Biology subjects

Anand, A.

Publications and source records attributed to Anand, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Asparagine couples group A Streptococcal metabolism to virulence

Streptococcus (GAS) is a highly adapted and human-restricted pathogen causing a wide variety of infections, some life-threatening1. This ability is linked to the expression of many virulence factors, whose transcription is regulated by the two-component system, CovR/S2-5. Here, we show that genome transcription of GAS cultured in a chemically defined medium (CDM) is globally affected when supplemented with asparagine (Asn), including increased expression of many virulence genes. For the first time, we report that GAS solely depends on asparagine synthetase (AsnA) for Asn synthesis, on the ABC transporter (GlnPQ) to import Asn, and on the asparaginase (AsnB) to maintain a precisely balanced intracellular Asn concentration. Furthermore, we show that mutants defective in either asnA, glnP, or asnB express significantly lower levels of virulence factors in CDM and are severely attenuated in the sublethal murine model of human GAS soft-tissue infection. We further show that the synthesis and import of Asn in GAS are ATP-dependent and negatively regulated by intracellular Asn. Thus, Asn availability controls the intracellular ATP level. When ATP becomes limiting, CovR phosphorylation decreases. This augments GAS growth rate, virulence production, metabolism, and vice versa when the ATP level increases. Furthermore, excess Asn accumulates inside GAS in AsnB mutant, destroying the balance between Asn and ATP. We discuss the high similarity between these mechanistic principles of the Asn-mediated control of GAS virulence and metabolism to the Asn-mediated control of tumor growth6, indicating evolutionary significance.

microbiology↗

bioRxiv2024

The role of LEDGF in transcription is exploited by HIV-1 to position integration

HIV-1 integration occurs across actively transcribed genes due to the interaction of integrase with host chromatin factor LEDGF. Although LEDGF was originally isolated as a co-activator that stimulates promoter activity in purified systems, this role is inconsistent with LEDGF-mediated integration across gene bodies and with data indicating LEDGF is a histone chaperone that promotes transcriptional elongation. We found LEDGF is enriched in pronounced peaks that match the enrichments of H3K4me3 and RNA Pol II at transcription start sites (TSSs) of active promoters. Our genome-wide chromatin mapping revealed that MLL1 had a dominant role in recruiting LEDGF to promoters and the presence of LEDGF recruits RNA Pol II. Enrichment of LEDGF at TSSs correlates strongly with levels of integration across the transcribed sequences, indicating that LEDGF at TSSs contributed to integration across gene bodies. Although the N-terminal Pro-Trp-Trp-Pro (PWWP) domain of LEDGF interacts with nucleosomes containing H3K36me3, a modification thought to recruit LEDGF to chromatin, we found H3K36me3 does not contribute to gene specificity of integration. These data support a dual role model of LEDGF where it is tethered to promoters by MLL1 and recruits RNA Pol II. Subsequently, LEDGF travels across genes to effect HIV-1 integration. Our data also provides a mechanistic context for the contribution made by LEDGF to MLL1-based infant acute leukemia and acute myeloid leukemia in adults.

genomics↗

bioRxiv2024

Microbiota metabolized Bile Acids accelerate Gastroesophageal Adenocarcinoma via FXR inhibition

BackgroundThe incidence of Barrett esophagus (BE) and Gastroesophageal Adenocarcinoma (GEAC) correlates with obesity and a diet rich in fat. Bile acids (BA) support fat digestion and undergo microbial metabolization in the gut. The farnesoid X receptor (FXR) is an important modulator of the BA homeostasis. The capacity of inhibiting cancer-related processes when activated, make FXR an appealing therapeutic target. In this work, we assess the role of diet on the microbiota-BA axis and evaluate the role of FXR in disease progression. ResultsHere we show that high fat diet (HFD) accelerated tumorigenesis in L2-IL1B mice (BE- and GEAC- mouse model) while increasing BA levels and enriching gut microbiota that convert primary to secondary BA. While upregulated in BE, expression of FXR was downregulated in GEAC in mice and humans. In L2-IL1B mice, FXR knockout enhanced the dysplastic phenotype and increased Lgr5 progenitor cell numbers. Treatment of murine organoids and L2-IL1B mice with the FXR agonist obeticholic acid (OCA) deacelerated GEAC progression. ConclusionWe provide a novel concept of GEAC carcinogenesis being accelerated via the diet-microbiome-metabolome axis and FXR inhibition on progenitor cells. Further, FXR activation protected with OCA ameliorated the phenotype in vitro and in vivo, suggesting that FXR agonists have potential as differentiation therapy in GEAC prevention. Statement of significanceIf its inhibition is linked to disease progression and its activation to cancer prevention, exploring the potential of FXR as a therapeutic target has great clinical relevance in GEAC context.

cancer biology↗

bioRxiv2024

Endogenously produced hydrogen cyanide serves as a novel mammalian gasotransmitter

Small, gaseous molecules, known as gasotransmitters (NO, CO, H2S), are produced endogenously in mammalian cells and serve important biological roles. Hydrogen cyanide, traditionally considered a cytotoxic molecule in mammals, serves as an endogenous mediator in several plants and bacterial species. Here we show that low concentrations of cyanide are generated endogenously in mouse liver and human hepatocytes. Cyanide production is stimulated by glycine, occurs at the low pH of lysosomes and requires peroxidase activity. Cyanide, in turn, is detectable in several cellular compartments. Cyanide is also detectable basally in the blood of mice; its levels increase after treatment of the animals with glycine. Rhodanese activity regulates endogenous cyanide levels. Cyanide, when generated endogenously at an optimal level, exerts stimulatory effects on mitochondrial bioenergetics, cell metabolism and cell proliferation. Dysregulation of endogenous cyanide, either below or above optimal levels, impairs cellular bioenergetics. The regulatory effects of cyanide are in part mediated by posttranslational modification of cysteine residues via protein cyanylation; cyanylated protein residues can be detected basally, and increase after treatment with glycine. Controlled low-dose cyanide supplementation exhibits cytoprotective effects, as demonstrated in hypoxia and reoxygenation models in vitro and in vivo. However, pathologically elevated cyanide production, as demonstrated in nonketotic hyperglycinemia - an autosomal recessive disease of glycine metabolism - is deleterious to the cells.

biochemistry↗

bioRxiv2024

Combinatorial characterization of bacterial taxa-driven differences in the microbiome of oyster reefs

Oyster reefs are invaluable ecosystems that provide a wide array of critical ecosystem services, including water filtration, coastal protection, and habitat provision for various marine species. However, these essential habitats face escalating threats from climate change and anthropogenic stressors. To combat these challenges, numerous oyster restoration initiatives have been undertaken, representing a global effort to preserve and restore these vital ecosystems. A significant, yet poorly understood, component of oyster reefs is the microbial communities. These communities account for a substantial proportion of marine reefs and are pivotal in driving key biogeochemical processes. Particularly, the environmental microbiome plays a crucial role in supporting the health and resilience of oyster populations. In our study, we sought to shed light on the microbiome within oyster reef ecosystems by characterizing the abundance, and diversity of microorganisms in the soil, biofilm, and oysters in 4 sites using a combinatorial approach to identify differentially abundant microbes by sample type and by sampling location. Our investigation revealed distinct microbial taxa in oysters, sediment and biofilm. The maximum Shannon Index indicated a slightly increased diversity in Herons Head (5.47), followed by Brickyard park (5.35), Dunphy Park (5.17) and Point Pinole (4.85). This is likely to be driven by significantly higher oyster mortality observed at Point Pinole during routine monitoring and restoration efforts. Interestingly Ruminococcus, Streptococcus, Staphylococcus, Prevotella, Porphyromonas, Parvimonas, Neisseria, Lactococcus, Haemophilus, Fusobacterium, Dorea, Clostridium, Campylobacter, Bacteroides, and Akkermansia were positively associated with the biofilm. Yet we have limited understanding of their beneficial and/or detrimental implications to oyster growth and survival. By unraveling the intricate relationships in microbial composition across an oyster reef, our study contributes to advancing the knowledge needed to support effective oyster reef conservation and restoration efforts.

microbiology↗

bioRxiv2024

KRas, in addition to Tp53 is a driver for early carcinogenesis and a molecular target in a mouse model of invasive gastro-esophageal adenocarcinoma

ObjectiveThe incidence of gastro-esophageal adenocarcinoma (GEAC) has increased dramatically and is associated with Barretts Esophagus (BE). Gastric cardia progenitors are the likely origin for BE and GEAC. Here we analyze p53, Rb1 and Kras alterations in Lgr5 progenitor cells during carcinogenesis. DesignWe introduced single and combined genetic alterations (p53, Rb1 and Kras) in Lgr5-expressing progenitor cells at the inflamed gastroesophageal junction in the L2-IL1b (L2) mouse model crossed to Lgr5-CreERTmice. For in-vitro treatment we utilized mouse and human 3D organoids. ResultsInactivation of Tp53 or Rb1 alone (L2-LP and L2-LR mice) resulted in metaplasia, and mild dysplasia, while expression of KrasG12D (L2-LK) accelerated dysplasia in L2-IL1b mice. Dual induction of genetic alteration in L2-LPR, L2-LKP and L2-LKR mice confirmed the accelerating role of mutant Kras, with the development of invasive cancer in mice with combined Tp53 and Kras alteration. All three genetic events in cardia progenitor cells generated invasive cancer at 6 months of age, with chromosomal instability (CNV). The dominant role of Kras prompted us to treat with a SHP2 inhibitor in combination with an ERK or MEK inhibitor, leading to reduced growth in Kras mutant organoids. SHP2 and MEK inhibition in-vivo reduced Kras dependent tumor formation. ConclusionIn the first invasive GEAC mouse model, Kras mutation in combination with loss of tumor suppressor genes Tp53 or Rb1 emerges as a key player in GEAC and with importance of p53 and Rb1 in promoting metaplasia. Targeting this SHP2/MEK/KRAS pathway represents a promising therapeutic option for Kras altered GEAC. What is already known on this topicThe increased incidence of GEAC is challenging current screening and surveillance strategies. Therapeutic and preventive options are limited due to a lack of knowledge on the role of genetic alterations commonly associated with GEAC and their function during progression to dysplasia. What this study addsWe generate the first invasive GEAC model and show that KRAS at least in combination with a second genetic alterations (Tp53 and/or Rb1) may be a driver of tumorigenesis, and targeting KRAS alterations could be a promising now treatment substitution. How this study might affect research, practice or policyTargeting KRAS alterations will be important for GEAC, especially as specific KRAS inhibitor are on the horizon. In addition, a concept of single genetic alteration inducing metaplasia as an adaptation to chronic inflammation might emerge as an important factor for surveillance.

cancer biology↗

bioRxiv2024

A Scalable Adaptive Quadratic Kernel Method for Interpretable Epistasis Analysis in Complex Traits

Our knowledge of the contribution of genetic interactions (epistasis) to variation in human complex traits remains limited, partly due to the lack of efficient, powerful, and interpretable algorithms to detect interactions. Recently proposed approaches for set-based association tests show promise in improving power to detect epistasis by examining the aggregated effects of multiple variants. Nevertheless, these methods either do not scale to large numbers of individuals available in Biobank datasets or do not provide interpretable results. We, therefore, propose QuadKAST, a scalable algorithm focused on testing pairwise interaction effects (also termed as quadratic effects) of a set of genetic variants on a trait and quantifying the proportion of phenotypic variance explained by these effects. We performed comprehensive simulations and demonstrated that QuadKAST is well-calibrated. Additionally, QuadKAST is highly sensitive in detecting loci with epistatic signal and accurate in its estimation of quadratic effects. We applied QuadKAST to 53 quantitative phenotypes measured in {approx} 300, 000 unrelated white British individuals in the UK Biobank to test for quadratic effects within each of 9, 515 protein-coding genes (after accounting for linear additive effects). We detected 32 trait-gene pairs across 17 traits that demonstrate statistically significant signals of quadratic effects ([Formula] accounting for the number of genes and traits tested). Our method enables the detailed investigation of epistasis on a large scale, offering new insights into its role and importance.

genetics↗

bioRxiv2024

Targeting glioblastoma tumor hyaluronan to enhance therapeutic interventions that regulate metabolic cell properties

Despite extensive advances in cancer research, glioblastoma (GBM) still remains a very locally invasive and thus challenging tumor to treat, with a poor median survival. Tumor cells remodel their microenvironment and utilize extracellular matrix to promote invasion and therapeutic resistance. We aim here to determine how GBM cells exploit hyaluronan (HA) to maintain proliferation using ligand-receptor dependent and ligand-receptor independent signaling. We use tissue engineering approaches to recreate the three-dimensional tumor microenvironment in vitro, then analyze shifts in metabolism, hyaluronan secretion, HA molecular weight distribution, as well as hyaluronan synthetic enzymes (HAS) and hyaluronidases (HYAL) activity in an array of patient derived xenograft GBM cells. We reveal that endogenous HA plays a role in mitochondrial respiration and cell proliferation in a tumor subtype dependent manner. We propose a tumor specific combination treatment of HYAL and HAS inhibitors to disrupt the HA stabilizing role in GBM cells. Taken together, these data shed light on the dual metabolic and ligand - dependent signaling roles of hyaluronan in glioblastoma. SignificanceThe control of aberrant hyaluronan metabolism in the tumor microenvironment can improve the efficacy of current treatments. Bioengineered preclinical models demonstrate potential to predict, stratify and accelerate the development of cancer treatments.

bioengineering↗

bioRxiv2023

Ultrasound-Assisted Hydrolysis of Food Waste using Glucoamylase: Statistical Optimization and Mechanistic Analysis with Molecular Simulations

Food waste represents a promising and cost-effective resource for synthesizing value-added products through a fermentative pathway. Preceding fermentation, the hydrolysis of food waste into monomeric sugars is a crucial step. This study presents a comprehensive investigation of food waste hydrolysis, encompassing experimental and computational approaches, using glucoamylase (GLCM) enzyme. Initial optimization of hydrolysis parameters was conducted through the Box-Behnken design of experiments, resulting in a total reducing sugar yield (TRS) of 263.4 mg/g biomass under optimized conditions within 42 hours. Sonication of hydrolysis mixture at 35 kHz at 20% duty cycle, yielded a 4x reduction in hydrolysis time with 22% enhancement in TRS yield (320 mg/g biomass). Analysis of GLCMs secondary structure revealed sonication-induced changes through FTIR spectra deconvolution in both control and test experiments. Sonication led to a reduction in -helix content and an increase in random coil content. Molecular dynamics simulations, including molecular docking, unveiled the majority of amino acid residues associated with the GLCM binding pocket in the -helix and random coil regions. Consequently, sonication widened the binding pockets, facilitating easier transport of substrate and product. This effect translated into improved reaction kinetics in food waste hydrolysis. Research HighlightsO_LIStatistical optimization of food waste hydrolysis: TRS yield = 263.4 mg/g in 42 h C_LIO_LI4x reduction of hydrolysis time, 22% rise in TRS yield with 35 kHz sonication C_LIO_LISonication reduced -helix content & increased random coil content of glucoamylase C_LIO_LIMolecular docking simulation to deduce mechanism of ultrasound-assisted hydrolysis C_LIO_LIMD simulations reveal widening of binding pockets and enhancing catalytic efficiency C_LI

biochemistry↗

bioRxiv2023

Genomic plasticity is a blueprint of diversity in Salmonella lineages

The flexible genome, organised in regions of genomic plasticity (RGP), serves as a potent facilitator of the dynamic evolution of bacterial genomes through gene acquisition and loss. Here we explored the genomic plasticity across Salmonella lineages, revealing a purposeful, non-random integration pattern of pathogenicity-related gene clusters into specific RGP. Noteworthy examples include the correlation between the type I-E CRISPR-Cas system, gold tolerance, and specific RGP. The scattered prevalence of RGP across Salmonella lineages profoundly shapes the pathogenicity makeup of Salmonella strains. The preferences of RGP seem guided by conserved flanking genes that likely share regulatory and functional coordination. For example, metal resistance genes are predominant in RGP positioned near stress resistance genes, indicating a regulatory network to efficiently counter stressors. Additionally, we observed that different plasmid incompatibility types and prophage genera carry distinct pathogenicity genes. Similar to RGP, their distribution across Salmonella lineages plays a critical role in defining pathogenicity. By uncovering these intricate connections among gene clusters, RGP, mobile genetic elements, and pathogenic attributes, our study offers novel insights into the evolutionary trajectory of Salmonella and holds promise for predicting future adaptations and developing targeted interventions against infections.

microbiology↗

bioRxiv2023

Default mode network spatio-temporal electrophysiological signature and causal role in creativity

The default mode network (DMN) is a widely distributed, intrinsic brain network thought to play a crucial role in internally-directed cognition. It subserves self-referential thinking, recollection of the past, mind wandering, and creativity. Knowledge about the electrophysiology underlying DMN activity is scarce, due to the difficulty to simultaneously record from multiple distant cortical areas with commonly-used techniques. The present study employs stereo-electroencephalography depth electrodes in 13 human patients undergoing monitoring for epilepsy, obtaining high spatiotemporal resolution neural recordings across multiple canonical DMN regions. Our results offer a rare insight into the temporal evolution and spatial origin of theta (4-8Hz) and gamma signals (30-70Hz) during two DMN-associated higher cognitive functions: mind-wandering and alternate uses. During the performance of these tasks, DMN activity is defined by a specific pattern of decreased theta coupled with increased gamma power. Critically, creativity and mind wandering engage the DMN with different dynamics: creativity recruits the DMN strongly during the covert search of ideas, while mind wandering displays the strongest modulation of DMN during the later recall of the train of thoughts. Theta band power modulations, predominantly occurring during mind wandering, do not show a predominant spatial origin within the DMN. In contrast, gamma power effects were similar for mind wandering and creativity and more strongly associated to lateral temporal nodes. Interfering with DMN activity through direct cortical stimulation within several DMN nodes caused a decrease in creativity, specifically reducing the originality of the alternate uses, without affecting creative fluency or mind wandering. These results suggest that DMN activity is flexibly modulated as a function of specific cognitive processes and supports its causal role in creative thinking. Our findings shed light on the neural constructs supporting creative cognition and provide causal evidence for the role of DMN in the generation of original connections among concepts.

neuroscience↗

bioRxiv2023

A biobank-scale test of marginal epistasis reveals genome-wide signals of polygenic epistasis

The contribution of epistasis (interactions among genes or genetic variants) to human complex trait variation remains poorly understood. Methods that aim to explicitly identify pairs of genetic variants, usually single nucleotide polymorphisms (SNPs), associated with a trait suffer from low power due to the large number of hypotheses tested while also having to deal with the computational problem of searching over a potentially large number of candidate pairs. An alternate approach involves testing whether a single SNP modulates variation in a trait against a polygenic background. While overcoming the limitation of low power, such tests of polygenic or marginal epistasis (ME) are infeasible on Biobank-scale data where hundreds of thousands of individuals are genotyped over millions of SNPs. We present a method to test for ME of a SNP on a trait that is applicable to biobank-scale data. We performed extensive simulations to show that our method provides calibrated tests of ME. We applied our method to test for ME at SNPs that are associated with 53 quantitative traits across {approx} 300 K unrelated white British individuals in the UK Biobank (UKBB). Testing 15, 601 trait-loci associations that were significant in GWAS, we identified 16 trait-loci pairs across 12 traits that demonstrate strong evidence of ME signals (p-value [Formula]). We further partitioned the significant ME signals across the genome to identify 6 trait-loci pairs with evidence of local (within-chromosome) ME while 15 show evidence of distal (cross-chromosome) ME. Across the 16 trait-loci pairs, we document that the proportion of trait variance explained by ME is about 12x as large as that explained by the GWAS effects on average (range: 0.59 to 43.89). Our results show, for the first time, evidence of interaction effects between individual genetic variants and overall polygenic background modulating complex trait variation.

genetics↗

bioRxiv2023

Insight into spatial intratumoral genomic evolution in glioblastoma

Glioblastoma undergoes a complex and dynamic evolution involving genetic and epigenetic changes. Understanding the mechanisms underlying this evolution is vital for the development of efficient therapeutic strategies. Although treatment resistance is associated with intratumoral heterogeneity in glioblastoma, it remains uncertain whether hypometabolic and hypermetabolic lesions observed through positron emission tomography (PET) imaging are influenced by spatial intratumoral genomic evolution. In this study, we precisely isolated autologous hypometabolic and hypermetabolic lesions from glioblastoma using advanced neurosurgical and brain tumor imaging technologies, followed by comprehensive whole-genome exome and transcriptome analyses. Our findings revealed that hypermetabolic lesions evolved from hypometabolic lesions, harbored shrewd focal amplifications and deletions, and exhibited a higher frequency of critical genomic alterations linked to increased aggressiveness, upregulated APOBEC3 and hypoxic genes, and downregulated putative tumor suppressors. This study highlights spatial genomic evolution with diagnostic implications and unveils the obstacles and possibilities that should be considered in the development of novel therapeutic strategies. Statement of significanceGlioblastoma is a multifaceted disease that is difficult to treat, and insights into the metabolic gradient observed in imaging and the underlying role of genomic evolution are lacking. This study is the first to investigate the molecular basis of hypermetabolic tumor lesions in glioblastoma using precise three-dimensional biopsy isolation, whole genome/exome, and mRNA sequencing. These findings have diagnostic significance, provide insights into therapeutic resistance, and shed light on the obstacles encountered by precision therapeutics for glioblastoma.

cancer biology↗

bioRxiv2023

CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

Glioblastoma is the most prevalent and aggressive malignant tumor of the central nervous system. With a median overall survival of only one year, glioblastoma patients have a particularly poor prognosis, highlighting a clear need for novel therapeutic strategies to target this disease. Transcriptional cyclin-dependent kinases (tCDK), which phosphorylate key residues of RNA polymerase II (RNAPII) c-terminal domain (CTD), play a major role in sustaining aberrant transcriptional programs that are key to development and maintenance of cancer cells. Here, we show that either pharmacological inhibition or genetic ablation of the tCDKs, CDK12 and CDK13, markedly reduces both the proliferation and migratory capacity of glioma cells and patient-derived organoids. Using a xenograft mouse model, we demonstrate that CDK12/13 inhibition not only reduces glioma growth in vivo. Mechanistically, inhibition of CDK12/CDK13 leads to a genome-wide abrogation of RNAPII CTD phosphorylation, which in turn disrupts transcription and cell cycle progression in glioma cells. In summary, the results provide proof-of-concept for the potential of CDK12 and CDK13 as therapeutic targets for glioblastoma. Significance statementGlioblastoma is a common, aggressive, and invasive type of brain tumor that is usually fatal. The standard treatment for glioblastoma patients is surgical resection, radiotherapy, and chemotherapy with DNA-alkylating agents, and unfortunately current treatments only extend overall survival by a few months. It is therefore critical to identify and target additional biological processes in this disease. Here, we reveal that targeting a specific transcriptional addiction for glioma cells by inhibition of CDK12/CDK13 disrupts glioma-specific transcription and cell cycle progression and has potential to provide a new therapeutic strategy for glioblastoma.

cancer biology↗

bioRxiv2023

A novel, precise and high-throughput technology for viroid detection in cannabis (MFDetectTM)

Hop latent viroid (HLVd) is a severe disease of cannabis, causing substantial economic losses in plant yield and crop value for growers worldwide. The best way to control the disease is early detection to limit the spread of the viroid in grow facilities. This study describes MFDetect as a rapid, highly sensitive, and high-throughput tool for detecting HLVd in the early stages of plant development. Furthermore, we compared MFDetect with RT-PCR in a time course experiment using different plant tissue, leaf, petiole, and roots at different plant development stages to demonstrate both technologies are comparable. Our study found leaf tissue is a suitable plant material for HLVd detection, with the viroid titer increasing in the infected leaf tissue with the age of plants. The study showed that other tissue types, including petiole and roots, were equally sensitive to detection via MFDetect. The assay developed in this research allows the screening of thousands of plants in a week. The assay can be scaled easily to provide the growers with a quick turnaround and cost-effective diagnostic tool for screening many plants and tissue types at different stages of development.

plant biology↗

bioRxiv2023

Opposing roles for TGFbeta and BMP-signaling during nascent alveolar differentiation in the developing human lung

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGF{beta}- and BMP-signaling, where inhibition of TGF{beta}- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGF{beta}-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGF{beta}- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

developmental biology↗

bioRxiv2023

Resolving exit strategies of mycobacteria by combining high-pressure freezing with 3D-correlative light and electron microscopy

The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of vacuolar and cytosolic mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well-established light and advanced electron microscopy techniques in this system, a correlative approach that integrates both methodologies with almost native ultrastructural preservation is still lacking at the moment. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D-correlative light and electron microscopy. This approach includes high-pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens.

cell biology↗

bioRxiv2023

ER-dependent membrane repair of mycobacteria-induced vacuole damage

Several intracellular pathogens, such as Mycobacterium tuberculosis, damage endomembranes to access the cytosol and subvert innate immune responses. The host counteracts endomembrane damage by recruiting repair machineries that retain the pathogen inside the vacuole. Here, we show that the endoplasmic reticulum (ER)-Golgi protein oxysterol binding protein (OSBP) and its Dictyostelium discoideum homologue OSBP8 are recruited to the Mycobacterium-containing vacuole (MCV) after ESX-1-dependent membrane damage. Lack of OSBP8 causes a hyperaccumulation of phosphatidylinositol-4-phosphate (PI4P) on the MCV and decreased cell viability. OSBP8-depleted cells had reduced lysosomal and degradative capabilities of their vacuoles that favoured mycobacterial growth. In agreement with a function of OSBP8 in membrane repair, human macrophages infected with M. tuberculosis recruited OSBP in an ESX-1 dependent manner. These findings identified an ER-dependent repair mechanism for restoring MCVs in which OSBP8 functions to equilibrate PI4P levels on damaged membranes. ImportanceTuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the ER-Golgi lipid transfer protein OSBP8 in the D. discoideum/ M. marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defence mechanism against intracellular pathogens such as M. tuberculosis.

cell biology↗