Biology subjects

Singh, P.

Publications and source records attributed to Singh, P..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

A climate and population dependent diffusion model forecasts the spread of Aedes Albopictus mosquitoes in Europe

Vectors of Dengue, Chikungunya, Zika, and Yellow Fever are emerging in new areas, posing increasing public health risks. Aedes albopictus, a key vector for these diseases, is expanding its range beyond its tropical and subtropical origins, driven by suitable climate, population mobility, trade, and urbanization. Since its introduction to Europe, Ae. albopictus has rapidly spread and triggered recurrent outbreaks. Past model attempts have handled vector suitability and vector introduction as independent drivers. Here we develop a novel, highly predictive spatio-temporal vector diffusion model based on minimum temperature, median temperature, relative humidity and human population as predictors. The model predicts areas of presence or absence with an accuracy of 99% and 79% for new established vector populations. The model explains how short- and long-range spread of Ae. albopictus interacts with vector suitability. These results show that the expansion of Ae. albopictus in Europe is predictable and closely linked to climate suitability, and human population. The new model integrates in one simultaneous model framework the climate and mobility drivers, providing a better basis for anticipating future outbreaks in situations of dependent interacting co-drivers. Significance statementThe Tiger mosquito, Aedes albopictus, which spreads diseases like Dengue, Chikungunya, Zika and Yellow fever, has accelerated its presence in Europe over the past decades. This poses a public health risk as Europe face an upsurge of autochthonous arbovirus transmission events. The change is partly due to conducive environments, climate change and human mobility and trade. We develop a novel model explaining the spread of the mosquito over the years 2010-2023, observing a change in recorded mosquito presence areas from 138 regions in 2010 to 537 regions in 2023. The model incorporates patterns in both space and time, capturing with high accuracy how the mosquito spreads from region to region based on climate suitability, geographical diffusion and human population density.

molecular biology↗

bioRxiv2024

Disclosing temperature sensitivity of West Nile virus transmission: novel computational approaches to mosquito-pathogen trait responses

Temperature influences the transmission of mosquito-borne pathogens with significant implications for disease risk in the context of climate change. Mathematical models of mosquito-borne infections rely on functions that capture mosquito-pathogen interactions in response to temperature to better estimate transmission dynamics. To derive these functions, experimental studies provide valuable data on the temperature sensitivity of mosquito life-history traits and pathogen transmission. However, the scarcity of experimental data and inconsistencies in methodologies for analysing and comparing temperature responses across mosquito species and pathogens present challenges to accurately modelling mosquito-borne infections. In this study, we investigated the thermal biology of West Nile virus (WNV), a major mosquito-borne infection. We critically reviewed existing experimental studies, obtaining temperature responses for eight mosquito-pathogen traits across 15 mosquito species. Using this data, we employed Bayesian hierarchical models to estimate temperature response functions for each trait and estimate their variation between species and experiments. We incorporate the resulting functions into mathematical models to estimate the temperature sensitivity of WNV transmission, focusing on six competent mosquito species of the genus Culex. Our study finds similarities in the temperature response among Culex species, with a general optimal transmission temperature around 24{degrees}C. We demonstrate that differing mechanistic assumptions in published models can result in a temperature optimum variation exceeding 3{degrees}C, underscoring the need for model scrutiny. Additionally, we identify substantial variability between trait temperature responses across experiments on the same mosquito species, indicating significant intra-species variation in trait performance. We provide recommendations for future experimental studies, emphasizing the critical need for additional data on all mosquito-pathogen traits, except for mosquito larva-to-adult development rate and survival, which are relatively well-documented. Incorporating additional data into our multi-species approach enhances the accuracy of temperature trait response estimates. These improvements are critical for forecasting future shifts in mosquito-borne disease risk due to climate change.

ecology↗

bioRxiv2024

E. coli associated with Crohn's disease exhibit distinct strategies to colonize macrophages

Patients with Crohns disease exhibit abnormal colonization of the intestine by Proteobacteria, particularly the adherent-invasive Escherichia coli (AIEC) group. These bacteria are predominant in the mucus, adhere to epithelial cells, colonize them, and survive inside macrophages. We recently demonstrated that the acclimation of strain LF82 to phagolysosomal stress occurs in two distinct steps: first, a replication halt producing stress- tolerant persisters, and second, a replication phase that leads to the formation of Intracellular Bacterial Communities (IBC) organized with a biofilm-like matrix. Given the significant genomic diversity among strains with the AIEC phenotype, we conducted a comparative analysis of the genomes and macrophage colonization characteristics of 13 AIEC strains collected from patients during a clinical study conducts by the CHU of Clermont-Ferrand. Our results demonstrate that IBCs serve as replicative niches for all AIEC strains within macrophages. However, these strains form IBCs using different strategies, including varying levels of phagosome detoxification, distinct biofilm characteristics, and diverse macrophage responses. Our study reveals a strong positive correlation between vacuole acidification and persister induction that explains intracellular survival of the different strains. In addition, we revealed distinct AIEC dissemination strategies outside macrophages, which may contribute to the propagation of inflammation in the human host. These findings highlight that research on pathogens and pathobionts with plastic genomes should not rely solely on a few laboratory models.

microbiology↗

bioRxiv2024

Life stage- and sex-specific sensitivity to nutritional stress in a holometabolous insect

O_LIOver the course of their lives, organisms can be repeatedly exposed to stress, which shapes their phenotype. At certain life stages, known as sensitive phases, individuals might be more receptive to such stress than at others. One of these stresses is nutritional stress, such as food limitation. However, little is known about how plastic responses differ between individuals experiencing nutritional stress early versus later in life or repeatedly, particularly in species with distinct ontogenetic niches. Moreover, there may be sex-specific differences due to distinct physiology. C_LIO_LIThe turnip sawfly, Athalia rosae (Hymenoptera: Tenthredinidae), is a holometabolous herbivore, whose larvae consume leaves and flowers, while the adults take up nectar. We examined the effects of starvation experienced at different life stages on life-history traits as well as adult behavioural and metabolic traits to determine which life-stage may be more sensitive to nutritional stress and how specific these traits respond. We exposed individuals to four distinct nutritional regimes, no, larval, or adult starvation, or starvation periods during both larval and adult stage. C_LIO_LILarvae exposed to starvation had a prolonged developmental time, and starved females reached a lower initial adult body mass than non-starved individuals. However, males did not differ in initial adult body mass regardless of larval starvation, suggesting the ability to conform well to poor nutritional conditions, possibly through changes in development and metabolism. C_LIO_LIAdult behaviour, measured as activity, was not significantly impacted by larval or adult starvation in either sex. Individuals starved as larvae had similar carbohydrate and lipid (i.e. fatty acid) contents as non-starved individuals, potentially due to building up energy reserves during their prolonged development, while starvation during adulthood or at both stages led to reduced energy reserves in males. C_LIO_LIThis study indicates that the sensitivity of a life stage to nutritional stress depends on the specific trait under consideration. Life-history traits were mainly affected by larval nutritional stress, while activity appeared to be more robust and metabolism mostly impacted by the adult nutritional conditions. Individuals differed in their ability to conform to the given environment, with the responses being life stage- and sex-specific. C_LI

ecology↗

bioRxiv2024

Micro-C reveals MORC/ApiAP2-mediated links between distant, functionally related genes in the human malaria parasite

Genome organization plays a significant role in silencing heterochromatinized genes in the most virulent human malaria parasite, Plasmodium falciparum. However, it remains unclear how heterochromatinized genes spatially cluster or if active genes are also organized in a specific manner. We used Micro-C to achieve a near-nucleosome resolution DNA-DNA contact map, which revealed new inter- and intrachromosomal heterochromatic and euchromatic structures in the blood stage parasite. We observed subtelomeric fold structures that facilitate interactions amongst heterochromatinized genes involved in antigenic variation. In addition, we identified long-range intra- and interchromosomal interactions amongst active, stage-specific genes. Both structures are mediated by AP2-P, an ApiAP2 transcription factor, and a putative MORC chromatin remodeler, and functional specificity is achieved via combinatorial binding with other sequence-specific DNA-binding factors. This study provides unprecedented insight into the organizational machinery used by this medically important eukaryotic parasite to spatially coordinate genes underlying antigenic variation and to co-activate stage-specific genes.

molecular biology↗

bioRxiv2024

A consortium-based approach to adaptive laboratory evolution of Acinetobacter baylyi ADP1 for lignin valorization

The utility of Acinetobacter baylyi ADP1 (ADP1) for lignin valorization has yet to be sufficiently investigated compared to other organisms such as Pseudomonas, Rhodococcus, etc. In this study, a two-step Adaptive Laboratory Evolution (ALE) process was used to evolve a unique ADP1 strain (A. baylyi SAG_185). Initially, several ADP1 strains were evolved for substrate tolerance to specific lignin-related aromatics (LRAs). Subsequently, a consortium of these strains was adaptively evolved in a mixture of LRAs, which resulted in the evolution of SAG_185. This strain was capable of simultaneous utilization of multiple LRAs at higher concentrations as well as grow on a depolymerized lignin-rich residue obtained from enzymatic hydrolysis of pre-treated corncob. This is the first report on such an evolutionary strategy. Whole-genome sequence analysis of all the evolved strains revealed large-scale mutations involving insertion sequences (IS). In particular, SAG_185 revealed a critical mutation in the vanR repressor gene, resulting in the up-regulation of vanAB genes required to convert vanillate to the key intermediate, protocatechuate (PCA). Additionally, there were two large deletions of 9kb and 38kb DNA segments, including genes for putative transcriptional regulators of LysR, MarR and AraC family. The evolved strains also showed mutations in the hcaE gene, responsible for the uptake of LRAs. The vast number of mutations in hypothetical proteins, transporter and regulatory sequences indicate the underlying effects of these regions on the uptake of multiple LRAs. Overall, our findings provide potential targets for reverse engineering of A. baylyi ADP1 for lignin valorization. IMPORTANCEThis study shows a novel strategy for adaptive laboratory evolution, which can be generically adopted to evolve bacterial strains for taking up multiple substrates which are toxic at higher concentrations. We developed a two-step evolutionary strategy to evolve a unique strain, A. baylyi SAG_185, which could take up multiple lignin-related aromatic monomers at higher concentrations as well as grow on depolymerized lignin. Initially, individual strains were adapted to utilize single aromatic monomers at higher concentrations. After many unsuccessful attempts to adapt these strains for utilizing multiple monomers, a consortium of the five evolved strains were grown on a mixture of aromatics and adapted to utilize all the monomers at high concentrations. The adapted consortia resulted in the evolution of SAG_185. Whole-genome sequence analysis of all these strains gave rise to many interesting insights on potential genetic targets for reverse engineering of A. baylyi ADP1 for lignin valorization.

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

Kinome-wide RNAi screen in Caenorhabditis elegans reveals new modulators of insulin signaling and longevity

The insulin/IGF-I-like signaling (IIS) pathway is a highly conserved signaling cascade that plays a crucial role in regulating longevity across species. Given its significance in aging, identifying novel kinases interacting with the IIS pathway can provide deeper insights into the mechanisms governing longevity. In this study, we performed a targeted RNAi screen of the Caenorhabditis elegans kinome, utilizing dauer formation as a phenotypic readout. We identified several known and novel kinase modulators of the IIS pathway. These hits were enriched with both previously documented as well as undocumented lifespan regulators. Thermotolerance assays revealed mixed trends, with some kinase inhibitions increasing while others decreasing protection. We observed a positive correlation between thermotolerance and lifespan extension, as well as between dauer formation and lifespan extension, with thermotolerance proving to be a better predictor of longevity. Our findings offer a valuable resource for researchers exploring the IIS pathway and highlight novel, unannotated kinases as potential new therapeutic targets for aging interventions.

molecular biology↗

bioRxiv2024

Rapid, high-resolution, non-destructive assessments of metabolic and morphological homogeneity uniquely identify high-grade cervical precancerous lesions

PurposeTwo-photon microscopy (2PM) is an emerging clinical imaging modality with the potential to non-invasively assess tissue metabolism and morphology in high-resolution. This study aimed to assess the translational potential of 2PM for improved detection of high-grade cervical precancerous lesions. Experimental Design2P images attributed to reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and oxidized flavoproteins (FP) were acquired from the full epithelial thickness of freshly excised human cervical tissue biopsies (N = 62). Fifteen biopsies harbored high-grade squamous intraepithelial lesions (HSILs), 14 biopsies harbored low-grade SILs (LSILs), and 33 biopsies were benign. Quadratic discriminant analysis (QDA) leveraged morphological and metabolic functional metrics extracted from these images to predict the presence of HSILs. We performed gene set enrichment analysis (GSEA) using datasets available on the Gene Expression Omnibus (GEO) to validate the presence of metabolic reprogramming in HSILs. ResultsIntegrating metabolic and morphological 2P-derived metrics from finely sampled, full-thickness epithelia achieved a high 90.8 {+/-} 6.1% sensitivity and 72.3 {+/-} 11.3% specificity of HSIL detection. Notably, sensitivity (91.4 {+/-} 12.0%) and specificity (77.5 {+/-} 12.6%) were maintained when utilizing metrics from only two images at 12- and 72-{micro}m from the tissue surface. Upregulation of glycolysis, fatty acid metabolism, and oxidative phosphorylation in HSIL tissues validated the metabolic reprogramming captured by 2P biomarkers. ConclusionLabel-free 2P images from as few as two epithelial depths enable rapid and robust HSIL detection through the quantitative characterization of metabolic and morphological reprogramming, underscoring the potential of this tool for clinical evaluation of cervical precancers. Translational Relevance StatementThe colposcopy and biopsy paradigm for cervical pre-cancer detection leads to an excessive number of unnecessary biopsies, with significant economic and psychological costs. This study highlights the potential of label-free, high-resolution two photon imaging to improve this paradigm by introducing real-time morphofunctional tissue assessments. In an extensive dataset comprising freshly excised high-grade and low-grade cervical intraepithelial lesions, along with benign metaplastic and inflamed human cervical tissue biopsies, we successfully characterize a loss of morphofunctional heterogeneity indicative of high-grade precancerous changes. Leveraging a combination of two-photon imaging-derived quantitative morphofunctional metrics, our findings showcase a substantial improvement in both sensitivity and specificity of high-grade lesion detection compared to the current gold standard of colposcopy followed by a single biopsy. The demonstrated enhancement in sensitivity and specificity highlights the prospect of integrating non-invasive, label-free metabolic imaging into clinical practice, offering a more effective and efficient approach to identify and manage cervical precancerous lesions.

bioengineering↗

bioRxiv2024

ATTRv-V30M Type A amyloid fibrils from the heart and nerves exhibit structural homogeneity.

ATTR amyloidosis is a systemic disease characterized by the deposition of amyloid fibrils made of transthyretin, a protein integral to transporting retinol and thyroid hormones. Transthyretin is primarily produced by the liver and circulates in blood as a tetramer. The retinal epithelium also secretes transthyretin, which is secreted to the vitreous humor of the eye. Because of mutations or aging, transthyretin can dissociate into amyloidogenic monomers triggering amyloid fibril formation. The deposition of transthyretin amyloid fibrils in the myocardium and peripheral nerves causes cardiomyopathies and neuropathies, respectively. Using cryo-electron microscopy, here we determined the structures of amyloid fibrils extracted from cardiac and nerve tissues of an ATTRv-V30M patient. We found that fibrils from both tissues share a consistent structural conformation, similar to the previously described structure of cardiac fibrils from an individual with the same genotype, but different from the fibril structure obtained from the vitreous humor. Our study hints to a uniform fibrillar architecture across different tissues within the same individual, only when the source of transthyretin is the liver. Moreover, this study provides the first description of ATTR fibrils from the nerves of a patient and enhances our understanding of the role of deposition site and protein production site in shaping the fibril structure in ATTRv-V30M amyloidosis.

biophysics↗

bioRxiv2024

Multi-organ structural homogeneity of amyloid fibrils in ATTRv-T60A amyloidosis patients, revealed by Cryo-EM

ATTR amyloidosis is a degenerative disorder characterized by the systemic deposition of the protein transthyretin. These amyloid aggregates of transthyretin (ATTR) can deposit in different parts of the body causing diverse clinical manifestations. Our laboratory aims to investigate a potential relationship between the different genotypes, organ of deposition, clinical phenotypes, and the structure of ATTR fibrils. Using cryo-electron microscopy, we have recently described how the neuropathic related mutations ATTRv-I84S and ATTRv-V122{Delta} can drive structural polymorphism in ex vivo fibrils. Here we question whether the mutation ATTRv-T60A, that commonly triggers cardiac and neuropathic symptoms, has a similar effect. To address this question, we extracted and determined the structure of ATTR-T60A fibrils from multiple organs (heart, thyroid, kidney, and liver) from the same patient and from the heart of two additional patients. We have found a consistent conformation among all the fibril structures, acquiring the "closed-gate morphology" previously found in ATTRwt and others ATTRv related to cardiac or mixed manifestations. The closed-gate morphology is composed by two segments of the protein that interact together forming a polar channel, where the residues glycine 57 to isoleucine 68 act as a gate of the polar cavity. Our study indicates that ATTR-T60A fibrils present in peripheral organs adopt the same structural conformation in all patients, regardless of the organ of deposition.

molecular biology↗

bioRxiv2024

Quinolinic acid links kidney injury to brain toxicity

Kidney dysfunction often leads to neurological impairment, yet the complex kidney-brain relationship remains elusive. We employed spatial and bulk metabolomics to investigate a mouse model of rapid kidney failure induced by mouse double minute 2 (Mdm2) conditional deletion in the kidney tubules to interrogate kidney and brain metabolism. Pathway enrichment analysis of focused plasma metabolomics panel pinpointed tryptophan metabolism as the most altered pathway with kidney failure. Spatial metabolomics showed toxic tryptophan metabolites in the kidneys and brains, revealing a novel connection between advanced kidney disease and accelerated kynurenine degradation. In particular, the excitotoxic metabolite quinolinic acid was localized in ependymal cells adjacent to the ventricle in the setting of kidney failure. These findings were associated with brain inflammation and cell death. A separate mouse model of acute kidney injury also had an increase in circulating toxic tryptophan metabolites along with altered brain inflammation. Patients with advanced CKD similarly demonstrated elevated plasma kynurenine metabolites and quinolinic acid was uniquely correlated with fatigue and reduced quality of life in humans. Overall, our study identifies the kynurenine pathway as a bridge between kidney decline, systemic inflammation, and brain toxicity, offering potential avenues for diagnosis and treatment of neurological issues in kidney disease.

molecular biology↗

bioRxiv2024

Olive phenolic compounds, potent tankyrase 1 inhibitor exhibit anti colon cancer effects by blocking Wnt/β-catenin pathway

Colon cancer (CC) needs special attention to develop novel targets and therapeutic approaches, as the total number of morbidity and mortality is growing rapidly. Although Olive has been long reported for its anti-CC activities but the mode is not completely understood yet. Here, we targeted tankysare 1 (TNKS), an important mediator of the Wnt/{beta}-catenin pathway, crucial in colon carcinogenesis. Bioinformatics analysis revealed that the mutation and copy number alterations of TNKS is found to be 12% whereas the proteins expression of TNKS is moderate to high in tumor groups vs normal in CC datasets. On the more, TNKS and its positively associated genes are found to be concomitant with several carcinogenesis related biological processes and signaling pathways. Further, three olive phytochemicals (apigenin, luteolin, and quercetin), which were shortlisted by employing filters like drug likeness and toxicity screening, molecular docking and finally molecular dynamics simulations, can target tankyrase 1. These ployphenols demonstrated cytotoxic effect in CC (HT-29) cells either alone or in combination. Further, these compounds successfully reduced {beta}-catenin level and its target genes (CCND1, CDK4, and MYC). Overall, inhibition of the Wnt/{beta}-catenin pathway by targeting tankyrase 1 was revealed as one of the anti-CC mechanisms of olive polyphenols.

cancer biology↗

bioRxiv2024

Amyloid fibril polymorphism in the heart of an ATTR amyloidosis patient with polyneuropathy attributed to the V122Δ variant

ATTR amyloidosis is a phenotypically heterogeneous disease characterized by the pathological deposition of transthyretin in the form of amyloid fibrils into various organs. ATTR amyloidosis may result from mutations in variant (ATTRv) amyloidosis, or aging in wild-type (ATTRwt) amyloidosis. ATTRwt generally manifests as cardiomyopathy, whereas ATTRv may present as polyneuropathy, cardiomyopathy, or mixed, in combination with many other symptoms deriving from multisystem organ involvement. Over 220 different mutational variants of transthyretin have been identified, many of them being linked to specific disease symptoms. Yet, the role of these mutations in explaining differential disease manifestations remains unclear. Using cryo-electron microscopy, here we structurally characterized fibrils from the heart and the liver of an ATTRv patient carrying the V122{Delta} mutation, which is predominantly associated with polyneuropathy. Our results show that these fibrils are polymorphic, presenting as both single and double filaments. Our study alludes to a structural connection contributing to phenotypic variation in ATTR amyloidosis, as polymorphism in ATTR fibrils may manifest in patients with predominantly polyneuropathic phenotypes. SignificanceATTR amyloidosis is a systemic, clinically diverse disease that results in organ failure due to the accumulation of transthyretin amyloid fibrils. ATTR patients present with varied symptoms, yet the root of this phenotypic heterogeneity remains unclear. Previous studies suggest an association between phenotype and fibril structure polymorphism. Here we describe the cryo-electron microscopy structure of variant transthyretin amyloid fibrils associated with a predominantly polyneuropathy phenotype. We have found polymorphism within these fibrils, a phenomenon we have thus far only observed in polyneuropathic associated transthyretin mutations. Our results signify an association between fibril structure and phenotype in ATTR amyloidosis.

molecular biology↗

bioRxiv2024

Genetic architecture underlying response to the fungal pathogen Dothistroma septosporum in lodgepole pine, jack pine, and their hybrids

In recent decades, Dothistroma needle blight (DNB), a pine tree disease caused by the fungal pathogen Dothistroma septosporum, has severely damaged lodgepole pine (Pinus contorta Dougl. ex. Loud.) in British Columbia, Canada, and raised health concerns for jack pine (Pinus banksiana Lamb.). The pathogen has already shown signs of host shift eastward to the hybrid populations between lodgepole pine and jack pine (Pinus contorta x P. banksiana), and possibly into pure jack pine. However, we have little knowledge about mechanisms of resistance to D. septosporum, especially the underlying genetic basis of variation in pines. In this study, we conducted controlled inoculations to induce infection by D. septosporum and performed a genome-wide case-control association study with pooled sequencing (pool-seq) data to dissect the genetic architecture underlying response in lodgepole pine, jack pine, and their hybrids. We identified candidate genes associated with D. septosporum response in lodgepole pine and in hybrid samples. We also assessed genetic structure in hybrid populations and inferred how introgression may affect the distribution of genetic variation involved in D. septosporum response in the studied samples. These results can be used to develop genomic tools to evaluate DNB risk, guide forest management strategies, and potentially select for resistant genotypes.

plant biology↗

bioRxiv2024

DAB-APT: a Fluorescence-Based Assay for Determining Aminopropyl Transferase Activity and Inhibition

Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), promising targets in antimicrobial, antineoplastic and antineurodegenerative therapies. A major limitation, however, is the lack of high-throughput assays to measure their activity. We developed the first fluorescence-based assay, DAB-APT, for measurement of APT activity using 1,2-diacetyl benzene, which forms fluorescent conjugates with putrescine, spermidine and spermine with fluorescence intensity increasing with increasing carbon chain length. The assay has been validated using APT enzymes from S. cerevisiae and P. falciparum and is suitable for high-throughput screening of large chemical libraries. Given the importance of APTs in infectious diseases, cancer and neurobiology, our DAB-APT assay has broad applications, holding promise for advancing research and drug discovery efforts.

biochemistry↗

bioRxiv2024

A salmon fish model reveals genetic signals in brain associated with early maturation

Emerging evidence suggests a link between adiposity and early maturation, potentially impacting hormonal signaling pathways governing puberty timing. Fish models have proven invaluable in studying these processes, given their genetic and physiological similarities to humans. In Atlantic salmon, in addition to being linked with environmental shifts and lipid reserves, the timing of sexual maturation also has a strong genetic basis. A gene encoding a co-factor of Hippo pathway, vgll3, is a major determinant of maturation timing in salmon, and the same gene was found to be associated with maturation timing in humans. Intriguingly, vgll3 also inhibits adipogenesis in mice. Recent studies in salmon revealed correlations between vgll3 genotypes and reproductive axis gene expression, possibly modulated through the Hippo signaling pathway. The Hippo pathway is known for its role in sexual maturation and adipogenesis and responds to environmental cues such as dietary fat and temperature. In this study, we employed a custom gene expression panel in male Atlantic salmon with different vgll3 early (E) and late (L) maturation genotypes testing components of this pathway in brain at immature and mature stages. We found increased brain expression of a major Hippo pathway kinase (lats1b) and melanocortin receptor encoding genes (mc4ra and mc4rc) in individuals with early (E) maturation genotypes of vgll3 before maturation development of testes. Moreover, we found components and interacting partners of Hippo pathway showing differential expression in brain of individuals with early (E) and late (L) vgll3 genotypes prior to maturation. This could indicate extensive and complex roles of Hippo pathway in brain processes required for preparing for [the onset of] maturation at upstream of reproductive axis. This study elucidates molecular mechanisms underpinning early maturation, and for the first time in fish, offering insights into detecting it at molecular level in brain before visible gonadal changes occur.

genetics↗

bioRxiv2024

Nuclear compression-mediated DNA damage drives ATR-dependent Lamin expression and mouse ESC differentiation

Embryonic stem cells (ESCs) which are susceptible to DNA damage depend on a robust and highly efficient DNA damage response (DDR) mechanism for their survival. However, the implications of physical force-mediated DNA damage on ESC fate remains unclear. We show that stiffness-dependent spreading of mouse ESCs (mESCs) induces DNA damage through nuclear compression, with DNA damage causing differentiation through early induction of Lamin A/C expression. Interestingly, differentiation is associated with rescue of DNA damage and activation of the DDR factor ATR. While ATR is typically known to play roles in DDR pathway, its role during stiffness-mediated nuclear compression and mESC differentiation is unknown. Nuclear enrichment of activated ATR on stiff substrates and reduction of Lamin A/C expression upon ATR inhibition suggests that mESC differentiation is driven by nuclear compression-mediated DNA damage and involves ATR-dependent modulation of Lamin A/C.

cell biology↗