Biology subjects

Singh, A.

Publications and source records attributed to Singh, A..

At least 523 records · Page 29Linked to original sources

bioRxiv2021

Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its GEF (guanine nucleotide exchange factor) Syt22 was required for both Arf6 node localization and proper size at division. In arf6{Delta} mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6{Delta} was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

cell biology↗

bioRxiv2021

Data science competition for cross-site delineation and classification of individual trees from airborne remote sensing data

Delineating and classifying individual trees in remote sensing data is challenging. Many tree crown delineation methods have difficulty in closed-canopy forests and do not leverage multiple datasets. Methods to classify individual species are often accurate for common species, but perform poorly for less common species and when applied to new sites. We ran a data science competition to help identify effective methods for delineation of individual crowns and classification to determine species identity. This competition included data from multiple sites to assess the methods ability to generalize learning across multiple sites simultaneously, and transfer learning to novel sites where the methods were not trained. Six teams, representing 4 countries and 9 individual participants, submitted predictions. Methods from a previous competition were also applied and used as the baseline to understand whether the methods are changing and improving over time. The best delineation method was based on an instance segmentation pipeline, closely followed by a Faster R-CNN pipeline, both of which outperformed the baseline method. However, the baseline (based on a growing region algorithm) still performed well as did the Faster R-CNN. All delineation methods generalized well and transferred to novel forests effectively. The best species classification method was based on a two-stage fully connected neural network, which significantly outperformed the baseline (a random forest and Gradient boosting ensemble). The classification methods generalized well, with all teams training their models using multiple sites simultaneously, but the predictions from these trained models generally failed to transfer effectively to a novel site. Classification performance was strongly influenced by the number of field-based species IDs available for training the models, with most methods predicting common species well at the training sites. Classification errors (i.e., species misidentification) were most common between similar species in the same genus and different species that occur in the same habitat. The best methods handled class imbalance well and learned unique spectral features even with limited data. Most methods performed better than baseline in detecting new (untrained) species, especially in the site with no training data. Our experience further shows that data science competitions are useful for comparing different methods through the use of a standardized dataset and set of evaluation criteria, which highlights promising approaches and common challenges, and therefore advances the ecological and remote sensing field as a whole.

ecology↗

bioRxiv2021

Evaluating the feasibility and efficacy of a dual-modality nanoparticle contrast agent (Nanotrast-CF800) for image-guided sentinel lymph node mapping in the oral cavity of healthy dogs

A combination of pre- and intraoperative sentinel lymph node (SLN) mapping techniques has been suggested to optimize SLN detection. A novel liposomal nanoparticle, Nanotrast-CF800 (CF800), utilizes computed tomography lymphography (CTL) and near infrared fluorescence imaging (NIRF) for image-guided surgery and SLN mapping. This novel tracer agent has not been evaluated in companion animals. The objective of this study was to evaluate the feasibility and efficacy of CF800 for SLN mapping in the oral cavity of healthy dogs and to report any local adverse effects. Six healthy adult purpose-bred research dogs randomly received either 1 mL (group 1) or 2 mL (group 2) of CF800 injected into the submucosa at the level of the right canine maxillary tooth. CTL and percutaneous NIRF were performed at 1, 3, and 10 minutes, then 1, 2, 4, 7, and 10 days post-injection (p.i). Overall, both CTL and NIRF identified SLNs in all dogs. The overall peak mean contrast enhancement of the SLNs was 73.98 HU (range 63.45-86.27 HU) at 2 days p.i. Peak fluorescence of the SLN occurred at 1 day p.i. The agent was retained within the SLN for at least 7 days for CTL and 4 days for NIRF. No adverse effects were observed. Local administration of CF800 was simple and feasible for the detection of SLNs using CTL+NIRF in the head and neck of healthy dogs and was not associated with significant local adverse events.

cancer biology↗

bioRxiv2021

A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting Fas signaling

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, while long non-coding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between lncRNA HOTTIP and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP-seq., (genome wide HOTTIP binding analysis), ChIP-seq., and ATAC-seq., we found that miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a and miR-10b), located cis & trans, were most dramatically regulated and significantly decreased in HOTTIP-/- AML cells. HOTTIP bound to the miR-196b promoter, and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster associated miRNAs in AML cells, while reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival compared to wild-type cells. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription, consequently repressing tumor suppressors and promoting leukemogenesis.

biochemistry↗

bioRxiv2021

Stochastic comparison of synchronization in activator- and repressor-based coupled gene oscillators

Inside living cells, proteins or mRNA can show oscillations even without a periodic driving force. Such genetic oscillations are precise timekeepers for cell-cycle regulations, pattern formation during embryonic development in higher animals, and daily cycle maintenance in most organisms. The synchronization between oscillations in adjacent cells happens via intercellular coupling, which is essential for periodic segmentation formation in vertebrates and other biological processes. While molecular mechanisms of generating sustained oscillations are quite well understood, how do simple intercellular coupling produces robust synchronizations are still poorly understood? To address this question, we investigate two models of coupled gene oscillators - activator-based coupled oscillators (ACO) and repressor-based coupled oscillators (RCO) models. In our study, a single autonomous oscillator (that operates in a single cell) is based on a negative feedback, which is delayed by intracellular dynamics of an intermediate species. For the ACO model (RCO), the repressor protein of one cell activates (represses) the production of another protein in the neighbouring cell after a intercellular time delay. We investigate the coupled models in the presence of intrinsic noise due to the inherent stochasticity of the biochemical reactions. We analyze the collective oscillations from stochastic trajectories in the presence and absence of explicit coupling delay and make careful comparison between two models. Our results show no clear synchronizations in the ACO model when the coupling time delay is zero. However, a non-zero coupling delay can lead to anti-phase synchronizations in ACO. Interestingly, the RCO model shows robust in-phase synchronizations in the presence and absence of the coupling time delay. Our results suggest that the naturally occurring intercellular couplings might be based on repression rather than activation where in-phase synchronization is crucial.

systems biology↗

bioRxiv2021

Lightsheet Optical Tweezer (LOT) for Optical Manipulation of Microscopic Particles and Live Cells

A light sheet based optical tweezer (LOT) is developed to trap microscopic dielectric particles and live HeLa cells. The technique requires the generation of a tightly focussed diffraction-limited light sheet which is realized by a combination of cylindrical lens and high NA objective lens. The field pattern generated at the geometrical focus is a tightly focussed line (along x-axis) perpendicular to the beam propagation direction (z-axis). Spherical beads undergoing Brownian motion in the solution are trapped by the gradient potential, and the time (to reach trap-center under the influence of gradient potential) is estimated from the fast CMOS camera (operating at 230frames/sec). High-speed imaging of beads at varying laser power shows a steady increase in the stiffness of LOT with a maximum of 0.00118 pN/nm at 52.5 mW. This is an order less than traditional optical point-traps. The trapped beads displayed free movement along the light-sheet axis (x-axis), exhibiting a single degree of freedom. Subsequently, LOT is used to optically trap and pattern dielectric beads and HeLa cells in a line. We could successfully pattern 8 dielectric beads and 3 HeLa cells in a straight line. We anticipate that LOT can be used to study the 1D-physics of microscopic particles and help understand the patterned growth of live cells.

biophysics↗

bioRxiv2021

CaProDH2-mediated modulation of proline metabolism confers tolerance to Ascochyta in chickpea under drought

Drought and leaf blight caused by the fungus Ascochyta rabiei often co-occur in chickpea (Cicer arietinum)-producing areas. While the responses of chickpea to either drought or A. rabiei infection have been extensively studied, their combined effect on plant defense mechanisms is unknown. Fine modulation of stress-induced signaling pathways under combined stress is an important stress adaptation mechanism that warrants a better understanding. Here we show that drought facilitates resistance against A. rabiei infection in chickpea. The analysis of proline levels and gene expression profiling of its biosynthetic pathway under combined drought and A. rabiei infection revealed the gene encoding proline dehydrogenase (CaProDH2) as a strong candidate conferring resistance to A. rabiei infection. Transcript levels of CaProDH2, pyrroline-5-carboxylate (P5C) quantification, and measurement of mitochondrial reactive oxygen species (ROS) production showed that fine modulation of the proline-P5C cycle determines the observed resistance. In addition, CaProDH2-silenced plants lost basal resistance to A. rabiei infection induced by drought, while overexpression of the gene conferred higher resistance to the fungus. We suggest that the drought-induced accumulation of proline in the cytosol helps maintain cell turgor and raises mitochondrial P5C contents by a CaProDH2-mediated step, which results in ROS production that boosts plant defense responses and confers resistance to A. rabiei infection. Our findings indicate that manipulating the proline-P5C pathway may be a possible strategy for improving stress tolerance in plants suffering from combined drought and A. rabiei infection.

plant biology↗

bioRxiv2021

Characterizing non-exponential growth and bimodal cell size distributions in Schizosaccharomyces pombe: an analytical approach

Unlike many single-celled organisms, the growth of fission yeast cells within a cell cycle is not exponential. It is rather characterized by three distinct phases (elongation, septation and fission), each with a different growth rate. Experiments also show that the distribution of cell size in a lineage is often bimodal, unlike the unimodal distributions measured for the bacterium Escherichia coli. Here we construct a detailed stochastic model of cell size dynamics in fission yeast. The theory leads to analytic expressions for the cell size and the birth size distributions, and explains the origin of bimodality seen in experiments. In particular our theory shows that the left peak in the bimodal distribution is associated with cells in the elongation phase while the right peak is due to cells in the septation and fission phases. We show that the size control strategy, the variability in the added size during a cell cycle and the fraction of time spent in each of the three cell growth phases have a strong bearing on the shape of the cell size distribution. Furthermore we infer all the parameters of our model by matching the theoretical cell size and birth size distributions to those from experimental single cell time-course data for seven different growth conditions. Our method provides a much more accurate means of determining the cell size control strategy (timer, adder or sizer) than the standard method based on the slope of the best linear fit between the birth and division sizes. We also show that the variability in added size and the strength of cell size control of fission yeast depend weakly on the temperature but strongly on the culture medium. Author summaryAdvances in microscopy enable us to follow single cells over long timescales from which we can understand how their size varies with time and the nature of innate strategies developed to control cell size. This data shows that in many cell types growth is exponential and the distribution of cell sizes has one peak, namely there is a single characteristic cell size. However data for fission yeast shows remarkable differences: growth is non-exponential and the distribution of cell sizes has two peaks, meaning two characteristic cell sizes exist. Here we construct the first mathematical model of this organism; by solving the model analytically we show that it is able to predict the two peaked distributions of cell size seen in data and provides an explanation for each peak in terms of the various growth phases of the single-celled organism. Furthermore by fitting the model to the data, we infer values for the rates of all microscopic processes in our model. This method is shown to provide a much more reliable inference than current methods and sheds light on how the strategy used by fission yeast cells to control their size varies with external conditions.

systems biology↗

bioRxiv2021

Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardised modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fibre system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

synthetic biology↗

bioRxiv2021

Mechanistic insights into the Japanese Encephalitis Virus RNA dependent RNA polymerase protein inhibition by bioflavonoids from Azadirachta indica

Japanese encephalitis (JE) virus, belongs to the genus flavivirus, is a major cause of viral encephalitis, which results in neurological damage. RNA-dependent RNA polymerase (RdRp) protein is the sole enzyme responsible for viral genome replication in RNA viruses and serves as an excellent target for anti-viral therapeutic development, as its homolog is not present in humans. In this study, the crystal structure of JE RNA-dependent RNA polymerase (jRdRp) protein was obtained from protein data bank while 43 by bioflavonoids reported in Azadirachta indica were retrieved from the PubChem database. Following, structure based virtual screening was employed using MTiOpenScreen server and top four compounds, viz. Gedunin, Nimbolide, Ohchinin acetate, and Kulactone, with the most negative docking scores (> -10 kcal/mol) conformations were redocked using AutoDock Vina; these complexes showed mechanistic interactions with Arg474, Gly605, Asp668, and Trp800 residues in the active site of jRdRp protein against reference ligand, i.e., Guanosine-5-Triphosphate. Furthermore, 100 ns classical molecular dynamics simulation and binding free energy calculation showed considerable stability (via hydrogen bonding and hydrophobic interactions) of docked bioflavonoids in the active pocket of jRdRp and significant contribution of van der Waals interactions for docked complex stability during simulation, respectively. Therefore, the outcome of this study predicted the substantial anti-viral activity of Gedunin, Nimbolide, Ohchinin acetate, and Kulactone against jRdRp protein and can be considered for further antiviral drug development.

bioinformatics↗

bioRxiv2021

Protein sensors of bacterial kinase activity reveal antibiotic-dependent kinase activation in single cells

A lack of direct single-cell readouts for bacterial kinase activity remains a major barrier to our understanding of most signaling systems. At the single-cell-level, protein kinase activity is typically inferred by the activity of downstream transcriptional reporters. Complicating this approach in vivo, promoters are often co-regulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we have designed and constructed new, direct and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor that responds to phosphorylation. We demonstrate the utility of these reporters in measuring kinase activity in population-based and single-cell assays during various growth phases and antibiotic treatments. These sensors respond to a highly conserved bacterial Ser/Thr kinase, PrkC that has no known dedicated transcription factor and whose regulon is known to be convolved with an essential signaling system. We used these new sensors to measure PrkC activity in colonies, bulk culture, and single cells. Together these new sensors provide evidence for considerable heterogeneity in PrkC activity in actively growing populations. We further demonstrate that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). This is consistent with a model where PrkC senses and responds to blocks in the extracellular steps in cell wall synthesis. As the design of these phosphorylation sensors is modular, we anticipate that this work may have broad applications to other bacterial signaling systems in the future.

synthetic biology↗

bioRxiv2021

Density Fluctuations Yield Distinct Growth and Fitness Effects in Single Bacteria

Single-cells grow by increasing their biomass and size. Here, we report that while mass and size accumulation rates of single Escherichia coli cells are exponential, their density fluctuates during growth. As such, the rates of mass and size accumulation of a single-cell are generally not the same, but rather cells differentiate into increasing one rate with respect to the other. This differentiation yields a previously unknown density homeostasis mechanism, which we support mathematically. Further, growth differentiation challenges ongoing efforts to predict single-cell reproduction rates (or fitness-levels), through the accumulation rates of size or mass. In contrast, we observe that density fluctuations can predict fitness, with only high fitness individuals existing in the high density fluctuation regime. We detail our imaging approach and the invisible microfluidic arrays that critically enabled increased precision and throughput. Biochemical production, infections, and natural communities start from few, growing, cells, thus, underscoring the significance of density-fluctuations when considering non-genetic variability.

biophysics↗

bioRxiv2021

Human fetal neural stem cell-derived astrocytes maintain glutamate transport after hypoxic injury in vitro

Astrocytes are the most abundant glial cells that play many critical roles in the central nervous system physiology including the uptake of excess glutamate from the synapse by Excitatory Amino Acid Transporters (EAATs). Among the EAATs, EAAT2 are predominantly functional, astrocyte-specific glutamate transporters in the forebrain. Hypoxic brain injury is a pathological phenomenon seen in various clinical conditions including stroke and neonatal hypoxic ischemic encephalopathy. Glutamate excitotoxicity is an important cause of neuronal cell death in disorders involving hypoxic brain injury. As findings from rodent models cannot always be reliably extrapolated to humans, we aimed to develop a homogenous population of primary human astrocytes to study the effect of hypoxic injury on astrocyte function, especially glutamate uptake. We successfully isolated, established and characterized cultures of human fetal neural stem cells (FNSCs) from aborted fetal brains. FNSCs were differentiated into astrocytes, and characterized by increased expression of the astrocyte markers, glial fibrillary acidic protein (GFAP), EAAT1 and EAAT2. A concomitant decrease in neural stem cell marker, Nestin, was observed. Differentiated astrocytes were exposed to various oxygen concentrations mimicking normoxia (20% and 6%), moderate and severe hypoxia (2% and 0.2% respectively). Interestingly, no change was observed in the expression of glutamate transporter, EAAT2 and glutamate uptake by astrocytes, even after exposure to hypoxia. Our novel model of human FNSC derived astrocytes exposed to hypoxic injury, establishes that astrocytes are able to maintain glutamate uptake even after exposure to severe hypoxia for 48 hours, and thus provides evidence for the neuroprotective role of astrocytes in hypoxic injury.

neuroscience↗

bioRxiv2021

Optimal Chemotherapy Scheduling for Non-Genetic Drug Resistance

One of the most difficult challenges in cancer therapy is the emergence of drug resistance within tumors. Sometimes drug resistance can emerge as the result of mutations and Darwinian selection. However, recently another phenomenon has been discovered, in which tumor cells switch back and forth between drug-sensitive and pre-resistant states. Upon exposure to the drug, sensitive cells die off, and pre-resistant cells become locked in to a state of permanent drug resistance. In this paper, we explore the implications of this transient state switching for therapy scheduling. We propose a model to describe the phenomenon and estimate parameters from experimental melanoma data. We then compare the performance of continuous and alternating drug schedules, and use sensitivity analysis to explore how different conditions affect the efficacy of each schedule. We find that for our estimated parameters, a continuous therapy schedule is optimal. However we also find that an alternating schedule can be optimal for other, hypothetical parameter sets, depending on the difference in growth rate between pre-drug and post-drug cells, the delay between exposure to the drug and emergence of resistance, and the rate at which pre-resistant cells become resistant relative to the rate at which they switch back to the sensitive state.

systems biology↗

bioRxiv2021

The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties

Osteosarcoma (OS) is the most common type of primary bone cancer affecting children and adolescents. OS has a high propensity to spread, meaning the disease is often incurable and fatal. There have been no improvements in survival rates for decades. This highlights an urgent need for development of novel therapeutic strategies. In this study, we have produced in vitro and in vivo data that demonstrates the role of purinergic signalling, specifically, the B isoform of the purinergic receptor P2RX7 (herein termed " P2RX7B"), in OS progression and metastasis. Our data shows that P2RX7B expression confers a survival advantage in TE85+P2RX7B and MNNG-HOS+ P2RX7B human OS cell lines in vitro that is minimised following treatment with A740003, a specific P2RX7 antagonist. P2RX7B expression reduced cell adhesion and P2RX7B activation promoted invasion and migration in vitro, suggesting a probable metastatic phenotype. Using an in vivo OS xenograft model, MNNG-HOS+P2RX7B tumours exhibited ectopic bone formation that was abrogated with A740003 treatment. An increased metastatic phenotype was further demonstrated in vivo as expression of P2RX7B in primary tumour cells increased the propensity of the tumour to metastasise to the lungs. RNA-seq identified a novel gene axis, FN1/LOX/PDGFB/IGFBP3/BMP4, downregulated in response to A740003 treatment. In conclusion, our data indicates for the first time a role for P2RX7B in OS tumour growth, progression and metastasis. We show that P2RX7B is a potential therapeutic target in human OS. Novelty and ImpactWe provide evidence for the pro-tumorigenic role of the B isoform of the P2RX7 purinergic receptor in osteosarcoma (OS). In addition to increasing proliferation, P2RX7B increases the cancerous properties of OS cells, reducing adhesion and increasing migration and invasion. In vivo, P2RX7B does not affect primary tumour growth, but does lead to an increased propensity to metastasize. RNA-seq revealed a new axis of oncogenic genes inhibited by the P2RX7 antagonist and this data could potentially lead to new targets for OS treatment.

cancer biology↗

bioRxiv2021

SARS-CoV-2 Entry Protein TMPRSS2 and Its Homologue, TMPRSS4 Adopts Structural Fold Similar to Blood Coagulation and Complement Pathway Related Proteins

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes TMPRSS2 receptor to enter target human cells and subsequently causes coronavirus disease 19 (COVID-19). TMPRSS2 belongs to the type II serine proteases of subfamily TMPRSS, which is characterized by the presence of the serine-protease domain. TMPRSS4 is another TMPRSS member, which has a domain architecture similar to TMPRSS2. TMPRSS2 and TMPRSS4 have been shown to be involved in SARS-CoV-2 infection. However, their normal physiological roles have not been explored in detail. In this study, we analyzed the amino acid sequences and predicted 3D structures of TMPRSS2 and TMPRSS4 to understand their functional aspects at the protein domain level. Our results suggest that these proteins are likely to have common functions based on their conserved domain organization. Furthermore, we show that the predicted 3D structure of their serine protease domain has significant similarity to that of plasminogen which dissolves blood clot, and of other blood coagulation related proteins. Additionally, molecular docking analyses of inhibitors of four blood coagulation and anticoagulation factors show the same high specificity to TMPRSS2 and TMPRSS4 3D structures. Hence, our observations are consistent with the blood coagulopathy observed in COVID-19 patients and their predicted functions based on the sequence and structural analyses offer avenues to understand better and explore therapeutic approaches for this disease.

bioinformatics↗

bioRxiv2021

Identification of oxalyl-CoA synthetase gene (LsAAE3) and its regulatory role in β-ODAP biosynthesis in grasspea (Lathyrus sativus L.)

Grasspea is a popular pulse crop due to its hardiness and low cost of production. Presence of anti-nutritive factor {beta}-ODAP in its seeds and other plant parts hinder its widespread cultivation and usage. Oxalyl-CoA synthetase is one of the key enzyme of {beta}-ODAP biosynthesis pathway, catalyses the conversion of oxalate to oxalyl-CoA. ACYL ACTIVATING ENZYME 3 (AAE3) gene has been characterised to encode an oxalyl-CoA synthetase enzyme in many plant species. We report here the isolation of full length AAE3 homolog in grasspea with a combination of PCR based strategy and in silico analysis. We first identified AAE3 homolog by PCR using degenerate primers. The partial LsAAE3 sequence showed 88% amino acids sequence identity with the characterised AAE3 gene of M. truncatula. We then predicted the full length AAE3 sequence using the publically available transcriptome datasets of grasspea. Determination of LsAAE3 gene and protein structure and phylogenetic relationship analysis strongly suggested that LsAAE3 is a true homolog of AAE3 gene. Expression profiling of LsAAE3 in grasspea varieties with contrast in {beta}-ODAP content revealed its inverse relationship with the {beta}-ODAP content, LsAAE3 thus negatively regulates the synthesis of {beta}-ODAP. Involvement of AAE3 encoded oxalyl-CoA synthetase in a CoA-dependent pathway of oxalate degradation is well proven in many plant species. We also identified the CoA-dependent pathway of oxalate degradation in grasspea. Based on these observations, we hypothesized that LsAAE3 may regulate {beta}-ODAP content, possibly, by CoA-dependent pathway of oxalate degradation in grasspea. If this hypothesis is substantiated, genetic manipulation of LsAAE3 presents viable option for reducing {beta}-ODAP content in grass pea.

molecular biology↗

bioRxiv2021

Protein noise and distribution in a two-stage gene-expression model extended by an mRNA inactivation loop

Chemical reaction networks involving molecular species at low copy numbers lead to stochasticity in protein levels in gene expression at the single-cell level. Mathematical modelling of this stochastic phenomenon enables us to elucidate the underlying molecular mechanisms quantitatively. Here we present a two-stage stochastic gene expression model that extends the standard model by an mRNA inactivation loop. The extended model exhibits smaller protein noise than the original two-stage model. Interestingly, the fractional reduction of noise is a non-monotonous function of protein stability, and can be substantial especially if the inactivated mRNA is stable. We complement the noise study by an extensive mathematical analysis of the joint steady-state distribution of active and inactive mRNA and protein species. We determine its generating function and derive a recursive formula for the protein distribution. The results of the analytical formula are cross-validated by kinetic Monte-Carlo simulation.

systems biology↗