Biology subjects

Chakraborty, A.

Publications and source records attributed to Chakraborty, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

CTCF directly binds G-quadruplex structures to regulate genome topology and gene expression

DNA G-quadruplexes (G4s) are non-B form secondary DNA structures that are highly conserved across evolution. G4 structures occupy key regulatory sites in the mammalian genomes and are implicated in several cellular processes. However, the mechanisms by which G4s contribute to distinct facets of genome function are not well understood. Here, we conduct a proteomics screen with G4s of diverse topologies to uncover novel G4 binding activities in genomic regulators of nucleosome remodeling, paraspeckle assembly, RNA splicing, and 3D genome organization. Among the most prominent hits, we identify the genomic architectural protein, CTCF, as one of the strongest G4 binders. Building on this discovery, we perform extensive biochemical validation of CTCF-G4 interaction and identify CTCF mutants, with pronounced affinity for G4s over its consensus DNA motif. By implementing well-established approaches and developing new G4 mapping tools, we define a comprehensive catalog of genomic G4s and investigate their association with CTCF binding. Using genetic reconstitution of mouse embryonic stem cells with a G4-specific CTCF mutant, we ascribe genomic functions to CTCF-G4 interactions in regulation of CTCF occupancy, chromatin looping and gene expression. Interestingly, our studies reveal a subset of G4-linked chromatin loop anchors that form "persistent loops" which are retained even upon CTCF depletion. Collectively, our work establishes the G4 binding activity of CTCF and provides new insights into the functional significance of G4 structures.

molecular biology↗

bioRxiv2025

Quorum Sensing Regulators and Non-ribosomal Peptide Synthetases Govern Antibacterial Secretions in Xenorhabdus szentirmaii

The decades-long gap in antibiotic discovery has led to a significant health crisis due to antimicrobial resistance (AMR). The bacterial genus Xenorhabdus, which forms symbiotic relationships with the soil nematode Steinernema, are known to secrete a variety of antimicrobial compounds with potential effectiveness against AMR. These antimicrobial compounds are primarily bio-synthesized by non-ribosomal peptide synthetases (NRPS) and polyketide synthetase (PKS) genes. In this study, we report that X. szentirmaii produces high levels of antibiotic activity during the stationary phase against diverse bacteria including known antibiotic resistant pathogens. It possesses 17 operons to encode predicted NRPS and PKS enzymes, designated as ste1 through ste17. The ste15-ste16 and ste17 operons are predicted to produce the known antibiotics Pax peptide and Fabclavine, respectively. Additionally, the newly identified operons ste3, ste4, ste5, ste8, ste9, and ste14 consist of single genes, each containing two or more NRPS genes. The ste13 operon harbors two NRPS genes, while the ste7 and ste12 operons contain three NRPS genes each. Further, RNA-seq analysis showed that lsrF that encodes a quorum sensing autoinducer-2 (AI-2) aldolase was expressed at high levels during stationary phase. These findings provide evidence that X. szentirmaii uses quorum sensing (QS) to synchronize the expression of multiple NRPS and PKS enzymes responsible for synthesizing various antimicrobial compounds. This study underscores the potential to leverage these regulatory insights for maximizing commercial applications of novel antibiotics combating AMR, as well as broader industrial uses.

microbiology↗

bioRxiv2025

12-Lipoxygenase inhibition improves glucose homeostasis and obesity-associated inflammation in human gene replacement mice

Obesity-associated inflammation is characterized by macrophage infiltration into peripheral tissues, contributing to the progression of prediabetes and type 2 diabetes (T2D). The enzyme 12-lipoxygenase (12-LOX) catalyzes the formation of pro-inflammatory eicosanoids and is known to promote the migration of macrophages, yet its role in obesity-associated inflammation remains incompletely understood. Furthermore, differences between mouse and human orthologs of 12-LOX have limited efforts to study existing pharmacologic inhibitors of 12-LOX. In this study, we utilized a human gene replacement mouse model in which the gene encoding mouse 12-LOX (Alox15) is replaced by the human ALOX12 gene. As a model of obesity and dysglycemia, we administered these mice a high-fat diet. We subsequently investigated the effects of VLX-1005, a potent and selective small molecule inhibitor of human 12-LOX. Oral administration of VLX-1005 resulted in improved glucose homeostasis, decreased {beta} cell dedifferentiation, and reduced macrophage infiltration in islets and adipose tissue. Analysis of the stromal vascular fraction from adipose tissue showed a reduction in myeloid cells and cytokine expression with VLX-1005 treatment, indicating decreased adipose tissue inflammation. In a distinct mouse model in which Alox15 was selectively deleted in myeloid cells, we observed decreased {beta} cell dedifferentiation and reduced macrophage infiltration in both islets and adipose tissue, suggesting that the effects of VLX-1005 may relate to the inhibition of 12-LOX in macrophages. These findings highlight 12-LOX as a key factor in obesity-associated inflammation and suggest that 12-LOX inhibition could serve as a therapeutic strategy to improve glucose homeostasis and peripheral inflammation in the setting of obesity and T2D.

physiology↗

bioRxiv2024

A new mechanochemical vertex model with Ca2+ signalling, for apical constriction in neural tube closure

Apical constriction during neural tube closure is driven by cell contractions which are preceded by asynchronous and cell-autonomous Ca2+ flashes, as demonstrated in recent experiments. Disruption of these Ca2+ signals and contractions leads to neural tube defects, such as anencephaly and spina bifida. A good understanding of the two-way mechanochemical coupling of Ca2+ signalling and mechanics remains elusive, while live-cell imaging is difficult. Thus, mathematical modelling is essential but existing models do not exhibit good agreement with experiments. We present two new mechanochemical vertex models of apical constriction during neural tube closure and simulate them using CelluLink, a new user-friendly open-source Python package for vertex modelling. The first, one-way mechanochemical model only studies the effect of Ca2+ signalling on cell mechanics. It improves previous models, reproducing some key experimental observations, such as the reduction of the neural plate size to 2%-8% of its initial area. Other novel features of the one-way model is the incorporation of the surface ectoderm and of the experimental amplitude and frequency profiles of the Ca2+ flashes. Furthermore, guided by experiments, the damping coefficient of the vertices and cell-cell adhesion are modelled as functions of the actomyosin concentration and cell size. Furthermore, we develop a two-way model which improves the one-way model by capturing the two-way coupling between Ca2+ signalling and cell mechanics, through the incorporation of stretch-sensitive Ca2+ channels. These channels enable cells to sense mechanical stimuli and encode them into Ca2+ signals. In the two-way model, the Ca2+ flash frequency and amplitude profiles are model outputs and are not inputs as in the one-way model. Finally, we use both models to propose a series of hypotheses for future experiments. Author summaryAs a baby is growing in the womb, its neural tube closes to form the brain and the spinal cord. During neural tube closure, cells are contracting in a ratchet-like way while experiencing a choreography of Ca2+ flashes. If the Ca2+ flashes or the contractions go wrong, serious birth defects like spina bifida and anencephaly may arise. Understanding how Ca2+ flashes and contractions work together is complex, especially since studying living cells is challenging. To address this challenge, we developed two new mathematical models. The first model captures how Ca2+ flashes affect contractions, improving previous models and accurately capturing some experimental results. For example, it incorporates recent experimental measurements of the amplitude of Ca2+ flashes (brightness) and their frequency (how frequently the flashes appear). The second model builds on the first model by additionally capturing the effect contractions have on the Ca2+ flashes. We capture this two-way coupling by enabling cells to sense mechanical stimuli through stretch-sensitive Ca2+ channels. In this case, the amplitude and frequency of the Ca2+ flashes arise as outputs. Both models inform future experiments that will further elucidate embryo malformations.

systems biology↗

bioRxiv2024

A High-quality Genome Assembly of Annona squamosa (Custard Apple) Provides Functional Insights into an Emerging Fruit Crop

Annona squamosa, also known as custard apple, is an emerging fruit crop with medicinal significance. We constructed a high-quality genome of A. squamosa along with transcriptome data to gain insights into its phylogeny, evolution and demographic history. The genome has an N50 value of 93.2 Mb assembled into seven pseudochromosomes. The demographic history showed a continuous decline in the effective population size of A. squamosa. Phylogenetic analysis revealed that magnoliids were sister to eudicots, whereas ASTRAL gene trees showed discordance due to incomplete lineage sorting (ILS). Genome syntenic and Ks distribution analyses confirmed the absence of a recent whole genome duplication event in the A. squamosa. Gene families related to photosynthesis, oxidative phosphorylation and plant thermogenesis were found to be highly expanded in the genome. Comparative analysis with other magnoliids revealed the adaptative evolution in the genes of flavonoid biosynthesis pathway, amino sugar, nucleotide sugar and sucrose metabolism, conferring medicinal value and enhanced hexose sugar accumulation. In addition, we performed genome-wide identification of SWEET genes. Our high-quality genome and evolutionary insights of this emerging fruit crop, thus, serve as a valuable resource for advancing studies in functional genomics, evolutionary biology, and crop improvement.

genomics↗

bioRxiv2024

Fructose-2,6-bisphosphate restores TDP-43 pathology-driven genome repair deficiency in motor neuron diseases

TAR DNA-binding protein 43 (TDP-43) proteinopathy plays a critical role in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We recently reported that TDP-43 plays an essential role in DNA double-strand break (DSB) repair via the non-homologous end-joining (NHEJ) pathway. Here, we provide evidence that the brain of patients with ALS exhibit persistent DNA damage in the transcribed regions of the genome. While investigating the mechanistic basis, we found that the activity of polynucleotide kinase 3-phosphatase (PNKP) was severely impaired in the nuclear extracts of patient brains and TDP-43-depleted cells. PNKP is a key player in DSB repair within the transcribed genome, where its 3-phosphate termini processing activity is crucial for preventing persistent DNA strand breaks and neuronal death. The inactivation of PNKP was due to the reduced level of its interacting partner, phosphofructo-2-kinase fructose 2,6 bisphosphatase (PFKFB3), and its biosynthetic product, fructose-2,6-bisphosphate (F2,6BP), an allosteric modulator of glycolysis. Recently, we have demonstrated that F2,6BP acts as a positive modulator of PNKP activity in vivo. Furthermore, F2,6BP supplementation in cultured ALS patient-derived neural progenitor stem cells (NPSCs) reduced the toxic aggregation of polyubiquitinated TDP-43 and cytosolic pTDP-43 (S409/410). Notably, F2,6BP supplementation restored the PNKP activity in the nuclear extracts from autopsied ALS/FTD brain tissues and patient iPSC-derived NPSCs harboring TDP-43 mutations. Importantly, F2,6BP administration significantly restored the genome integrity and motor phenotypes in a Drosophila model of ALS-TDP-43. Collectively, these findings underscore the therapeutic potential of F2,6BP in TDP-43 pathology-associated motor neuron diseases.

neuroscience↗

bioRxiv2024

F2,6BP restores mitochondrial genome integrity in Huntingtons Disease

Several reports have indicated that impaired mitochondrial function contributes to the development and progression of Huntingtons disease (HD). Mitochondrial genome damage, particularly DNA strand breaks, is a potential cause for its compromised functionality. Here we show that the activity of polynucleotide kinase 3-phosphatase (PNKP), a critical DNA end-processing enzyme, is significantly decreased in the mitochondrial extract of HD patients brains due to a lower level of fructose-2,6 bisphosphate (F2,6BP), a biosynthetic product of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Such decrease in PNKP activity leads to persistent DNA strand breaks that are refractory to subsequent steps for repair completion. Both PFKFB3 and F2,6BP, an allosteric modulator of glycolysis, are also present in the mitochondria and PFKFB3 is part of a mitochondrial DNA repair complex containing HTT, PNKP, DNA Pol {gamma} (POLG) and Lig III. Notably, PNKP binds F2,6BP (Kd= 525{+/-}25 nM) and utilizes it as a cofactor. The levels of both F2,6BP and PFKFB3 are significantly decreased in the mitochondrial extract of HD mouse striatal neuronal cells and patients brain. Activity of PNKP is thus severely decreased in the mitochondrial extract; however, addition of F2,6BP restored its activity. Moreover, supplementation of F2,6BP in HD cells restored PFKFB3 level, mitochondrial genome integrity and partially restored mitochondrial membrane potential, mitochondrial respiration and prevented pathogenic aggregate formation. We also observed that supplementation with F2,6BP restored mitochondrial genome integrity in an HD Drosophila model. Our findings, therefore, suggest that F2,6BP-mediated restoration of PNKP activity could have a profound impact in ameliorating neurodegenerative symptoms in HD. SignificanceWe reported earlier the loss of PNKP activity in the nuclear extracts from HD patients brain. However, a glycolytic metabolite, F2,6BP, can restore PNKP activity and rescue organismal phenotypes in HD fly models. As PNKP is present in mitochondria and several reports indicate that mitochondrial dysfunction contributes to HD, we therefore analyzed PNKP activity in the mitochondrial extract. Surprisingly, we found that PFKFB3 and its product, F2,6BP are present in mitochondria, but significantly low in patients brains. Exogenous addition of F2,6BP restored PNKP activity in patients brain mitochondrial extract. Moreover, supplementing F2,6BP in HD cells and fruit flies restored mitochondrial genome integrity suggesting maintaining adequate intracellular F2,6BP levels is critical for proper functionality of PNKP and thereby of brain health.

neuroscience↗

bioRxiv2024

LSD1 inhibition attenuates targeted therapy-induced lineage plasticity in BRAFV600E colorectal cancer

BRAF activating mutations occur in approximately 10% of metastatic colorectal cancer (CRCs) and are associated with worse prognosis due to an inferior response to standard chemotherapy. Standard of care for patients with refractory metastatic BRAFV600E CRC is treatment with BRAF and EGFR inhibitors. However, responses are not durable. Lineage plasticity to neuroendocrine cancer is an emerging mechanism of targeted therapy resistance in several cancer types. Enteroendocrine cells (EECs), the neuroendocrine cell of the intestine, are uniquely present in BRAFV600ECRC as compared to BRAF wildtype CRC. Here, we demonstrated that combined BRAF and EGFR inhibition enriches for EECs in several models of BRAFV600ECRC. Additionally, EECs and other secretory cell types were enriched in a subset of BRAFV600E CRC patient samples following targeted therapy. Importantly, inhibition of the lysine demethylase LSD1 with a clinically relevant inhibitor attenuated targeted therapy-induced EEC enrichment through blocking the interaction of LSD1, CoREST2 and STAT3. Statement of SignificanceOur findings that BRAF plus EGFR inhibition induces lineage plasticity in BRAFV600E CRC represents a new paradigm for how resistance to BRAF plus EGFR inhibition occurs and our finding that LSD1 inhibition blocks lineage plasticity has the potential to improve responses to BRAF plus EGFR inhibitor therapy in patients.

cancer biology↗

bioRxiv2024

Hypusinated and unhypusinated isoforms of eIF5A exert distinct effects in models of pancreas development and function

Hypusination of eukaryotic translation initiation factor 5A (eIF5A) is essential for its role in translation elongation and termination. Although the function of hypusinated eIF5A (eIF5AHyp) in cellular proliferation is well-characterized, the role of its unhypusinated form (eIF5ALys) remains unclear. We hypothesized that eIF5ALys exerts independent effects on cellular replication and metabolism distinct from the loss of eIF5AHyp. To test this hypothesis, we utilized zebrafish and mouse models with inducible knockdowns of deoxyhypusine synthase (DHPS) and eIF5A to investigate their roles in cellular growth. Gene expression analysis via RNA sequencing and morphometric measurements of pancreas and {beta}-cell mass were performed to assess phenotypic changes and identify affected biological pathways. Loss of DHPS in zebrafish resulted in significant defects in pancreatic growth, accompanied by the dysregulation of mRNA translation, neurogenesis, and stress pathways. By contrast, knockdown of eIF5A had minimal impact on pancreas development, suggesting that the effects of DHPS loss are not solely due to the lack of eIF5AHyp. In mice, {beta} cell-specific deletion of DHPS impaired {beta} cell mass expansion and glucose tolerance, while eIF5A deletion had no statistically significant effects. These findings reveal an independent role for eIF5ALys in regulating developmental and functional responses and that a balance in levels of the hypusinated and unhypusinated isoforms of eIF5A may be pivotal in cellular phenotypes in health and disease.

cell biology↗

bioRxiv2024

Wag31, a membrane tether, is crucial for lipid homeostasis in mycobacteria

The mycobacterial cytoskeletal protein Wag31 is necessary for maintaining cell shape and directing cellular growth and elongation. Wag31 has a characteristic N-terminal DivIVA-domain and a C-terminal coiled-coil domain. While the role of Wag31 in polar elongation is known, there is limited mechanistic insight on how it orchestrates growth and elongation. In this report, we delineate roles of the N- and C-terminal domains of Wag31 using genetics, state-of-the-art multi-omics, biochemical, and imaging approaches. We show that Wag31 predominantly interacts with several membrane-associated proteins involved in lipid metabolism, cell wall synthesis and division. Native levels of Wag31 are critical for the maintenance and distribution of membrane lipids. Both depletion and overexpression of Wag31 perturbs lipid homeostasis, leading to the formation of intracellular lipid inclusions (ILIs). Protein-lipid crosslinking and imaging studies reveal that purified Wag31 can bind and effectively tether Cardiolipin (CL)-containing liposomes. We further show that the tethering activity lies in the DivIVA-domain containing N-terminal of Wag31 while the C-terminal mediates protein-protein interactions of Wag31. Despite retaining its ability to interact with partner proteins, the DivIVA domain-deleted Wag31 mutant shows defects in liposome tethering in vitro and non-polar localization of CL in vivo, which eventually causes lethality. Our study suggests that membrane tethering licenses Wag31 to form scaffolds that help orchestrate protein-lipid and protein-protein interactions necessary for mycobacterial growth and survival.

microbiology↗

bioRxiv2024

Emergence of Dip2-mediated Specific DAG-based PKC Signalling Axis in Eukaryotes

Diacylglycerols (DAGs) are used for metabolic purposes and are tightly regulated secondary lipid messengers in eukaryotes. DAG subspecies with different fatty-acyl chains are proposed to be involved in the activation of distinct PKC isoforms, resulting in diverse physiological outcomes. However, the molecular players and the regulatory origin for fine-tuning the PKC pathway are unknown. Here, we show that Dip2, a conserved DAG regulator across Fungi and Animalia, has emerged as a modulator of PKC signalling in yeast. Dip2 maintains the level of a specific DAG subpopulation, required for the activation of PKC-mediated cell wall integrity pathway. Interestingly, the canonical DAG-metabolism pathways, being promiscuous, are decoupled from PKC signalling. We demonstrate that these DAG subspecies are sourced from a phosphatidylinositol pool generated by the acyl-chain remodelling pathway. Furthermore, we provide insights into the intimate coevolutionary relationship between the regulator (Dip2) and the effector (PKC) of DAG-based signalling. Hence, our study underscores the establishment of Dip2-PKC axis about 1.2 billion years ago in Opisthokonta, which marks the rooting of the first specific DAG-based signalling module of eukaryotes.

cell biology↗

bioRxiv2024

Motif distribution in genomes gives insights into gene clustering and co-regulation

We read the genome as proteins in the cell would - by studying the distributions of 5-6 base motifs of DNA in the whole genome or smaller stretches such as parts of, or whole chromosomes. This led us to some interesting findings about motif clustering and chromosome organisation. It is quite clear that the motif distribution in genomes is not random at the length scales we examined: 1kbps to entire chromosomes. The observed to expected (OE) ratios of motif distributions show strong correlations in pairs of chromosomes that are susceptible to translocations. With the aid of examples, we suggest that similarity in motif distributions in promoter regions of genes could imply co-regulation. A simple extension of this idea empowers us with the ability to construct gene regulatory networks. Further, we could make inferences about the spatial proximity of genomic fragments using these motif distributions. Spatially proximal regions, as deduced by Hi-C or pcHi-C, were [~]3.5 times more likely to have their motif distributions correlated than non-proximal regions. These correlations had strong contributions from the CTCF protein recognizing motifs which are known markers of TADs. In general, correlating genomic regions by motif distribution comparisons alone is rife with functional information. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/613605v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1cf4193org.highwire.dtl.DTLVardef@1da47f5org.highwire.dtl.DTLVardef@1aa4172org.highwire.dtl.DTLVardef@aeaa60_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

High-speed 4D fluorescence light field tomography of whole freely moving organisms

Volumetric fluorescence imaging techniques, such as confocal, multiphoton, light sheet, and light field microscopy, have become indispensable tools across a wide range of cellular, developmental, and neurobiological applications. However, it is difficult to scale such techniques to the large 3D fields of view (FOV), volume rates, and synchronicity requirements for high-resolution 4D imaging of freely behaving organisms. Here, we present reflective Fourier light field computed tomography (ReFLeCT), a new high-speed volumetric fluorescence computational imaging technique. ReFLeCT synchronously captures entire tomograms of multiple unrestrained, unanesthetized model organisms over multi-millimeter 3D FOVs at 120 volumes per second. In particular, we applied ReFLeCT to reconstruct 4D videos of fluorescently labeled zebrafish and Drosophila larvae, enabling us to study their heartbeat, fin and tail motion, gaze, jaw motion, and muscle contractions with nearly isotropic 3D resolution while they are freely moving. As a novel approach for snapshot tomographic capture, ReFLeCT is a major advance towards bridging the gap between current volumetric fluorescence microscopy techniques and macroscopic behavioral imaging.

animal behavior and cognition↗

bioRxiv2024

E-cigarettes increase the risk of adenoma formation in murine colorectal cancer model.

BackgroundE-cigarettes (E.cigs) cause inflammation and damage to human organs, including the lungs and heart. In the gut, E.cig vaping promotes inflammation and gut leakiness. Further, E.cig vaping increases tumorigenesis in oral and lung epithelial cells by inducing mutations and suppressing host DNA repair enzymes. It is well known that cigarette (cig) smoking increases the risk of colorectal cancer (CRC). To date, it is unknown whether E.cig vaping impacts CRC development. MethodsA mouse model of human familial adenomatous polyposis (CPC-APC) was utilized wherein a mutation in the adenomatous polyposis coli (APC) gene, CDX2-Cre-APCMin/+, leads to the development of colon adenomas within 16 weeks. Mice were exposed to air (controls), E.cig vaping, cig, or both (dual exposure). After 4 weeks of 2-hour exposures per day (1 hour of each for dual exposures), the colon was collected and assessed for polyp number and pathology scores by microscopy. Expression of inflammatory cytokines and cancer stem cell markers were quantified. DNA damage such as double-strand DNA breaks was evaluated by immunofluorescence, western blot and gene-specific long amplicon qPCR. DNA repair enzyme levels (NEIL-2, NEIL-1, NTH1, and OGG1) were quantified by western blot. Proliferation markers were assessed by RT-qPCR and ELISA. ResultsCPC-APC mice exposed to E.cig, cig, and dual exposure developed a higher number of polyps compared to controls. Inflammatory proteins, DNA damage, and cancer stemness markers were higher in E-cig, cig, and dual-exposed mice as well. DNA damage was found to be associated with the suppression of DNA glycosylases, particularly with NEIL-2 and NTH1. E.cig and dual exposure both stimulated cancer cell stem markers (CD44, Lgr-5, DCLK1, and Ki67). The effect of E.cigs on polyp formation and CRC development was less than that of cigs, while dual exposure was more tumorigenic than either of the inhalants alone. ConclusionE.cig vaping promotes CRC by stimulating inflammatory pathways, mediating DNA damage, and upregulating transcription of cancer stem cell markers. Critically, combining E.cig vaping with cig smoking leads to higher levels of tumorigenesis. Thus, while the chemical composition of these two inhalants, E.cigs and cigs, is highly disparate, they both drive the development of cancer and when combined, a highly common pattern of use, they can have additive or synergistic effects.

cancer biology↗

bioRxiv2024

Local Synthesis of Reticulon-1C Lessens the Outgrowth of Injured Axons by Controlling Spastin Activity

The regenerative potential of developing cortical axons depends on intrinsic mechanisms, such as axon-autonomous protein synthesis, that are still not fully understood. An emerging factor in this regenerative response is the bi-directional interplay between microtubule dynamics and the axonal endoplasmic reticulum (ER). We hypothesize that locally synthesized ER proteins regulate microtubule dynamics and the regeneration of cortical axons. RNA data-mining identified the ER-shaping protein Reticulon-1 as a relevant candidate across eight axonal transcriptomes. Using microfluidic chambers, we demonstrate that local knockdown of Reticulon-1 synthesis increases the outgrowth of injured cortical axons while reducing their distal tubulin levels. Furthermore, we show by live-cell imaging that axonal Reticulon-1 knockdown restores the microtubule growth rate and track length modified by injury. Interestingly, local inhibition of the microtubule- severing protein Spastin prevents the axonal knockdown-mediated effects over outgrowth and tubulin levels. We demonstrate that the Reticulon-1C isoform is locally synthesized within axons and support its isoform-specific role in attenuating Spastin-mediated microtubule severing. These findings uncover a mechanism by which axonal protein synthesis finely controls microtubule dynamics and outgrowth upon injury.

neuroscience↗

bioRxiv2024

Get to know your neighbors with a SNAQ™: A framework for single cell spatial neighborhood analysis in immunohistochemical images

MotivationAnalyzing the local microenvironment of tumor cells can provide significant insights into their complex interactions with their cellular surroundings, including immune cells. By quantifying the prevalence and distances of certain immune cells in the vicinity of tumor cells through a neighborhood analysis, patterns may emerge that indicate specific associations between cell populations. Such analyses can reveal important aspects of tumor-immune dynamics, which may inform therapeutic strategies. This method enables an in-depth exploration of spatial interactions among different cell types, which is crucial for research in oncology, immunology, and developmental biology. ResultsWe introduce an R Markdown script called SNAQTM (Single-cell Spatial Neighborhood Analysis and Quantification), which conducts a neighborhood analysis on immunofluorescent images without the need for extensive coding knowledge. As a demonstration, SNAQTM was used to analyze images of pancreatic ductal adenocarcinoma. Samples stained for DAPI, PanCK, CD68, and PD-L1 were segmented and classified using QuPath. The resulting CSV files were exported into RStudio for further analysis and visualization using SNAQTM. Visualizations include plots revealing the cellular composition of neighborhoods around multiple cell types within a customizable radius. Additionally, the analysis includes measuring the distances between cells of certain types relative to others across multiple regions of interest. Availability and implementationThe R Markdown files that comprise the SNAQTM algorithm and the input data from this paper are freely available on the web at https://github.com/AryehSilver1/SNAQ. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/606539v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1cda240org.highwire.dtl.DTLVardef@157280forg.highwire.dtl.DTLVardef@1072cf8org.highwire.dtl.DTLVardef@1964e0_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com.

bioinformatics↗

bioRxiv2024

12-Lipoxygenase Inhibition Suppresses Islet Immune and Inflammatory Responses and Delays Autoimmune Diabetes in Human Gene Replacement Mice

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing {beta} cells and involves an interplay between {beta} cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in {beta} cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our finding demonstrated that VLX-1005, a potent 12-LOX inhibitor, effectively delayed the onset of autoimmune diabetes in human gene replacement non-obese diabetic mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecule PD-L1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets and polarized proinflammatory macrophages revealed significant alteration of cytokine-responsive pathways and a reduction in interferon response after VLX-1005 treatment. Our studies demonstrated that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

molecular biology↗

bioRxiv2024

Diet breadth in two polyphagous Spodoptera moths in a wide range of host and non-host plants and the potential for range expansion

O_LISeveral polyphagous moths are severe crop pests. Diet breadth patterns and mechanisms among polyphagous insects provide an excellent system to study ecological and evolutionary processes in herbivores, driving dietary specialization. However, studies of diet breadth on more than a handful of crops are scarce. C_LIO_LIHere, we estimated the diet breadth in two species of lepidopteran herbivores from the genus Spodoptera: S. littoralis (SL), with host range including both mono- and dicotyledonous plants and S. frugiperda (SF) Corn strain, primarily adapted to different grass species. C_LIO_LILarval performance on 23 crop and wild plant species from 17 families from terrestrial and wetland habitats was compared to an artificial diet in no-choice feeding bioassays. SL survived and performed better on most tested plants, particularly on the family level, except on two monocot plants (maize and leek), where SF performed well. There were five wild non-host plants where both generalists failed to survive. Nutrition indices assay corroborates the findings on a subset of plants. C_LIO_LIIn a subset of plants, larval feeding preference correlated partly, and larval attraction correlated well with larval performance. Female oviposition choice showed a weak correlation with larval performance. This weak correlation implies that these traits are decoupled, and other factors are crucial for female host plant selection. C_LIO_LIDuring larval dispersal greenhouse experiments, SL and SF larvae strongly tended to migrate onto their suitable host plants, indicating that this is one factor that modulates female host plant selection. C_LIO_LIIn summary, SL has a broader diet breath compared to SF, surviving on wild plants with no previous exposure. The present study provides the first comprehensive data on the diet breadth of two range-expanding and highly invasive polyphagous herbivores. C_LI

ecology↗