Biology subjects

Deshpande, S.

Publications and source records attributed to Deshpande, S..

36 records · Page 2Linked to original sources

bioRxiv2024

Fatty acid desaturation guides cellular decisions between ferroptosis and cellular senescence

When subject to damage or stress, cells develop responses in order to maintain tissue homeostasis. Two such decisions are ferroptosis and cellular senescence, but how cells decide between these outcomes remains unclear. Here we show that senescent cells increase levels of multiple membrane-bound polyunsaturated fatty acids (PUFAs), but a specific PUFA, dihomo-gamma-linolenic acid (DGLA, 20:3{omega}-3) is reduced. Exogenous repletion of DGLA or inhibition of delta-5-desaturase, the enzyme that metabolizes DGLA, instead results in cell death by ferroptosis. Senescent cells had elevated levels of other fezzroptosis sensitizers, including labile iron and expression of lipoxygenases - but also increased Gpx4 levels to prevent ferroptosis. Oral DGLA lowered senescent cell burden in aged mice and improved age-related functional outcomes. Finally, obese humans with lowered DGLA desaturation rates showed lower markers of adipose tissue senescence. Together, our data implicate DGLA and its desaturation as a major driver of decisions between senescence and ferroptosis.

cell biology↗

bioRxiv2023

Sex-specific Proximal Tubular Cell differentiation pathways identified by single-nucleus RNA

BackgroundPostpartum kidney growth is substantial but proliferation and differentiation pathways underpinning nephron elongation are not well defined. Here we performed sequential characterization of mouse kidney transcriptomics at the single cell level to address this. MethodsSingle nuclear RNA sequencing (snRNA-seq) was performed on kidney tissue from male and female mice at 1, 2, 4 and 12 weeks of age using the 10x Chromium platform. ResultsUnbiased clustering was performed on 68,775 nuclei from 16 animals. 31 discrete cellular clusters were seen, which were identified through comparison of their gene expression profiles to canonical markers of kidney cell populations. High levels of proliferation were evident at early time points in some cell types, especially tubular cells, but not in other cell types, for example podocytes. Proliferation was especially evident in Proximal Tubular Cells (PTCs) which are the most abundant cell type in the adult kidney. Uniquely when compared to other kidney cell types, PTCs demonstrated sex-specific expression profiles at late, but not early, time points. Mapping of PTC differentiation pathways using techniques including trajectory and RNA Velocity analyses delineated increasing PTC specialization and sex-specific phenotype specification. ConclusionOur single-cell transcriptomics data characterise cellular states observed during kidney growth. We have identified PTC differentiation pathways that lead to sex-specific tubular cell phenotypes.

cell biology↗

bioRxiv2023

BioModelsML: Building a FAIR and reproducible collection of machine learning models in life sciences and medicine for easy reuse

Machine learning (ML) models are widely used in life sciences and medicine; however, they are scattered across various platforms and there are several challenges that hinder their accessibility, reproducibility and reuse. In this manuscript, we present the formalisation and pilot implementation of community protocol to enable FAIReR (Findable, Accessible, Interoperable, Reusable, and Reproducible) sharing of ML models. The protocol consists of eight steps, including sharing model training code, dataset information, reproduced figures, model evaluation metrics, trained models, Dockerfiles, model metadata, and FAIR dissemination. Applying these measures we aim to build and share a comprehensive public collection of FAIR ML models in the BioModels repository through incentivized community curation. In a pilot implementation, we curated diverse ML models to demonstrate the feasibility of our approach and we discussed the current challenges. Building a FAIReR collection of ML models will directly enhance the reproducibility and reusability of ML models, minimising the effort needed to reimplement models, maximising the impact on the application and significantly accelerating the advancement in the field of life science and medicine.

bioinformatics↗

bioRxiv2023

Phase Separation and Ageing of Glycine-Rich Protein from Tick Adhesive

Hard ticks feed on their host for multiple days. To ensure firm attachment, they secrete a protein-rich saliva that eventually forms a solid cement cone. The underlying mechanism of this liquid-to-solid transition is not yet understood. This study focuses on the phase transitions of a disordered glycine-rich protein (GRP) that is prominent in tick saliva. We show that GRP undergoes liquid-liquid phase separation via simple coacervation to form biomolecular condensates in salty environments. Cation-pi and pi-pi interactions near the C-terminus promote coacervation while a negatively charged N-terminus prolongs its onset through electrostatic repulsion. Interestingly, GRP condensates exhibit ageing and undergo liquid-to-gel transition to form viscoelastic networks as well as solid-like condensates. Lastly, we provide evidence for protein-rich condensates in natural tick saliva. Our findings provide a starting point to gain insights into the bioadhesion of ticks, develop novel tick control strategies, and towards biomedical applications such as tissue sealants.

biochemistry↗

bioRxiv2022

Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay

Millions of people are infected by the Dengue and Zika viruses each year, which can result in serious illness, permanent disability or death. There are currently no FDA-approved antivirals for treating infection by these viruses. Galidesivir is an adenosine nucleoside analog which can attenuate flavivirus replication in cell-based and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases, and subsequently incorporated into viral RNA by viral RNA-dependent RNA polymerases, leading to the termination of RNA synthesis via an unknown mechanism. Here we report the direct in vitro testing of the effects of Galidesivir triphosphate on RNA synthesis by the polymerases of Dengue-2 and Zika virus. Galidesivir triphosphate was chemically synthesized and inhibition of RNA synthesis followed using a continuous fluorescence-based assay. Galidesivir triphosphate was equipotent against the polymerase activity of Dengue-2 and Zika, with IC50 values of 42 {+/-} 12 M and 47 {+/-} 5 M, respectively. This modest potency in vitro is consistent with results previously obtained in cell-based antiviral assays and suggests that the binding affinity for Galidesivir triphosphate is similar to the natural ATP substrate that it closely mimics. The inhibition assay we have developed will allow the rapid screening of Galidesivir and related compounds against other flavivirus polymerases, and the availability of Galidesivir triphosphate will allow detailed analysis of its mechanism of action. HighlightsO_LIGalidesivir triphosphate was chemically synthesized. C_LIO_LIA continuous assay detecting double-stranded RNA formation was optimized for polymerase inhibition studies. C_LIO_LIGalidesivir triphosphate has moderate potency against DENV2 and ZIKA polymerase activity. C_LIO_LIThe availability of Galidesivir triphosphate will facilitate study of its mechanism of action. C_LI

microbiology↗

bioRxiv2022

High resolution cryo-EM structures of two potently SARS-CoV-2 neutralizing monoclonal antibodies of same donor origin that vary in neutralizing Omicron variants

While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency. HighlightsO_LITwo potent human mAbs obtained from a single donor differ binding to Omicron spikes C_LIO_LIPattern of binding and conformation of these mAbs bound to full length spike differs C_LIO_LIAntibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms. C_LIO_LICryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs. C_LI

biophysics↗

bioRxiv2022

Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. ImportanceDevelopment of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.

immunology↗

bioRxiv2021

A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 delta variant

Although efficacious vaccines have significantly reduced the morbidity and mortality due to COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of two highly potently neutralizing mAbs (THSC20.HVTR04 and THSC20.HVTR26) from an Indian convalescent donor, that neutralize SARS-CoV-2 VOCs at picomolar concentrations including the delta variant (B.1.617.2). These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein thereby preventing the virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness. HighlightsO_LIIdentification of an Indian convalescent donor prior to emergence of SARS-CoV-2 Delta variant whose plasma demonstrated neutralization breadth across SARS-CoV-2 variants of concern (VOCs). C_LIO_LITwo (THSC20.HVTR04 and THSC20.HVTR26) monoclonal antibodies isolated from peripheral memory B cells potently neutralize SARS-CoV-2 VOCs: Alpha, Beta, Gamma, Delta and VOIs: Kappa and Delta Plus. C_LIO_LITHSC20.HVTR04 and THSC20.HVTR26 target non-competing epitopes on the receptor binding domain (RBD) and represent distinct germline lineages. C_LIO_LIPassive transfer of THSC20.HVTR04 and THSC20.HVTR26 mAbs demonstrated protection against Delta virus challenge in K18-hACE2 mice at low antibody doses. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/474152v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f1b55corg.highwire.dtl.DTLVardef@1b9b438org.highwire.dtl.DTLVardef@e6d2a6org.highwire.dtl.DTLVardef@f92cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

TIAToolbox: An End-to-End Toolbox for Advanced Tissue Image Analytics

Computational Pathology (CPath) has seen rapid growth in recent years, driven by advanced deep learning (DL) algorithms. These algorithms typically share the same sequence of steps. However, due to the sheer size and complexity of handling large multi-gigapixel whole-slide images, there is no open-source software library that provides a generic end-to-end API for pathology image analysis using best practices for CPath. Most researchers have designed custom pipelines from the bottom-up, restricting the development of advanced CPath algorithms to specialist users. To help overcome this bottleneck, we present TIAToolbox, a Python toolbox designed to make CPath more accessible to new and advanced CPath scientists and pathologists alike. We provide a usable and adaptable library with efficient, cutting-edge and unit-tested tools for data loading, pre-processing, model inference, post-processing and visualization. This enables all kinds of users to easily build upon recent DL developments in the CPath literature. TIAToolbox provides a user-friendly modular API to enable seamless integration of advanced DL algorithms. We show with the help of examples how state-of-the-art DL algorithms can be streamlined using TIAToolbox.

bioengineering↗

bioRxiv2021

Actinosomes: condensate-templated proteinaceous containers for engineering synthetic cells

Engineering synthetic cells has a broad appeal, from understanding living cells to designing novel biomaterials for therapeutics, biosensing, and hybrid interfaces. A key prerequisite to creating synthetic cells is a functional three-dimensional container capable of orchestrating biochemical reactions. In this study, we present an easy and effective technique to make cell-sized porous containers crafted using the interactions between biomolecular condensates and actin cytoskeleton - we coin them actinosomes. This approach uses polypeptide/nucleoside triphosphate condensates and localizes actin monomers on their surface. By triggering actin polymerization at the expense of sequestered ATP and using osmotic gradients, the condensates are structurally transformed into containers with the boundary made up of actin filaments and polylysine polymers. We show that the GTP-to-ATP ratio is a crucial parameter for forming actinosomes: insufficient ATP prevents condensate dissolution while excess ATP leads to undesired crumpling. The surface of actinosomes lacks any structural order and is porous. We show the functionality of the actinosomes by using them as bioreactors capable of protein synthesis. Actinosomes are a handy addition to the synthetic cell platform, with appealing properties like ease-of-production, inherent encapsulation capacity, and an active surface which holds the potential to trigger signaling cascades and form multicellular assemblies, with potential for medical and biotechnological applications.

bioengineering↗

bioRxiv2021

Designing Biological Micro-Sensors with Chiral Nematic Liquid Crystal Droplets

Biosensing using liquid crystals has a tremendous potential by coupling the high degree of sensitivity of their alignment to their surroundings with clear optical feedback. Many existing set-ups use birefringence of nematic liquid crystals, which severely limits straightforward and frugal implementation into a sensing platform due to the sophisticated optical set-ups required. In this work, we instead utilize chiral nematic liquid crystal micro-droplets, which show strongly reflected structural colour, as sensing platforms for surface active agents. We systematically quantify the optical response of closely related biological amphiphiles and find unique optical signatures for each species. We detect signatures across a wide range of concentrations (from M to mM), with fast response times (from seconds to minutes). The striking optical response is a function of the adsorption of surfactants in a non-homogeneous manner and the topology of the liquid crystal orientation at the interface requiring a scattering, multidomain structure, which we observe to be different between molecules. We show lab-on-a-chip capability of our method by drying droplets in high-density two-dimensional arrays and simply hydrating the chip to detect dissolved analytes. Finally, we show proof-of-principle in vivo biosensing in the intestinal tracts of live zebrafish larvae, demonstrating CLC droplets show a clear and differential optical response between healthy and inflamed tissues. Our unique approach has great potential in developing on-site detection platforms and detecting biological amphiphiles in living organisms.

bioengineering↗

bioRxiv2021

Sorghum pan-genome explores the functional utility to accelerate the genetic gain

Sorghum (Sorghum bicolor L.) is one of the most important food crops in the arid and rainfed production ecologies. It is a part of resilient farming and is projected as a smart crop to overcome the food and nutritional challenges in the developing world. The development and characterisation of the sorghum pan-genome will provide insight into genome diversity and functionality, supporting sorghum improvement. We built a sorghum pan-genome using reference genomes as well as 354 genetically diverse sorghum accessions belonging to different races. We explored the structural and functional characteristics of the pan-genome and explain its utility in supporting genetic gain. The newly-developed pan-genome has a total of 35,719 genes, a core genome of 16,821 genes and an average of 32,795 genes in each cultivar. The variable genes are enriched with environment responsive genes and classify the sorghum accessions according to their race. We show that 53% of genes display presence-absence variation, and some of these variable genes are predicted to be functionally associated with drought traits. Using more than two million SNPs from the pan-genome, association analysis identified 398 SNPs significantly associated with important agronomic traits, of which, 92 were in genes. Drought gene expression analysis identified 1,788 genes that are functionally linked to different conditions, of which 79 were absent from the reference genome assembly. This study provides comprehensive genomic diversity resources in sorghum which can be used in genome assisted crop improvement.

bioinformatics↗

bioRxiv2020

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-a-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The thermostable 1PGE-THIVC Env displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. While the specificity of antibodies elicited in rabbits associated with neutralizing autologous viruses were distinct to those developed in the elite neutralizer, EMPEM analysis demonstrated significant changes to Env conformations when incubated with polyclonal antibody sera from the elite neutralizer, suggesting these antibodies lead to the destabilization of Env trimers. Our study not only shows distinct mechanisms associated with potent neutralization of sequence matched and unmatched autologous viruses by antibodies induced in rabbits and in the elite neutralizer, but also highlights how neutralizing antibodies developed during the course of natural infection can impact viral Env conformations. Author SummaryThe interplay between circulating virus variants and broadly cross neutralizing polyclonal antibodies developed in a subset of elite neutralizers is widely believed to provide strategies for rational immunogen design. In the present study, we studied the structural, antigenic and immunogenic properties of a thermostable soluble trimeric protein with near native pre-fusion conformation prepared using the primary sequence of an HIV-1 clade C env isolated from the broadly cross neutralizing plasma of an elite neutralizer. This novel SOSIP Env trimer demonstrated comparable antigenic, structural and immunogenic properties that favoured several ongoing subunit vaccine design efforts. The novel clade C SOSIP induced polyclonal neutralizing antibody response developed in rabbits not only differed in its epitope specificity compared to that elicited in natural infection in presence of pool of viral quasispecies but also showed how they differ in their ability to influence Env structure and conformation. A better understanding of how vaccine-induced polyclonal neutralizing antibody responses compares to responses that developed in natural infection will improve our knowledge in designing better vaccine design strategies.

immunology↗

bioRxiv2020

Geospatial HIV-1 subtype C gp120 sequence diversity and its predicated impact on broadly neutralizing antibody sensitivity

Evolving diversity in globally circulating HIV-1 subtypes presents formidable challenge in defining and developing neutralizing antibodies for prevention and treatment. HIV-1 subtype C is responsible for majority of global HIV-1 infections. Broadly neutralizing antibodies (bnAbs) capable of neutralizing distinct HIV-1 subtypes by targeting conserved vulnerable epitopes on viral envelope protein (Env) are being considered as promising antiviral agents for prevention and treatment. In the present study, we examined the diversity in genetic signatures and attributes that differentiate region-specific global HIV-1 subtype C gp120 sequences associated with virus neutralization outcomes to key bnAbs having distinct epitope specificities. A total of 1814 full length HIV-1 subtype C gp120 sequence from 37 countries were retrieved from Los Alamos National Laboratory HIV database (www.hiv.lanl.gov). The amino acid sequences were assessed for their phylogenetic association, variable loop lengths and prevalence of potential N-linked glycosylation sites (pNLGS). Responses of these sequences to bnAbs were predicted with a machine learning algorithm bNAb-ReP and compared with those reported in the CATNAP database. Phylogenetically, sequences from Asian countries including India clustered together however differed significantly when compared with pan African subtype C sequences. Variable loop lengths within Indian and African clusters were distinct from each other, specifically V1, V2 and V4 loops. Furthermore, V1V2 and V2 alone sequences were also found to vary significantly in their charges. Pairwise analyses at each of the 25 pNLG sites indicated distinct country specific profiles. Highly significant differences (p<0.001***) were observed in prevalence of four pNLGS (N130, N295, N392 and N448) between South Africa and India, having most disease burden associated with subtype C. Our findings highlight that the distinctly evolving clusters within global intra-subtype C gp120 sequences are likely to influence the disparate region-specific sensitivity of circulating HIV-1 subtype C to bnAbs.

bioinformatics↗

bioRxiv2020

Oxylipin metabolism is controlled by mitochondrial b-oxidation during bacterial inflammation.

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial {beta}-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin {beta}-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-{gamma}. Last, dampening oxylipin levels by {beta}-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial {beta}-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

biochemistry↗

bioRxiv2019

An integrated microfluidic platform for quantifying drug permeation across biomimetic vesicle membranes

The low membrane permeability of candidate drug molecules is a major challenge in drug development and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging since one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological salt and pH levels, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin across biomimetic liposomes.

biophysics↗

bioRxiv2019

Microhomology-mediated end joining drives complex rearrangements and over expression of MYC and PVT1 in multiple myeloma

MYC is a widely acting transcription factor and its deregulation is a crucial event in many human cancers. MYC is important biologically and clinically in multiple myeloma, but the mechanisms underlying its dysregulation are poorly understood. We show that MYC rearrangements are present in 36.0% of newly diagnosed myeloma patients, as detected in the largest set of next generation sequencing data to date (n=1267). Rearrangements were complex and associated with increased expression of MYC and PVT1, but not other genes at 8q24. The highest effect on gene expression was detected in cases where the MYC locus is juxtaposed next to super-enhancers associated with genes such as IGH, IGK, IGL, TXNDC5/BMP6, FAM46C and FOXO3. We identified three hotspots of recombination at 8q24, one of which is enriched for IGH-MYC translocations. Breakpoint analysis indicates primary myeloma rearrangements involving the IGH locus occur through non-homologous end joining, whereas secondary MYC rearrangements occur through microhomology-mediated end joining. This mechanism is different to lymphomas, where non-homologous end joining generates MYC rearrangements. Rearrangements resulted in over-expression of key genes and ChIP-seq identified that HK2, a member of the glucose metabolism pathway, is directly over-expressed through binding of MYC at its promoter.

cancer biology↗

bioRxiv2018

Genome-wide Population Structure Analyses of Three Minor Millets: Kodo Millet, Little Millet, and Proso Millet

Millets are a diverse group of small-seeded grains that are rich in nutrients but have received relatively little advanced plant breeding research. Millets are important to smallholder farmers in Africa and Asia because of their short growing season, good stress tolerance, and high nutritional content. To advance the study and use of these species, we present a genome-wide marker datasets and population structure analyses for three minor millets: kodo millet (Paspalum scrobiculatum), little millet (Panicum sumatrense), and proso millet (Panicum miliaceum). We generated genome-wide marker data sets for 190 accessions of each species with genotyping-by-sequencing (GBS). After filtering, we retained between 161 and 165 accessions of each species, with 3461, 2245, and 1882 single-nucleotide polymorphisms (SNPs) for kodo, proso, and little millet, respectively. Population genetic analysis revealed 7 putative subpopulations of kodo millet and 8 each of proso millet and little millet. To confirm the accuracy of this genetic data, we used public phenotype data on a subset of these accessions to estimate the heritability of various agronomically relevant phenotypes. Heritability values largely agree with the prior expectation for each phenotype, indicating that these SNPs provide an accurate genome-wide sample of genetic variation. These data represent one of first genome-wide population genetics analyses, and the most extensive, in these species and the first genomic analyses of any sort for little millet and kodo millet. These data will be a valuable resource for researchers and breeders trying to improve these crops for smallholder farmers.

genetics↗