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Biology subjects

Roy, J.

Publications and source records attributed to Roy, J..

At least 37 records · Page 2Linked to original sources

Simpler predictive models provide higher accuracy for ovarian cancer detection

Ovarian cancer remains a danger to womens health, and accurate screening tests would likely increase survival. Two established protein biomarkers, CA125 and HE4, have been shown to work well in isolation, but achieve even higher accuracy when combined using logistic regression (LR). We show here that this LR-based combination of protein concentrations achieves high accuracy when distinguishing healthy samples from cancer samples (AUC = 0.99) and benign masses from cancer (AUC = 0.86). This approach exhibits superior performance on an external validation cohort compared to a more complex method, which was published with the dataset we use here and tested on the same data. While our method only uses proteins, the more complex method also uses features derived from cell-free DNA (cfDNA). We show that many of that methods cfDNA features are affected by confounding technical variation, which impacts the previously reported results. Our results are in line with the principle that simpler machine learning models will tend to exhibit better generalizability on new data.

cancer biology↗

Analysis of Categorical Data with Logistic Regression and the Cochran-Mantel-Haenszel Tests in Biological Experiments

The choice of statistical test is a fundamentally important one when analyzing experimental data. Here, we consider the question of categorical data, defined by their properties (for example color) rather than by continuous numbering. Using simple and complex example datasets generated from Caenorhabditis elegans research, we conduct a statistical analysis of (1) a rare cellular event involving the formation of a neuronal extrusion called an exopher, and of (2) a variable behavioral response across a timescale. Two tests we use here are the Cochran- Mantel-Haenszel (CMH) test and logistic regression. These two tests pose practical challenges to researchers that include lack of easy access to statistical software and the need for prior programming knowledge. To this end we provide step-by-step tutorials and example code. We emphasize the flexibility of logistic regression in handling both simple and complex datasets, emphasizing the capacity of logistic regression to provide more comprehensive insights into experimental outcomes than simpler tests like CMH. By analyzing real biological examples and demonstrating their analysis with R code, we provide a practical guide for biologists to enhance the rigor and reproducibility of categorical data analysis in experimental studies.

genetics↗

Imaging the interactions of chimeric antigen receptor-expressing T-cells with colorectal cancer cells

Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, anti-CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting primary CAR T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We found that CRC cells glycocalyx is much deeper than that of the CAR T cell causing delayed activation. Oscillating calcium fluxes, indicative of non-sustained CAR T cell activation and reduced cytotoxicity, were observed when CAR T cells interacted with CRC cells, which increased with increasing cell-seeding time. Imaging revealed that this effect correlated with a progressive loss of accessible CEACAM5 antigen on the CRC cell surface, possibly due to their sequestration in the intercellular junction, rendering CAR T cell engagement less effective. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T cell calcium fluxes, increasing the killing of CRC cells. Similar enhanced interaction was observed after treatment of CRC cell monolayer with hyaluronidase, approved for use in humans. We observed limited availability of CEACAM5 on human colorectal cancer tissues, whereas treatment with trypsin or hyaluronidase increased accessibility. Our results reveal why CAR T cells targeting CEACAM5 are ineffective and suggest possible routes to improved therapy for CRC.

immunology↗

A Novel Approach for Using Thermal Imaging to Study Behavioural Responses to Environmental Stressors in Passerines

Infrared thermography is a promising tool for assessing proxy for stress response in animals. With the advancement of longer recording capabilities, this technique allows the monitoring of the dynamic nature of the temperature variation which is highly correlated to the stress faced by the animal. The current method of extraction of data from thermal videos involves manual identification of the beginning and end of the activity (feeding bout in our case) and extracting the temperature data into CSV files. In this paper, we aim to identify the beginning and ending of the feeding bout by the Identification of Temperature Change (ITC) that happens when a bird arrives and leaves the frame. By identifying this we can monitor longer IRT videos and extract the thermal imaging data for stress monitoring in a faster manner. The identification of the beginning and end frames, also allows us to get behavioural data such as feeding bout length and feeding frequency which also act as a proxy for perceived stress. This method was developed to simplify the data analysis process of thermal imaging videos and proved to be an effective method with wider applications in behavioural monitoring based on temperature change.

animal behavior and cognition↗

Urbanization Affects Nest Material Composition and Use of Anthropogenic Materials in Blue Tit Nests

Avian nests are complex structures that protect eggs and nestlings. Nest-building behaviour varies between species and habitats, and recent work has highlighted that in areas with high human activity and low availability of natural nest material, birds may use anthropogenic material to construct nests. However, we still know relatively little about how nest composition is affected by human presence, including along urban gradients. Here we present the results of a study where we examined how nest composition differed between an urban and a forest population of blue tits, and the impact that variation in nest composition had on reproductive success. We found a significant decrease in the amount of natural materials and an increase in anthropogenic materials in urban compared to forest nests. Earlier urban nests showed a higher amount of anthropogenic materials compared to late urban nests. The amount of moss incorporated in a nest was the strongest predictor of fledgling success, suggesting that the use of anthropogenic material by urban birds might be a maladaptation, and/or that urban birds are constrained in the amount of moss they can use to build their nests. Future studies should aim at quantifying the availability of natural materials to distinguish between these two hypotheses.

ecology↗

Single-embryo metabolomics reveals developmental metabolism in the early Drosophila embryo

Early embryonic development is characterized by the transition from maternal factor reliance to zygotic control. These processes set the stage for the embryos basic structure and cellular differentiation. While relatively detailed knowledge exists of the transcriptional events during early development, little is known about the concurrent metabolic processes. Understanding these processes, however, is important since they are linked to cell fate determination and organ and tissue formation. The primary reasons for the limited progress in the field are technical limitations due to the small amount of material available during early embryonic time windows. Here, we introduce a novel single-embryo methodology that places us in an exciting position to analyze the early embryos metabolome and transcriptome in an integrated manner and at high temporal resolution. The resulting data allow us to map concomitant metabolic and transcriptional programs in early Drosophila embryonic development. Our results reveal that a substantial number of metabolites exhibit dynamic patterns with some changing even before the onset of zygotic transcription. dNTPs for example show a temporal pattern that correlates with cell division patterns in the early embryo. In summary, here we present an operationally simple single-embryo metabolomics methodology and provide a detailed picture of early developmental metabolic processes at unprecedented temporal resolution.

developmental biology↗

T and Z, Partial Seed Coat Patterning Genes in Common Bean, Provide Insight into the Structure and Protein Interactions of a Plant MBW Complex

Flavonoids are secondary metabolites associated with seed and flower color. A ternary MBW protein complex consisting of interfacing MYB, beta-helix-loop-helix (bHLH), and WD40 repeat (WDR) proteins controls the expression of late biosynthetic genes in the flavonoid pathway. P, the master regulator gene of flavonoid expression in common bean (Phaseolus vulgaris L.) was recently determined to encode a bHLH protein. Two other genes, T and Z, are also historically considered regulators since they determine the distribution of color in seeds and flowers. These two genes were cloned using reverse genetics based on genetic mapping, candidate gene selection, phylogenetic analysis, and mutant analysis. Domain and AlphaFold2 structure analyses determined T encoded a seven-bladed {beta}-propeller WDR protein, while Z encoded a R2R3 MYB protein. Modeling of the Z MYB/P bHLH/T WDR MBW complex identified interfacing sequence domains and motif in all three genes that are conserved in dicots. One Z MYB motif is a possible {beta}-MoRF that only appears in a structured state when Z MYB is modeled in a MBW complex. Complexes containing mutant T and Z proteins changed the interaction of members of the complex in ways that would alter their role in regulating expression of genes in the flavonoid pathway. HighlightThe MBW complex of bean comprised of the classic Z, P, and T genes associated with seed coat patterning defines critical proteins interactions for flavonoid gene expression.

genetics↗

A sucrose non-fermenting-1-related protein kinase 1 gene from wheat, TaSnRK1α regulates starch biosynthesis by modulating AGPase activity

Major portion of wheat grain consist of carbohydrate, mainly starch. The proportion of amylose and amylopectin in starch greatly influence the end product quality. Advancement in understanding starch biosynthesis pathway and modulating key genes has enabled the genetic modification of crops resulting in enhanced starch quality. However, the regulation of starch biosynthesis genes still remains unexplored. So, to expand the limited knowledge, here, we characterized a Ser/Thr kinase, SnRK1 in wheat and determined its role in regulating starch biosynthesis. SnRK1 is an evolutionary conserved protein kinase and share homology to yeast SNF1. Yeast complementation assay suggest TaSnRK1 restore growth defect and promotes glycogen accumulation. Domain analysis and complementation assay with truncated proteins suggest the importance of ATP-binding and UBA domain in TaSnRK1 activity. Sub-cellular localization identified nuclear and cytoplasmic localization of TaSnRK1 in tobacco leaves. Further, heterologous over-expression (O/E) of TaSnRK1 in Arabidopsis not only led to increase in starch content but also enlarges the starch granules. TaSnRK1 was found to restore starch accumulation in Arabidopsis kin10. Remarkably, TaSnRK1 O/E increase the AGPase activity suggesting the direct regulation of rate limiting enzyme AGPase involved in starch biosynthesis. Furthermore, in vitro and in vivo interaction assay reveal that TaSnRK1 interacts with AGPase large sub-unit. Overall, our findings indicate that TaSnRK1 plays role in starch biosynthesis by regulating AGPase activity. HighlightsO_LITaSnRK1 is Ser/Thr kinase in wheat and show dual localization in nucleus and cytoplasm. C_LIO_LIOverexpression of TaSnRK1 increases starch content and enlarges starch granules in Arabidopsis. C_LIO_LITaSnRK1 enhances AGPase activity thereby regulating starch biosynthesis. C_LIO_LITaSnRK1 directly interact with AGPase large subunit in vivo and in vitro. C_LI

molecular biology↗

The circular RNA Ataxia-telangiectasia mutated (cATM) regulates oxidative stress in smooth muscle cells in expanding abdominal aortic aneurysms

An abdominal aortic aneurysm (AAA) is a pathological widening of the aortic wall characterized by loss of smooth muscle cells (SMCs), extracellular matrix degradation, and local inflammation. This condition is often asymptomatic until rupture occurs, leading to high morbidity and mortality rates. Diagnosis is often accidental, and for now, the only available treatment option remains surgical intervention. Circular RNAs (circRNAs) are RNA loops that originated from backsplicing, which have received increasing attention as a novel class of functional non-coding RNAs contributing to cardiovascular physiology and disease. Their high structural stability, combined with a remarkable enrichment in body fluids, make circRNAs promising disease biomarkers. We aimed to investigate the contribution of circRNAs to AAA pathogenesis and their potential application as biomarkers for AAA diagnosis. Combined circRNA array and quantitative real-time PCR analysis revealed the presence of differentially expressed circular transcripts stemming from AAA-relevant gene loci. Among these, the circRNA to the Ataxia-telangiectasia mutated gene (cATM) was upregulated in human AAA tissue specimens, in AAA patient-derived SMCs, and serum samples collected from aneurysm patients. In control primary aortic SMCs, cATM increased upon angiotensin II stimulation, while its silencing triggered apoptosis. Furthermore, doxorubicin could induce cATM expression, supporting a link with acute stress response in SMCs. Constitutively higher cATM expressing AAA patient-derived SMCs were less vulnerable to oxidative stress-induced cell death and survival pathways enriched when compared to control SMCs. Taken together, this data supports the role of cATM in adapting SMCs to oxidative stress in the vascular AAA micromilieu. This molecular signature provides an additional parameter to be included in procedures for AAA screening in combination with already established practices.

molecular biology↗

Evolution of Delta variant by non-Spike signature co-appearing mutations: trailblazer of COVID-19 disease outcome

The high transmissibility and infectivity of a SARS-CoV-2 variant is usually ascribed to the Spike mutations, while emerging non-spike mutations might be a serious threat to the current Spike-recombinant vaccines. In addition to mutations in structural Spike glycoprotein, rapid accumulation of mutations across non-structural genes is leading to continuous virus evolution, altering its pathogenicity. We performed whole genome sequencing of SARS-CoV-2 positive samples collected from different clinical groups from eastern India, during the second pandemic wave (April-May, 2021). In addition to the several common spike mutations in Delta variant, two mutually explicit signature constellations of non-spike co-appearing mutations were identified, driving symptomatic and asymptomatic infections. We attempted to correlate these unique signatures of non-Spike co-appearing mutations to COVID-19 disease outcome. Results revealed that the Delta strains harboring a unique constellation of 9 non-spike co-appearing mutations could be the wheeler and dealer of symptomatic infection, even post vaccination. The strains predominantly driving asymptomatic infection possessed 7 non-spike co-appearing mutations, which were mutually exclusive in contrast to the set of mutations causing symptomatic disease. Phylodynamic analysis depicted high probability of emergence of these unique sub-clusters within India, with subsequent spread worldwide. Interestingly, some mutations of this signature were selected in Omicron and IHU variants, which suggest that gradual accumulation of such co-existing mutations may lead to emergence of more "vaccine-evading variants" in future. Hence, unfaltering genome sequencing and tracking of non-Spike mutations might be significant in formulation of any future vaccines against emerging SARS-CoV-2 variants that might evade the current vaccine-induced immunity.

genomics↗

Palmitoylation targets the Calcineurin phosphatase to the Phosphatidylinositol 4-kinase complex at the plasma membrane

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNA{beta}1. We show that unlike canonical cytosolic calcineurin, CNA{beta}1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNA{beta}1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNA{beta}1.

biochemistry↗

The VE-cadherin/AmotL2 mechanosensory pathway suppresses aortic inflammation and the formation of abdominal aortic aneurysms

Arterial endothelial cells (ECs) have the ability to respond to mechanical forces exerted by fluid shear stress. This response is of importance, as it is protective against vascular diseases such as atherosclerosis and aortic aneurysms. Mechanical forces are transmitted at the sites of adhesion to the basal membrane as well as cell-cell junctions where protein complexes connect to the cellular cytoskeleton to relay force into the cell. Here we present a novel protein complex that connects junctional VE-cadherin and radial actin filaments to the LINC complex in the nuclear membrane. We show that the scaffold protein AmotL2 is essential for the formation of radial actin filaments and the flow-induced alignment of aortic and arterial ECs. The deletion of endothelial AmotL2 alters nuclear shape as well as subcellular positioning. Molecular analysis shows that VE-cadherin is mechanically associated with the nuclear membrane via binding to AmotL2 and Actin. Furthermore, the deletion of AmotL2 in ECs provokes a pro-inflammatory response and abdominal aortic aneurysms (AAA) in the aorta of mice on a normal diet. Remarkably, transcriptome analysis of AAA samples from human patients revealed a negative correlation between AmotL2 expression and aneurysm diameters, as well as a positive correlation between AmotL2 and YAP expression. These findings provide a conceptual framework regarding how mechanotransduction in the junctions is coupled with vascular disease.

cell biology↗

Linkage disequilibrium and population structure in a core collection of Brassica napus (L.)

Estimation of genetic diversity in rapeseed/canola is important for sustainable breeding program to provide an option for the development of new breeding lines. The objective of this study was to elucidate the patterns of genetic diversity within and among different structural groups, and measure the extent of linkage disequilibrium (LD) of 383 globally distributed rapeseed/canola germplasm using 8,502 single nucleotide polymorphism (SNP) markers. The germplasm accessions were divided into five subpopulations (P1 to P5) with obvious geographic and growth habit-related patterns. All subpopulations showed moderate genetic diversity (average H = 0.22 and I = 0.34). The pairwise Fst comparison revealed a great degree of divergence (Fst > 0.24) between most of the combinations. The rutabaga type showed highest divergence with spring and winter types. Higher divergence was also found between winter and spring types. Overall, mean linkage disequilibrium was 0.03 and it decayed to its half maximum within < 45 kb distance for whole genome. The LD decay was slower in C genome (< 93 kb), relative to the A genome (< 21 kb) which was confirmed by availability of larger haplotype blocks in C genome than A genome. To maximize genetic gain, hybridization between rutabaga and other types are potentially the best option. Hybridization between spring and winter, semi-winter type is also helpful to maximize the diversity in subsequent populations. Low genetic differentiation between both spring type subpopulations (P4 and P3) will accelerate favorable allele accumulation for specific traits in elite lines. The Neighbor-Joining tree and kinship matrix will assist to identify distantly related genotypes from subpopulations to utilize in hybridization. The low levels of LD and population structure make the core collection an important resource for association mapping efforts to identify genes useful in crop improvement as well as for selection of parents for hybrid breeding.

genomics↗

Neural effects of propofol-induced unconsciousness and its reversal using thalamic stimulation

The specific circuit mechanisms through which anesthetics induce unconsciousness have not been completely characterized. We recorded neural activity from the frontal, parietal, and temporal cortices and thalamus while maintaining unconsciousness in non-human primates (NHPs) with the anesthetic propofol. Unconsciousness was marked by slow frequency (~1 Hz) oscillations in local field potentials, entrainment of local spiking to Up states alternating with Down states of little spiking, and decreased coherence in frequencies above 4 Hz. Thalamic stimulation "awakened" anesthetized NHPs and reversed the electrophysiologic features of unconsciousness. Unconsciousness is linked to cortical and thalamic slow frequency synchrony coupled with decreased spiking, and loss of higher-frequency dynamics. This may disrupt cortical communication/integration.

neuroscience↗

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins - structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

cell biology↗

Functional composition rather than species richness drive carbon gain and allocation in experimental grasslands

Numerous experiments have shown positive diversity effects on plant productivity, but little is known about related processes of carbon gain and allocation. We investigated these processes in a controlled environment (Montpellier European Ecotron) applying a continuous 13CO2 label for three weeks to 12 soil-vegetation monoliths originating from a grassland biodiversity experiment (Jena Experiment) and representing two diversity levels (4 and 16 sown species). Plant species richness did not affect community- and species-level 13C abundances neither in total biomass nor in non-structural carbohydrates (NSC). Community-level 13C excess tended to be higher in the 16-species than in the 4-species mixtures. Community-level 13C excess was positively related to canopy leaf nitrogen (N), i.e. leaf N per unit soil surface. At the species level shoot 13C abundances varied among plant functional groups and were larger in legumes and tall herbs than in grasses and small herbs and correlated positively with traits as leaf N concentrations, stomatal conductance and shoot height. The 13C abundances in NSC were larger in transport sugars (sucrose, raffinose-family oligosaccharides) than in free glucose, fructose and compounds of the storage pool (starch) suggesting that newly assimilated carbon is to a small portion allocated to storage. Our results emphasize that the functional composition of communities is key in explaining carbon assimilation in grasslands.

ecology↗

Discovering novel calcineurin inhibitors through quantitative mapping of protein-peptide affinity landscapes

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and therapeutically perturbing signaling networks, but is challenging due to their weak affinities. We present a powerful technology, MRBLE-pep, that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using computational modeling and MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We establish that flanking residues and post- translational modifications critically contribute to PxIxIT-CN affinity, and discover CN-inhibitory peptides with unprecedented affinity and therapeutic potential. The quantitative measurements provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

bioengineering↗

Evidence for subsoil specialization in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungal communities are now known to vary with depth in arable land. Here we use two previously published high-throughput Illumina sequencing data sets, and compare a 52 year long chronosequence of recultivated agriculture fields after a topsoil and subsoil mixing event, with a set of undisturbed topsoil and subsoil samples from a similar field. We show that AM taxa identified as subsoil indicators are exclusively present in early stages of the chronosequence, whereas topsoil indicator taxa can be found across the chronosequence, and that similarities from the chronosequence fields to the subsoil communities decrease with time. Our results provide evidence on the ecological specialization of certain AM fungal taxa to deep soil layers.

ecology↗