Biology subjects

Nguyen, T.

Publications and source records attributed to Nguyen, T..

At least 163 records · Page 9Linked to original sources

bioRxiv2023

Comparison of commercial DNA extraction kits for whole metagenome sequencing of human oral, vaginal, and rectal microbiome samples

IntroductionAdvancements in DNA extraction and sequencing technologies have been fundamental in deciphering the significance of the microbiome related to human health and pathology. Whole metagenome shotgun sequencing (WMS) is gaining popularity in use compared to its predecessor (i.e., amplicon-based approaches). However, like amplicon-based approaches, WMS is subject to bias from DNA extraction methods that can compromise the integrity of sequencing and subsequent findings. The purpose of this study was to evaluate systematic differences among four commercially available DNA extraction kits frequently used for WMS analysis of the microbiome. MethodsOral, vaginal, and rectal swabs were collected in replicates of four by a healthcare provider from five participants and randomized to one of four DNA extraction kits. Two extraction blanks and three replicate mock community samples were also extracted using each extraction kit. WMS was completed with NovaSeq 6000 for all samples. Sequencing and microbial communities were analyzed using nonmetric multidimensional scaling and compositional bias analysis. ResultsExtraction kits differentially biased the percentage of reads attributed to microbial taxa across samples and body sites. The PowerSoil Pro kit performed best in approximating expected proportions of mock communities. While HostZERO was biased against gram-negative bacteria, the kit outperformed other kits in extracting fungal DNA. In clinical samples, HostZERO yielded a smaller fraction of reads assigned to Homo sapiens across sites and had a higher fraction of reads assigned to bacterial taxa compared to other kits. However, HostZERO appears to bias representation of microbial communities and demonstrated the most dispersion by site, particularly for vaginal and rectal samples. ConclusionsSystematic differences exist among four frequently referenced DNA extraction kits when used for WMS analysis of the human microbiome. Consideration of such differences in study design and data interpretation is imperative to safeguard the integrity of microbiome research and reproducibility of results.

microbiology↗

bioRxiv2023

The sequence of regional structural disconnectivity due to multiple sclerosis lesions

ObjectivePrediction of disease progression is challenging in multiple sclerosis (MS) as the sequence of lesion development and retention of inflammation within a subset of chronic lesions is heterogeneous among patients. We investigated the sequence of lesion-related regional structural disconnectivity across the spectrum of disability and cognitive impairment in MS. MethodsIn a full cohort of 482 patients, the Expanded Disability Status Scale was used to classify patients into (i) no or mild vs (ii) moderate or severe disability groups. In 363 out of 482 patients, Quantitative Susceptibility Mapping was used to identify paramagnetic rim lesions (PRL), which are maintained by a rim of iron-laden innate immune cells. In 171 out of 482 patients, Brief International Cognitive Assessment was used to identify subjects with cognitive impairment. Network Modification Tool was used to estimate the regional structural disconnectivity due to MS lesions. Discriminative event-based modeling was applied to investigate the sequence of regional structural disconnectivity due to all representative lesions across the spectrum of disability and cognitive impairment. ResultsStructural disconnection in the ventral attention and subcortical networks was an early biomarker of moderate or severe disability. The earliest biomarkers of disability progression were structural disconnections due to PRL in the motor-related regions. Subcortical structural disconnection was an early biomarker of cognitive impairment. InterpretationMS lesion-related structural disconnections in the subcortex is an early biomarker for both disability and cognitive impairment in MS. PRL-related structural disconnection in the motor cortex may identify the patients at risk for moderate or severe disability in MS.

neuroscience↗

bioRxiv2023

Molecular mechanisms of coronary artery disease risk at the PDGFD locus

Platelet derived growth factor (PDGF) signaling has been extensively studied in the context of vascular disease, but the genetics of this pathway remain to be established. Genome wide association studies (GWAS) for coronary artery disease (CAD) have identified a risk locus at 11q22.3, and we have verified with fine mapping approaches that the regulatory variant rs2019090 and PDGFD represent the functional variant and putative functional gene. Further, FOXC1/C2 transcription factor (TF) binding at rs2019090 was found to promote PDGFD transcription through the CAD promoting allele. Employing a constitutive Pdgfd knockout allele along with SMC lineage tracing in a male atherosclerosis mouse model we mapped single cell transcriptomic, cell state, and lesion anatomical changes associated with gene loss. These studies revealed that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype and vascular calcification. This is in contrast to protective CAD genes TCF21, ZEB2, and SMAD3 which we have shown to promote the fibroblast-like cell transition or perturb the pattern or extent of transition to the chondromyocyte phenotype. Further, Pdgfd expressing fibroblasts and pericytes exhibited greater expression of chemokines and leukocyte adhesion molecules, consistent with observed increased macrophage recruitment to the plaque. Despite these changes there was no effect of Pdgfd deletion on SMC contribution to the fibrous cap or overall lesion burden. These findings suggest that PDGFD mediates CAD risk through promoting SMC expansion and migration, in conjunction with deleterious phenotypic changes, and through promoting an inflammatory response that is primarily focused in the adventitia where it contributes to leukocyte trafficking to the diseased vessel wall.

genetics↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗

bioRxiv2022

Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium

Obligate intracellular bacteria of the order Rickettsiales include numerous arthropod-borne human pathogens. However, our understanding of the basic biology of Rickettsia species is limited by technical challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed quantitative methods to assess the cell wall composition, intracellular growth, and morphology of Rickettsia parkeri, a human pathogen in the Spotted Fever Group of the Rickettsia genus. Analysis of the cell wall composition of R. parkeri revealed unique features including a high M3 monomer fraction and absence of LD-crosslinks. Using a novel fluorescence microscopy approach, we quantified the cell morphology of R. parkeri in live host cells and found that bacterial morphology is maintained stably during exponential growth in two different epithelial cell lines. To assess population growth kinetics in a high-throughput and high-resolution manner, we developed an imaging-based growth assay and applied this to determine the growth rate of up to 24 infected cultures at a time. We also sought to gain insight into the cell cycle regulation of R. parkeri. To this end, we developed methods to quantify the fraction of the population preparing to divide as well as those undergoing active constriction. These approaches permitted a quantitative analysis of cell cycle status across a population of R. parkeri. Finally, as a proof of concept, we applied the above tools to quantitatively determine how MreB, a bacterial actin homolog, contributes to the growth and morphogenesis of R. parkeri. Inhibition of MreB with the small molecule MP265 led to cell rounding and slowed growth, suggesting that MreB is required for the growth and shape maintenance of R. parkeri. Collectively, we developed a toolkit of high-throughput, quantitative tools to understand intracellular growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria. AUTHOR SUMMARYThe obligate intracellular lifestyle of members of the bacterial order Rickettsiales, which includes important human pathogens, has hindered our progress in understanding their biology. Here we developed and applied high-throughput, quantitative tools to analyze essential features of rickettsial cell biology such as morphology and growth in living host cells. By applying these tools in a proof of concept, we showed that the bacterial actin homolog, MreB is required for the regulation of rod shape and intracytoplasmic growth.

microbiology↗

bioRxiv2022

Umbilical cord blood-derived cell therapy for perinatal brain injury: A systematic review & meta-analysis of preclinical studies - Part B.

IntroductionWe previously described preclinical literature, which supports umbilical cord blood-derived cell (UCBC) therapy use for perinatal brain injury. However, efficacy of UCBCs may be influenced by different patient populations and intervention characteristics. ObjectivesTo systematically review effects of UCBCs on brain outcomes in animal models of perinatal brain injury across subgroups to better understand contribution of model type (preterm versus term), brain injury type, UCB cell type, route of administration, timing of intervention, cell dosage and number of doses. MethodsA systematic search of MEDLINE and Embase databases was performed to identify studies using UCBC therapy in animal models of perinatal brain injury. Subgroup differences were measured by chi2 test where possible. ResultsDifferential benefits of UCBCs were seen in a number of subgroup analyses including intraventricular haemorrhage (IVH) vs. hypoxia ischaemia (HI) model (apoptosis white matter (WM): chi2=4.07; P=0.04, neuroinflammation-TNF-: chi2=5.99; P=0.01), UCB-derived mesenchymal stromal cells (MSCs) vs. UCB-derived mononuclear cells (MNCs) (oligodendrocyte WM: chi2=5.01; P=0.03, neuroinflammation-TNF-: chi2=3.93; P=0.05, apoptosis grey matter (GM), astrogliosis WM) and intraventricular/intrathecal vs. systemic routes of administration (microglial activation GM: chi2=7.51; P=0.02, astrogliosis WM: chi2=12.44; P=0.002). We identified a serious risk of bias and overall low certainty of evidence. ConclusionsPreclinical evidence suggests greater efficacy for UCBCs in IVH compared to HI injury model, use of UCB-MSCs compared to UCB-MNCs, and use of local administrative routes compared to systemic routes in animal models of perinatal brain injury. Further research is needed to improve certainty of evidence found and address knowledge gaps. SIGNIFICANCE STATEMENTIn neonatal medicine there is a clear need for the development of new therapies that can provide neuroregenerative benefits for infants with brain injuries. This review offers a unique and comprehensive resource to inform the development of future preclinical and clinical studies. In part A of this review, we systematically reviewed the preclinical literature surrounding UCBCs as a therapy for perinatal brain injury. In part B of this review, we investigated the effect variables, such as UCB cell type, timing of administration and dosage, have on the efficacy of UCB-derived cell therapy in animal models of perinatal brain injury. We identified UCBCs to show greater efficacy in the brain injury model of IVH compared to HI, the use of UCB-derived MSCs compared to MNCs and the use of local administrative routes compared to systemic routes. In addition to this, we identified knowledge gaps such as the limited preclinical literature surrounding the effect of dose number and sex.

neuroscience↗

bioRxiv2022

Umbilical cord blood-derived cell therapy for perinatal brain injury: A systematic review & meta-analysis of preclinical studies - Part A

IntroductionPerinatal brain injury is a major contributor to long-term adverse neurodevelopment. There is mounting preclinical evidence for use of umbilical cord blood (UCB)-derived cell therapy as potential treatment. ObjectivesTo systematically review and analyse effects of UCB-derived cell therapy on brain outcomes in preclinical models of perinatal brain injury. MethodsMEDLINE and Embase databases were searched for relevant studies. Brain injury outcomes were extracted for meta-analysis to calculate standard mean difference (SMD) with 95% confidence interval (CI), using an inverse variance, random effects model. Outcomes were separated based on grey matter (GM) and white matter (WM) regions where applicable. Risk of bias was assessed using SYRCLE, and GRADE was used to summarise certainty of evidence. ResultsFifty-five eligible studies were included (7 large, 48 small animal models). UCB-derived cell therapy significantly improved outcomes across multiple domains, including decreased infarct size (SMD 0.53; 95%CI (0.32, 0.74), P<0.00001), apoptosis (WM, SMD 1.59; 95%CI (0.86, 2.32), P<0.0001), astrogliosis (GM, SMD 0.56; 95%CI (0.12, 1.01), P=0.01), microglial activation (WM, SMD 1.03; 95%CI (0.40, 1.66), P=0.001), neuroinflammation (TNF-, SMD 0.84; 95%CI (0.44, 1.25), P<0.0001); as well as improved neuron number (SMD 0.86; 95%CI (0.39, 1.33), P=0.0003), oligodendrocyte number (GM, SMD 3.35; 95%CI (1.00, 5.69), P=0.005) and motor function (cylinder test, SMD 0.49; 95%CI (0.23, 0.76), P=0.0003). Risk of bias was determined as serious, and overall certainty of evidence was low. ConclusionsUCB-derived cell therapy is an efficacious treatment in pre-clinical models of perinatal brain injury, however findings are limited by low certainty of evidence. SIGNIFICANCE STATEMENTPerinatal brain injury can lead to significant long-term neurodevelopmental deficits. There are limited treatment options available, and new interventions are urgently required. Through assessment of preclinical studies, this systematic review and meta-analysis shows that umbilical cord blood-derived cell therapy is an efficacious treatment for perinatal brain injury across a wide range of neuropathological and functional domains, albeit with low certainty of evidence. It also identified knowledge gaps, including that future studies should focus on non-hypoxic ischemic models, preterm models, large animal models and should explore the heterogeneity that exists in treatment protocols.

neuroscience↗

bioRxiv2022

Internal needs drive the cephalic phase response in Drosophila melanogaster

Hungry animals consistently show a desire to obtain food. Even a brief sensory detection of food can trigger bursts of physiological and behavioral changes. However, the underlying mechanisms by which the sensation of food triggers the acute behavioral response remain elusive. We have previously shown in Drosophila that hunger drives a preference for low temperature. Because Drosophila is a small ectotherm, a preference for low temperature implies a low body temperature and a low metabolic rate. Here, we show that taste sensing triggers a switch from a low to a high temperature preference in hungry flies. We show that taste stimulation by artificial sweeteners or optogenetics triggers an acute warm preference, but is not sufficient to reach the fed state. Instead, nutrient intake is required to reach the fed state. The data suggest that starvation recovery is controlled by two components: taste-evoked and nutrient-induced warm preferences, and that taste and nutrient quality play distinct roles in starvation recovery. Animals are motivated to eat based on time of day or hunger. We found that clock genes and hunger signals profoundly control the taste-evoked warm preferences. Thus, our data suggest that the taste-evoked response is one of the critical layers of regulatory mechanisms representing internal energy homeostasis and metabolism.

neuroscience↗

bioRxiv2022

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein AAV transgene expression is silenced using the clinically validated modality of chemically modified short interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

genomics↗

bioRxiv2022

Phenotypic switch and reduced the growth of melanoma spheroids in the presence of mast cell-conditioned medium: potential impact of nutrient starvation effects

Mast cells are abundant in melanoma tumors, and studies suggest that they can be either detrimental or protective for melanoma growth. However, the underlying mechanisms are not fully understood. Here, we adopted a hanging drop-established spheroid system to investigate how mast cells can influence melanoma growth and phenotype in a 3-D context. In the presence of mast cells or mast cell-conditioned medium, melanoma spheroid growth was profoundly reduced. To address the underlying mechanism, we conducted a transcriptomic analysis, which revealed that mast cell-conditioned medium had extensive effects on the melanoma gene expression patterns. Pathway analyses revealed profound effects on the expression of genes related to amino acid and protein metabolism. The conditioned medium also induced an upregulated expression of cancer-related genes, including adhesion molecules implicated in metastatic spreading. In line with this, after transfer to a Matrigel extracellular matrix milieu, spheroids that had been developed in the presence of mast cell-conditioned medium displayed enhanced elevated growth and adhesive properties. However, when assessing for possible effects of nutrient starvation, i.e., reduced nutrient content in mast cell-conditioned medium, we found that the observed effects on melanoma spheroid growth potentially could be explained by such effects. Hence, it cannot be excluded that the observed phenotypic alterations of melanoma spheroids grown in the presence of mast cells or mast cell-conditioned media are, at least partly, due to nutrient starvation rather than to the action of factors secreted by mast cells. Instead, our findings may provide insight into the effects on gene expression events that occur in melanoma tumors under nutrient stress.

immunology↗

bioRxiv2022

Your turn, my turn. Neural synchrony in mother-infant proto-conversation.

Even before infants utter their first words, they engage in highly coordinated vocal exchanges with their caregivers. During these so-called proto-conversations, caregiver-infant dyads use a presumably universal communication structure - turn-taking, which has been linked to favourable developmental outcomes. However, little is known about potential mechanisms involved in early turn-taking. Previous research pointed to interpersonal synchronisation of brain activity between adults and preschool-aged children during turn-taking. Here, we assessed caregivers and infants at 4-6 months of age (N=55) during a face-to-face interaction. We used functional-near infrared spectroscopy hyperscanning to measure dyads brain activity and microcoded their turn-taking. We also measured infants inter-hemispheric connectivity as an index for brain maturity and later vocabulary size and attachment security as developmental outcomes potentially linked to turn-taking. The results showed that more frequent turn-taking was related to interpersonal neural synchrony, but the strength of the relation decreased over the course of the proto-conversation. Importantly, turn-taking was positively associated with infant brain maturity and later vocabulary size, but not with later attachment security. Taken together, these findings shed light on mechanisms facilitating preverbal turn-taking and stress the importance of emerging turn-taking for child brain and language development.

neuroscience↗

bioRxiv2022

Mediator 1 ablation induces enamel-to-hair lineage conversion through enhancer dynamics

Postnatal cell fate has been postulated to be primarily determined by the local tissue microenvironment. Here, we found that Mediator 1 (Med1) dependent epigenetic mechanisms dictate tissue-specific lineage commitment and progression of dental epithelia. Deletion of Med1, a key component of the Mediator complex linking enhancer activities to gene transcription, provokes a tissue extrinsic lineage shift, causing hair generation in the dental environment. Med1 deficiency gives rise to unusual hair growth via primitive cellular aggregates on incisors. Mechanistically, we found that Med1 establishes super-enhancers that control enamel lineage transcription factors in dental stem cells and their progenies. However, Med1 deficiency reshapes the enhancer landscapes and causes a switch from the dental epithelial transcriptional program towards hair and epidermis on incisors in vivo, and in dental epithelial stem cells in vitro. Med1 loss also provokes an increase in the number and size of enhancers. Interestingly, control dental epithelia already exhibit enhancers for hair and epidermal key transcription factors; these expand in size and transform into active super-enhancers upon Med1 loss suggesting that these epigenetic mechanisms cause the transcriptomic and phenotypic shift towards epidermal and hair lineages. Thus, we propose a role for Med1 in safeguarding lineage specific enhancers, highlight the central role of enhancer accessibility and usage in lineage reprogramming and provide new insights into ectodermal regeneration.

developmental biology↗

bioRxiv2022

Global Protein-Turnover Quantification in Escherichia coli Reveals Cytoplasmic Recycling under Nitrogen-Limitation

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the degradation rates of [~]3.2k E. coli proteins under 12 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we introduce broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to decipher the control of proteostasis.

systems biology↗

bioRxiv2022

Canonical Wnt Signaling Maintains Human Mesenchymal Progenitor Cell Multipotency During Adipose Tissue Development

Tissue development and repair throughout life depends on the availability of multipotent mesenchymal stem/progenitor cells capable of differentiating into specialized cell types. How an appropriately sized pool of such multipotent progenitors is maintained under varied signals for tissue growth and repair is unknown. We addressed this question by monitoring fate trajectories of human adipose tissue-derived multipotent progenitor cells using single-cell transcriptomics. Homogenous multipotent progenitors underwent two distinct fate trajectories rapidly upon induction of adipose differentiation- one toward the adipocyte fate, and the other towards a distinct, non-differentiated state characterized by up-regulation of canonical Wnt target genes. Upon isolation, this latter cell population was able to resume proliferation and display multipotency. Using canonical Wnt agonists and antagonists we find Wnt signaling is required for the maintenance of this multipotent pool under differentiation stimulus. In vivo, these cells are retained in adipose tissue developed from human multipotent progenitor cells in immunocompromised mice, and their transcriptomic signature is detected in human adult adipose tissue. Our study reveals a previously unrecognized mechanism for maintaining a functional pool of human mesenchymal progenitor cells under conditions of differentiation pressure, driven by Wnt signaling.

developmental biology↗

bioRxiv2022

A 96-WELL VALVED MICROFLUIDIC DEVICE FOR TESTING OF LIVE INTACT TUMOR CUBOIDS

There is a pressing need for functional testing platforms that use human, live tumor tissue to better predict traditional and immunotherapy responses. Such platforms should also retain as much of the native tumor microenvironment (TME) as possible, as many cancer drug actions rely on TME-dependent mechanisms. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, micro-dissected tumor tissue "spheroids" that retain an intact TME have shown promising responses to immunomodulators acting on native immune cells. Here we demonstrate a microfluidic 96-well platform designed for drug treatment of hundreds of similarly-sized, cuboidal micro-tissues ("cuboids") produced from a single tumor sample. Four cuboids per well are automatically arrayed into the platform using hydrodynamic trapping. The microfluidic device, entirely fabricated in thermoplastics, features microvalves that fluidically isolate each well after the cuboid loading step. Since the platform effectively makes the most of scarce tumor tissue, we believe it could ultimately be applied to human biopsies for drug discovery and personalized oncology, altogether bypassing animal testing.

bioengineering↗

bioRxiv2022

Knockout of AMD-associated gene POLDIP2 reduces mitochondrial superoxide in human retinal pigment epithelial cells

Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2. We conducted functional studies on the POLDIP2 knockout cell line and showed that they retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 causes a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2. In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.

neuroscience↗

bioRxiv2022

In Vitro Evaluation and Mitigation of Niclosamide Liabilities as a COVID-19 Treatment.

Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1.1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future. ImportanceThere is still an urgent need for effective anti-SARS-CoV-2 therapeutics due to waning vaccine efficacy, the emergence of variants of concern, and limited efficacy of existing antivirals. One potential therapeutic option is niclosamide, an FDA approved anthelmintic compound that has shown promising anti-SARS-CoV-2 activity in cell-based assays. Unfortunately, there are significant barriers for the clinical utility of niclosamide as a COVID-19 therapeutic. Our work emphasizes these limitations by showing that niclosamide has high cytotoxicity at antiviral concentrations, variable potency against variants of concern, and significant polypharmacology as a result of its activity as a nonspecific protonophore. Some of these clinical limitations can be mitigated, however, through structural modifications to the niclosamide scaffold, which we demonstrate through a preliminary structure activity relationship analysis. Overall, we show that niclosamide is not a suitable candidate for the treatment of COVID-19, but that structural analogs with improved drug properties may have higher clinical-translational potential.

microbiology↗

bioRxiv2022

A kinesin mediates VEGFR2 recycling and regulates VE-cadherin phosphorylation, essential for vascular permeability

Excessive vascular endothelial growth factor-A (VEGF-A) signaling induces vascular leakage and angiogenesis in diseases. VEGFR2 trafficking to the cell surface, mediated by kinesin-3 family protein KIF13B, is essential to respond to VEGF-A in inducing angiogenesis. However, the precise mechanism of how KIF13B regulates VEGF-induced signaling and endothelial permeability is unknown. Here we show that KIF13B-mediated recycling of internalized VEGFR2 through Rab11-positive recycling vesicle regulates VE-cadherin phosphorylation and endothelial permeability. Phosphorylated VEGFR2 at the cell-cell junction was internalized and associated with KIF13B in Rab5-positive early endosomes. KIF13B mediated VEGFR2 recycling through Rab11-positive recycling vesicle, and inhibition of this recycling attenuated phosphorylation of VEGFR2 at Y951, Src, and VE-cadherin at Y685, which are necessary for endothelial permeability. Failure of VEGFR2 trafficking to the cell surface induced accumulation and degradation of VEGFR2 in lysosomes. Furthermore, in the animal model of wet age-related macular degeneration (AMD), inhibition of KIF13B-mediated VEGFR2 trafficking also mitigated vascular leakage. Thus, the present results identify the fundamental role of VEGFR2 recycling to the cell surface in mediating vascular permeability, suggesting a promising strategy for mitigating vascular leakage associated with inflammatory diseases.

cell biology↗