Biology subjects

Ansari, S.

Publications and source records attributed to Ansari, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Multivalent adaptor networks generate nanoscale organisation within T cell signalling condensates

Ligand engagement of the T cell receptor (TCR) triggers phosphorylation-dependent assembly of signalling complexes at the plasma membrane, yet how these assemblies are organised to coordinate signal propagation remains unclear. Here, using quantitative super-resolution imaging in primary human T cells, we show that phosphorylated TCR (pTCR) and linker for activation of T cells (pLAT) reside within micrometre-scale signalling condensates that exhibit defined nanoscale organisation. Within these condensates, pTCR and pLAT are spatially interleaved with characteristic spacing of [~]70-100 nm and display short-range dispersion rather than aggregation. Spatial cross-correlation analysis reveals that pLAT is organised around pTCR sites, consistent with a model in which activated TCRs act as local sources of LAT phosphorylation that nucleate condensate assembly. This organisation suggests that multivalent interactions and steric constraints generate structured, yet dynamic, reaction environments that couple receptor activation to downstream signalling. Together, our findings establish that TCR signalling condensates are not amorphous assemblies but possess intrinsic nanoscale architecture and support a general framework in which membrane-proximal signalling is regulated by spatially organised, multivalent assemblies.

immunology↗

bioRxiv2026

Physiological Characterization under the Influence of Drought Stress and Salicylic Acid in Valeriana wallichii DC

Valeriana wallichii DC, a high-altitude medicinal plant, is highly sensitive to drought stress, which adversely impacts its physiological functions and root biomass. This study aimed to evaluate salicylic acids role in mitigating drought stress by assessing physiological and biochemical responses in V. wallichii. Plants were treated with foliar applications of SA at concentrations of 0.25, 0.50, 0.75, and 1.0 mM during vegetative and flowering stages. Physiological parameters such as photosynthetic rate, stomatal conductance, water use efficiency (WUE), intrinsic WUE (iWUE), carboxylation efficiency, and biochemical traits including chlorophyll and carotenoid content were measured, alongside fresh and dry root biomass.Drought stress significantly reduced photosynthetic performance (Pn) (3.95 mol CO2m{square}2s{square}1 at 25% FC), stomatal conductance, relative water content, and root yield. SA application notably improved these traits. At 25% FC, 1.0 mM SA enhanced WUE (28.78), iWUE (642.37 mol/mol), and fresh root weight (8.41 g). Under moderate drought (50% FC), 0.50 mM SA improved net photosynthesis (12.5 mol CO2 m{square}2s{square}1), dry root weight (2.31 g), and chlorophyll content. SA treatments also stabilized membrane integrity and maintained higher chlorophyll fluorescence efficiency (Fv/Fm).These findings demonstrate that foliar application of SA enhances drought tolerance in V. wallichii by improving photosynthetic efficiency, water relations, and root biomass. The study supports the strategic use of SA to improve the resilience and productivity of medicinal plants under drought stress conditions. Highlights of the studyO_LIDrought lowers growth and yield by reducing photosynthesis and water use. C_LIO_LISalicylic acid reduces drought effects and improves plant performance. C_LIO_LIAt severe drought, 1- 0.50 mM SA raised WUE, conductance, and root yield. C_LIO_LIUnder mild drought, 0.75- 0.50 mM SA improved gas exchange and yield. C_LIO_LISA doses 0.25-1 mM differ in effectiveness across drought stress stages. C_LI

plant biology↗

bioRxiv2025

A Physically-Realistic Simulator and Cosine-Based Decoder for MERFISH Spatial Transcriptomics

Imaging-based spatial transcriptomics technologies have opened new avenues for studying cellular organization and gene expression within intact tissues. However, the accuracy of downstream analyses depends critically on the decoding step that reconstructs barcodes from fluorescence patterns and maps them to gene identities. Despite a growing number of decoding methods, systematic benchmarking has been limited. Here, we introduce Serval, a modular framework for developing and benchmarking decoding methods across diverse spatial transcriptomics platforms. Serval separates key decoding stages into independently configurable modules, enabling flexible integration of alternative algorithms. Using this framework, we develop the Cosine decoder, a novel method that improves transcript recovery by optimizing cosine similarity to known barcodes. We evaluate Cosine and baseline methods on synthetic and real MERFISH datasets, showing that Cosine achieves higher transcript recovery and superior correlation with expression references compared to existing methods. Furthermore, we demonstrate that the Serval framework generalizes beyond MERFISH. By extending to the DART-FISH platform, we show that Cosine improves transcript recovery, clustering stability and supports more direct annotation of complex biological structures such as the human primary motor cortex. These results establish that modular decoding frameworks facilitate robust, platformagnostic benchmarking, ultimately supporting more accurate spatial transcriptomics analysis across diverse biological samples.

bioinformatics↗

bioRxiv2024

In cell NMR reveals cells selectively amplify and structurally remodel amyloid fibrils

Amyloid forms of -synuclein adopt different conformations depending on environmental conditions. Although advances in structural biology have accelerated fibril characterization, it remains unclear which conformations predominate in biological settings because current approaches typically require fibrils to be isolated from their native environments. In addition, these approaches provide limited information about flanking flexible regions. Here, using a defined polymorphic seed population and quantitative in-cell NMR, we show that propagation in intact cellular environments--but not in vitro buffer or crowded cellular lysates--reshapes the conformational ensemble of -synuclein fibrils. In vitro and in cellular lysates, both amyloid-core structure and the conformational preferences of flanking intrinsically disordered regions of the seed are faithfully propagated. In contrast, propagation inside intact cells selectively amplifies the amyloid-core conformer that is minor in vitro, increases its molecular order, and remodels conformational preferences in the flanking disordered region. These results demonstrate that cellular organization plays a decisive role in determining amyloid structure and that biologically relevant conformations cannot be inferred from purified or lysate systems alone.

biophysics↗

bioRxiv2024

Network controllability analysis reveals the antiviral potential of Etravirine against Hepatitis E Virus infection

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in lower- and middle-income countries. HEV infection may lead to acute liver failure, chronic liver disease and high mortality in pregnant women. Antiviral therapy is not a standard treatment for HEV patients. Computational biology tools promise to revolutionize the antiviral drug discovery. Here, we analyzed the transcriptome data of HEV infected primary human hepatocyte (PHH)-cells through connectivity map database and applied control theory on functional network to identify antiviral targets against HEV. The above analyses predicted PKC{beta}, PKB/AKT and CK1{varepsilon} as potential antiviral targets against HEV. The antiviral function of PKB/AKT and CK1{varepsilon} was experimentally validated by using respective biochemical inhibitors in g3 (genotype 3)-HEV replicon and Huh7 cell-based model of g3 and g1-HEV infection. Further, knockdown of CK1{varepsilon} showed a similar effect. These data confirmed that CK1{varepsilon} is an antiviral target for HEV. At present, there are no FDA approved drugs targeting CK1{varepsilon}. Etravirine is an FDA approved non-nucleoside reverse transcriptase inhibitor drug, used for the treatment of Human immunodeficiency virus type 1 (HIV-1) infected patients. An in silico study predicted Etravirine to be a potent inhibitor of CK1{varepsilon}. Our experiments revealed potent antiviral activity of Etravirine against HEV, which was mediated via its ability to inhibit the activity of CK1{varepsilon}. Taken together, the current study demonstrates that PKB/AKT and CK1{varepsilon} are bonafide antiviral targets for HEV and paves the way for repurposing Etravirine for the treatment of HEV infected patients. ImportanceAntiviral treatment is not the standard care for acute viral hepatitis E patients. Unbiased identification of antiviral targets or large-scale screening of antiviral compounds against the hepatitis E virus (HEV) has not been reported. Here, computational biology approach was followed to unbiasedly identify antiviral targets of HEV. Transcriptome data of HEV infected primary human hepatocyte (PHH) cells were analyzed to identify modulators of the network and generate directional networks. Network controllability analysis identified PKC{beta}, PKB/AKT and CK1{varepsilon} as potential antiviral targets against HEV. Antiviral function of PKB/AKT and CK1{varepsilon} was confirmed using cell-based models of genotype 1 (g1)- and g3-HEV infection. Further experiments demonstrated the antiviral activity of Etravirine against HEV, mediated via its ability to inhibit the CK1{varepsilon} activity. Etravirine is an FDA approved non-nucleoside reverse transcriptase inhibitor, used for the treatment of Human immunodeficiency virus type-1 (HIV-1)-infected patients. This study reveals the potential of repurposing Etravirine for treatment of HEV patients and illustrate the importance of computational biology in antiviral drug discovery.

microbiology↗

bioRxiv2023

Quantifying yeast microtubules and spindles using the Toolkit for Automated Microtubule Tracking (TAMiT)

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that is solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work, and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

cell biology↗

bioRxiv2023

Towards development of serum substitute medium to induce osteoclast differentiation of human peripheral blood derived monocytes

Fetal bovine serum (FBS) is a widely used supplement in cell culture media despite its known drawbacks, including ethical, safety, and scientific issues. To overcome the drawbacks of using FBS in cell culture, a defined serum substitute medium needs to be developed. The development of such a medium depends on the cell type, which makes it impossible to use one universal serum substitute medium for all cells. Osteoclasts are large, multinucleated cells originated from the hematopoietic stem cell lineage that play an important role in regulating bone mass and quality. To date, no defined serum substitute medium formulations have been reported for osteoclast differentiation of monocytes derived from peripheral blood mononuclear cells (PBMCs). Here, we have attempted to develop such a serum substitute medium for the osteoclastogenesis process in a stepwise approach. Essential components were added to the medium while monocytes were cultured in 96-well plates and in Osteo-Assay well plates to analyze the formation of tartrate resistant acid phosphatase (TRAP) expressing multinucleated osteoclasts with distinct actin ring and to analyze the resorption activity of mature osteoclasts for 21 days, respectively. The serum substitute medium was aimed at supporting monocyte and later osteoclast survival, differentiation of monocytes towards multinucleated osteoclasts, and the resorption of mineralized matrix as a measure of functionality. All points were achieved after 21 days of culture in the developed serum substitute medium. This serum substitute medium could potentially replace FBS in osteoclastogenesis studies eliminating its debated use. Moreover, the well-defined serum substitute environment simplifies the study of factors released by the cells that were so far overwhelmed by the complexity of FBS.

bioengineering↗

bioRxiv2023

Matrix vesicle isolation from a three-dimensional in vitro bone model

Extracellular vesicles (EV) are nano-sized bilayer vesicles that are involved in biological functions and secreted by a wide variety of cells. Osteoblasts, the bone forming cells, can release a subset of EVs known as matrix vesicles (MtVs) which are believed to be involved in matrix mineralization and feature bone forming properties. Osteoblast-derived EVs or MtVs have been mostly isolated from conditions which are still far from nature, i.e. mesenchymal stromal cells (MSCs), or osteoblast cell lines cultured in two-dimensional (2D) tissue culture flasks. In our study, we aimed at investigating whether MtVs could also be isolated from an environment which better resembles the complex in vivo situation. This study investigated the EVs secretion during osteogenic differentiation of human bone marrow MSCs (hBMSCs) in the most advanced human three-dimensional (3D) in vitro woven bone constructs previously developed by our group. hBMSCs were cultured in spinner flask bioreactors which induced wall shear stress on cells and directed the cells to differentiate towards osteoblasts and osteocytes. The EVs secreted into the culture medium were isolated and characterized based on their morphological, biological, and functional properties. The characteristics of a part of isolated EVs shared similarities with MtVs. These vesicles were electron-dense and electron-lucent, showed alkaline phosphatase (ALP) activity, increased the amount of released free phosphate into the culture medium, and increased the amount of deposited phosphate within the ECM. The results indicate that a complex 3D environment mimicking bone development is favorable to stimulate MtV-producing cells to produce targeted MtVs in vitro. These MtVs potentially could be used as a biological agent for bone regeneration and fracture healing through, for instance, integration with biomaterials to target bone formation locally.

bioengineering↗

bioRxiv2022

Development of serum substitute medium for bone tissue engineering

In tissue engineering, cells are grown often on scaffolds and subjected to chemical/mechanical stimuli. Most such cultures still use fetal bovine serum (FBS) despite its known disadvantages including ethical concerns, safety issues, and variability in composition, which greatly influences the experimental outcomes. To overcome the disadvantages of using FBS, chemically defined serum substitute medium needs to be developed. Development of such medium depends on cell type and application - which makes it impossible to define one universal serum substitute medium for all cells in any application. Here, we developed a serum substitute medium for bone tissue engineering (BTE) in a step-by-step process. Essential components were added to the medium while human bone marrow mesenchymal stromal cells (hBMSCs, osteoblast progenitor cells) were cultured in two-dimensional (2D) and three-dimensional (3D) substrates. In a 3-week culture, the developed serum substitute medium worked equally well as FBS containing medium in term of cell attachment to the substrate, cell survival, osteoblast differentiation, and deposition of extracellular matrix. In the next step, the use of serum substitute medium was evaluated when culturing cells under mechanical loading in the form of shear stress. The outcomes showed that the application of shear stress is essential to improve extracellular matrix formation while using serum substitute medium. The developed serum substitute medium could pave the way in replacing FBS for BTE studies eliminating the use of controversial FBS and providing a better-defined chemical environment for BTE studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/511271v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@7bc741org.highwire.dtl.DTLVardef@5689fcorg.highwire.dtl.DTLVardef@17d1a3corg.highwire.dtl.DTLVardef@456844_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Human endogenous retrovirus-R envelope is a host restriction factor against severe acute respiratory syndrome-coronavirus-2

Coronavirus induced disease-19 (COVID-19), caused by the SARS-CoV-2 remains a major global health challenge. Human endogenous retroviruses (HERVs) represent retroviral elements that got integrated into the ancestral human genome. HERVs are important in development and diseases, including cancer, inflammation and viral infections. Here, we analyzed the expression of several HERVs in SARS-CoV-2 infected cells and observed increased activity of HERV-E, HERV-V, HERV-FRD, HERV-MER34, HERV-W and HERV-KHML2. In contrast, HERV-R-envelope was downregulated in cell-based models and COVID-19 patient PBMCs. HERV-R overexpression inhibited SARS-CoV-2 replication, suggesting its antiviral action. Further studies demonstrated the role of extracellular signal-regulated kinase (ERK) in regulating HERV-R antiviral activity. Cross-talk between the ERK and p38 MAPK controls HERV-R envelope synthesis, which in turn modulates the replication of SARS-CoV-2. These findings establish the importance of HERV-R envelope as a host restriction factor against SARS-CoV-2 and illustrate the advantage of integration and evolutionary maintenance of retroviral-elements in the human genome.

microbiology↗

bioRxiv2022

Kinesin-5/Cut7 moves bidirectionally on fission-yeast spindles with activity that increases in anaphase

Kinesin-5 motors are essential to separate mitotic spindle poles and assemble a bipolar spindle in many organisms. These tetrameric motors crosslink and slide apart antiparallel microtubules via microtubule plus-end-directed motility. However, kinesin-5s typically accumulate more at spindle poles than in the center of the spindle where antiparallel microtubule overlaps are most numerous. While the relevance of this localization has remained unclear, increasing evidence suggests that it occurs due to bidirectional motility or trafficking of kinesin-5 motors. The kinesin-5 motor Cut7p from fission yeast has been shown to move bidirectionally in reconstituted systems. However, bidirectional movement has not been shown in cells and the funtion of the minus-end-directed movement remains unclear. Here, we characterized the motility of kinesin-5/Cut7 on bipolar and monopolar spindles in fission yeast and observed movement both toward plus and minus ends of microtubules. Notably, we found that the activity of the motor increases at the onset on anaphase B. Perturbations to microtubule dynamics did not significantly change the observed Cut7p movement, while Cut7p mutation reduced or abolished observable movement. These results suggest that the directed movement of Cut7p was due to the motility of the motor itself. Mutations of Cut7p that decreased plus-end-directed motility enhanced its spindle-pole localization. In contrast, abolishing Cut7 motility or replacing it with plus-end-directed human Eg5 eliminates the pole localization. Our results suggest a new hypothesis for the function of minus-end-directed motility and spindle-pole localization of kinesin-5s in spindle assembly.

cell biology↗

bioRxiv2021

Alkaline phosphatase activity present in serum influences osteogenic differentiation cultures

Fetal bovine serum (FBS) is a widely used supplement in cell culture medium, despite its known variability in composition which greatly affects cellular function and consequently the outcome of studies. In bone tissue engineering, the deposited mineralized matrix is one of the main outcome parameters, but using different brands of FBS can result in large variations. Alkaline phosphatase (ALP) is present in FBS. Not only is ALP used to judge the osteogenic differentiation of bone cells, it may affect deposition of mineralized matrix. The present study focused on the enzymatic activity of ALP in FBS of different suppliers and its contribution to mineralization in osteogenic differentiation cultures. It was hypothesized that culturing cells in a medium with high intrinsic ALP activity of FBS will lead to higher mineral deposition compared to media with lower ALP activity. The used FBS types were shown to have significant differences in enzymatic ALP activity. Our results indicate that the ALP activity of the medium not only affected the deposited mineralized matrix but also the osteogenic differentiation of cells as measured by a changed cellular ALP activity of human bone marrow derived mesenchymal stromal cells (hBMSC). In media with low inherent ALP activity, the cellular ALP activity was increased and played the major role in the mineralization process; while, in media with high intrinsic ALP activity contribution from the serum, less cellular ALP activity was measured and the ALP activity of the medium also contributed to mineral formation substantially. Our results highlight the diverse effects of ALP activity intrinsic to FBS on osteogenic differentiation and matrix mineralization and how FBS can determine the experimental outcomes, in particular for studies investigating matrix mineralization. Once again, the need to replace FBS with more controlled and known additives is highlighted. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/462693v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@902078org.highwire.dtl.DTLVardef@43b6fborg.highwire.dtl.DTLVardef@1a244fborg.highwire.dtl.DTLVardef@5ef819_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Recombinant binder of sperm protein1 (rec-BSP1) as a potent fertility factor mediates its effect on embryo production

ObjectiveTo understand the effect of recombinant BSP1 (rec-BSP1) on in vitro capacitation of sperm and fertilization study Method(s)Articles were screened for reports including rec-BSP1, Capacitation, in vitro fertilization InterventionNone Main Outcome Measure(s)Reproductive outcomes, effect on gametes and embryos Result(s)Here we report an optimization of condition for rec-BSP1 production which was used for in vitro capacitation and enhancement of buffalo embryo production. The sequence of the protein was used for multiple sequence alignment which has 99% similarity with PDC 109 protein. The expression of rec-BSP1 was carried out successfully with 1 mM IPTG at 160 C for 22 hrs and purified it in soluble form. The structure of rec-BSP1 was generated using 3D modelling and analysed its mode of binding with heparin and PC by molecular docking and the structural stability of rec-BSP1-PC and rec-BSP1-heparin complexes by using molecular dynamic (MD) simulation. The effect of rec-BSP1 was observed on in vitro capacitation of spermatozoa and buffalo blastocyst production. It was found that the rec-BSP1 enhanced the sperm motility at a concentration of 50 g/ml for 1 h of incubation without having any detrimental effect on the sperm morphology and a significant increase in blastocyst production at concentration of 50 g/ml rec-BSP1. Hence this finding represents a new insight and advance the prospective approach to develop a potential fertility factor in reproduction. Conclusion(s)The purified rec-BSP1 may enhance on male fertility and mediated its effect on in vitro blastocyst production in buffalo.

bioengineering↗

bioRxiv2021

An integrated strategy reveals complex glycosylation of erythropoietin using top-down and bottom-up mass spectrometry

The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

biochemistry↗

bioRxiv2021

Large portion of essential genes is missed by screening either fly or beetle indicating unexpected diversity of insect gene function

Most gene functions were detected by screens in very few model organisms but it has remained unclear how comprehensive these data are. Here, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes and we compared the gene sets involved in several processes between beetle and fly. We find that around 50 % of the gene functions are detected in both species while the rest was found only in fly (~10%) or beetle (~40%) reflecting both technical and biological differences. We conclude that work in complementary model systems is required to gain a comprehensive picture on gene functions documented by the annotation of novel GO terms for 96 genes studied here. The RNAi screening resources developed in this project, the expanding transgenic tool-kit and our large-scale functional data make T. castaneum an excellent model system in that endeavor.

genetics↗

bioRxiv2020

A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC+ phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple division inhibition pathways exist during the stress-induced SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism. ImportanceFilamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

microbiology↗

bioRxiv2020

An Organoid for woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel both cellular and matrix formation processes. Stem cell-based organoids are promising as functional, self-organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self-organizing co-culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in vivo osteocytes the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under influence of external triggers (mechanical stimulation, drug administration).

bioengineering↗

bioRxiv2019

Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling

The essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood, despite extensive study. To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle, we developed a computational model of fission-yeast mitosis. Robust chromosome biorientation requires progressive restriction of attachment geometry, destabilization of misaligned attachments, and attachment force dependence. Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long. The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis, while interkinetochore stretch becomes important after biorientation. The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset. This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation, spindle length regulation, and force generation in the spindle.

cell biology↗