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Metrics details The delivery of genetic cargo remains one of the largest obstacles to the successful translation of experimental therapies in large part due to the absence of targetable delivery vectors Enveloped delivery modalities use viral envelope proteins which determine tropism and induce membrane fusion Here we develop DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design) a modular platform that consists of separate fusion and targeting components To achieve high modularity and programmable cell type specificity we develop multiple strategies to recruit or immobilize antibodies on the viral envelope including a chimeric antibody binding protein and a SNAP-tag enabling the use of antibodies or other proteins as targeting molecules we show that fusogens from multiple viral families are compatible with DIRECTED and that DIRECTED components can target multiple delivery chassis (e.g. lentivirus and MMLV gag) to specific cell types including primary human T cells in PBMCs and whole blood these studies highlight the potential of separating the cell entry and cell targeting functions of fusogens we describe DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design) a modular platform for achieving programmable cell targeting that separates the fusion and targeting functions of fusogens and can be used with various packaging chassis DIRECTED encompasses both natural and engineered cell fusion components as well as multiple strategies for cellular targeting We show that these components can be used with lentiviral particles which offer the capacity to deliver integrating genetic information The modular nature of DIRECTED enables precise cellular targeting across diverse contexts and substantially expands the landscape of targetable cell types a Schematic of lentiviral vector production and lentivector-mediated delivery to target cells b Schematic to expand tropism by co-expressing VSV-G and a variant of protein AG (pAG) that can recruit an antibody on virions (left) Transduction efficiency of HEK293FT cells (as a percentage of mCherry+ cells) of lentiviral particles with various ratios of VSV-G and pAG in the presence or absence of αHLA-A2 (right c Schematic to reduce VSV-G-mediated tropism by mutating residues that interact with low-density lipoprotein receptors (LDL-R) (left) Quantification of titers for VSV-G mutants (middle; N = 18 for WT; 12 for H8A Transduction efficiency of HEK293FT cells (as a percentage of mCherry+ cells) of lentiviral particles with VSV-G point mutations (right; N = 6 for WT; 4 for H8A d Schematic showing separate fusion and targeting components by combining VSV-G K47Q/R354Q (VSV-Gdm) with pAG (left) Transduction efficiency of HEK293FT wild-type cells or B2M knockout cells as a percentage of mCherry+ cells (right N = 4 for all conditions) of lentiviral particles with VSV-Gdm and pAG in the presence or absence of αHLA-A2 at different MOI a two-sided Welch’s t-test with Bonferroni correction was used not significant; * p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; Data are presented as mean ± standard deviation Source data are provided as a Source Data file] tropism can be expanded through the addition of a second targeting moiety These results suggested that VSV-Gdm confers efficient cell entry but abolishes inherent tropism providing the basis for DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design) a modular platform for cell type-specific delivery wherein fusion and targeting functions are separated a Titration of αCD3 antibody amount for transduction of Jurkat E6 cells (N = 3 for all conditions) Data was analyzed by an ANOVA followed by a Dunnett’s post-hoc test with a Bonferroni correction b Transduction efficiency (as a percentage of mCherry+ cells) of Jurkat E6 cells (CD3+) and K562 cells (HLA-A2+) in a co-culture with either αCD3-DIRECTED (N = 3 for each condition) αHLA-A2-DIRECTED (N = 3 for each condition) N = 2 for each condition) lentiviral particles A two-sided Welch’s t-test with Bonferroni correction was used not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; Data are presented as mean ± standard deviation a (Top) Schematic representation of antibody-tethering strategies and (bottom) relative transduction efficiency of Jurkat+surface-HA cells sorted into four bins of different expression levels (low high) or WT cells (no) using DIRECTED lentiviral particles with the scFv strategy (left) with excess antibody or after removal of excess antibody) targeting surface-HA (N = 4 for each condition) b (Top) Representative microscopy images of HEK293FT cells engineered to express different synthetic surface receptors (surface-HA and surface-V5) after delivery of an H2B-mCherry transgene using pAG-DIRECTED lentiviral particles with the indicated targeting antibodies (no antibody (Bottom) Transduction efficiency (as a percentage of mCherry+ cells) for the synthetic cell lines with the indicated targeting antibodies at different MOI (N = 4 for each condition) c (Top) Representative microscopy images of the same cell lines as in (b) using SNAP-DIRECTED lentiviral particles in the presence of an excess of the indicated BG-labeled targeting antibodies (no antibody (Bottom) Transduction efficiency (as a percentage of mCherry+ cells) for the synthetic cell lines with an excess of the indicated targeting antibodies at different MOI (N = 4 for each condition) Data are presented as the mean with the error bars indicating the sample standard deviation Source data are provided as a Source Data file For panel (a) a two-sided Welch’s t-test with Bonferroni correction was used 0.016; SNAP strategy (excess antibody)—no antibody 0.524 1; SNAP strategy (excess antibody)—αHA-BG 1.55E-05 3.77E-07; SNAP strategy (excess antibody removed)—no antibody 1 1; SNAP strategy (excess antibody removed)—αHA-BG 0.000412 not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; scale bar is 100 µm; SecSig secretion signal PDGFRb is not endogenously expressed in Jurkat cells which may interfere with the internalization and turnover rate of this receptor on Jurkat cells As DIRECTED relies on receptors that can efficiently be endocytosed this could result in inefficient internalization and baculovirus GP64 (hydrophobic residues in yellow) Cyan: SecSig; magenta: DIII; orange: DII; red: DI fusion domain; blue: DIV; green: DV; gray: MP; cyan: TM; yellow: CTD f Transduction efficiency (%mCherry+) of WT and ΔB2M HEK293FT cells for pAG-DIRECTED particles using GP64 as the fusogen with an αHLA-A2 antibody at different MOI (N = 4 for each condition) g Schematic showing the modular features of DIRECTED particles highlighting that the fusion and targeting components can be combined in a plug-and-play manner not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001] Data are presented as mean ± standard deviation a (Left) Schematic showing the architecture of αCD5-targeting SNAP-DIRECTED-CreVLPs that package Cre protein (Right) Efficiency of Cre protein delivery as determined by percentage of GFP+ cells upon application of αCD5-targeting SNAP-DIRECTED-CreVLPs without the removal of excess antibody on Jurkat E6 Cre reporter cells at different doses (N = 2 for each condition b (Left) Schematic showing the elements of Cas9-RNP delivering pAG-DIRECTED particles (Right) Loss of B2M protein expression on Jurkat E6 cells upon delivering of Cas9-sgRNA using pAG-DIRECTED particles in the absence of an antibody or with a CD5-targeting antibody for VSV-Gdm+pAG (p-values: 0.0408 0.324) at different doses (N = 3 for each condition) c (Left) Histogram of CD3 or CD5 surface expression on Jurkat E6 cells by flow cytometry (Middle) Loss of B2M surface expression upon treatment of Jurkat E6 cells with VSV-Gdm+pAG-DIRECTED particles in the absence of antibody or upon targeting of CD3 or CD5 0.0001704] (Right) Analysis of the surface level of B2M after using VSV-Gdm+SNAP-DIRECTED particles without removal of excess antibody in the same conditions as before 1.14E-05] (N = 3 for each condition) For panels (a) not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001] a Cell type composition [in percent] of PBMCs as determined by flow cytometry after staining for specific surface markers b Delivery efficiency of H2B-mCherry transgene to primary human T cells in PBMCs from three donors by VSV-G and VSV-Gdm+SNAP lentiviral vectors in the absence of antibody (VSV-Gdm) or functionalized with B cell targeting ligands (αCD19-BG Shown is the percent of mCherry+ CD4+ (left other variants: MOI of 250 (N = 2 infections per donor for 3 donors) c Delivery efficiency of H2B-mCherry transgene to primary human B cells in PBMCs from three donors by WT VSV-G VSV-G+SNAP and VSV-Gdm: MOI of 3500; VSV-Gdm-Bcell (N = 2 independent infections per donor for 3 donors 0.627) d (left) Schematic highlighting defense mechanisms in whole blood (right) Cell type composition of whole blood after removal of red blood cells e Delivery efficiency of H2B-mCherry transgene to primary human T cells in whole blood from two donors by VSV-G and VSV-Gdm+SNAP lentiviral vectors in the absence of antibody (VSV-Gdm) or functionalized with T cell targeting ligands (VSV-Gdm-Tcell) Shown is the percent of mCherry+ CD4+ T cells (left VSV-G and VSV-Gdm: MOI of 1000; VSV-Gdm-Bcell MOIs were calculated estimating 5000 leukocytes per μl of whole blood (N = 2 infections per donor for 2 donors) Data are presented as mean ± standard deviation two-sided Welch’s t-test with BH correction was used these experiments show that DIRECTED allows the incorporation of cell type-specific targeting ligands or combinations thereof which can increase transduction efficiency when combined with wild-type VSV-G or completely redirect tropism when using VSV-Gdm Achieving the potential of gene therapy will require a comprehensive array of delivery modalities that can be tailored to specific cargoes and target cells Here we demonstrate that targeting various delivery modalities can be reprogrammed using a suite of modular components We capitalized on the ability to separate the cell entry mechanisms of these delivery vehicles into independent fusion and targeting functions to generate the DIRECTED platform which combines interchangeable fusogenic and targeting components A key advantage of DIRECTED is the compatibility with commercial antibodies (via both pAG-mediated antibody recruitment or SNAP-mediated covalent linkage) which substantially expands the range of targetable cell types including cell types that have been refractory to transduction which showed highly efficient on-target delivery could be used in co-cultures or organoids to deliver transgenes to specific cell types pAG may not be suitable due to the competition of antibodies in serum but the scFv and SNAP strategies can be deployed in these situations although reducing the off-target uptake of DIRECTED particles by macrophages will be required to achieve efficient delivery in vivo our in vivo experiments show lower delivery efficiency to monocytes and B cells for CD5-DIRECTED particles when compared to CreVLPs pseudotyped with wild-type VSV-G the achieved transduction efficiency does represent a biologically meaningful proportion of T cells which in the case of in vivo CAR-T therapy would further expand upon antigen encounter and could enable the generation of CAR-T cells in vivo using a single-step protocol we identified several principles for choosing cell surface receptors that are compatible with DIRECTED particles: (1) higher cell surface expression level of a receptor favors more effective delivery; (2) the targeted receptor needs to undergo endocytosis to enable the DIRECTED particles to get to the endosomal compartment and experience low pH where the fusogen becomes activated; and (3) to increase the set of potential receptors the SNAP strategy affords the most flexibility presumably due to the reduced complexity of virus-receptor binding as the targeting antibody and virus are covalently linked the use of NHS ester chemistry for the functionalization of antibodies with benzylguanine necessitates individual optimization of the labeling step Future research will be needed to determine the quantitative relationship between the biophysical properties of receptors endogenous ligands or agonistic antibodies that increase receptor internalization may boost delivery efficiency Future studies to optimize the efficiency of antibody-tethering on the virion surface evaluate additional mutations that could help to reduce off-target delivery and identify receptors that allow specific targeting of unique cell types in vitro and in vivo will further advance the DIRECTED technology and help to address the critical unmet need for cell type-specific delivery of molecular cargoes the presented data suggest that SNAP-DIRECTED particles allow the integration of multiple targeting ligands This approach potentially enables the manipulation of cell fate and cell state of specific target cells in situ and may allow for the delivery of therapeutic payloads such as replacement genes or genome editing tools both in a transient and integrating manner All experiments were performed in compliance with all relevant ethical regulations as approved by the Institutional Biosafety Committee (IBC) of the Broad Institute (Protocol # IBC-2017-00146) Gibson assembly was carried out using Gibson assembly Master Mix (NEB # E2611L) in 10-µl reaction volume using 50 ng of backbone per reaction and a threefold molar excess of other fragments Reactions were incubated for at least 1 h at 50 °C before 2 µl were transformed into chemically competent Stbl3 cells (Thermo Fisher Scientific # C737303) and plated on agar plates containing the corresponding antibiotic for selection (Concentrations: Ampicillin 100 µg/ml; Kanamycin 50 µg/ml) The next day single colonies were picked into TB containing the corresponding antibiotic (Concentrations: Ampicillin 100 µg/ml; Kanamycin 50 µg/ml) and allowed to grow overnight before plasmids were extracted by miniprep (Qiagen All plasmid sequences were subsequently confirmed by sequencing All antibodies used in this study can be found in Supplementary Data 3 BG-labeled antibodies were used at the same dilutions as unlabeled antibodies for DIRECTED tropism All cell lines were confirmed to be mycoplasma free All cells were cultured in a humidified incubator at 37 °C and 5% CO2 FBS was heat inactivated before use for 30 min at 56 °C Buffy coat preparations containing peripheral blood mononuclear cells (PBMCs) were diluted 1:1 in preparation buffer (PBS with 0.1% BSA and 2 mM EDTA) and mononuclear cell separation was performed by density centrifugation (Ficoll-Paque Premium 1.084 Cytiva) with diluted peripheral blood cells (centrifugation 30 min without brakes 600 × g) in 50-mL Leucosep(R) tubes with porous barrier (Greiner Bio Cells were carefully aspirated and washed with preparation buffer (centrifugation 5 min at 500 × g) Red blood cells were lysed using ACK Lysis Buffer (Gibco and cells were washed (centrifugation 5 min 500 × g) and resuspended in preparation buffer 1 × 10e7 cells were frozen per aliquot in 90% FCS (VWR # 97068-085) supplemented with 10% DMSO (Sigma-Aldrich # D2438-5X10ML) and stored in liquid nitrogen until further use HEK293FT cells were seeded at a cell density of 8.3E4 cells per cm2 in DMEM with 10% FBS and 1x Pen/Strep cells were transfected with a mix of envelope plasmid (27%) and a plasmid encoding the desired transfer genome (44%) using PEI (Polysciences the amount of envelope plasmid was split to account for the envelope and the helper envelope (i.e. or scFv) without changing the amounts of the lentiviral helper plasmid (29%) or the transfer genome (44%) the media was exchanged for fresh DMEM with 10% FBS and Pen/Strep and filtered through a 0.45-µm filter (Millipore Sigma the viral supernatant was concentrated by ultracentrifugation at 26,000 × rpm (~90,000 × g at raverage) for 2 h at 18 °C over 2 ml of a 20% sucrose cushion in an AH-629 or SW 28 rotor The pellet was then resuspended in 1/50th to 1/200th of the initial volume of PBS before being used for titration To remove excess antibody from antibody-BG SNAP click reactions (performed with 250× concentrated virus) the mixture was resuspended in 25 ml of PBS before being concentrated by ultracentrifugation at 26,000 × rpm (~90,000 × g at raverage) for 2 h at 18 °C over 2 ml of a 20% sucrose cushion in an AH-629 or SW 28 rotor To determine the number of viral genomes in lentiviral vector preps the supernatant or concentrated viral stock was incubated with benzonase (Sigma-Aldrich # E1014-25KU) to remove free nucleic acids such as plasmids and RNA resulting from cell death by incubating up to 100 µl of viral vector suspension with 0.5 µl of benzonase and 0.4 µl 1 M magnesium chloride (Thermo Fisher Scientific If the volume of viral vector suspension was lower than 100 µl the rest of the volume was filled up with ultrapure water (Thermo Fisher Scientific the reaction was incubated for at least 2 h at 37 °C before viral RNA was extracted using a viral RNA extraction kit (Zymo # R1041 or R1035) according to the manufacturer’s instructions The viral genome copy number was determined using an RT-qPCR kit (Lenti-X Quant All reactions were run on a BioRad CFX Opus 384 RT-PCR machine (Cat Target cells were seeded at 5000 or 10,000 cells per well of a 96-well plate and directly incubated with viral particles at the indicated multiplicities of infection (MOI) in their cognate media (in the presence of FBS) and cells were incubated for at least another 72 h before analysis 8 µg/ml of Polybrene (Santa Cruz Biotechnology Cat # sc-134220) or 10 µg/ml of vectofusin (Miltenyi Biotec Spinoculations were performed at 1500 × g for 90 min at 33 °C the indicated antibody amounts were used unless otherwise stated For the in vitro transduction of Kasumi-1 cells lentiviral particles were co-incubated with αCD117 or αCD20 antibody for 30 min at room temperature before excess antibody was removed using Amicon Ultra-0.5 Centrifugal Filter Units (Millipore Sigma # UFC510096) by washing three times with excess PBS 150,000 cells were seeded per well of a 96-well plate in TCM with 50U/ml rhIL-2 (PeproTech Cat DIRECTED particles were prepared by incubating 80 μl of 100x concentrated VSV-Gdm+SNAP with either 6 μg αCD3-BG (VSV-Gdm+αCD3) or 2.5 μg αCD19-BG and 0.5 μg MegaCD40L-BG (VSV-Gdm-Bcell) overnight at 4 °C For DIRECTED particles with expanded tropism 80 μl of 100x concentrated VSV-G+SNAP were incubated with 6μg αCD3 overnight at 4 °C The next day unreacted antibody was removed by harvesting the functionalized particles by ultracentrifugation through a 20% sucrose cushion and the pellet was resuspended in 80 μl of PBS VSV-Gdm+SNAP and VSV-G were used without additional purification media on cells was exchanged with fresh TCM with 50U/ml rhIL-2 8 µg/ml Polybrene (Santa Cruz Biotechnology Cat # sc-134220) and 2 μl per well of Fc block (BioLegend Cat Fifteen minutes later virus was added at an MOI of 500 (for VSV-Gdm+SNAP and VSV-G) or 250 (for VSV-Gdm-Bcell VSV-Gdm-αCD3) in two wells each and cells were spinoculated for 1.5 h at 1000 × g and 33 °C media was exchanged with fresh TCM (supplemented with 50U/ml rhIL-2) and cells were incubated for an additional 5 days with a media change at day 3 post-transduction cells were collected and half of the cells were used for analysis by flow cytometry while the other half was used to stimulate T cell outgrowth by adding αCD3 and αCD28 T cell activator beads (Thermo Scientific Cat # 11131D) in fresh T cell media supplemented with 50U/ml rhIL-2 and cells were debeaded and analyzed by flow cytometry on day 14 post-infection DIRECTED particles were prepared by incubating 80 μl of 100x concentrated VSV-Gdm+SNAP or VSV+SNAP with 2.5 μg αCD19-BG and 0.5 μg MegaCD40L-BG (VSV-Gdm-Bcell and the pellet was resuspended in 80 ml of PBS and VSV-G were used without additional purification 100,000 freshly thawed PBMCs were seeded per well of a 96-well plate in TCM with 50U/ml rhIL-2 and 8 µg/ml Polybrene and incubated with viral vectors at an MOI of 3500 for VSV-Gdm or VSV-G and an MOI of 1750 for VSV-Gdm-Bcell or VSV-G-Bcell in two wells each and cells were spinoculated for 1.5 h at 1000 × g and 33 °C 2 μl of αCD3 and αCD28 T cell activator beads were added per well media was exchanged with fresh TCM (supplemented with 50U/ml rhIL-2 and cells were incubated for an additional 5 days with a media change at day 3 post-transduction Fresh blood from two human donors was obtained from Research Blood Components in CPT-NaCitrate tubes 100 μl of blood was transferred to a 96-well deep-well plate and incubated with 2 μl of Fc block (BioLegend Cat viral vectors (prepared as described in “Transduction of PBMCs with T cell and B cell targeting DIRECTED lentiviral vectors”) were added at an MOI of 1000 for VSV-Gdm and VSV-G or at an MOI of 500 for VSV-Gdm-Tcell and VSV-Gdm-αCD3 MOIs were calculated based on an assumption of 5000 leukocytes per μl blood The cells were incubated for 6 h shaking in a humidified incubator at 37 °C and 5% CO2 red blood cells were lysed using RBC lysis buffer (BioLegend monocytes and leukocytes) were transferred to TCM (supplemented with 50U/ml rhIL-2 and 2 μl of T cell activator beads were added per well The next day media was replaced with fresh TCM (supplemented with 50U/ml rhIL-2 and cells were incubated for another 5 days with media changes every other day and half of the cells were incubated with fresh T cell activator beads (2 μl per well) whereas the other half was used for analysis Media was exchanged every other day on the cultured cells washed and analyzed on day 14 post-transfection cells were seeded at 50,000 cells/well in 6-well plates and transduced with 1 ml of viral supernatant antibiotic selection was started using 1 µg/ml Puromycin (Thermo Fisher Scientific Cells were not maintained under Puromycin selection for in vitro infection experiments To establish the Jurkat+surface-HA cell line Jurkat E6 wild-type cells were seeded at 5E5 cells/ml and incubated with 3 ml of viral supernatant in 10 ml total media and the next day antibiotic selection was started using 1 µg/ml Puromycin (Thermo Fisher Scientific To establish a Jurkat E6 Cre Reporter cell line 5E5 cells/ml wild-type Jurkat E6 cells were transduced with 3 ml lentiviral supernatant containing a Cre-responsive GFP reporter (pBS-loxP-GFP) in 10 ml total media The next day media was exchanged for fresh media and the day after antibiotic selection with Blasticidin S HCl (Thermo Fisher Scientific and surviving cells were plated by limited dilution into 96-well plates to grow out clones where one plate was tested using 100 µl of VSV-G CreVLPs to identify clones that show high switching frequency The clone showing the highest switching capacity (~90%) was expanded and used for in vitro DIRECTED-CreVLP experiments wild-type cells were seeded at 50,000 cells/well in 6-well plates before treating them with 1 ml of VSV-G pseudotyped eVLPs delivering Cas9-RNPs with a B2M targeting sgRNA The cells were subsequently expanded and evaluated for the loss of B2M surface expression by flow cytometry HEK293FT ΔB2M cells were stained with αB2M-FITC antibody (BioLegend) at a dilution of 1:100 for 45 min at 4 °C cells were washed twice and sorted for B2M negative cells on a Sony SH800 sorter Jurkat E6 + surface-HA cells were stained with aHA-PB450 (1:100) for 45 min at 4 °C washed twice with Flow buffer (PBS+2% FBS+5 mM EDTA) and sorted into four bins of different expression levels on a Sony MA900 sorter Antibodies were desalted using Zebaspin Desalting Columns (7 kDa MWCO # 89882) and diluted to a concentration of 0.5 mg/ml before incubation with a 40-fold molar excess of BG-GLA-NHS (New England Biolabs or 80-fold molar excess of BG-GLA-NHS was added to the antibodies The reaction was allowed to continue for 30 min at room temperature before it was stopped by removing the unreacted NHS-BG substrate using Zebaspin Desalting Columns (7 kDa MWCO) # ALX-522-110-C010) was labeled with a 10-fold molar excess of NHS-GLA-BG due to the high number of surface exposed lysines # 302202) were each labeled with a 40-fold molar excess of NHS-GLA-BG # 302933) was labeled with a 20-fold molar excess of NHS-GLA-BG due to significant precipitation at 40-fold molar excess All BG-labeled antibodies were resuspended in PBS and their concentration was determined using the IgG function on a Nanodrop 2000 device BG-antibodies were incubated with a twofold molar excess of purified SNAP protein (NEB The samples were mixed with Bolt Sample Buffer (Thermo Fisher Scientific # B0008) supplemented with reducing agent (Thermo Fisher Scientific # B0009) and boiled for 5 min at 95 °C before loading onto Bolt 4-12% Bis-Tris gradient gels (Thermo Fisher Scientific # NW04125BOX) before proceeding to western blot analysis with an additional incubation of cells with an antibody after the first wash in the Flow Buffer The used antibodies and corresponding dilutions are listed in the table of antibodies unstained and single-stained samples were acquired and used to calculate the necessary compensation in FlowJo or murine splenocytes cell suspensions were incubated with Fc block (for human: BioLegend Cat # 553142) for 15 min at room temperature before proceeding with the antibody stainings as described above fixable viability dye 780 (Thermo Scientific Cat # 65-0865-18) was used and staining was performed in PBS 8E5 HEK293FT cells were seeded in 6-well plates the day before transfection The next day cells were transfected with pHCMV-VSVgSS-pAG-TM (3 µg DNA) cells were incubated with a mouse-IgG-FITC antibody (BioLegend # 407406) at a dilution of 1:100 for 15 min before washing the cells once in PBS and immediately imaging the cells on an EVOS M5000 system Protein structure was predicted using AlphaFold2 via the Google Colab repository (https://github.com/sokrypton/ColabFold/ and https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb) The algorithm was run using the protein sequences with the following parameters deviating from the standard options: Sampling options; num_models: 3; max_recycles: 12 The VSV-G competitor (recombinant diCR2 protein) was produced by transfection of 30 ml Expi-HEK suspension cells with pHCMV-diCR2 using a commercial transfection kit (Thermo Fisher Scientific # A14524) according to the manufacturer’s instructions Cells were allowed to produce protein for 5 days before media was collected filtered through a 0.45-µm filter (Millipore Sigma and crude supernatant was stored at 4 °C until use (up to 1 week) HEK293FT cells were seeded at a cell density of 8.3E4 cells per cm2 in DMEM with 10% FBS and Pen/Strep cells were transfected using PEI with the following plasmids: for eVLPs retroviral helper plasmid containing MLV gagpol (35%) and plasmid containing an sgRNA expression cassette (45%); for CreVLPs containing Cre recombinase retroviral helper plasmid containing MLV gagpol (60%) To generate DIRECTED-eVLPs or DIRECTED-CreVLPs the amount of envelope plasmid was split to account for the envelope (i.e. VSV-G K47Q/R354Q) and the helper envelope (i.e. or scFv) without changing the amounts of the other plasmids the viral supernatant was concentrated by ultracentrifugation at 26,000×rpm (~90,000 × g at raverage) for 2 h at 18 °C over 2 ml of a 20% sucrose cushion in an AH-629 or SW 28 rotor The pellet was then resuspended in PBS at 1/50th to 1/200th of the initial volume Protein samples were diluted with 4x Bolt Sample Buffer (Thermo Fisher Scientific # B0009) before boiling at 95 °C for 5 min samples were allowed to cool to room temperature before being loaded onto Bolt 4-12% Bis-Tris gradient gels (Thermo Fisher Scientific Gels were run at 200 V for 24 min in an MES running buffer (Thermo Fisher Scientific gels were rinsed in distilled water before transfer onto a PVDF membrane (Thermo Fisher Scientific 7 min) on an iBlot2 device (Thermo Fisher Scientific the PVDF membrane was blocked using TBS-T Blocking Buffer (Licor # 927-60001) for at least 30 min at room temperature before the addition of primary antibody and incubation at 4 °C overnight on a shaker device blots were rinsed twice with TBS-T (Thermo Fisher Scientific # 28360) and then incubated with secondary fluorescently labeled antibody (LiCor or 926-32211) at a 1:2500 to 1:10,000 dilution for at least 45 min at room temperature blots were washed three times with PBS-T for at least 5 min per wash before being imaged on a BioRad ChemiDoc Imaging System Images were exported as raw tif files for all subsequent analyses All densitometry analyses were performed in Fiji software different amounts of purified Cre recombinase (NEB # M0298S) or dilutions of CreVLPs were incubated with pLox2+ (25 ng/reaction # N0416SVIAL) in Cre reaction buffer supplemented with 0.25% NP-40 (Thermo Fisher Scientific # 85124) in a total reaction volume of 5 µl To test the background levels of Cre recombinase in CreVLP preparation Reactions were incubated for 45 min at 37 °C before heat inaction (70°C samples were diluted 1:10 in ultrapure water and reactions were run on an Agilent TapeStation device using D5000 ScreenTape (Agilent HEK293FT + surface-Spot were seeded at 10,000 cells per well of a 96-well plate in DMEM+10%FBS+Pen/Strep before transduction with lentiviral vectors in the presence or absence of antibody at different MOIs washed once in PBS and stained with Fixable Viability Dye eFluor 780 (Thermo Scientific cells were washed and stained with Annexin V-PB450 (BioLegend # 640926) for 15 min at room temperature in Annexin V binding buffer according to the manufacturer’s instructions Thereafter an equal volume of Annexin V binding buffer was added and cells were immediately analyzed by flow cytometry on a Beckman Coulter CytoFLEX S device Jurkat E6 cells were seeded at 25,000 cells per well in 50 µl per well in a 96-well plate in RPMI+10%FBS+Pen/Strep + 8 µg/ml Polybrene (Santa Cruz Biotechnology Cat the cells were transduced with lentiviral vectors at the indicated MOIs αCD3-SNAP-DIRECTED particles were purified by ultracentrifugation through a sucrose cushion as described above to remove excess unreacted antibody and cells were resuspended in 100 µl RPMI+10%FBS+Pen/Strep cells were stained as described above and immediately analyzed by flow cytometry on a Beckman Coulter CytoFLEX S device All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute (Protocol ID 0017-09-14-2) Animal maintenance complied with all relevant ethical regulations and were consistent with local state and federal regulations as applicable including the National Institutes of Health Guide for the Care and Use of Laboratory Animals Animals were kept on a 12-h light/dark cycle between 68 °F and 79 °F and 30–70% humidity Mice had ad libitum access to standard rodent diet and water 4–6-week-old female Ai14 mice (N = 17) were ordered from Jackson Laboratories (Strain # 007914) and injected with CreVLPs via tail vein injection Each animal received ~50 µl of the purified CreVLP suspension Four animals were injected with VSV-Gdm+SNAP+αCD5-BG CreVLPs (αCD5) five animals with VSV-Gdm+SNAP CreVLPs (no Antibody) and six animals were injected with VSV-G CreVLPs (VSV-G) animals were euthanized by CO2 asphyxiation and cervical dislocation before perfusion with 25 ml PBS by cardiac puncture Half of the spleens of the animals were used to isolate cells as follows: Spleens were minced through a 70-µm cell strainer and the cell strainers were washed with 15 ml of PBS then cells were collected by centrifugation (500 × g The cell pellets were resuspended in 10 ml of RBC lysis buffer (BioLegend Cat and RBC lysis was performed for 5 min at room temperature before the reaction was quenched with 10 ml of PBS Cells were pelleted and washed twice before proceeding with the blocking of Fc receptors using Fc block (BD Biosciences 100-µl aliquots of the cell suspension were stained with 2 μl of αCD3-BV421 (BioLegend Cat # 101236) and fixable viability dye eFluor 780 (1:1000 # 65-0865-18) in individual wells of a 96-well plate for 30 min at 4 °C 100 µl of fresh PBS was added to the wells cells were collected by centrifugation and washed twice with 200 µl PBS cells were resuspended in 100 µl Flow Buffer (PBS+2% FBS+5 mM EDTA) and analyzed on a Beckman Coulter CytoFLEX S device two-sided Welch’s t-test with BH correction All reported test values are provided in the Source Data Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Therapeutic in vivo delivery of gene editing agents Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector Genome editing in primary cells and in vivo using viral-derived nanoblades loaded with Cas9-sgRNA ribonucleoproteins Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery Pseudotyped lentiviral vectors: one vector Altering the tropism of lentiviral vectors through pseudotyping Structural insights into membrane fusion mediated by convergent small fusogens LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein The effects of N-terminal insertion into VSV-G of an scFv peptide Antigen identification and high-throughput interaction mapping by reprogramming viral entry Engineered retroviruses map ligand–receptor interactions Targeted and highly efficient gene transfer into CD4+ cells by a recombinant human immunodeficiency virus retroviral vector Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs Altering entry site preference of lentiviral vectors into neuronal cells by pseudotyping with envelope glycoproteins Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A Antibody-directed targeting of retroviral vectors via cell surface antigens Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells Receptor-targeted Nipah virus glycoproteins improve cell-type selective gene delivery and reveal a preference for membrane-proximal cell attachment An optimized measles virus glycoprotein-pseudotyped lentiviral vector production system to promote efficient transduction of human primary B cells Identification of a pH-sensitive switch in VSV-G and a crystal structure of the G pre-fusion state highlight the VSV-G structural transition pathway Engineered cell entry links receptor biology with single-cell genomics Generation of high-titer pseudotyped lentiviral vectors Distinct requirements for HIV-1 accessory proteins during cell coculture and cell-free infection High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes Infectivity enhancement of different HIV-1-based lentiviral pseudotypes in presence of the cationic amphipathic peptide LAH4-L1 The physico-chemical factors that govern retrovirus-mediated gene transfer Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo Platelet-derived growth factor receptor beta activates Abl2 via direct binding and phosphorylation VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses Use of heterologous vesiculovirus G proteins circumvents the humoral anti-envelope immunity in lentivector-based in vivo gene delivery Cocal-pseudotyped lentiviral vectors resist inactivation by human serum and efficiently transduce primate hematopoietic repopulating cells Structures of human-infecting Thogotovirus fusogens support a common ancestor with insect baculovirus Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state Evidence that rabies virus forms different kinds of fusion machines with different pH thresholds for fusion Mutations in the glycoprotein of viral haemorrhagic septicaemia virus that affect virulence for fish and the pH threshold for membrane fusion Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes Highly accurate protein structure prediction with AlphaFold ColabFold: making protein folding accessible to all Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64 A tool with many applications: vesicular stomatitis virus in research and medicine Programmable extracellular vesicles for macromolecule delivery and genome modifications CAR T cells produced in vivo to treat cardiac injury Kupffer cells and not liver sinusoidal endothelial cells prevent lentiviral transduction of hepatocytes The toxicity of metallic nanoparticles on liver: the subcellular damages Four challenges to CAR T cells breaking the glass ceiling Optimizing commercial manufacturing of tisagenlecleucel for patients in the US: a 4-year experiential journey Combining T-cell–specific activation and in vivo gene delivery through CD3-targeted lentiviral vectors Assessment of SARS-CoV-2 specific CD4+ and CD8+ T cell responses using MHC class I and II tetramers Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection SARS-CoV-2 hijacks folate and one-carbon metabolism for viral replication Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo Lineage-specific laminar organization of cortical GABAergic interneurons Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation Optimized large-scale production of high titer lentivirus vector pseudotypes high-affinity streptavidin monomer for protein labeling and monovalent biotin detection Multiple input sensing and signal integration using a split Cas12a system A SARS-CoV-2 protein interaction map reveals targets for drug repurposing Dynamics of epigenetic regulation at the single-cell level Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex Improved vectors and genome-wide libraries for CRISPR screening The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines Download references Suberski for help with plasmid sequencing; and all members of the Zhang lab for support and discussions was supported by fellowships from the Swiss National Science Foundation (P400PB_199261 and P2ELP3_187926) is supported by the Howard Hughes Medical Institute; Milky Way Research Foundation; K Tan Molecular Therapeutics Center at MIT; K Lisa Yang Brain–Body Center at MIT; Broad Institute Programmable Therapeutics Gift Donors; The Pershing Square Foundation and Neri Oxman; James and Patricia Poitras; BT Charitable Foundation; Asness Family Foundation; Kenneth C the Phillips family; David Cheng; and Robert Metcalfe Department of Brain and Cognitive Sciences Department of Electrical Engineering and Computer Science conceived the project and designed experiments carried out the computational analysis of fusogens supervised the research and experimental design with support from R.M wrote the manuscript with input from all authors are coinventors on a pending patent application (PCT/US22/52871) related to this work filed by the Broad Institute and MIT reports receiving speaker’s bureau honoraria from Pfizer is a scientific advisor and cofounder of Editas Medicine The remaining authors declare no competing interests Nature Communications thanks Enrico Mastrobattista Emiliano Ricci and the other anonymous reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Reprints and permissions Download citation DOI: https://doi.org/10.1038/s41467-023-40788-8 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Diálogo Américas Puerto Rico and the Dominican Republic are joining efforts to stop transnational criminal groups that attempt to move drugs to the United States or Europe on Caribbean waters together they seized 2,723 kilograms of cocaine at different locations in their territorial seas the Puerto Rico Police Joint Forces of Rapid Action (FURA in Spanish) seized 450 kg of cocaine and detained two people 3 nautical miles off Punta Figuras Authorities found the drug in 15 bales on a vessel sailing with its lights off both Puerto Rico and the Dominican Republic hit narcotrafficking structures Puerto Rico Police’s Intelligence Division and Maritime Division seized 1,600 kg of cocaine and detained a Dominican and a Venezuelan national at El Cocal beach with bundles that they suspected were drugs so they proceeded to inspect it and later confirmed that it was cocaine,” the Dominican website El Caribe reported the Dominican National Drug Control Directorate (DNCD in Spanish) seized 515 kg of cocaine in an interdiction operation carried out at a home in the Los Cacicazgos sector “The residence was used by the criminal network as a center for the collection and preparation of drugs for export to Europe through the country’s ports,” the DNCD said in a statement the DNCD and the Dominican Navy dismantled a criminal organization that used speedboats to smuggle drugs between the Dominican Republic and Puerto Rico Authorities seized 158 kg of cocaine and detained 16 people five firearms (two pistols and three shotguns) For more on security and defense issues around the globe CoCal Landscape and its owner Jesus "Chuy" Medrano were recently profiled in The Washington Post Tracy Jan reported the story in Colorado and in Mexico and tells Colorado Matters that CoCal lost about $1.7 million in business this year because it didn't get as many H-2B visas as the company wanted early in the season Jan says landscaping is the industry that relies most on H-2Bs; others include ski resorts but they arrived late in the summer season Colorado Postcards are snapshots of our colorful state in sound. They give brief insights into our people and places, our flora and fauna, and our past and present, from every corner of Colorado. Listen now. © 2025 Colorado Public Radio. All Rights Reserved. Privacy Policy Become a navigator of the tobacco cessation process through this free Read our simple and effective tips for protecting you and your family from the dangers of air pollution Our key findings add to the evidence that a changing climate is making it harder to protect human health Share your voice and advocate for policies that will save lives Step up for lung health by participating in a Fight For Air Climb event near you Matching gifts are a great way to stretch your donation Pneumococcal pneumonia is a potentially serious bacterial lung disease you shouldn't ignore It can disrupt your life for weeks and even land you in the hospital Many people think pneumococcal pneumonia is a cold or the flu Pneumococcal pneumonia is caused by bacteria that live in the upper respiratory tract and it can spread to others through coughing or close contact Severe cases of pneumococcal pneumonia can lead to hospitalization and can even be life threatening Pneumococcal pneumonia can strike any time so now is the time to talk to a healthcare provider about vaccination You can't get pneumococcal pneumonia from getting vaccinated because pneumococcal vaccines do not contain live bacteria.  The CDC recommends adults 19-49 with certain chronic health conditions and all adults 50 or older to talk to a healthcare provider about pneumococcal vaccination Pneumococcal vaccines are available today at many doctor's offices local pharmacies and at some local health departments Pneumococcal vaccination helps protect against two additional serious infections, meningitis, an infection of the lining of the brain and spinal cord and bacteremia The immune system naturally weakens with age being 50 or older puts you at increased risk for pneumococcal pneumonia Other factors like certain chronic health conditions further increase pneumococcal pneumonia risk in adults 19 and older compared with healthy adults in the same age range With each chronic condition your risk increases further Having asthma can sometimes restrict me from doing things I love like riding my bike hiking or playing frisbee with my border collies I really try to prioritize taking proactive steps to help keep myself healthy I’ve also learned that I am at increased risk of getting pneumococcal pneumonia because I have asthma I thought I was too young to get a pneumococcal pneumonia vaccination but when my pulmonologist recommended it I like knowing it’s helping to protect me against this potentially serious lung disease It’s important to be an active part of your healthcare team If you are 65 years of age or older or 19 or older and have certain chronic health conditions like asthma ask your healthcare provider if pneumococcal pneumonia vaccination is right for you Adults 19 or older with chronic health conditions such as COPD and chronic heart disease face greater risk for pneumococcal pneumonia Adults 50+ are at 6.4x greater risk for pneumococcal pneumonia compared to adults aged 18–49 Adults 65+ are over 10x more likely to be hospitalized with pneumococcal pneumonia than adults aged 18-49 Answer these three questions to learn more about your risk chronic health conditions and lifestyle factors may increase your risk for getting pneumococcal pneumonia If you’re 19 or older with a chronic health condition or 50 or older Even if you are active and otherwise healthy an important thing to remember is: If you're 50 or over our immune systems aren’t as effective at fighting off infections and helping to protect us from vaccine-preventable disease certain chronic health conditions can make the body more vulnerable to serious illnesses such as pneumococcal pneumonia and smoking history can contribute to an increased risk for getting pneumococcal pneumonia Talk to your healthcare provider about your risk factors and how vaccines may help protect you Certain Chronic Conditions Increase Risk in Adults Aged 50+* Your age and health conditions may put you at increased risk for getting pneumococcal pneumonia Your age and smoking history may put you at increased risk for getting pneumococcal pneumonia Adults aged 50 or older are at increased risk due to age alone Although you haven't selected any health conditions or behavior choices that would increase your risk age is still a key risk factor for pneumococcal pneumonia due in large part to the natural Although your age and health conditions are not risk factors your smoking history may increase your risk for getting pneumococcal pneumonia Although your age and behaviors are not risk factors you have health conditions that may increase your risk for getting pneumococcal pneumonia Certain Chronic Conditions Increase Risk in Adults Aged 19-49* the risk of pneumococcal pneumonia increases with the presence of certain chronic conditions Although you're not currently in an elevated-risk age group your health conditions and smoking history may put you at increased risk for pneumococcal pneumonia Your answers to these three questions indicate that you are not in an elevated risk group it's always important to recognize your risks as you age Developed by the American Lung Association in partnership with Pfizer Inc.The health information contained herein is provided for educational purposes only and is not intended to replace discussions with a healthcare provider All decisions regarding patient care must be made with a healthcare provider considering the unique characteristics of the patient The American Lung Association does not endorse products Your gift will help people with asthma breathe easier—at school Donate now Join over 700,000 people who receive the latest news about lung health The American Lung Association is a 501(c)(3) charitable organization This website uses cookies to improve content delivery Talk to our lung health experts at the American Lung Association Our service is free and we are here to help you This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Lentiviral vectors (LVs) used to transfect cells for gene expression can withstand shear rates that are twice as high as those normally used and still maintain comparable productivity That insight from researchers at University College London removes some of the hurdles that have constrained gene therapies from advancing in clinical trials Reporting in Biotechnology & Bioengineering evaluated the manufacturing processes various vectors experience and the conditions that lead to vector loss They assessed a three-plasmid system and a four-plasmid system (both pseudotyped with the RDpro and VSV-G envelope proteins) in terms of process shear and the impact of chromatography buffers on the lentiviral vectors To evaluate shear forces during diafiltration the researchers used an ultra-scale-down (USD) tangential flow filtration (TFF) mimic Many researchers perform TFF at flow rates of less than 500 s−1 and shear rates of less than 2000 s−1 began with a flow rate of approximately 800 s−1 and a shear rate of 1000 RPM the overall functional titers appear to increase with higher shear VSV-g envelope protein has a mild decrease from initial to 3,000 RPM but titers from this point onwards are maintained,” the authors noted maintains high permeate flux rates and reduces membrane fouling clarification by membrane filtration had the greatest effect on the lentiviral vectors They found “a weak correlation of lower functional titers with pump flow.” Of the four filter materials tested—polyvinylidene difluoride (PVDF) mixed cellulose esters (MCE) and nylon—the PVDF filters consistently recovered more lentiviral vector than the others have demonstrated comparable results to the USD system,” Rayat’s team reported “These results reinforce the need to…test new LV products in terms of their robustness for manufacture,” and to apply rational bioprocess development to lentiviral production may be like those experienced by the rational design of recombinant protein production Copyright © 2025 Sage Publications or its affiliates including those for text and data mining and training of large language models As a designated “sanctuary city,” Denver does not cooperate with the federal government on immigration enforcement “We’re not going to sell out those values to anyone,” Johnston added “We’re not going to be bullied into changing them.” The response from Tom Homan, a hardliner Trump appointed as his “border czar” after winning the election Appearing alongside Texas Governor Greg Abbott — a staunch ally of Trumpism — on Sean Hannity’s Fox News show Homan warned that he would not let Johnston hinder his plans we agree on one thing; he’s willing to go to jail “It’s a felony if you knowingly harbor and conceal an illegal alien from immigration authorities It’s also a felony to impede a federal law enforcement officer This standoff is the latest chapter in an escalating battle Since Trump’s election victory less than a month ago Democratic governors and mayors nationwide have vowed to resist cooperating with his immigration agenda and other members of the president-elect’s circle have issued stark warnings to the officials who have spoken out against the Republican’s plan to carry out “the largest deportation operation in American history.” Threats include imprisonment as suggested by Homan in Johnston’s case; severe cuts to federal funding for defiant states and cities which could cripple local budgets; and the deployment of thousands of federal agents to enforce immigration laws directly in noncompliant communities Mayor Johnston reiterated his stance in a more measured statement we respect the law and enforce it without fear or favor across every inch of our city If Donald Trump tries to break the law and abuse his power and we will do everything in our power to protect those who live here We are considering a number of options to strengthen protections for all our residents and we continue to provide education about the rights of our immigrant community so they can best protect themselves from any unlawful actions,” the statement said A spokesperson for the mayor added that Denver would withhold further comment on a federal policy that has yet to be detailed However, Denver is far from the only city resisting the Republican’s plans Los Angeles officially declared itself a “sanctuary city.” Although California’s largest city has operated under sanctuary policies for decades this legal codification strengthens its stance by explicitly prohibiting both direct and indirect sharing of data with federal immigration authorities This move by Los Angeles sparked a strong response from Homan “If I’ve got to send twice as many officers to L.A then that’s what we’re going to do,” he said This is going to happen with or without you.” Similar tensions were seen on the other side of the country has vowed to fight the mass deportation effort “We are not cooperating with those efforts that actually threaten the safety of everyone by causing widespread fear and having large-scale economic impact,” she said Massachusetts’s largest city has reinforced its commitment through the Trust Act which prohibits police from stopping or arresting individuals solely for immigration enforcement purposes in keeping with his and Trump’s combative tone lashed out at Wu during an interview on Newsmax describing her as “not very smart” and reiterating that harboring undocumented immigrants is a federal crime “Either she helps us [or] she gets the hell out of the way because we’re going to do it,” Homan said “To those people who say they’re going to stop us from what they’re doing they will not [...] You’re not going to stop us The consequences Homan alluded to include cuts in federal funding to states and cities a common leverage tactic in negotiations between Washington and local governments I guarantee President Trump will do that.” The potential withholding of federal funds could have significant financial consequences California received approximately $163 billion in federal grants While these figures were unusually high due to pandemic-related assistance federal funding remains a critical revenue source for states ranking as the second-largest revenue stream in fiscal year 2022 Trump’s deportation plan, however, is fraught with uncertainty. While the president-elect and his team have made their goals clear, the mechanisms for executing them remain ambiguous, especially given the resistance from states and cities refusing to cooperate. States like Texas and Florida have pledged full support for Trump’s immigration agenda but a significant number of other jurisdictions are pushing back intensifying the already heated political climate But with control over all three branches of government Trump and his team appear determined to press forward with their agenda Sign up for our weekly newsletter to get more English-language news coverage from EL PAÍS USA Edition ¿Quieres añadir otro usuario a tu suscripción ¿Por qué estás viendo esto? cambia tu suscripción a la modalidad Premium Cada uno accederá con su propia cuenta de email lo que os permitirá personalizar vuestra experiencia en EL PAÍS ¿Tienes una suscripción de empresa? Accede aquí para contratar más cuentas En el caso de no saber quién está usando tu cuenta, te recomendamos cambiar tu contraseña aquí. Si decides continuar compartiendo tu cuenta, este mensaje se mostrará en tu dispositivo y en el de la otra persona que está usando tu cuenta de forma indefinida, afectando a tu experiencia de lectura. Puedes consultar aquí los términos y condiciones de la suscripción digital. .st1{fill-rule:evenodd;clip-rule:evenodd;fill:#2a2a2a}By Gary Ridley | gridley@mlive.comFLINT MI -- A Flushing couple is out on bond after the federal government claims that they purposely hid profits from millions in foreign investments from tax officials are accused of purposely failing to claim their foreign bank accounts that were funded by profits from the millions the couple invested in Costa Rican property on their federal tax returns which was unsealed July 10 in Flint federal district court the couple has an ownership interest in two condominiums and Gray owns and operates Hotel Cocal and Casino Gray explained in an email he sent to a federal informant that he does not report capital gains taxes on his condominium activities in the United States you have capital gains and would have to pay tax in Costa Rica no capital gains,” Gray wrote in the email A revolving credit application uncovered by the government during its investigation shows that the condominium assets owned by the couple are valued at $5.68 million and the hotel and casino is valued at $2.86 million During a face-to-face meeting with Gray in May 2011 an undercover federal agent posing as an investor asked Gray how to report property owned outside of the United States on a federal tax return “You don’t,” Gray told the agent according to the complaint Gray allegedly told the agent that he began moving his bank accounts from Panama to Costa Rica after the U.S signed a treaty with Panama making tax information for U.S citizens with more than $10,000 in Panamanian bank accounts available to the federal government Gray told the agent that he would then place all the profits from the condominiums into the foreign accounts and use a bank-issued debit card so the money wouldn’t have to be moved through the U.S a search warrant was executed at the couple’s Flushing home business records and copies of emails that show the couple’s foreign properties and financial accounts Three firearms were also seized from the residence The complaint alleges Gray was in violation of federal law for possessing the firearms because he is a convicted felon Gray was convicted of conspiracy to possess with intent to distribute cocaine in federal court in 1986 Amerling-Gray was released on a $100,000 unsecured bond while Gray was released on a $250,000 unsecured bond The couple does not currently have an attorney after their former attorney Amerling-Gray declined to comment on case when contacted by The Flint Journal A preliminary exam is scheduled for August 6 Use of and/or registration on any portion of this site constitutes acceptance of our User Agreement, (updated 8/1/2024) and acknowledgement of our Privacy Policy, and Your Privacy Choices and Rights (updated 1/1/2025) © 2025 Advance Local Media LLC. All rights reserved (About Us) The material on this site may not be reproduced except with the prior written permission of Advance Local Community Rules apply to all content you upload or otherwise submit to this site YouTube's privacy policy is available here and YouTube's terms of service is available here Ad Choices and some debris separate the coastal bluff in the Barrio Obrero community of Arecibo from the ocean-view car wash where Christian works who has lived in this community all his life said how the neighbors have tried to find options to tackle the accelerated coastal erosion rate in recent years The result has been a makeshift landfill made with discarded construction materials he says that municipal politicians rarely stop by the area and that these sporadic visits almost always happen during electoral campaigns “Because this is a small neighborhood almost no one cares,” he tells the Center for Investigative Journalism (CPI while looking for the liquid to shine car tires In the past, the Arecibo municipal government said there were no resources to implement a mitigation plan in this coastal area Former Mayor Carlos Molina said the only solution for residents in vulnerable zones was to find another house somewhere else the residents of Barrio Obrero throw objects such as brick fragments pipes and pieces of tile onto the eroded slope with the hope of forming a breakwater that protects their properties for a while but as long as it’s not garbage,” Christian said when discussing how the people in his neighborhood struggle to try to protect themselves from erosion and from the episodes of strong tides that threaten the existence of their residences of this coastal community in Arecibo with the arrival of a new administration in Arecibo an ordinance was signed to create the town’s first Office of Planning and Territorial Management saying that in her newly created office they will work on strategies to manage the coastal erosion problem in Barrio Obrero and in nearby communities Like other areas west of the Río Grande de Arecibo, the Barrio Obrero community has faced the erosion of its coastal slope, according to “The study of the beaches of Puerto Rico Post-María” research study One of the main researchers on the project describes the problem in some of these areas of Arecibo as one of coastal bluff erosion It is an erosion process that is just starting to be examined in Puerto Rico and which is different from beach erosion and wearing away of other areas of the flat coast If you have that exposed to a coast with regular waves with hurricanes that are becoming increasingly [stronger] due to the effect of climate change Added to that is coastal mismanagement in the past so you have the perfect equation to speed it up,” Barreto explained to the CPI was one that marine geologist and her work team from the University of Puerto Rico (UPR) noticed while doing field work to learn how much Puerto Rico’s beaches had changed after Hurricane Maria Although the research was not intended to study the phenomenon of coastal bluff erosion coastal bluff erosion is more difficult to identify through aerial photos and her research in Puerto Rico has been more limited when compared to flat coast erosion El Cocal beach in the town of Yabucoa, in southeastern Puerto Rico, presents another significant case of coastal bluff erosion, the second part of the study reveals the CPI found that municipal officials said they were aware of the problem and the danger to properties in high areas but admitted that specific mitigation strategies for coastal bluff erosion have not been implemented “We don’t have much information about the elevated coast,” Luis Rivera the area of El Negro beach also shows a case of coastal bluff erosion From El Cocal beach dozens of properties that are part of El Cocal Country & Beach Club can be seen on the slope which includes units available for rent through the Airbnb platform A visit to the area reveals how vulnerable the slope that divides this complex from the popular beach has become The sturdy natural wall that once separated the residential complex from the beach has narrowed as a result of erosion and landslides further undermining the roots of the trees and palms trees there When Rivera was asked about what measures the municipality has taken to address the dangers faced by those who stay in these housing and rental units since it is a “private property.” “El Cocal [Country & Beach Club] is a plot of land where several people bought and got titles but it’s a plot of land that hasn’t been segregated We can see the effects of erosion from the public area [of the beach] we would have to go in there at the request [of the residents],” Rivera explained the area adjacent to Kikita Beach and the Ojo del Buey reserve in the Mameyal neighborhood of the northern municipality of Dorado also face a coastal bluff erosion problem that includes the area where there are properties for rent through Airbnb Although his reasons are different from those outlined by Yabucoa municipal officials the mayor of Dorado has not gotten involved with managing and mitigating the coastal bluff erosion that is happening in his town He held the central government accountable “This is really the responsibility of the Department of Natural and Environmental Resources (DNER) [the agency] has refused to take any responsibility there’s an area that could affect residences in the future have been trying to qualify for to solve the problem and we weren’t able to get Department [of Natural and Environmental Resources] to help at this time,” Dorado Mayor Carlos Lopez told the CPI Regarding the case of Mameyal and the area of the El Ojo del Buey reserve where there are areas of coastal bluff erosion which is a type of metal mesh rectangular box filled with rocks used as a barrier He also said he will request federal funds to be able to conduct ocean studies to design a protective structure although he did not explain why he waited until now to do this The CPI contacted the DRNA for a reaction to the mayor’s statements While it is being decided whether the responsibility of addressing coastal bluff erosion falls on the town of Dorado or the DRNA the Atlantic Ocean continues reclaiming its space The episodes of strong swells caused by cyclones or cold fronts along Puerto Rico’s northern region push the ocean into the Mameyal’s narrow streets Business and apartment owners in elevated areas near Kikita Beach also toss rocks at the shoreline area to try to mitigate erosion which the Mayor himself claims to be unaware of at times Although these practices respond to people in fear of losing their business or property their consequences are not always favorable since it represents a type of informal mitigation that does not respond to the coastal studies required in these processes That’s why mitigation decisions cannot be made without following the processes but what people might think of as an option may be the worst medicine,” Barreto warned “The mitigation strategies have to be based on the behavior of the coast It has to go along with community participation It has to be an analysis among the community They cannot be independent decisions,” said the also professor at the Graduate School of Planning of the UPR’s Río Piedras Campus several reports and videos have documented the progressive deterioration and the Paseo’s existing critical condition One of its sections was closed to the public due to the vulnerability of the slope on which the Paseo’s structure stands The unsafe condition of this recreational area that cost $32 million in public funds forced the House of Representatives’ Commission for the Development and Supervision of Public Funds of the Capital City Cataño and Guaynabo to launch an investigation into the controversial project in May it was revealed that it was not clear whether the Municipality of San Juan or the Department of Transportation and Public Works (DTOP in Spanish) was the entity that should assume the main control over the Paseo the Infrastructure Financing Authority has stated on several occasions that its responsibility for the project ended once construction was completed While the Municipality of San Juan and government agencies point fingers at each other without assuming responsibility the erosion of the coastal bluff that supports the Paseo continues to gain ground putting the people who pass through the area on a daily basis at risk Some open sections of the Paseo are supported by ground that is increasingly prone to landslide events chairman of the College of Engineers and Surveyors of Puerto Rico’s Earthquake Commission “The issue there [in Puerta de Tierra] is that the sea is very close to that sloping terrain where it’s about 30 feet high between where the street is and the square and down to the sand,” Rivera explained to the CPI “Soil studies and recommendations were made to put some meshes that didn’t work It is completely steep and that’s a very serious problem,” added the engineer who also warned that any cyclonic event that occurs this year could accelerate the landslides prompt the shutdown of more sections of the Paseo The firm GeoCim was contracted in April 2015 to carry out the geotechnical study for the Paseo Lineal in Puerta de Tierra which is supposed to have included measures aimed at controlling coastal erosion While both types of beach erosion happen in a coastal context their characteristics and processes of change are different This is why mitigation and adaptation strategies and the development of public policy have to be specific boosting research that contributes to the understanding of coastal bluff erosion and the main reasons why it happens is necessary Many of these coastal slopes in Puerto Rico feature a combination of different types of sedimentary rock And each combination poses a different density Geomorphologist Banery Mujica explained to the CPI when addressing one of the reasons for the vulnerability in areas where there are elevated coasts in Puerto Rico the material comes mostly from the mountainous central region Especially along Puerto Rico’s northern area there’s a combination of sedimentary material that includes different types of sedimentary material,” said the scientist speaking of the convergence of rocks such as limestone and that of volcanic origin in some parts of the north of the island especially in the northern part of the Capitol [building] This material comes from the Rio Grande de Loíza which was pulled by the basins and the marine currents which were collecting all this material in the north of Puerto Rico.” A tour of the area under the Paseo Lineal in Puerta de Tierra not only shows the fragility of the slope but also allows the identification of the presence of two types of sediments that converge in this coastal formation “In the lower part it’s more limestone and in the upper part it’s more sedimentary material of igneous origin,” said the also professor of the Department of Geography of the UPR’s Río Piedras Campus The problem of accelerated erosion in Puerto Rico must be addressed by combining several scientific disciplines Mujica recommends that for construction projects in coastal areas soil studies carried out by geologists are not the only reference for decision-making but that they must integrate geomorphological analysis something with which renowned Geomorphologists Maritza Barreto and José Molinelli agree all that coast of San Juan is a combination It’s exactly the same mix that you will see in Vega Alta and Vega Baja since the Puerta de Tierra coast in San Juan is a bluff erosion behaves differently,” Mujica noted the study of coastal erosion in Puerto Rico should not only take into consideration the current climate crisis and the rise in sea levels but also aspects such as the geomorphological processes that happen in the island’s interior and the problem of mishandling of rivers Díaz Torres is a member of Report for America Maria: The Money Trail is a project of the Center for Investigative Journalism focused on putting the spotlight on the recovery process in Puerto Rico after Hurricanes Irma and María in 2017 This initiative is possible with the support of the Puerto Rico Foundations Network Si tiene una solicitud de investigación, queja, aclaración, ‘orejita’, prueba, inquietud, u observación sobre alguna información publicada por el Centro de Periodismo Investigativo, escriba al correo electrónico [email protected] El CPI reconoce que el requisito fundamental para una verdadera democracia es que la ciudadanía esté bien informada y que existan entidades independientes con la capacidad de fiscalizar los poderes que accionan en la sociedad speaking at the closing ceremony of the 39th Congress of the Federation of Organizing Entities of Congresses of Latin America (COCAL) stressed the importance of betting on tourism as a sector that generates the income the country needs to continue improving the living conditions of the people Cuba is not alone as the enemies of the Revolution pretend said the Head of Government in this seaside resort city In the presence of more than 330 professional organizers from 20 countries Marrero commented that tourism in the largest of the Antilles is managed under complex conditions compared to other destinations in the region because of the sanctions of the U.S the activity continues to develop with the efforts of all and innovation Cuba has the potential to establish itself as a destination for event tourism hotel facilities with infrastructure and trained personnel in addition to the nation’s historical About what happened during these days in Cuba’s sun and beach destination par excellence he highlighted the relevance of the exhibitions marketing and sustainability in the activity of meetings and professional events He urged that after this event the sector in the archipelago should take advantage of what it has learned; so much experience and knowledge must be put to good use which was also attended by Juan Carlos García Granda Marrero Cruz praised the fact that the organization of the meeting included the pre-congress session IMEX COCAL Future Leaders Forum a unique opportunity for young people from several Cuban universities and other Latin American nations to exchange and learn about Congress He recommended COCAL’s directors to take into account the university students from the largest of the Antilles so that they can bring their experiences to the 40th edition of the Congress and thanked them for having shared and “left a mark that Cuban tourism will know how to take advantage of” The MICE segment is a travel modality that is carried out for professional reasons and is of great importance for the so-called smokeless industry as it generates a high economic contribution de-seasonalizes demand and positions the destination.  Escambray reserves the right to publish comments and website in this browser for the next time I comment Jun 20 (ACN) The Plaza America Convention Center in Varadero will host from July 3 to 6 the 39th Congress of the Federation of Organizing Entities of Congresses of Latin America (COCAL).Pilar Alvarez Azze general director of marketing and communications of the Ministry of Tourism (MINTUR) said that this is the time to strengthen the positioning of the resort which is being held for the third time on the island inclusion and development.A sign of the confidence and prestige gained by Cuba is the attendance of many countries of the region and others that are not COCAL member states and the non-state sector will have presence and participation.At the International Press Center in Havana informed that they are still working on the accreditation process of the participants in the Congress who come from Mexico Argentina and the Dominican Republic as the countries with the highest attendance as well as recreation and leisure for visitors to get to know the destination for its natural said the directive.COCAL is a community that creates value in the meetings industry is part of a network of professionals and unites them in Latin America were damaged by heavy rain and gusty winds on Thursday night and Friday morning Cocal/Mafeking councillor Renelle Kissoon lives in the village and told Newsday several roofs were blown off near her home “We had a lot of roofs that were blown completely away and that’s been our biggest concern.” A home on Bel Air Road which is near to Grand Lagoon Village lost its roof after heavy rain and gusty winds on Friday morning Kissoon said there were also reports of roofs being blown off in other parts of the district of Cocal/Mafeking "We’ve also had some fallen trees and we’ve had power outages in a couple of streets because power lines were blown down.” When Newsday spoke with Kissoon shortly after 9am on Friday she said it was still raining and that repairs to the damaged homes in her immediate area would have to be done when it stops raining the roofs of several homes in Grand Lagoon Kissoon said residents were given tarpaulins to cover their houses Coca-Cola’s commitment to investing in our planet and in its packaging has earned the company multiple awards at the Environmental Sustainability Conference organised by ImpactReports Africa and Brand Communicator environmental sustainability goes beyond one-off philanthropic interventions to long-term commitments the maiden edition of ECOSEA saw the convergence of public and private sector stakeholders in the environmental sustainability space deliberate on Smarter Waste Management for Safer Cities The Role of Communication in Environmental Sustainability and Net Zero Emissions: The Journey to 2060 described it as an annual platform that champions the cause of environmentally sustainable actions in Africa With the evolution of corporate sustainability practices from corporate philanthropy into an integral part of business models and operations Coca-Cola Nigeria is blazing trails and transforming communities Coca-Cola emerged as the winner in both categories namely ‘Award for the Most Outstanding Beverage Company in Environmental Sustainability’ and ‘Award for the Eco-Friendly Bottled Water Brand of the Year’ Nwamaka Onyemelukwe emerged ‘Environmental Sustainability Professional of the Year’ themed “Smart Waste Management for Safer Cities” Onyemelukwe explained that Coca-Cola’s World Without Waste initiative is anchored by three fundamental goals: making 100 per cent of its packaging recyclable globally by 2025— and using at least 50 per cent recycled material in its packaging by 2030 (Design); collecting and recycling a bottle or can for each one it sells by 2030 (Collect); and bringing people together to support a healthy Coca-Cola announced an industry-leading reusable packaging goal which states that by 2030 the organization aims to have at least 25% of its beverages worldwide by volume sold in refillable/returnable glass or plastic bottles or in fountain dispensers with reusable packaging This builds on its already strong track record with refillable packaging especially in Africa and Nigeria in particular Another packaging innovation by Coca-Cola is the Sprite packaging transition one of its largest global sparkling soft drink brands transitioned from its iconic green bottles to clear PET to enable easier conversion into new bottles by increasing the supply of high-value recycled plastic in the after-use market in its journey towards achieving “A Safer Environment” through investments in recycling interventions and innovative packaging Coca-Cola Nigeria’s sustainability actions are well-deserving of these accolades Nigeria is ranked 168 out of 180 countries in the Environment Performance Index (EPI) 2022 The country had a score of 28.32 out of 100 for “environment and sustainability,” placing it 41st out of 46 sub-Saharan nations This was published in a 2022 report by researchers from Columbia University’s Center for International Earth Science Information Network and Yale Center for Environmental Law & Policy This indicates that the most populous country in Africa struggles to safeguard its people from environmental health concerns that include poor air quality It is within this context that Coca-Cola Nigeria is working with social enterprises and organisations with a focus on the circular economy to deal with plastic waste and contribute towards improving Nigeria’s environmental sustainability performance © 2025 Leadership Media Group - All Rights Reserved © 2025 Leadership Media Group - All Rights Reserved the carpark of S&S Supermarket in Mayaro will be transformed into a farmers’ market where over 30 vendors and small businesses are expected to ply their trade Funds raised from the market will go towards helping over 100 families in Mayaro Leading the effort is 24-year-old Cocal/Mafeking councillor Renelle Kissoon it’s very hard to think about yourself without thinking about everyone else You want everyone to be just as comfortable as you are.” Kissoon came up with the idea in early November while thinking of activities to help constituents during the Christmas season she approached farmers in Mayaro who agreed to sell their extra produce and donate all the proceeds from those sales Kissoon said several small business owners also stepped forward to help and pledged part of their proceeds to the initiative Visitors can also expect entertainment and giveaways Kissoon said any funds raised from the market will go a long way “I know these families (assisted with funds from the market) will appreciate whatever we can help them with especially around Christmas time I always look for families I can help because I see myself in them.” She said the proceeds will help provide constituents with food hampers and home building materials Past national censuses by the Central Statistical Office have shown the Mayaro area to have a high poverty rate — something with which the councillor can relate there were always things we (my siblings and I) wanted that we couldn’t have because of our financial circumstances that’s why I’m in this field (as a councillor) to play a social role in my community.” Regardless of the socio-economic challenges in Mayaro Kissoon doesn't want residents to feel ashamed about their circumstances but rather to continue to work together to developing the area among them owner of S&S Persad Supermarket Sunita Persad “Sunita’s idea was to do something to give back to the community “When I pitched my idea (for the farmers’ market) Persad is also providing Kissoon with sponsorship and tents among other things Other sponsors include BpTT and Rotoplastics Trinidad Ltd whose bins will be placed throughout the market for people to donate canned items Kissoon is calling on people anywhere to reach out and assist in any way they can She said people from other areas such as Princes Town and Sangre Grande have offered to be a part of the initiative because I know how much help I got growing up.” People visiting the market are reminded that covid19 protocols will be in place which include the wearing of masks Anyone interested in supporting or participating in the farmers’ market can contact Kissoon at 778-8026 and S&S Persad Supermarket Visit the event’s Facebook page at 12 Days to Christmas- Christmas Village The element requested is either not valid or does not exist (Official Criminal Complaint Enclosed) Well it looks like another business is about to become under extreme scrutiny by the US government Also is the fact that it has been recently raided by authorities to prevent it from being a brothel as well as a place that owners are presenting as a family hotel  I do not know about you but I would not want to take my wife and kids to a place where there are prostitutes hanging out both around the pool and exiting rooms of clients After a recent interview with the chief of police in Jaco it was discovered that the Hotel Cocal and Casino would be subject to raids on a regular basis  As a person that used to visit Costa Rica for much of the year having police in heavy armor and assault rifles coming in to ask me for my passport and not letting people leave until they checked everyone is not how I want to potentially spend an evening I would rather go to The Beatle Bar to pick up a woman than this place A Michigan couple is out on bond after the federal government claims that they purposely hid profits of millions in foreign investments from tax officials the couple has an ownership interest in two condominiums and Gray owns and operates Hotel Cocal and Casino Official Criminal Complaint 2012 by admin · Leave a Comment  […] Wow they must be really competing with the Beatle Bar Why do I care what taxes they are paying or not paying to to the US This is clearly an attempt to harass them because they are offering a better option in Jaco Oh and by the way anyone going to Jaco to take the wife and kids on a “Family Vacation ” is and idiot because everyone knows its not that type of destination the rumor is that they may have to close down for a while because the US and CR banks are freezing their accounts and they will not be able to use their merchant accounts for credit cards etc. just trying to let people know whats up… it would really suck if you had a reservation and they were closed and you couldn’t get back your cc deposit…the other rumor story is that the main owner cannot come back to CR while all this is going on – so it can affect the condos and his business…and he is facing jail time for not completing some tax form and some gun charge because he is an ex-con […] If you are not aware of the above or have not filed this information in the past then you are subject to both civil penalties which can add up to substantial Just look at recent article published about the owner of the Cocal Casino in Jaco You must be logged in to post a comment @2017 - Costa Rican Times. All Right Reserved Hotel Cocal is the main attraction and is currently the center of Jaco at night at any given day of the week When the hotel got renovated it added a tower of posh condo units that offers a more relaxed ambiance compared to the hotel itself The hotel boasts of 42 rooms that are either facing the ocean or the poolside and have amenities like safes and fridges It also has a small casino that serves its guests after seven in the evening for games like rummy slot machines and Caribbean stud and upstairs there is a poker room that also serves as a sports book where you can place your bets directly The main focal where all the steamy transactions happen is in the poolside bar of the hotel At night ladies swarm the place looking to meet generous men looking for company at a price If you come in earlier than 7pm make sure to wait until later that night before choosing the girl who would accompany you for the night as you might miss your chance of meeting a hotter and more gorgeous lady if you think you have seen it all then you are mistaken Hotel Cocal women who throng the poolside come dressed to impress with their sexy dresses makeup and the likes that you would mistake most of them for models or actresses which some of them are blondes and redheads in all shapes and sizes; athletic fair skinned and all shades in between; you name it You will find all bra cups and sizes either enhanced or natural in this place so be warned that you may find yourself a little overwhelmed with all the delicious morsel presented to you By 10:30 pm all girls frequenting the joint have already arrived so you can start choosing the most beautiful and sexiest girl that you would like to spend a couple of hours with they vary depending on how good your negotiation skills are but they usually start at $150 this is business for the women and you got to make sure that important details like prices services offered and length of stay should be agreed on before you leave the place or you might find yourself in a difficult situation; prostitution is legal in Costa Rica and women working in this industry can call the police for help if you do not pay the price they are expecting you would or if not they will get their friends involved so be smart with your transactions be smart and make sure that your valuables and money are in the safe to avoid getting outsmarted by the woman you decided to take home with you If you are interested in having your own Costa Rica Guys Trip or Bachelor Party in Costa Rica get your free E-Book to help you plan your get away Where to pick up Costa Rican Women & Nighttime Company You must be logged in to post a comment @2017 - Costa Rican Times. All Right Reserved. he talks Jaco nightlife and names his favorite places but no one seems to talk about it is this: Tell me about the nightlife in Jaco As a travel consultant in Jaco, who specializes in fishing Costa Rica is famous for its natural beauty and outside of the capital city of San Jose Jaco’s nightlife is by far the most vibrant Nowadays Jaco is one of the best beach destinations in Costa Rica But for all you night owls and party people Jaco also offers a great party scene for those looking for excellent nightlife I’ll break the following suggestions into two categories Jaco Blu is one of my favorite Jaco beach bars but is also super popular at night as a club sitting right on the beach with pools in the center Please note that this place is currently closed for a refurb Republik Lounge only opens on Thursday A post shared by Republik Lounge (@republikcr) Every night is ladies’ night at XTC every night except for Mondays and Tuesdays On the second floor on the east side of the main street (not the beach side) in the center of town – you’ll find it A post shared by XTC Jaco Beach (@xtcjaco) Orange Pub could be the largest club in Jaco – the space is massive and go-go dancers around the bar and dance areas They also have some nice VIP sections that make for excellent people watching A post shared by Orange Pub (@orange.pub) Le Loft is the granddaddy of clubs in Jaco the go-to spot for amazing all-nighters that don’t stop until the sun comes up The perfect place to finish your Jaco club crawl ice-cold beer and plenty of beautiful people to hang out with party like a rockstar” is what they say here Located at the north end of town, right off the main road as you come to the main strip. GSpot calls itself a “fusion between the best of a bar and has a two-pole dance floor in the center A post shared by Gspot Jaco Beach (@gspotjacobeach) Centerfolds is above La Loft in the center of Jaco after moving from elsewhere in town during the pandemic It’s reopening and rebirth as part of the Le Loft family ensures a well-run gentleman’s club for Jaco So there is no real way to describe this property, but it would be churlish to keep it out of any guide to Jaco nightlife. Years ago, Cocal was a great beachfront family hotel but has since changed to THE place to go for guys looking to mingle with beautiful women Cocal has some of the best bar food around with great drinks and endless eye candy to view or socialize with If you’re a guy traveling with your family or with a girlfriend/spouse what I’ve listed above – in my opinion – are the best places for party people in Jaco to check out I’ll keep looking around and update where necessary Justin DeBoom lives in Jaco, Costa Rica where he works as a travel consultant and fishing specialist at Namu Travel where he helps people plan trips to Costa Rica and Panama He can be contacted at justindeboom@namutravel.com Justin DeBoom first came to Costa Rica in 2005 with friends and fell in love with the country As an avid angler in his home state of Florida he quickly decided Costa Rica would be his primary destination for travel until he made the permanent move in 2009 traveling and fishing throughout Costa Rica Costa Rica and own a fishing charter called Caribsea Sportfishing Justin also works for Costa Rica Vacations helping people plan amazing vacations Contact him at justindeboom@namutravel.com Costa Rica tourism faces challenges as high season ends with declining visitor numbers amid concerns over rising prices and competitiveness What are the top things to do in Placencia Find out from someone who lives in one of Belize’s most vibrant beach communities Balancing investment opportunities with social responsibility in areas affected by Costa Rican gentrification where tourism has created a ‘Gringo Market’ pricing locals out of their communities We’ll provide you with invaluable info and resources for an unforgettable Central American experience