external="" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41598-022-05670-5?format=refman&amp;flavour=citation">Download citation</a>
DOI: https://doi.org/10.1038/s41598-022-05670-5
&#8221; is seen in this Norwegian Coastal Administration handout picture in Oygarden
Jakob Ostheim/Norwegian Coastal Administration/Handout vis REUTERS
By Nerijus Adomaitis and Terje Solsvik OSLO
Nov 13 (Reuters) &#8211; A Norwegian navy frigate that collided with an oil tanker last week was almost completely submerged on Tuesday despite efforts to salvage the sinking vessel
pictures taken by the Norwegian Coastal Administration showed
The ship&#8217;s plight off the Norwegian coast is
not disrupting the nearby Sture crude oil export terminal
&#8220;We are in normal operations,&#8221; said a spokeswoman for the plant&#8217;s operator
<a href="https://gcaptain.com/video-ais-animation-shows-collision-between-oil-tanker-and-norwegian-frigate/">See an AIS animation of the collision</a>
The Norwegian military has been working since Thursday to salvage the ship by tethering it with several cables to the shore
we realized it was not safe for our staff to carry on the work further,&#8221; said Haavard Mathiesen
the head of the salvage operation for the Norwegian Defence Materiel Agency
more wires broke and the ship sank further
It is now in deep water and stable,&#8221; he told a news conference
The ship was stranded off Norway&#8217;s west coast early last Thursday after it collided with the tanker that had left the Sture terminal
The facility was shut for several hours as a result
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President Donald Trump's administration is considering softening its proposed fee on China-linked ships visiting U.S
ports after a flood of negative feedback from industries that said the idea could be economically devastating
By Dimitri Rhodes Nov 7 (Reuters) &#8211; Belgian oil tanker company CMB Tech says it will focus on the fast growing market in India as it reported third quarter results..
In a bold move amidst Russia&#8217;s intensified rocket attacks on civilian ships and Ukrainian ports
Maersk has launched a new weekly container service into Ukraine
signaling resilience in the country&#8217;s..
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Essential news coupled with the finest maritime content sourced from across the globe
announced the launch of their Scraper parakite on 16 January
saying their mission is to push the boundaries of parakite performance and create “exceptional flying experiences” for advanced and skilled soarers
He aimed to create a “high performance” wing to bridge gaps in the Netherlands’ low coastal dunes
and 20m2 sizes and comes in one colour combination (purple/white/black)
It weighs between 3.3 and 4.2kg with flat aspect ratios from 6.2-7.0
A wingtip steering option will “deliver extreme roll control” for expert pilots wanting that “roller coaster ride”
Dune Rider say the Scraper is for expert level pilots who “crave the ultimate dune flying adventure”
Current delivery time is approximately 3-4 months for pre-orders made through the Dune Rider website
<a href="https://www.dune-rider.com/">dune-rider.com</a>
Everything at KitePower revolves around that kite in the distance
It is a product of intuition and of science
How do design and research complement each other
KitePower designs and makes huge kites for generating electricity
is one of astronaut and professor Wubbo Ockels&#8217; (1946-2014) dream projects that took off quite well
After three successful deployments and nine improved versions of the kite
the Delft start-up declared itself ready for the next phase: bringing its first 100-kilowatt mobile kite power unit to market
The crowdfunding launched with that goal in August has now raised over a million euros &#8211; 1.5 times more than requested
Kitepower develops so-called airborne wind technology
The start-up does not use conventional wind turbines but designs kites to generate energy
As the kite spins its figure eights through the air
the Dyneema® line reels out further and further
The force on the line (3.5 tonnes) can lift three passenger cars
The drum is connected to a generator that generates an average of 100 kilowatts of power
the kite flips over and is winched back in for the next round
Reeling in is four times faster than reeling out
the KitePower Falcon produces enough electricity to power 150 households
Inside KitePower&#8217;s workshop in the old cable factory on Schieweg
one can see just how immense the kite actually is
Kite designer Bryan van Ostheim (35) talks about 60 square metres &#8211; the wall of a sports hall
the kite is not much bigger than a bungalow tent and weighs 60 kilograms
which is why people are working here in the hall
Then the call of the sea becomes irresistible
That is when you&#8217;ll find the staff on Scheveningen beach where they flash over the waves as kite surfers
Kite surfing brought Bryan van Ostheim into kite design (Photo: private collection)
Roland Schmehl leads the research on airborne wind energy systems (AWES) at the aerospace engineering faculty
a researcher at the Faculty of Aerospace Engineering
was one of KitePower&#8217;s founders in 2016
along with the current CEO Johannes Peschel
Schmehl had come to TU Delft to explore the possibilities of power generation with kites: airborne wind energy
Schmehl chose to stay involved in teaching and research at TU Delft
“KitePower is our outdoor laboratory,” he explains
they fly and collect data that we use in our research
they get information from us on how they can further improve their kites
Van Ostheim&#8217;s claim to fame was a light-weather kite for kitesurfing
The trailing edge was a single layer to save weight
But the leading edge (the nose) was inflated
making it easy for the kite to come back off the water
“I made a small six square metre prototype and it worked immediately
The KiteMobile platform made a video of it which generated publicity.”
That&#8217;s how KitePower spotted Bryan van Ostheim
who had learned the kite making craft in New Zealand from Peter Lynn &#8211; a legend in the kite community
“I was uncertain about it at first,” Van Ostheim recalls
“I thought that I would then be among all these TU Delft boffins and I wasn’t sure how I would deal with that
You just have to make sure you complement each other and listen well to each other
Everyone does what he or she is best at.” 
Bryan van Ostheim in front of and on top of a 60 m2 kite
so when we fly we can watch live,” Van Ostheim says
A kite like that is full of markings and attachment points
so you can trace any deformation back to a precise position
You can then try to reproduce the deformation in the simulation so that you can correct it
The deformations make kite simulations very difficult
The kite’s shape can be aerodynamically perfect on the computer
but if it doesn&#8217;t look right in the air you still have to start changing things.”
“Including deformations in your calculations is the basis of kite simulations,” Schmehl responds
“I call that a fluid structure interaction problem
The shape of the wing affects the aerodynamics around the wing and this distributes pressure on the wing
There is a cycle of shape that affects force that affects shape
you have to solve shape and force simultaneously and that is one of the most challenging problems we have here at the Faculty.”
as do the optimisations and the scaling up
KitePower will work on scaling up to kites of 200
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A top-of-the-line Norwegian warship collided with a tanker last week
causing the crew to intentionally run the ship aground to try to save it
The 5,000-ton frigate was part of a NATO fleet operating in the Atlantic prior to the collision
Source: <a href="http://www.nationmultimedia.com/detail/asean-plus/30358485" rel=" nofollow" target="_blank" data-track-click="{&quot;click_type&quot;:&quot;other&quot;,&quot;element_name&quot;:&quot;body_link&quot;,&quot;event&quot;:&quot;outbound_click&quot;}">AFP</a>
Source: <a href="https://www.washingtonpost.com/world/europe/norwegian-frigate-sinks-after-it-was-rammed-by-tanker/2018/11/13/61eb753e-e73c-11e8-8449-1ff263609a31_story.html?utm_term=.54a3a127cde7" rel=" nofollow" target="_blank" data-track-click="{&quot;click_type&quot;:&quot;other&quot;,&quot;element_name&quot;:&quot;body_link&quot;,&quot;event&quot;:&quot;outbound_click&quot;}">Associated Press</a>
Source: <a href="https://www.defensenews.com/naval/2018/11/11/warnings-and-confusion-preceded-norwegian-frigate-disaster-heres-what-we-know/" rel=" nofollow" target="_blank" data-track-click="{&quot;click_type&quot;:&quot;other&quot;,&quot;element_name&quot;:&quot;body_link&quot;,&quot;event&quot;:&quot;outbound_click&quot;}">Defense News</a>
Australia&#039;s best free military news site
Share the post "Inspired by grandfather’s sacrifice"
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it was only natural for this young sailor to join the military – but unlike her father and grandfather
she found her choice of career in the Navy
CAPTION: Leading Seaman Elizabeth McCallum in front of HMAS Collins at Fleet Base West in Rockingham
Leading Seaman Elizabeth McCallum grew up in a family of Army officers with her grandfather Alec Weaver
Inspired by her grandfather’s rich history of service and sacrifice
Leading Seaman McCallum decided the Navy was where she could fulfil her dreams
and to make a difference and serve the country I love,” she said
“I joined the Navy because I love to travel and I wanted to become a submariner in particular for the challenge and passion which all submariners share for their jobs.”
Now a maritime logistic support operator submariner
Leading Seaman McCallum spends her days at sea supporting operations on board a submarine and delivering hospitality services
“I really enjoy the diversity my role offers and having the resources and support to learn beyond my core role,” Leading Seaman McCallum said
“I am qualified to drive the helm of a submarine
I have assisted on the combat system with track motion analysis and I have learnt how to be a minor war vessel health care provider supporting the medics at sea
“I even had the opportunity to be the first submariner ever who served as the Personal Assistant to the Chief of Navy in 2019.”
Leading Seaman McCallum also conducts training and mentors new submarine sailors
With three generations having served in the ADF
and a time when I can reflect on my family’s rich military history and the service they gave to the country,” Leading Seaman McCallum said
“I am proud to wear my uniform on Anzac Day and stand proudly by the men and women whom I serve with
Leading Seaman McCallum will watch the dawn service
light her candle and share some stories with her veteran neighbours
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JOHNSTOWN (AP) — At least some of the miners who spent 77 hours in a mine pit 240 feet underground won't be going back to their old line of work
"You don't think about the hazards of the job as much when you're doing it ..
what everybody went through," miner Randy Fogle said at a news conference Monday
"I don't know if too many of us will go back to what we did do
It put our families through a lot; it was hard on us and it was I think harder on them," said Fogle
Some of the miners had asked for chew even before they were pulled from the mine early Sunday
All the men praised the efforts of rescue workers
who quickly pumped air into the pocket where they were trapped
and said they relied on each other to stay alive
They even found nourishment when Thomas Foy happened upon fellow miner Blaine Mayhugh's lunch inside a dry bucket — a corned beef sandwich and some Mountain Dew — and later some Pepsi
"One guy took a bite and passed it around," Thomas Foy said
"I figured we were good for another couple days."
Asked whether they were still being paid by the mining company
"I haven't heard from them — haven't got a call or a visit from them."
miner John Phillippi returned to the mine site with his wife
for an hourlong visit Monday afternoon as crews removed one of the two rigs used in the rescue operation.Phillippi
shook hands with all of the workers and posed for a group photograph with some of the men
I was just one that contributed to saving nine lives," Hamilton said
A steady stream of visitors also started appearing at the mine site Monday
"With so much negativity and uncertainty since September 11th
it's nice to see something come out well," said Craig Ostheim
the Quecreek Mine in rural western Pennsylvania after water from an abandoned mine flooded the shaft where they were working
A desperate rescue operation with tons of heavy equipment and 18 medical helicopters finally paid off when rescuers reached the miners Sunday morning and pulled them up a narrow shaft
As more than 150 rescue workers and neighbors above ground struggled to reach them
the miners fought to keep their heads above the cold water
"What took you guys so long?" the miners said when they spoke to rescuers for the first time
They reportedly asked for chewing tobacco and beer — which doctors wouldn't allow
where the families of the men had been gathering
burst into celebration at the news they were all alive
through the narrow hole in a 7-foot-tall yellow cage
Rescuers greeted them with applause and shouted out nicknames
Mayhugh described a nightmarish scene: the men tied themselves together so they would "live or die as a group."
All nine were immediately taken to local hospitals; six were released on Sunday
Two of the three men remaining in the hospital would be discharged later Monday
Russell Dumire of Conemaugh Memorial Medical Center
The one miner who would be staying in the hospital was successfully treated with medication for a racing heartbeat but was having severe heartburn that has made him unable to eat
Foy told family members "he'll never go underground again," said Foy's daughter
Though they were covered in coal dust and their heavy-duty clothes were soaked through
the miners surprised medical personnel who had prepared to treat them for symptoms of hypothermia or the bends
ambulances and helicopters were at the scene 55 miles southeast of Pittsburgh — the same rural area where the hijacked Flight 93 crashed on Sept
close-knit community still fresh from that earlier tragedy
this happy ending brought smiles across town and crowds to the site
Restaurant and gas station signs trumpeted "Nine Alive!" and "Prayers Answered."
when they inadvertently broke into an abandoned
water-filled mine that maps showed to be 300 feet away
Mayhugh said a 4-foot wall of water — as many as 60 million gallons — came crashing through the breached wall
there'd be dead bodies," said mine worker Doug Custer
The trapped miners spent roughly five hours in the water
at one point attempting to break through another wall to try to bring the water level down
forcing them to swim in their heavy miners' clothes
The miners also huddled around a pipe funneling down warm air
Drilling a rescue shaft to the men began more than 20 hours after the accident when a drill rig arrived from West Virginia
Drilling was halted early Friday morning because a 1,500-pound drill bit broke after hitting hard rock about 100 feet down
and it wasn't until Saturday that measurable progress was being made on both shafts
secretary of the state Department of Environmental Protection
promised a joint federal-state investigation to help determine why underground maps apparently showed the abandoned Saxman Mine some 300 feet away from where the miners were working
but I was upbeat the whole time," said Randy Popernack
He was at home calling distant relatives with the good news
Share the post "Unity celebrated in Perth"
The crew of German Navy frigate FGS Bayern on October 3 celebrated Germany&#8217;s national day while in Perth during their seven-month Indo-Pacific deployment
CAPTION: Leading Seaman Kirsten Robinson sings the German National Anthem during a commemorative service held at the Western Australian State War Memorial in King’s Park
as part of FGS Bayern&#8217;s visit to Western Australia during their ongoing Indo-Pacific deployment
German Unity Day marks the anniversary of the country’s reunification in 1990
A wreath-laying ceremony at the State War Memorial in Kings Park to honour those who had perished in previous conflicts was one of the activities held to mark the occasion
Guests at the commemoration included the German Ambassador to Australia
the German Honorary Consul to Western Australia
as well as Commanding Officer Bayern Commander Tilo Kalski and Commanding Officer HMAS Stirling Captain Gary Lawton
“The ceremony further enhanced the relationship between our two countries and the values we share,” Captain Lawton said
“Although the nature of the commemoration was sombre
the occasion served to highlight the friendship and cooperation that exists between the Royal Australian Navy and the German Navy
“The event gave us the opportunity to pay our respects to those from both nations who paid the ultimate sacrifice in service of their country.”
Commander Kalski and his crew gathered on board Bayern to mark the 31st  anniversary of German Unity Day
The VIPs in attendance included Minister for Defence Industry Melissa Price and Assistant Minister for Defence Andrew Hastie
German Defence Minister Annegret Kramp-Karrenbauer remotely delivered a speech online
in which she emphasised the values shared by Germany and Australia
including defending the rule of law and protecting freedom
and recognised Bayern’s tangible support in that regard
She also expressed her gratitude for the warm hospitality the ship’s company had received in Australia
Commander Kalski said he was pleased to welcome aboard distinguished guests from different nations to celebrate the reunification of Germany with his ship’s company
“It gives me great pleasure and honour to welcome everyone and share this special day with them,” Commander Kalski said
but I can already say that Australia has welcomed us with open arms
which feels like a reunification in its own right for me and my crew.”
After Bayern leaves Fremantle and is sailing to Darwin for a refuelling stop
the ship’s company will commemorate the 1941 World War II battle between HMAS Sydney (II) and German auxiliary cruiser Kormoran off the coast of Geraldton
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Leading Seaman Suamili Nanai has been selected in the Australian weightlifting team to compete at the Commonwealth Games in Birmingham
CAPTION: Leading Seaman Suamili Nanai trains at the HMAS Stirling gym in preparation for the 2022 Commonwealth Games in Birmingham
The combat systems operator – air is an instructor at the School of Maritime Warfare – West at HMAS Stirling
my family and friends means a great deal to me,” Leading Seaman Nanai said
with integrity – watch and learn through competition and be the greatest in the sport around the world.”
Leading Seaman Nanai started weightlifting in 2020 and already has some impressive achievements to his name
but he has his eyes set on gold at Birmingham
“I am sitting in silver/bronze at the moment with my current lifts
and it would be dishonourable if that wasn’t my goal,” Leading Seaman Nanai said
“My success has been fast-tracked under the supervision of my coaches but also the assistance I have received from Navy.”
“Leading Seaman Nanai is an outstanding example of what dedication
single-minded focus and discipline can achieve,” Captain Lawton said
“He is an inspiration to all of Navy and it has been terrific to hear of his progress in setting and achieving his personal and sporting goals
“Suamili has worked incredibly hard for his success and
Leading Seaman Nanai will be competing on August 3
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