Biology subjects

Nandi, S.

Publications and source records attributed to Nandi, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Cyclic di-AMP regulates genome stability and drug resistance in Mycobacterium through RecA-dependent and -independent recombination

In Escherichia coli, RecA plays a central role in the rescue of stalled replication forks, double-strand break (DSB) repair, homologous recombination (HR) and induction of the SOS response. While the RecA-dependent pathway is dominant, alternative HR pathways that function independently of RecA do exist, but relatively little is known about the underlying mechanism. Several studies have documented that a variety of proteins act either as positive or negative regulators of RecA to ensure high-fidelity HR and genomic stability. Along these lines, we previously demonstrated that the second messenger cyclic di-AMP binds to mycobacterial RecA proteins, but not E. coli RecA, and inhibits its DNA strand exchange activity in vitro via the disassembly of RecA nucleoprotein filaments. Herein, we demonstrate that Mycobacterium smegmatis {Delta}disA cells, which lack c-di-AMP, exhibit increased DNA recombination, higher frequency of mutation and gene duplications during RecA-dependent and RecA-independent DSB repair. We also found that c-di-AMP regulates SOS response by inhibiting RecA-mediated self-cleavage of LexA repressor and its absence enhances drug resistance in M. smegmatis {Delta}disA cells. Together, our results uncover a role of c-di-AMP in the maintenance of genomic stability through modulation of DSB repair in M. smegmatis. SignificanceCyclic di-AMP is a second messenger present in bacteria and archaea and is implicated in a variety of functions in the cell, including DNA repair, cell wall metabolism, virulence, and gene expression. We show here that it maintains genome stability in Mycobacterium by regulating RecA-dependent and -independent DNA recombination pathways. It also regulates SOS response by inhibiting the self-cleavage of LexA by mycobacterial RecA. Absence of c-di-AMP leads to higher drug resistance in Mycobacterium.

molecular biology↗

bioRxiv2024

Ready, set, yellow! Color Preference of Indian Free-ranging Dogs

Most of the research on color vision related behaviors in dogs has involved training the dogs to perform visual discrimination tasks. We investigated the meaning of color to untrained Indian free-ranging dogs (FRDs). Using one-time multi-option choice tests for color preference in 134 adult dogs, we found the dogs to prefer yellow objects over blue or gray ones (p < 0.001, Cramers V = 0.433) while there was no preference between blue and gray (N = 102, p = 0.165). We next pitted a yellow object against a gray object that had food. Here, the dogs ignored the food to approach the yellow object first, both when the food was biscuit (N = 52, p < 0.001, Cramers V = 0.576) and chicken (N = 61, p < 0.001, Cramers V = 0.540), indicating the color preference to be quite strong. Color preference has previously been investigated in many other animals and has implications for behaviors like mate choice and foraging. Our study provides a new perspective into the ecology of Indian FRDs and might have implications for pet dogs as well, if they too show this preference. HighlightsO_LIIndian free-ranging dogs (FRDs) show preference toward the color yellow over blue and gray. C_LIO_LIIndian FRDs show no preference between blue and gray colors. C_LIO_LIAttraction towards a yellow object can be stronger than attraction towards food rewards for Indian FRDs. C_LI

animal behavior and cognition↗

bioRxiv2022

Anti-Biofilm Potential of Nanonized Eugenol against Pseudomonas aeruginosa

This study dealt with nanonization of eugenol, a major phytochemical present in basil leaf, which has pharmacological potential as an anti-bacterial agent. Eugenol nanoparticle (ENP) was synthesized by simple ultrasonic cavitation method through emulsification of hydrophobic eugenol into hydrophilic gelatin. Thus, the nanonization process made the water-insoluble eugenol to water-soluble nano-eugenol, making the nano-form bioavailable. The average size of the ENPs was 20-30 nm. Entrapment efficiency of eugenol within gelatin cap was about 80% of the eugenol, that was used as precursor in the nanonization reaction. In vitro release of eugenol from gelatin cap was slow and sustained over a period of five days. The ENP had higher anti-biofilm potency than eugenol for both formation and eradication of biofilm, formed by clinically relevant pathogen Pseudomonas aeruginosa. Minimal biofilm inhibitory concentration and minimal biofilm eradication concentration of ENPs were 2.0 and 4.0 mM respectively. In addition, the measurement of P. aeruginosa biofilm biomass, biofilm pellicle formation, biofilm thickness, amount of biofilm-forming extra-polymeric substance, cell surface hydrophobicity, cell swarming and twitching efficiencies, cellular morphology and biofilm formation in catheter demonstrated that the anti-biofilm efficacy of nano-eugenol was 30-40% higher than that of bulk eugenol. Thus, ENP can be used as a potential drug against pneumonia, a chronic infection in lung caused by P. aeruginosa, which is difficult to treat with antibiotics, due to natural intrinsic resistance of biofilm-formed cells to most antibiotics. The overall actions of ENP have been presented in the figure 1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/521144v1_fig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@12445e2org.highwire.dtl.DTLVardef@6813aforg.highwire.dtl.DTLVardef@e4779borg.highwire.dtl.DTLVardef@1688a9a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Graphical abstract showing overall anti-biofilm action of ENP that is capable to inhibit each and every step of biofilm formation. ENP also carries the potential to destroy EPS and kill the biofilm residing cells by membrane disruption. Bacterial colonization has been found to be prevented significantly on ENP-coated catheter. C_FIG HighlightsO_LINano-formulation of eugenol, an important phytochemical, by ultrasonic cavitation method, which was simple, time-saving, low-cost and eco-friendly. C_LIO_LINanonization made water-insoluble eugenol into water-soluble form with enhanced therapeutic efficacy. C_LIO_LIThe eugenol nanoparticle (ENP) could inhibit formation of biofilm as well as facilitate eradication of pre-formed biofilm of P. aeruginosa. C_LIO_LIBiofilm formation was found to be prevented significantly on ENP-coated catheter. C_LIO_LINano-eugenol may be used as a potential drug against bacterial diseases, caused by pseudomonal biofilm, which are difficult to treat by antibiotics. C_LIO_LINano-formulated eugenol may also be used as an effective anti-fouling agent for biomedical devices like contact lens, pace-maker, materials for organ transplantation etc. to prevent bacterial colonization. C_LI

microbiology↗

bioRxiv2022

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed using an Indian field strain (LSDV/India/2019/Ranchi). The virus was attenuated by continuous passage (P=50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n=10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune response and exhibited complete protection upon virulent LSDV challenge. We observed a minimum Neethling response (0.018% animals; 5 out of 26940 animals) of the vaccine in field trials among 26940 animals. There was no significant reduction in the milk yield in lactating animals (n=10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n=2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

microbiology↗

bioRxiv2022

PRESGENE: A web server for PRediction of ESsential GENE using integrative machine learning strategies

Study of essential genes in disease-causing organisms has wide application in the prediction of therapeutic targets and exploring different clinical strategies. Predicting gene essentiality for large set of genes in non-model, less explored organisms is challenging. Computational methods that use machine learning (ML)-based strategies are popularly adopted for essential gene prediction as they provide key advantage of considering diverse biological features. Previous works from our group have demonstrated two ML-based pipelines for predicting essential genes with high accuracy that mitigates the problems of sufficient labeled imbalanced dataset and limited labeled datasets of essential genes. Here we present PRESGENE at https://presgene.ncl.res.in, a ML-based web server for prediction of essential genes in unexplored eukaryotic and prokaryotic organisms. Our algorithms mitigate the problems of training dataset imbalance and limited availability of experimentally labeled data for essential genes. PRESGENE with its user-friendly web interface and high accuracy will prove to be a seamless experience for biologists looking for an accurate essential gene prediction server with limited labeled data for novel organisms.

bioinformatics↗

bioRxiv2022

Combined promoter-capture Hi-C and Hi-C analysis reveals a fine-tuned regulation of 3D chromatin architecture in colorectal cancer

Hi-C is a widely used method for profiling chromosomal interactions in the 3-dimensional context. Due to limitations on the depth of sequencing, the resolution of most Hi-C datasets is often insufficient for scoring fine-scale interactions. We therefore used promoter-capture Hi-C (PCHi-C) data for mapping these subtle interactions. From multiple colorectal cancer (CRC) studies, we combined PCHi-C with Hi-C datasets to understand the dynamics of chromosomal interactions from cis regulatory elements to topologically associated domain (TAD)-level, enabling detection of fine-scale interactions of disease-associated loci within TADs. Our integrated analyses of PCHi-C and Hi-C datasets from CRC cell lines along with histone modification landscape and transcriptome signatures highlight significant genomic structural instability and their association with tumor-suppressive transcriptional programs. Such analyses also yielded nine dysregulated genes. Transcript profiling revealed a dramatic increase in their expression in CRC cell lines as compared to NT2D1 human embryonic carcinoma cells, supporting the predictions of our bioinformatics analysis. We further report increased occupancy of activation associated histone modifications H3K27ac and H3K4me3 at the promoter regions of the targets analyzed. Our study provides deeper insights into the dynamic 3D genome organization in CRC and identification of affected genes which may serve as potential biomarkers for CRC. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/515643v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@11a9fc8org.highwire.dtl.DTLVardef@f01eb4org.highwire.dtl.DTLVardef@6ff038org.highwire.dtl.DTLVardef@103fc24_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

Actomyosin cables position cell cohorts during Drosophila germband retraction by entraining their morphodynamic and mechanical properties

The unfolding and displacement of the germband during Drosophila germband retraction (GBR) accomplish the straightening of the embryonic anterior-posterior axis. The failure of GBR reduces embryonic viability and results in the mispositioning of the embryonic segments and the gastrointestinal tract. Despite its importance, the cellular, molecular and physical mechanisms that govern the unfolding of the germband and ensure the accurate positioning of cell fields within it remain poorly understood. Here, we uncover the requirement of planar polarized, supracellular, tensile actomyosin cables for entraining cellular morphodynamics, cell field positioning and retraction kinematics. Circumferential, non-constricting cables that form during early retraction ensure the coherence of placode-like cell cohorts, pattern medio-lateral gradients in cell shape and sidedness within it, and dampen retraction speed. Linear, constricting cables that power displacement at the end of retraction enable sequential, multi-tissue, collective T1 transitions to reposition medial cell fields to more posterior locations. Together, our results reveal how the spatiotemporally regulated deployment of actomyosin structures, functioning either as barricades or as purse strings, modulate the speed of tissue unfolding and enable cell field positioning by influencing the morphodynamic and mechanical properties of cell cohorts during morphogenesis.

cell biology↗

bioRxiv2022

Plant-based Production and Characterization of a Promising Fc-fusion Protein against Bone Mass Density Loss

Microgravity-induced bone loss is a main obstacle for long term space missions as it is difficult to maintain bone mass when loading stimuli is reduced. With a typical bone mineral density loss of 1.5% per month of microgravity exposure, the chances for osteoporosis and fractures may endanger astronauts health. Parathyroid Hormone or PTH (1-34) is an FDA approved treatment for osteoporosis, and may reverse microgravity-induced bone loss. However, PTH proteins requires refrigeration, daily subcutaneous injection, and have a short shelf-life, limiting its use in a resource-limited environment, like space. In this study, PTH was produced in an Fc-fusion form via transient expression in plants, to improve the circulatory half-life which reduces dosing frequency and to simplify purification if needed. Plant-based expression is well-suited for space medicine application given its low resource consumption and short expression timeline. The PTH-Fc accumulation profile in plant was established with a peak expression on day 5 post infiltration of 373 {+/-} 59 mg/kg leaf fresh weight. Once the PTH-Fc was purified, the amino acid sequence and the binding affinity to its target, PTH 1 receptor (PTH1R), was determined utilizing biolayer interferometry (BLI). The binding affinity between PTH-Fc and PTH1R was 2.30 x 10-6 M, similar to the affinity between PTH (1-34) and PTH1R (2.31 x 10-6 M). Its function was also confirmed in a cell-based receptor stimulation assay, where PTH-Fc was able to stimulate the PTH1R producing cyclic adenosine monophosphate (cAMP) with an EC50 of (8.54 {+/-} 0.12) x 10-9 M, comparable to the EC50 from the PTH (1-34) of 1.49 x 10-8 M. These results suggest that plant recombinant PTH-Fc exhibits a similar potency compared to PTH. Furthermore, it can be produced rapidly at high levels with minimal resources and reagents, making it ideal for production in low resource environments such as space.

synthetic biology↗

bioRxiv2022

Estrogen suppresses DMRT1 expression during ovarian development in the chicken

Primary sex determination is the choice between two different developmental fates, a testis or an ovary. This selection is thought to require the action of a master regulator gene that triggers expression of a gene cascade in the bipotential gonad precursor in one sex. The selection of a particular developmental pathway is also thought to suppress the alternative developmental pathway. In birds, where the male is the homogametic sex (ZZ) and females the heterogametic sex (ZW), the Z-linked transcription factor DMRT1 is considered the master regulator and has been shown to be essential for testis development, and to also inhibit the ovarian pathway. Here, we characterise in detail, DMRT1 transcription and protein levels during gonadal development in the chick. Our analysis suggests that DMRT1 protein levels are equivalent in male and female gonads during the bipotential phase of development, and that DMRT1 protein levels are reduced in the developing ovary during the differentiation phase. The reduction in DMRT1 protein levels in the somatic cells of the female medulla, coincides with the induction of aromatase expression and the initiation of estrogen synthesis. Analysis of sex-reversed gonads and mixed-sex chimeric gonads, suggests that the reduction in DMRT1 protein is due to inhibition of DMRT1 expression by estrogen. Our data suggests that estrogen signalling is involved in primary sex determination by regulation of DMRT1 protein expression.

developmental biology↗

bioRxiv2022

Whole-Genome Sequence of African Swine Fever Virus isolate from India provides insights into diversity and evolution

African swine fever (ASF) was first reported in 1921, and since then has posed a major threat to the world pig industry and still remains a major challenge as there is no vaccine or therapy available. In May 2020, the first incidence of ASF was recorded in India, followed by a number of outbreaks in the north eastern part of India. In this study, we report the first whole genome of an Indian isolate of ASF virus (ASF/IND/20/CAD/543) using next generation sequencing and compared with the other ASFV complete genome. On phylogenetic analysis, the virus was assigned to genotype II on the basis of p72 genotyping. However, the whole genome based phylogeny distinguished it from other genotype II isolates of clade 1.1.1. This study adds to our understanding of ASFVs genetic diversity and molecular evolution.

microbiology↗

bioRxiv2021

Production of novel Spike truncations in Chinese hamster ovary cells

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, Spike receptor binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg/L at day 7. In comparison, RBD titers peak at 54 mg/L at day 3. Next, we develop 8 Spike truncations (T1-T8) in pursuit of a truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 mg/L and 73 mg/L, respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest T1 is a promising Spike alternative for use in various applications.

bioengineering↗

bioRxiv2021

Functionalizing silica sol-gel with entrapped plant virus-based immunosorbent nanoparticles

Advancements in understanding and engineering of virus-based nanomaterials (VBNs) for biomedical applications motivate a need to explore the interfaces between VBNs and other biomedically-relevant chemistries and materials. While several strategies have been used to investigate some of these interfaces with promising initial results, including VBN-containing slow-release implants and VBN-activated bioceramic bone scaffolds, there remains a need to establish VBN-immobilized three dimensional materials that exhibit improved stability and diffusion characteristics for biosensing and other analyte-capture applications. Silica sol-gel chemistries have been researched for biomedical applications over several decades and are well understood; various cellular organisms and biomolecules (e.g., bacteria, algae, enzymes) have been immobilized in silica sol-gels to improve viability, activity, and form factor (i.e., ease of use). Here we present the immobilization of an antibody-binding VBN in silica sol-gel by pore confinement. We have shown that the resulting system is sufficiently diffuse to allow antibodies to migrate in and out of the matrix. We also show that the immobilized VBN is capable of antibody binding and elution functionality under different buffer conditions for multiple use cycles. The promising results of the VBN and silica sol-gel interface indicate a general applicability for VBN-based bioseparations and biosensing applications.

bioengineering↗

bioRxiv2021

Affinity sedimentation and magnetic separation with plant-made immunosorbent nanoparticles for therapeutic protein purification

The virus-based immunosorbent nanoparticle is a nascent technology being developed to serve as a simple and efficacious agent in biosensing and therapeutic antibody purification. There has been particular emphasis on the use of plant virions as immunosorbent nanoparticle chassis for their diverse morphologies and accessible, high yield manufacturing via crop cultivation. To date, studies in this area have focused on proof-of-concept immunosorbent functionality in biosensing and purification contexts. Here we consolidate a previously reported pro-vector system into a single Agrobacterium tumefaciens vector to investigate and expand the utility of virus-based immunosorbent nanoparticle technology for therapeutic protein purification. We demonstrate the use of this technology for Fc-fusion protein purification, characterize key nanomaterial properties including binding capacity, stability, reusability, and particle integrity, and present an optimized processing scheme with reduced complexity and increased purity. Furthermore, we present a coupling of virus-based immunosorbent nanoparticles with magnetic particles as a strategy to overcome limitations of the immunosorbent nanoparticle sedimentation-based affinity capture methodology. We report magnetic separation results which exceed the binding capacity of current industry standards by an order of magnitude.

bioengineering↗

bioRxiv2021

SARS-Cov-2 Spike binding to ACE2 is stronger and longer ranged with glycans

Highly detailed steered Molecular Dynamics simulations are performed on differently glycosylated receptor binding domains of the SARS-CoV-2 spike protein. The binding strength and the binding range increases with glycosylation. The interaction energy rises very quickly with pulling the proteins apart and only slowly drops at larger distances. We see a catch slip type behavior where interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode are hydrogen bonds but Lennard-Jones and electrostatic interactions are relevant as well. Statement of SignificanceGlycosylation of the receptor binding domain of the Spike protein of SARS-CoV-2 as well as the ACE2 receptor leads to stronger and longer ranged binding interactions between the proteins. Particularly, at shorter distances the interactions are between residues of the proteins themselves whereas at larger distances these interactions are mediated by the glycans. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/452507v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@7b9021org.highwire.dtl.DTLVardef@338aadorg.highwire.dtl.DTLVardef@1d5921eorg.highwire.dtl.DTLVardef@6c27e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Evaluating the cost of pharmaceutical purification for a long-duration space exploration medical foundry

There are medical treatment vulnerabilities in longer-duration space missions present in the current International Space Station crew health care system with risks, arising from spaceflight-accelerated pharmaceutical degradation and resupply lag times. Bioregenerative life support systems may be a way to close this risk gap by leveraging in situ resource utilization (ISRU) to perform pharmaceutical synthesis and purification. Recent literature has begun to consider biological ISRU using microbes and plants as the basis for pharmaceutical life support technologies. However, there has not yet been a rigorous analysis of the processing and quality systems required to implement biologically-produced pharmaceuticals for human medical treatment. In this work, we use the equivalent system mass (ESM) metric to evaluate pharmaceutical purification processing strategies for longer-duration space exploration missions. Monoclonal antibodies, representing a diverse therapeutic platform capable of treating multiple space-relevant disease states, were selected as the target products for this analysis. We investigate the ESM resource costs (mass, volume, power, cooling, and crew time) of an affinity-based capture step for monoclonal antibody purification as a test case within a manned Mars mission architecture. We compare six technologies (three biotic capture methods and three abiotic capture methods), optimize scheduling to minimize ESM for each technology, and perform scenario analysis to consider a range of input stream compositions and pharmaceutical demand. We also compare the base case ESM to scenarios of alternative mission configuration, equipment models, and technology reusability. Throughout the analyses, we identify key areas for development of pharmaceutical life support technology and improvement of the ESM framework for assessment of bioregenerative life support technologies.

bioengineering↗

bioRxiv2021

Altered synaptic connectivity and brain function in mice lacking microglial adapter protein Iba1

Growing evidence indicates that microglia impact brain function by regulating synaptic pruning and formation, as well as synaptic transmission and plasticity. Iba1 (Ionized Ca+2-binding adapter protein 1), encoded by the Allograft inflammatory factor 1 (Aif1) gene, is an actin-interacting protein in microglia. Although Iba1 has long been used as a cellular marker for microglia, its functional role remains unknown. Here, we used global Iba1-deficient (Aif1-/-) mice to characterize microglial activity, synaptic function and behavior. Microglial imaging in acute hippocampal slices and fixed tissues from juvenile mice revealed that Aif1-/- microglia display reductions in ATP-induced motility and ramification, respectively. Biochemical assays further demonstrated that Aif1-/- brain tissues exhibit an altered expression of microglial-enriched proteins associated with synaptic pruning. Consistent with these changes, juvenile Aif1-/- mice displayed deficits in excitatory synapse number and synaptic transmission assessed by neuronal labeling and whole-cell patch-clamp recording in acute hippocampal slices. Unexpectedly, microglial synaptic engulfment capacity was diminished in juvenile Aif1-/- mice. During early postnatal development when synapse formation is a predominant event in the hippocampus, excitatory synapse number was still reduced in Aif1-/- mice. Together these findings support an overall role of Iba1 in excitatory synaptic growth in juvenile mice. Lastly, postnatal synaptic deficits persisted in the adulthood and correlated with significant behavioral changes in adult Aif1-/- mice, which exhibited impairments in object recognition memory and social interaction. These results suggest that Iba1 critically contributes to microglial activity underlying essential neuro-glia developmental processes that may deeply influence behavior. SignificanceAbnormal microglia-neuron interaction is increasingly implicated in neurodevelopmental and neuropsychiatric conditions such as autism spectrum disorders and schizophrenia, as well as in neurodegenerative disorders such as Alzheimers disease. This study demonstrates that deletion of the microglia-specific protein Iba1, which has long been utilized as a selective microglial marker but whose role has remained unidentified, results in microglial structural and functional impairments that significantly impact synaptic development and behavior. These findings not only highlight the importance of microglia in brain function but may also suggest that modifying microglial function could provide a therapeutic strategy for treatment of neurodevelopmental, neuropsychiatric and neurodegenerative disorders.

neuroscience↗

bioRxiv2021

Analysis of variability of functionals of recombinant protein production trajectories based on limited data

We propose a method for analyzing the variability in smooth, possibly nonlinear, functionals associated with a set of product production trajectories measured under different experimental conditions. The key challenge is to make meaningful inference on these parameters across different experimental conditions when only a limited number of measurements in time are collected for each treatment, and when there are only a few, or no, replicates available. For this purpose we adopt a modeling approach by representing the production trajectories in a B-spline basis, and develop a bootstrap-based inference procedure for the parameters of interest, also accounting for multiple comparisons. The methodology is applied to study two types of quantities of interest - "time to harvest" and "maximal productivity" in the context of an experiment on the production of certain recombinant proteins under laboratory conditions. We complement the findings by extensive numerical experiments that look into the effects of different types of bootstrap procedures and associated schemes for computing p-values for tests of hypotheses.

bioengineering↗

bioRxiv2021

Bioprinting transgenic plant cells for production of a recombinant biodefense agent

Transgenic rice cells (Oryza sativa) producing recombinant butyrylcholinesterase (BChE) as a prophylactic/therapeutic against organophosphate nerve agent poisoning, cocaine toxicity, and neurodegenerative diseases like Alzheimers were immobilized in a polyethylene glycol-based hydrogel. The cells were sustained for 14 days in the semi-solid matrix, undergoing a growth phase from days 0-6, a BChE production phase in sugar-free medium from days 6-12, and a growth/recovery phase from days 12-14. Throughout this period, the cells maintained similar viability to those in suspension cultures and displayed analogous sugar consumption trends. The rice cells in the bioprintable hydrogel also produced a significant amount of active BChE, comparable to the levels produced in liquid cultures. A considerable fraction of this BChE was secreted into the media, allowing for easier product separation. Overall, we demonstrate a simple, efficient, robust, modular, and potentially field-deployable bioreactor system for the manufacture of biologics.

plant biology↗