Biology subjects

Kumar, S.

Publications and source records attributed to Kumar, S..

At least 649 records · Page 36Linked to original sources

bioRxiv2018

Quantitative mapping of triacylglycerol chain length and saturation using broadband CARS microscopy

Lipid droplets (LDs) are highly dynamic organelles that store neutral lipids, primarily triacylglycerols (TAGs), and are found in many cell types. While their primary function is to store excess energy, LDs are also modified in different disease states and during developmental processes. In many cases, not only the presence, but also the composition, of LDs can be equally important. In humans, LD composition has been linked to diseases such as type 2 diabetes; in plants and yeast, LD composition is relevant for engineering these organisms into biological factories in, e.g., algal bioenergy or food oil production. Therefore, lipid analysis of biological LDs with high speed and accuracy in situ is a very active area of research. Here we present an approach for in situ, quantitative TAG analysis using label-free, coherent Raman microscopy to decipher LD TAG composition in different biochemically complex samples. Our method allows direct visualization of inter-LD compositional heterogeneity of physiological quantities - TAG chain length and number of C=C bonds - with sub-micrometer spatial resolution within 5-100 milliseconds. Combined with virtually no sample preparation, this approach should enable rapid and accurate TAG LD analysis for a variety of applications.

bioengineering↗

bioRxiv2018

Nanopore sequencing enables high-resolution analysis of resistance determinants and mobile elements in the human gut microbiome

The analysis of information rich whole-metagenome datasets acquired from complex microbial communities is often restricted by the fragmented nature of assembly from short-read sequencing. The availability of long-reads from third-generation sequencing technologies (e.g. PacBio or Oxford Nanopore) can help improve assembly quality in principle, but high error rates and low throughput have limited their application in metagenomics. In this work, we describe the first hybrid metagenomic assembler which combines the advantages of short and long-read technologies, providing an order of magnitude improvement in contiguity compared to short read assemblies, and high base-pair level accuracy. The proposed approach (OPERA-MS) integrates a novel assembly-based metagenome clustering technique with an exact scaffolding algorithm that can efficiently assemble repeat rich sequences. Based on evaluations with defined in vitro communities and virtual gut microbiomes, we show that it is possible to assemble near complete genomes from metagenomes with as little as 9x long read coverage, thus enabling high quality assembly of lowly abundant species (<1%). Furthermore, OPERA-MSs fine-grained clustering is able to deconvolute and assemble multiple genomes of the same species in a single sample, allowing us to study the complex dynamics of the human microbiome at the sub-species level. Applying nanopore sequencing to gut metagenomes of patients undergoing antibiotic treatment, we show that long reads can be obtained from stool samples in clinical studies to produce more meaningful metagenomic assemblies (up to 200x improvement over short-read assemblies), including the closed assembly of >80 putative plasmid/phage sequences and a 263kbp jumbo phage. Our results highlight that high-quality hybrid assemblies provide an unprecedented view of the gut resistome in these patients, including strain dynamics and identification of novel plasmid sequences.

bioinformatics↗

bioRxiv2018

Biofilm forming capabilities and protein secretion systems distinguish ecologically diverse lineages of Xanthomonas from rice

Xanthomonas oryzae is a devastating pathogen of rice worldwide, however, X. sontii and X. maliensis are its non-pathogenic counterparts from the same host. So far, these non-pathogenic isolates were overlooked due to their less economic importance and lack of genomic information. We have carried out detailed ecological and evolutionary study focusing on diverse lifestyles of these strains. Phylogenomic analysis revealed two major lineages corresponding to X. sontii (ML-I) and X. oryzae (ML-II) species. Interestingly, one of the non-pathogenic Xanthomonas strains belonging to X. maliensis is intermediary to both the major lineages/species suggesting on-going diversification and selection. Accordingly, pangenome analysis revealed large number of lifestyle specific genes with atypical GC content indicating role of horizontal gene transfer in genome diversification. Our comprehensive comparative genomic investigation of major lineages has revealed that impact of recombination is more for X. sontii as compared to X. oryzae. Acquisition of type III secretion system and its effectome along with a type VI secretion system also seem to have played a major role in the pathogenic lineage. Other known key pathogenicity clusters or genes like biofilm forming cluster, cellobiohydrolase and non-fimbrial adhesin (yapH) are exclusive to pathogenic lineage. However, commonality of loci encoding exopolysacharide, rpf signalling molecule, iron-uptake, xanthomonadin pigment, etc. suggests their essentiality in host adaptation. Overall, this study reveals evolutionary history of pathogenic and non-pathogenic strains and will further open up a new avenue for better management of pathogenic strains for sustainable cultivation of a major staple food crop.

evolutionary biology↗

bioRxiv2018

Child Growth Predicts Brain Functional Connectivity and Future Cognitive Outcomes in Urban Bangladeshi Children Exposed to Early Adversities

BackgroundFaltered growth has been shown to affect 161 million children worldwide and derail cognitive development from early childhood. The neural pathways by which growth faltering in early childhood affects future cognitive outcomes remain unclear, which is partially due to the scarcity of research using both neuroimaging and sensitive behavioral techniques in low-income settings. We employed EEG to examine the association between growth faltering and brain functional connectivity and whether brain functional connectivity mediates the effect of early adversity on cognitive development.\n\nMethodsWe recruited participants from an urban impoverished neighborhood in Dhaka, Bangladesh. One sample consisted of 85 children whose EEG and growth measures (height for age, weight for age, and weight to height) were collected at 6 months and cognitive outcomes were assessed at 27 months. Another sample consisted of 115 children whose EEG and growth measures were collected at 36 months and IQ scores were assessed at 48 months. Path analysis was used to test the effect of growth measures on cognitive outcomes through brain functional connectivity.\n\nFindingsFaltered growth was found to be accompanied by overall increased functional connectivity in the theta and low-beta frequency bands for the 36-month-old cohort. For both cohorts, brain functional connectivity was negatively predictive of later cognitive outcomes at 27 and 48 months, respectively. Faltered growth was found to have a negative impact on childrens IQ scores in the older cohort, and this effect was found to be mediated by brain functional connectivity in the low-beta band.\n\nInterpretationThe association found between growth measures and brain functional connectivity may reflect a broad deleterious effect of malnutrition on childrens brain development. The mediation effect of functional connectivity on the relation between physical growth and later IQ scores provides the first experimental evidence that brain functional connectivity may mediate the effect of biological adversity on cognitive development.\n\nFundingBill and Melinda Gates Foundation (OPP1111625)

neuroscience↗

bioRxiv2018

Resumption of spermatogenesis in senescent goldfish Carassius auratus (Linnaeus, 1758) through spermatogonial cell therapy

In recent times, stem cell research has gained considerable prominence because of its applications in assisted reproductive technology and the treatment of deadly diseases. In teleost fishes, spermatogonial stem cells have been effectively used to produce progeny of difficult-to-breed fish species and/or commercially valuable species through the surrogacy technique. The present study is the first report of an innovative application of stem cell therapy in teleostean fish species for revitalising the reproductive competence of senescent individuals. Senescent male goldfish, Carassius auratus aged approximately 10 years were procured from an ornamental fish-breeding farm and were reared locally for an additional 2 years. The senescence of the fish was evaluated and confirmed using histological analysis, gonadal index assessment, and germ-cell specific vasa gene expression. Analyses revealed the absence of spermatogonial cells and other germ cells in the testes of the senescent fish (n = 5). Spermatogonial cells from a prepubertal C. auratus male donor were isolated using discontinuous percoll gradients, labelled with the fluorescent dye PKH-26, and transplanted into the gonads of senescent C. auratus males through the urogenital papilla. Six months after the therapy, spermatozoa from males were collected through applying gentle manual pressure on the abdomen and were observed under the microscope. All the senescent therapy-treated C. auratus males produced spermatozoa from the transplanted cells; this was confirmed by retention of PKH-26 in the spermatozoa and diagnostic SSR locus. The senescent males were crossed with gravid C. auratus females through artificial insemination and natural spawning, and viable progeny was produced. These observations suggest that the reproductive competence of senescent individuals of commercially valuable and/or endangered fish species can be revitalised and extended through spermatogonia stem cell therapy to produce functional gametes.

developmental biology↗

bioRxiv2018

An HIV-1 broadly neutralizing antibody from a clade C infected pediatric elite neutralizer potently neutralizes the contemporaneous and autologous evolving viruses

Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite-neutralizers are potential candidates for isolation of HIV-1 bNAbs and coexistence of bNAbs such as BG18 with neutralization susceptible autologous viruses in an HIV-1 infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1 infected children than adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral naive HIV-1 clade C chronically infected pediatric elite neutralizer AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, median inhibitory dilution (ID50) value of 1246 and presence of N160 and N332-supersite dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332-supersite dependent bNAb AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses; exhibited substantial indels despite limited somatic hypermutations; interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy and demonstrated high binding affinity. In addition, AIIMS-P01 potently neutralized the coexisting and evolving autologous viruses suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in AIIMS_330 pediatric elite neutralizer. Further studies on such pediatric elite-neutralizers and isolation of novel HIV-1 pediatric bNAbs may provide newer insights to guide vaccine design.\n\nImportanceMore than 50% of the HIV-1 infections globally are caused by clade C viruses. Till date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are underway to design immunogens that can elicit correlates of protection upon vaccination. Here we report the isolation and characterization of an HIV-1 N332-supersite dependent bNAb AIIMS-P01 from a clade C chronically infected pediatric elite neutralizer. The N332-supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibits substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation of bNAbs contributing to the plasma breadth in HIV-1 infected children may help to better understand their development and characteristics, which in turn may guide vaccine design.

immunology↗

bioRxiv2018

Sexually dimorphic gene expression and transcriptome evolution provides mixed evidence for a fast-Z effect in Heliconius

Sex chromosomes have different evolutionary properties as compared to the autosomes due to their hemizygous nature. In particular, recessive mutations are more readily exposed to selection, which can lead to faster rates of molecular evolution. Here, we report patterns of gene expression and molecular evolution in the sex chromosomes of a group of tropical butterflies. We first improved the completeness of the Heliconius melpomene reference annotation, a neotropical butterfly with a ZW sex determination system. Then we sequenced RNA from male and female whole abdomens and female ovary and gut tissue to identify sex and tissue specific gene expression profiles in H. melpomene. Using these expression profiles we compare sequence divergence and polymorphism, the strength of positive and negative selection and rates of adaptive evolution for Z and autosomal genes between two species of Heliconius butterflies, H. melpomene and H. erato.\n\nWe show that the rate of adaptive substitutions is higher for Z as compared to autosomal genes, but contrary to expectation it is also higher for male as compared to female biased genes. There is therefore mixed evidence that hemizygosity influences the rate of adaptive substitutions. Additionally, we find no significant increase in the rate of adaptive evolution or purifying selection on genes expressed in ovary tissue, a heterogametic specific tissue. Together our results provide limited support for fast-Z evolution. This contributes to a growing body of literature from other ZW systems that also provide mixed evidence for a fast-Z effect.

evolutionary biology↗

bioRxiv2018

Towards personalized computer simulation of breast cancer treatment: a multi-scale pharmacokinetic and pharmacodynamic model informed by multi-type patient data

Mathematical modeling and simulation have emerged as a potentially powerful, time and cost effective approach to personalized cancer treatment. The usefulness of mechanistic models to disentangle complex multi-scale cancer processes such as treatment response has been widely acknowledged. However, a major barrier for multi-scale models to predict the outcomes of therapeutic regimens in a particular patient lies in their initialization and parameterization which need to reflect individual cancer characteristics accurately. In this study we use multi-type routinely acquired measurements on a single breast tumor, including histopathology, magnetic resonance imaging, and molecular profiling to personalize parts of a complex multi-scale model of breast cancer treated with chemotherapeutic and anti-angiogenic agents. We model the dynamics of drugs in tissue (pharmacokinetics) and the corresponding effects on their targets (pharmacodynamics). We developed a open-source computer program that simulates cross-sections of tumors under 12-week therapy regimes and use it to individually reproduce and elucidate treatment outcomes of four patients. For two of the tumors that did not respond to therapy, we used model simulations to suggest alternative regimes, depending on their individual characteristics, with improved outcomes. We found that more frequent doses of chemothereapy reduce tumor burden in a low proliferative tumor while lower doses of anti-angiogenic agents improve drug penetration in a poorly perfused tumor. In addition to bridge multi-type clinical data to shed light on individual treatment outcomes, our approach identified a few tumor-related aspects that need to be clinically portraited better to allow for future model-driven personalized cancer therapy.

cancer biology↗

bioRxiv2018

HIV-1 vaccine design through minimizing envelope metastability

Overcoming envelope metastability is crucial to trimer-based HIV-1 vaccine design. Here, we present a coherent vaccine strategy by minimizing metastability. For ten strains across five clades, we demonstrate that gp41 ectodomain (gp41ECTO) is the main source of envelope metastability by replacing wild-type gp41ECTO with BG505 gp41ECTO of the uncleaved prefusion-optimized (UFO) design. These gp41ECTO-swapped trimers can be produced in CHO cells with high yield and high purity. Crystal structure of a gp41ECTO-swapped trimer elucidates how a neutralization-resistant tier 3 virus evades antibody recognition of the V2 apex. UFO trimers of transmitted/founder (T/F) viruses and UFO trimers containing a consensus-based ancestral gp41ECTO suggest an evolutionary root of the metastability. Gp41ECTO-stabilized trimers can be readily displayed on 24- and 60-meric nanoparticles, with incorporation of additional T cell help illustrated for a hyperstable 60-mer. In mice and rabbits, gp140 nanoparticles induced more effective tier 2 neutralizing antibody response than trimers with statistical significance.\n\nHIGHLIGHTSO_LIgp41 is the main source of HIV-1 envelope metastability\nC_LIO_LIBG505 gp41 of the UFO design stabilizes gp140 trimers of diverse subtypes\nC_LIO_LIgp41 stabilization facilitates gp140 nanoparticle assembly and improves production\nC_LIO_LINanoparticles elicit tier 2 neutralizing antibodies more effectively than trimers\nC_LI

microbiology↗

bioRxiv2018

Childhood cerebellar tumors mirror conserved fetal transcriptional programs

The study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. We used single-cell transcriptomics to study >60,000 cells from the developing murine cerebellum, and show that different molecular subgroups of childhood cerebellar tumors mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. Sonic Hedgehog medulloblastoma transcriptionally mirrors the granule cell hierarchy as expected, whereas Group 3 medulloblastoma resemble Nestin+ve stem cells, Group 4 medulloblastomas resemble unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumors demonstrates that many bulk tumors contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumors as a disorder of early brain development, and provide a proximate explanation for the peak incidence of cerebellar tumors in early childhood.

cancer biology↗

bioRxiv2018

Transcriptome Analysis of Bael (Aegle marmelos L.) a Member of Family Rutaceae

Aegle marmelos is a medicinally and horticulturally important tree member of the family Rutaceae. It is native to India where it is also known as Bael. Despite its importance; the genomic resources of this plant are scarce. This study presented the first-ever report of expressed transcripts in the leaves of Aegle marmelos. A total of 133,616 contigs were assembled to 46,335 unigenes with the minimum and maximum lengths of 201 and 14,853 bp. There were 7002 transcription factors and 94,479 simple sequence repeat (SSR) markers. The A. marmelos transcripts were also annotated based on information from other members of Rutaceae; namely Citrus clementine and Citrus sinensis. A total of 482 transcripts were annotated as cytochrome p450s (CYPs) and 314 transcripts were annotated as glucosyltransferases (GTs). In the A. marmelos leaves the monoterpenoid biosynthesis pathway was predominant. This study provides an important genomic resource along with useful information about A. marmelos.

genomics↗

bioRxiv2018

Pervasive correlation of molecular evolutionary rates in the tree of life

New species arise from pre-existing species and inherit similar genomes and environments. This predicts greater similarity of mutation rates and the tempo of molecular evolution between direct ancestors and descendants, resulting in autocorrelation of evolutionary rates within lineages in the tree of life. Surprisingly, molecular sequence data have not confirmed this expectation, possibly because available methods lack power to detect autocorrelated rates. Here we present a machine learning method to detect the presence evolutionary rate autocorrelation in large phylogenies. The new method is computationally efficient and performs better than the available state-of-the-art methods. Application of the new method reveals extensive rate autocorrelation in DNA and amino acid sequence evolution of mammals, birds, insects, metazoans, plants, fungi, and prokaryotes. Therefore, rate autocorrelation is a common phenomenon throughout the tree of life. These findings suggest concordance between molecular and non-molecular evolutionary patterns and will foster unbiased and precise dating of the tree of life.

evolutionary biology↗

bioRxiv2018

Late B lymphocyte action in dysfunctional tissue repair following kidney injury and transplantation

The mechanisms initiating the late immune response to allografts are poorly understood. Through transcriptome analysis of serial protocol biopsies in kidney transplant recipients, we found a tight correlation between the initial response to kidney injury and a late B lymphocyte signature associated with renal dysfunction and fibrosis, suggesting a link between dysfunctional repair and immunoreactivity. To specifically investigate the immunological consequences of dysfunctional repair, we followed the mouse kidney up to 18 months after ischemia/reperfusion. Even in the absence of foreign antigens we identified a sustained immune response in conjunction with the transition to chronic kidney damage. This tissue-driven immunological process involved both the innate and the adaptive immune system and eventually induced an antigen-driven proliferation, selection and maturation of B lymphocytes into broadly-reacting antibody secreting cells. These findings reveal an unappreciated role of dysfunctional tissue repair on local immunoregulation with a particular relevance for late transplantation immunobiology.

immunology↗

bioRxiv2018

Predicting clone genotypes from tumor bulk sequencing of multiple samples

MotivationAnalyses of data generated from bulk sequencing of tumors have revealed extensive genomic heterogeneity within patients. Many computational methods have been developed to enable the inference of genotypes of tumor cell populations (clones) from bulk sequencing data. However, the relative and absolute accuracy of available computational methods in estimating clone counts and clone genotypes is not yet known.\n\nResultsWe have assessed the performance of nine methods, including eight previously-published and one new method (CloneFinder), by analyzing computer simulated datasets. CloneFinder, LICHeE, CITUP, and cloneHD inferred clone genotypes with low error (<5% per clone) for a majority of datasets in which the tumor samples contained evolutionarily-related clones. Computational methods did not perform well for datasets in which tumor samples contained mixtures of clones from different clonal lineages. Generally, the number of clones was underestimated by cloneHD and overestimated by Phy-loWGS, and BayClone2, Canopy, and Clomial required prior information regarding the number of clones. AncesTree and Canopy did not produce results for a large number of datasets.\n\nConclusionsDeconvolution of clone genotypes from single nucleotide variant (SNV) frequency differences among tumor samples remains challenging, so there is a need to develop more accurate computational methods and robust software for clone genotype inference.\n\nAvailability and ImplementationCloneFinder is implemented in Python and is available from https://github.com/gstecher/CloneFinderAPI.\n\nContacts.kumar@temple.edu\n\nSupplementary informationSupplementary data are available at Bioinformatics online

cancer biology↗

bioRxiv2018

Computational enhancement of single-cell sequences for inferring tumor evolution

Motivation: Tumor sequencing has entered an exciting phase with the advent of single-cell techniques that are revolutionizing the assessment of single nucleotide variation (SNV) at the highest cellular resolution. However, state-of-the-art single-cell sequencing technologies produce data with many missing bases (MBs) and incorrect base designations that lead to false-positive (FP) and false-negative (FN) detection of somatic mutations. While computational methods are available to make biological inferences in the presence of these errors, the accuracy of the imputed MBs and corrected FPs and FNs remains unknown.\n\nResults: Using computer simulated datasets, we assessed the robustness performance of four existing methods (OncoNEM, SCG, SCITE, and SiFit) and one new method (BEAM). BEAM is a Bayesian evolution-aware method that improves the quality of single-cell sequences by using the intrinsic evolutionary information in the single-cell data in a molecular phylogenetic framework. Overall, BEAM and SCITE performed the best. Most of the methods imputed MBs with high accuracy, but effective detection and correction of FPs and FNs require sampling a large number of SNVs. Analysis of an empirical dataset shows that computational methods can improve both the quality of tumor single-cell sequences and their utility for biological inference.\n\nConclusions: Tumor cells descend from pre-existing cells, which creates evolutionary continuity in single-cell sequencing datasets. This information enables BEAM and other methods to correctly impute missing data and incorrect base assignments, but correction of FPs and FNs remains challenging when the number of SNVs sampled is small relative to the number of cells sequenced.\n\nAvailability: BEAM is available on the web at https://github.com/SayakaMiura/BEAM.\n\nContact: s.kumar@temple.edu

cancer biology↗

bioRxiv2018

ATRAID, a genetic factor that regulates the clinical action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFFs) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase (FDPS), resulting in defects in protein prenylation. Yet it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID. Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Lastly, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare non-synonymous coding variants in patients with ONJ or AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people. One Sentence SummaryATRAID is essential for responses to the commonly prescribed osteoporosis drugs nitrogen-containing bisphosphonates. OverlineBONE

genomics↗

bioRxiv2018

Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of Treponema pallidum subsp. pallidum, the syphilis spirochete.

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum (TPA), the syphilis spirochete, and identifying its surface-exposed {beta}-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of {beta}-barrel-encoding regions of tprC, tprD, and bamA, in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used tp0548 genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) {beta}-barrels in clinical strains could be grouped according to allelic variants in TPA reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored {beta}-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. Based upon structural models, non-conservative amino acid substitutions in predicted transmembrane {beta}-strands of TprC and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive TPA OMP diversity and that genetic exchange contributes to the evolutionary biology of TPA. They also set the stage for topology-based analysis of antibody responses against OMPs and help frame strategies for syphilis vaccine development.\n\nIMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum (TPA), little is known about how their surface-exposed, {beta}-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the {beta}-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in TPA from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within {beta}-barrel domains occurred predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function also were noted. Our findings suggest that selection pressures exerted by human populations drive TPA OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of TPA and frame strategies for vaccine development based upon conserved OMP extracellular loops.

microbiology↗

bioRxiv2018

A 3D Topographical Model of Parenchymal Infiltration and Perivascular Invasion in Glioblastoma

Glioblastoma (GBM) is the most common and invasive primary brain cancer. GBM tumors are characterized by diffuse infiltration, with tumor cells invading slowly through the hyaluronic acid (HA)-rich parenchyma toward vascular beds and then migrating rapidly along microvasculature. Progress in understanding local infiltration, vascular homing, and perivascular invasion is limited by an absence of culture models that recapitulate these hallmark processes. Here we introduce a platform for GBM invasion consisting of a tumor-like cell reservoir and a parallel open channel \"vessel\" embedded in 3D HA-RGD matrix. We show that this simple paradigm is sufficient to capture multi-step invasion and transitions in cell morphology and speed reminiscent of those seen in GBM. Specifically, seeded tumor cells grow into multicellular masses that expand and invade the surrounding HA-RGD matrices while extending long (10-100 {micro}m), thin protrusions resembling those observed for GBM in vivo. Upon encountering the channel, cells orient along the channel wall, adopt a 2D-like morphology, and migrate rapidly along the channel. Structured illumination microscopy reveals distinct cytoskeletal architectures for cells invading through the HA matrix versus those migrating along the vascular channel. Substitution of collagen I in place of HA-RGD supports the same sequence of events but with faster local invasion and a more mesenchymal morphology. These results indicate that topographical effects are generalizable across matrix formulations, but that mechanisms underlying invasion are matrix-dependent. We anticipate that our reductionist paradigm should speed the development of mechanistic hypotheses that could be tested in more complex tumor models.

bioengineering↗