Biology subjects

Singh, D.

Publications and source records attributed to Singh, D..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

De Novo Transcriptome Analysis Reveals Novel Insights into Secondary Metabolite Biosynthesis in Tylophora indica (Burm. f) Merrill.

Tylophora indica has been widely recognized for its therapeutic properties in traditional Indian medicine. Although its bioactive compounds are used extensively to treat a wide range of ailments, a comprehensive understanding of their genetic basis remains limited. In this study, we conducted a transcriptomic analysis of T indica leaf and root using the Illumina platform. High-quality RNA was isolated, and cDNA libraries were constructed for sequencing, generating4.67 GB and5.51 GB of data for leaf and root samples, respectively. 72,795 unigenes and 24,470 coding sequences (CDS) were predicted based on de novo assembly of reads, revealing a complex transcriptome landscape. Functional annotation and pathway analysis revealed biological processes and pathways associated with T indica. Based on the Gene Ontology (GO) mapping, the CDS was categorized into biological processes, cellular components, and molecular functions. An analysis of pathways using the KEGG database revealed involvement in critical metabolic pathways. Furthermore, SSRs contributed to the understanding of genetic diversity by identifying simple sequence repeats. In addition, differential gene expression analysis identified genes involved in secondary metabolite synthesis, among other physiological processes. The qRT-PCR validation of selected genes confirmed their differential expression profiles, with roots exhibiting higher expression than leaves. In this study, transcriptomics is conducted for the first time for T indica, which may be useful for future molecular research. The detailed findings help us understand T indicas biology, which can be used in biotechnology, and they also show how important it is to protect this species because it is used in medicine.

bioinformatics↗

bioRxiv2024

HY5 and COP1 function antagonistically in the light-dependent regulation of nicotine biosynthesis in tobacco

Nicotine constitutes approximately 90% of the total alkaloid content within the Nicotiana species, rendering it the most prevalent alkaloid. While the majority of genes responsible for nicotine biosynthesis express in root tissue, the influence of light on this process through shoot-to-root mobile ELONGATED HYPOCOTYL 5 (HY5) has been recognized. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a key regulator of light-associated responses, known for its role in modulating HY5 accumulation, remains largely unexplored in its relationship to light-dependent nicotine accumulation. Here, we identified NtCOP1, a COP1 homolog in Nicotiana tabacum, and demonstrated its ability to complement the cop1 mutant in Arabidopsis thaliana at molecular, morphological, and biochemical levels. Through the development of NtCOP1 overexpression (NtCOP1OX) plants, we observed a significant reduction in nicotine and flavonol content, inversely correlated with the down-regulation of nicotine and phenylpropanoid pathway. Conversely, CRISPR/Cas9-based knockout mutant plants (NtCOP1CR) exhibited an increase in nicotine levels. Further investigations, including yeast-two hybrid assays, grafting experiments, and western blot analyses, revealed that NtCOP1 modulates nicotine biosynthesis by targeting NtHY5, thereby impeding its transport from shoot-to-root. We conclude that the interplay between HY5 and COP1 functions antagonistically in the light-dependent regulation of nicotine biosynthesis in tobacco. HighlightCharacterization of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) overexpressing and CRISPR/Cas9-based mutant plants suggests the intricate role of COP1 in modulating nicotine biosynthesis in tobacco.

plant biology↗

bioRxiv2024

Steroidal glycoalkaloid biosynthesis and fungal tolerance are regulated by ELONGATED HYPOCOTYL 5, SlHY5, in tomato

Tomato (Solanum lycopersicum L.) is one of the highest consumable fruit crops, rich in nutrients, and has been an important target for enhancing the accumulation of various metabolites. Tomato also contains cholesterol-derived molecules, steroidal glycoalkaloids (SGAs), which contribute to pathogen defence but are toxic to humans and considered anti-nutritional compounds. Previous studies suggest the role of various transcription factors in SGA biosynthesis; however, the role of light and associated regulatory factors has not been studied in tomatoes. Here, we demonstrated that SGA biosynthesis is regulated by light through the ELONGATED HYPOCOTYL 5 homolog, SlHY5, by binding to light-responsive G-boxes present in the promoters of the structural and regulatory genes. Our analysis suggests that SlHY5 could complement the Arabidopsis thaliana and Nicotiana tabacum, hy5 mutants at molecular, morphological, and biochemical levels. We report the development of CRISPR/Cas9-based knockout mutant plants of tomato, slhy5CR, and show down-regulation of the SGA and phenylpropanoid pathway genes leading to a significant reduction in SGA (-tomatine and dehydrotomatine) and flavonol contents, whereas SlHY5 overexpression (SlHY5OX) plants show opposite effect. An enhanced SGA and flavonol levels in SlHY5OX lines provided tolerance against Alternaria solani fungus, while SlHY5CR was susceptible to the pathogen. This study advances our understanding of the HY5-dependent light-regulated biosynthesis of SGAs and flavonoids and their role in biotic stress in tomatoes. One Sentence SummaryLight-associated transcription factor, ELONGATED HYPOCOTYL 5, regulates biosynthesis of anti-nutrient molecules, steroidal glycoalkaloids, and fungal tolerance in tomato

molecular biology↗

bioRxiv2024

CanLncG4: A database curated for the assessment of G4s in the lncRNAs dysregulated in various human cancers

Long non-coding RNAs (lncRNAs) comprise a substantive part of the human genome and have emerged as crucial participants of cellular processes and disease pathogenesis. Dysregulated expression of lncRNAs in cancer contributes to various hallmarks of the disease, presenting novel opportunities for diagnosis and therapy. G-quadruplexes (G4s) within lncRNAs have gained attention, though their systematic evaluation in cancer biology is yet to be performed. In this work, we have formulated CanLncG4, a comprehensive database integrating experimentally validated associations between lncRNAs and cancer, and detailed predictions of their G4-forming potential. CanLncG4 categorizes predicted G4 motifs into anticipated G4 types and offers insights into the subcellular localization of the corresponding lncRNAs. It provides information on lncRNA-RNA and lncRNA-protein interactions, together with the RNA G4-binding capabilities of these proteins. To ensure the accuracy and validity of the data sourced from various databases, a meticulous examination of the output data was conducted to identify any discrepancies, including incorrect, missing, or duplicate entries. Additionally, scientific literature mining was performed to cross-validate the gathered information. Data from G4-prediction tools was generated using multiple parameter combinations to determine the parameters that yield more relevant and accurate predictions of the G4-forming potential. We validate our in silico G4-prediction pipeline through in vitro experiments, affirming the presence of G4s within specific cancer-dysregulated lncRNAs, thereby illustrating the predictive capability of CanLncG4. CanLncG4 represents a valuable resource for investigating G4-mediated lncRNA functions in diverse human cancers. It is expected to provide distinctive leads about G4-mediated lncRNA-protein interactions. CanLncG4 comprehensively documents 17,666 entries, establishing correlations between 6,408 human lncRNAs encompassing their transcript variants, and 15 distinct types of human cancers. The database is freely available at https://canlncg4.com/, offering researchers a valuable tool for exploring lncRNA and G4 biology towards cancer diagnosis and therapeutics.

bioinformatics↗

bioRxiv2024

RNF43 is a gatekeeper for colitis-associated cancer

Somatic mutations in the tumor suppressor Ring finger protein 43 (RNF43) were frequently found in colitis-associated cancer (CAC) and related to the duration of chronic inflammation, but their significance in inflammation and inflammation-associated carcinogenesis remained elusive. We assessed the onset of RNF43 mutations at different stages of human CAC development by exome sequencing, and comprehensively characterized RNF43 loss-of-function-driven malignant transformation in mice by RNA sequencing, flow cytometry, immunohistochemistry, computational transcriptome-microbiome associations, and determined the underlying mechanisms by performing functional stem-cell derived organoid studies and fecal microbiota transfers. Mutations in RNF43 were frequent (12.9 %) in precancerous lesions of ulcerative colitis (UC) patients and eventually detectable in 24.4 % of CAC patients. In a bacterial-induced colitis mouse model, Rnf43 mutations caused invasive colorectal carcinomas by aggravating and perpetuating inflammation due to impaired epithelial barrier integrity and pathogen control. We could demonstrate that Rnf43 loss-of-function-mutations were even sufficient to cause spontaneous intestinal inflammation, resulting in UC-typical pathological features and subsequent invasive carcinoma development. In detail, mutant Rnf43 impaired intestinal epithelial and particularly goblet cell homeostasis in a cell-intrinsic manner, and caused dysbiosis. The altered microbiota composition induced epithelial DNA damage and spontaneous mucosal inflammation characterized by TGF-{beta}-activating dendritic cells and pro-inflammatory (IL-17+, IL-22+, TNF+) T cells. Over time, the continuous epithelial and goblet cell dysfunction, combined with pro-tumorigenic and pro-inflammatory microbiota, resulted in accumulated epithelial damage with transformation into inflammation-associated cancer in the presence of constitutive WNT signaling activation. We identified mutant RNF43 as susceptibility gene for UC and bona fide driver of CAC.

cancer biology↗

bioRxiv2024

Identifying the genetic and epigenetic basis for asymmetric bZIP expression in temperature-stressed bread wheat

Asymmetric expression in the bread wheat (Triticum aestivum) genome refers to the differential expression of genes from A, B, and D parental genomes. Bread wheat is a hexaploid crop with six copies of each chromosome. This complexity can result in unequal expression of genes from each parental genome, leading to asymmetry in gene expression. In other polyploid crops like cotton, transcription factors (TF) exhibit genome-biased expression; however, there are no comparable studies for bread wheat. One of the most prominent TFs families in plants is the basic Leucine Zippers (bZIP) which are eukaryote-specific proteins and regulate various biological processes, including stress-related responses. bZIP proteins are dimeric and several heptads long. They exhibit typical coiled-coil structures with strategically placed amino acids in each heptad, responsible for their stability and specificity. Here, we aim to decipher the structural basis of the asymmetric expression of the bZIP TFs in wheat under low and high-temperature conditions. Furthermore, 19 highly expressed stress-related TabZIP TFs were analysed for their asymmetric expression profiles as plants were exposed to temperature-stress conditions. Two benchmarks were used to analyse the asymmetric gene expression of bZIPs, i.e., a) the promoters occupancy by the epigenetic marker histones, namely, H3K4me3, H3k9ac (active) and H3K27me3 (repressive), b) density and diversity of cis-regulatory elements in the promoters. Notably, the genetic basis of the differences in protein sequences of bZIP triads was explored, which may impart structural stability to a specific homeolog enabling the plant to endure the stress conditions better.

plant biology↗

bioRxiv2024

Tracing India's Canine Heritage through SNP-Based Haplotype Identification

Dog breeds/germplasm in India is mostly unexplored and the population structure of owned dogs has not been studied at all. The current study was designed to determine the haplotypes and explore the population structure among divergent breeds of dogs using genome-wide distributed SNPs, followed by validation of selected haplotypes through PCR-sequencing. The research employed custom ddRAD-GBS sequencing carried out using Illumina 150bp paired-end sequencing of 50 dog samples generated 2,18,433 high-quality SNPs meeting the screening criteria. The data was further analyzed for population structure assessment and haplotype identification using bash, and R-environment. Subsequently, three haplotypes (on AFAP1, CELSR1, and GBGT1 genes) were selected (based on SNP density and haplotype length) for validation via PCR followed by paired-end Sanger sequencing in seven different dog breeds (n=21). The results revealed notable connections between dog breeds from Punjab and Haryana, while the affiliation with Karnataka was found to be less pronounced. The sequencing results indicated that CELSR1 and GBGT1 genes contained SNPs, with the AFAP1 gene lacking SNPs. The results also provided insights into the molecular-level population structure, SNPs, and haplotypes of diverse dog breeds reared in India. This SNP variation could be used for molecular characterization of indigenous dogs.

evolutionary biology↗

bioRxiv2024

Integrative spatiotemporal map of nucleocytoplasmic transport

Nuclear Pore Complexes (NPCs) enable rapid, selective, and robust nucleocytoplasmic transport. To explain how transport emerges from the system components and their interactions, we used experimental data and theoretical information to construct an integrative Brownian dynamics model of transport through an NPC, coupled to a kinetic model of transport in the cell. The model recapitulates key aspects of transport for a wide range of molecular cargos, including pre-ribosomes and viral capsids. It quantifies how flexible phenylalanine-glycine (FG) repeat proteins raise an entropy barrier to passive diffusion and how this barrier is selectively lowered in facilitated diffusion by the many transient interactions of nuclear transport receptors with the FG repeats. Selective transport is enhanced by "fuzzy" multivalent interactions, redundant FG repeats, coupling to the energy-dependent RanGTP concentration gradient, and exponential dependence of transport kinetics on the transport barrier. Our model will facilitate rational modulation of the NPC and its artificial mimics.

cell biology↗

bioRxiv2023

Interaction of SCoV-2 NSP7 or NSP8 alone may cause constriction of the RNA entry channel in NSP12: Implications for novel RdRp inhibitor drug discovery

RNA-dependent RNA polymerase (RdRP) is a critical component of the RNA virus life cycle, including SCoV-2. Among the Coronavirus-encoded proteins, non-structural protein 12 (NSP12) exhibits polymerase activity in collaboration with one unit of NSP7 and two units of NSP8, constituting the RdRp holoenzyme. While there is abundant information on SCoV-2 RdRp-mediated RNA replication, the influence of interplay among NSP12, NSP7, and NSP8 on template RNA binding and primer extension activity remains relatively unexplored and poorly understood. Here, we recreated a functional RdRp holoenzyme in vitro using recombinant SCoV-2 NSP12, NSP7, and NSP8, and established its functional activity. Subsequently, molecular interactions among the NSPs in the presence of a variety of templates and their effects on polymerase activity were studied, wherein we found that NSP12 alone exhibited notable polymerase activity that increased significantly in the presence of NSP7 and NSP8. However, this activity was completely shut down, and the template RNA-primer complex was detached from NSP12 when one of the two cofactors was present. Through computational analysis, we found that the template RNA entry channel was more constricted in the presence of one of the two cofactors, which was relatively more constricted in the presence of NSP8 compared to that in the presence of NSP7. In conclusion, we report that NSP7 and NSP8 together synergise to enhance the activity of NSP12, but antagonise when present alone. Our findings have implications for novel drug development, and compounds that obstruct the binding of NSP7 or NSP8 to NSP12 can have lethal effects on viral RNA replication.

molecular biology↗

bioRxiv2023

MPK4 mediated phosphorylation of PIF4 controls thermosensing by regulation of H2A.Z deposition in Arabidopsis

Plants have the ability to perceive a slight upsurge in ambient temperature and respond by undergoing morphological changes, such as elongated hypocotyls and early flowering. The dynamic functioning of PHYTOCHROME INTERACTING FACTOR4 (PIF4) in thermomorphogenesis has been well established, although the regulatory pathway involved in thermosensing is not deciphered completely. In our study, we demonstrate that an increase in temperature from 22 to 28 induces the phosphorylation of PIF4 by MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) which shows high expression and activation at 28. Apparently, phosphorylated PIF4 represses the expression of ACTIN-RELATED PROTEIN 6 (ARP6) that is required for mediating histone variant H2A.Z deposition at its target gene loci. We demonstrate that variation of ARP6 expression in PIF4 phosphor -null and phosphor-mimetic seedlings affects hypocotyl growth and flowering at 22 and 28. Further, we show that change in ARP6 expression affects H2A.Z deposition at the loci of genes involved in hypocotyl elongation using PIF4 phosphor -null and phosphor-mimetic seedlings. Interestingly, the expression of MPK4 is also controlled by H2A.Z deposition in temperature dependent manner. Taken together, our findings highlight the cumulative molecular interplay between MPK4, PIF4, and chromatin modification by ARP6-mediated H2A.Z deposition as a regulatory mechanism of thermosensing.

plant biology↗

bioRxiv2023

A broadly protective CHO cell expressed recombinant spike protein subunit based vaccine (IMT-CVAX) against SARS-CoV-2

Protective immunity induced by COVID-19 vaccines is mediated mainly by spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report the development of a recombinant prefusion stabilized SARS-CoV-2 spike protein-subunit-based COVID-19 vaccine produced in the mammalian cell line. The gene encoding ectodomain (ECD) of the spike protein was engineered and cloned into Freedom pCHO 1.0, a mammalian expression vector, and subsequently expressed in the Chinese Hamster Ovary suspension cell line (CHO-S). The recombinant S protein ectodomain (hereafter referred to as IMT-CVAX) was purified using a combination of tangential flow filtration and liquid chromatography. Biochemical and biophysical characterization of IMT-CVAX was done to ensure its vital quality attributes. Intramuscular immunization of mice with two doses of adjuvanted IMT-CVAX elicited a strong anti-Spike IgG response. In pseudovirus-based assays, IMT-CVAX- immune mice sera exhibited a broad-spectrum neutralization of several SARS-CoV-2 variants of concern (VoCs). Golden Syrian Hamster immunized with IMT-CVAX provided excellent protection against SARS-CoV-2 infection, and, hamster immune sera neutralized the live SARS-CoV-2 virus. The adjuvanted IMT-CVAX induced robust Tfh-cells response and germinal center (GC) reaction in human ACE2 receptor-expressing transgenic mice. The findings of this study may pave the way for developing next-generation protein subunit-based vaccines to combat the existing SARS-CoV-2 and its emerging VoCs. The IMT-CVAX is produced using a scalable process and can be used for large-scale vaccine production in an industrial setup.

microbiology↗

bioRxiv2023

Dopamine neurons govern olfactory-gated infant begging behavior

Altricial young of many species beg parents for the nutrients required for healthy development. From human crying to begging chicks, young expend precious energy reserves to communicate their hunger. Despite repeated independent evolutionary origins, the neural basis of parent-directed communication by infants is unknown. Here, we examined the sensory and neural basis of begging behavior in tadpoles of the monogamous and biparental Mimetic poison frog (Ranitomeya imitator). In this species, tadpoles beg parents for egg meals by dancing. We used this robust motor display to determine that tadpoles use multimodal cues for caregiver recognition, where olfactory cues are necessary for caregiver recognition while visual cues provide an orientation goal. We found that dopamine related brain regions have higher neural activity in begging tadpoles and that dopamine signaling had opposing modulatory effects through D1 and D2 family receptors, similar to swimming behavior in other tadpole species. We then identified caudal posterior tuberculum dopamine neurons as more active during begging behavior and sensitive to caregiver olfactory cues. Projections of these dopaminergic neurons to the spinal accessory motor nucleus are required for begging displays. These findings support the idea that dopamine regulates olfactory-guided parental recognition in young and opens many avenues for studying how new communication behaviors can evolve from ancestral motor circuits.

neuroscience↗

bioRxiv2023

PandoraRLO: DQN and Graph convolution based method for optimized ligand pose

Predicting how proteins interact with small molecules is a complex and challenging task in the field of drug discovery. Two important aspects in this are shape complementarity and inter molecular interactions which are highly driven by the binding site and the ultimate pose of the ligand in which it interacts with the protein. Various state of the art methods exist which provide a range of ligand poses that are potentially a good fit for a given specific receptor, these are usually compute intensive and expensive. In this study, we have designed a method that provides a single optimized ligand pose for a specific receptor. The method is based on reinforcement learning where when exposed to a diverse protein ligand data set the agent is able to learn the underlying complex biochemistry of the protein ligand pair and provide an optimized pair. As a first study on usage of reinforcement learning for optimized ligand pose, the PandoraRLO model is able to predict pose within a range of 0.5[A] to 4[A] for a large number of test complexes. This indicates the potential of reinforcement learning in uncovering the inherent patterns of protein-ligand pair in 3D space.

bioinformatics↗

bioRxiv2023

System analysis of differentially expressed miRNAs in hexaploid wheat display tissue-specific regulatory role during Fe deficiency response

BackgroundIron (Fe) is an essential mineral element, and its deficiency in soil largely affects crop productivity. In plants, the molecular mechanisms underlying the genetic regulation of Fe deficiency responses have yet to be well understood. Specifically, microRNA (miRNA) mediated regulation of Fe deficiency response and its regulatory network is largely elusive. In the current work, we utilized a whole genome transcriptomic approach to identify the Fe deficiency-responsive miRNAs to understand the molecular mechanisms of Fe deficiency response in wheat seedlings. The study also identifies nine novel miRNAs putatively involved in Fe deficiency response. Further, the identified miRNAs showed tissue preferences relating them to differential mechanisms against Fe deficiency. ResultsIn the present study, we performed small RNA-targeted whole genome transcriptome analysis to identify the involvement of sRNAs in Fe deficiency response. The analysis identified 105 differentially expressed miRNAs corresponding to Fe deficiency response, among them, 9 miRNAs were found to be novel in this study. Interestingly, tissue-specific regulation of Fe deficiency response also participates through miRNA-mediated regulation. We identified 17 shoot specific miRNAs and 18 root-specific miRNAs with altered expression. We validated the tissue specificity of these miRNAs by stem-loop quantitative RT-PCR. Further, an attempt was made to predict their targets to speculate their participation in Fe deficiency response. This miRNA target prediction analysis suggested a few major targets of the identified miRNAs, such as multicopper oxidases, E3 ubiquitin ligases, GRAS family, and WRKY transcription factors previously known to play key roles in Fe homeostasis. Our analysis of selected miRNAs also confirmed a temporal regulation of the response. ConclusionThe first information generated here will classify the repository of wheat miRNAs (with few novel miRNAs) for their role in Fe deficiency response. Our work provides insights into miRNA-mediated regulatory pathways during Fe deficiency.

plant biology↗

bioRxiv2023

Dysregulated miR-3959-3p in response to Lumpy skin disease virus

Lumpy skin disease virus (LSDV), a member of the Capripoxvirus genus, causes substantial economic losses in the livestock industry and is rapidly spreading among various LSDV-free countries across the globe. Viral infections are known to alter the cellular miRNA expression profile of the host significantly.Besides being important biomarker candidates, circulating miRNAs have a significant role in controlling viral infection and antiviral immune responses, including several molecular mechanisms. miR-3959-3p, a significantly downregulated miRNA revealed in RNA-sequencing results of LSDV-infected LT cells, was selected to bedetected in the serum of LSDV-infected and uninfected cattle sera (40 LSDV-infected and 40 LSDV-uninfected). We optimized quantitative real-time PCR (qRT-PCR) for quantitative determination of miR-3959-3p in the bovine sera samples. The level of miR-3959-3p appears to be towards lower side in the LSDV-infected as compared to the uninfected animals. However, no significant correlation could be established between the two groups due to fluctuations in the miRNA levels in both groups. This is the first report on the detection of circulating miRNA in LSDV-infected cattle sera.The dysregulation pattern of miR-3959-3p appears to suggest that several other miRNAs need to be explored and may serve as biomarkers for LSDV infection. However, this needs further investigation by screening several other miRNAs and on large number of LSDV positive and negative animals. Author summaryMicroRNAs are the key regulators of viral infections. However, in LSDVinfection the miRNA response is greatly unknown. In this study, miRNA expression in Vero cell linepost LSDV infection was studied for the first time. One of the miRNAs identified in the RNA-sequencing results i.emiR-3959-3p, was shown to be downregulated LSDV infection. We detected the levels of mir-3959-3p in sera of LSDV-infected and uninfected cattle to explore its potential as a biomarker.

molecular biology↗

bioRxiv2023

A Novel Algorithm to Calculate Elementary Modes: Analysis of Campylobacter jejuni Metabolism

We describe a novel algorithm, LPEM, that given a steady-state flux vector from a (possibly genome-scale) metabolic model, decomposes that vector into a set of weighted elementary modes such that the sum of these elementary modes is equal to the original flux vector. We apply the algorithm to a genome scale metabolic model of the human pathogen Campylobacter jejuni. This organism is unusual in that it has an absolute growth requirement for oxygen, despite being able to operate the electron transport chain anaerobically. We conclude that 1) Microaerophilly in C. jejuni can be explained by the dependence of pyridoxine 5' -phosphate oxidase for the synthesis of pyridoxal 5' - phosphate (the biologically active form of vitamin B6), 2) The LPEM algorithm is capable of determining the elementary modes of a linear programming solution describing the simultaneous production of 51 biomass precursors, 3) Elementary modes for the production of individual biomass precursors are significantly more complex when all others are produced simultaneously than those for the same product in isolation and 4) The sum of elementary modes for the production of all precursors in isolation requires a greater number of reactions and overall total flux than the simultaneous production of all precursors.

systems biology↗

bioRxiv2022

LHX2 in germ cells control tubular organization in the developing mouse testis

In the gonads of mammalian XY embryos, the organization of cords is the hallmark of testis development. This organization is thought to be controlled by interactions of the Sertoli cells, endothelial and interstitial cells with little or no role of germ cells. Challenging this notion, herein we show that the germ cells play an active role in the organization of the testicular tubules. We observed that the LIM-homeobox gene, Lhx2 is expressed in the germ cells of the developing testis between E12.5-E15.5. In Lhx2 knockout-fetal testis there was altered expression of several genes not just in germ cells but also in the supporting (Sertoli) cells, endothelial cells, and interstitial cells. Further, loss of Lhx2 led to disrupted endothelial cell migration and expansion of interstitial cells in the XY gonads. The cords in the developing testis of Lhx2 knockout embryos are disorganized with a disrupted basement membrane. Together, our results show an important role of Lhx2 in testicular development and imply the involvement of germ cells in the tubular organization of the differentiating testis. HighlightO_LILhx2 is expressed in germ cells of developing mouse testis but is dispensable for germ cell and Sertoli cell specification C_LIO_LILoss of Lhx2 disrupts testicular vascularization, leads to the expansion of interstitial cells, and alters the tubular organization C_LIO_LIGerm cells govern the tubular organization in the developing testis C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/522214v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a1761dorg.highwire.dtl.DTLVardef@1519f12org.highwire.dtl.DTLVardef@1a263baorg.highwire.dtl.DTLVardef@4167f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapyinduced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, in particular LAG3. Functionally, the OX40hiGITRhi subset in the most highly suppressive ex vivo and Treg expression of OX40, GITR and LAG3, correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFN{gamma} and certain pro-inflammatory granzymes. Application of a gene score from the murine TAA-specific Th1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1 responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses. One-Sentence SummaryWe define 10 subsets of lung cancer-infiltrating regulatory T cells, one of which is highly suppressive and enriched in anti-PD-1 non-responders and the other is Th1-like and is enriched in PD-1 responders.

immunology↗