Biology subjects

Prasad, A.

Publications and source records attributed to Prasad, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Metal speciation in blood plasma

Metal speciation in blood plasma is heavily influenced by proteins and peptides including transferrin, albumin, and glutathione. Despite this, few studies have incorporated these large molecules in speciation calculations, probably due to a lack of experimental measurements. Additionally, there is increasing evidence that metal complexes of small molecules are bioavailable. Due to the limitations posed by analytical techniques, thermodynamic models can serve as an excellent alternative to experimental measurements of metal speciation. In this work, we predict metal speciation for several biologically relevant metals incorporating complexes with proteins, peptides and small molecules. We supplemented experimental measurements from the literature with linear free energy estimates to fulfill the inventory of stability constants required to perform these calculations. In addition to evaluating the speciation of naturally present metals, we also predict the speciation of metals used for therapeutic applications like anticancer drugs, antidiabetics and antacids. Our results indicate that metal speciation is heavily dependent on pH and chelator concentration and can change drastically as metals move from blood plasma to inside cells. Additionally, metal speciation can be dominated by proteins like transferrin and is subject to change as metals cross the blood-brain barrier. Our results corroborate many experimental measurements and can help design future experiments investigating the biological impact of metal-based drugs and metal-toxicity.

biochemistry↗

bioRxiv2025

Food-activated Microneedle Sensor for Real-time, Colorimetric Spoilage Monitoring of Pre-packaged Food

At a time of growing food insecurity, developing technologies to reduce food waste is critical. We report an inexpensive, colorimetric spoilage sensor for real-time food product assessment. The sensor is composed of dehydrated gelatin microneedles that exhibit high mechanical integrity in their base state. However, once exposed to fluid-rich food environments, they rapidly transition to a hydrogel sensing state. Food-derived anthocyanins embedded within these microneedles enable pH-based spoilage monitoring. When applied to sealed fish products, these microneedles non-destructively penetrate through packaging and are rehydrated by the underlying fish matrix. As the product ages, a defined colour shift occurs, demonstrating strong correlation with quantitative spoilage markers. When applied to unsealed fish products for rapid testing, the large microneedle sensing interface enables accelerated colorimetric sensing. Finally, successful fresh versus spoiled categorization of smartphone-acquired images of the sensor using machine learning removes readout ambiguity, empowering consumers with independent real-time product monitoring.

molecular biology↗

bioRxiv2025

A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, providing durable and even curative responses. However, most patients do not respond and current biomarkers (eg, programmed death ligand (PD-L1), mismatch repair deficiency (dMMR)/high microsatellite instability (MSI) and tumor mutational burden) lack predictive accuracy. Ex vivo profiling of patient-derived tumor fragments shows promise as a predictive biomarker but relies on substantial surgical tissue to mitigate intra-specimen heterogeneity. Innovations are needed that address these challenges, particularly where limited tissue is available in core needle biopsies (CNBs). MethodsLive tumor fragments (LTFs) were generated from 59 human tumor resections and 31 CNBs from patients enrolled in observational clinical trials (ClinicalTrials.gov identifiers: NCT05478538, NCT05520099, NCT06349642) to assess cytokine induction following ICI treatment. LTFs were encapsulated in hydrogel and cultured ex vivo for up to 72 hours. A sequential treatment strategy that applies control and treatment within the same well was used with response to ICI or CD3/CD28 assessed using a multiplex secretome assay. Viability was assessed using established metabolic assays and dynamic optical coherence microscopy. ResultsLTFs maintained viability and retained T cells responsive to stimulation throughout ex vivo culture. Multiplex immunofluorescence and immunohistochemistry showed key components of the tumor microenvironment, including relative proportions of CD4+ and CD8+ immune cell populations, were preserved. Specimens positive for PD-L1 or dMMR/MSI-high were enriched for cytokine upregulation, including T-cell response cytokines IFN{gamma} and CXCL10, after PD-1 treatment. To demonstrate clinical applicability of the sequential treatment strategy, CNBs from patients with lung, gastrointestinal or kidney cancer were profiled and differential cytokine induction in response to ICI treatment was observed. ConclusionsThe novel ex vivo platform presented is capable of detecting T-cell response to ICI treatment by using a sequential treatment strategy. This approach addresses challenges associated with cross-well heterogeneity in tissue composition and requires half as much tissue as a cross-well comparison, mitigating tissue limitations typically associated with non-surgical biopsies. Importantly, the platform is compatible with established functional assays as well as non-destructive spatial imaging, enabling researchers to characterize response to ICI longitudinally. Ongoing trials will enable clinicians to assess platform performance in predicting response to immunotherapy.

cancer biology↗

bioRxiv2025

Interleukin-3 as a Potential Bone Anabolic Agent in treating Postmenopausal Osteoporosis

Postmenopausal osteoporosis (PMO), a silent disorder caused due to estrogen deficiency, is characterized by loss of bone mass and low bone mineral density. Despite advancements in treatment, current medications often fail to fully restore bone integrity and prevent fragility fractures in the osteoporotic individuals. Thus, there is a high demand of novel therapeutic modalities in dealing with osteoporosis. Interleukin-3 (IL-3), a cytokine secreted by activated T cells, emerges as a promising therapeutic candidate. The dual role of IL-3 in inhibiting osteoclast differentiation and promoting osteoblast differentiation, offers protection against bone and joint degeneration in arthritic mice. However, its role in osteoporosis is not yet delineated. Therefore, our investigation focuses on elucidating the role of IL-3 in PMO, employing both prophylactic and therapeutic strategies in ovariectomized mice, mimicking human PMO. In our study, at the onset of osteoporosis, IL-3 treatment effectively preserved trabecular bone architecture and enhanced bone mineral density in OVX mice, particularly notable in therapeutic interventions where fully developed osteoporosis was addressed. Notably, in preventive measures, IL-3 inhibited osteoclast differentiation, thereby suppressing bone resorption, while in therapeutic approaches, it enhanced osteoblast differentiation, promoting bone formation. Irrespective of gender specific, microCT analysis of IL-3-/- (KO) mice showed reduced trabecular bone development compared to its respective wild type (WT) mice, highlighting essential role of IL-3 in skeletal integrity. Moreover, IL-3 increased Treg cells population while inhibiting B cell lymphopoiesis in ovariectomized mice with no adverse effects on hematopoiesis or vital organs. In conclusion, our findings collectively underscore the potential of IL-3 as a novel therapy for PMO, offering insights into its mechanisms of action and clinical applications. One Sentence SummaryIL-3 has a dual action on bone: anti-osteoclastic and pro-osteoblastic in OVX mice, thus can be a novel therapeutic drug in treating postmenopausal osteoporosis.

pathology↗

bioRxiv2025

Cyclo-stationary distributions of mRNA and Protein counts for random cell division times

There is a long history of using experimental and computational approaches to study noise in single-cell levels of mRNA and proteins. The noise originates from a myriad of factors: intrinsic processes of gene expression, partitioning errors during division, and extrinsic effects, such as, random cell-cycle times. Although theoretical methods are well developed to analytically understand full statistics of copy numbers for fixed or Erlang distributed cell cycle times, the general problem of random division times is still open. For any random (but uncorrelated) division time distribution, we present a method to address this challenging problem and obtain exact series representations of the copy number distributions in the cyclo-stationary state. We provide explicit cell age-specific and age-averaged results, and analyze the relative contribution to noise from intrinsic and extrinsic sources. Our analytical approach will aid the analysis of single-cell expression data and help in disentangling the impact of variability in division times.

systems biology↗

bioRxiv2025

Bacteriophage-Loaded Microneedle Patches for Targeted andMinimally Disruptive Foodborne Pathogen Decontamination

Antibacterial additive use has surged due to rising incidences of food contamination, despite concerns over antibiotic resistance. Bacteriophage (bacterial viruses) represent a unique and promising opportunity as antibacterial agents, offering targeted bacterial lysis while being food safe. However, their commercial success has been limited by the significant diffusion barriers they face within food, preventing effective delivery at contamination sites. Here, we introduce bacteriophage-loaded microneedle patches that enable targeted phage delivery directly within food, eliminating internal pathogens in a minimally disruptive manner. The application of microneedles within food is first explored. The platform is then substantiated by comparing performance in raw beef and cooked chicken, where we achieved up to 3-logs reduction in Escherichia coli, thus providing complete decontamination according to regulatory limits. In contrast, conventional surface application of the same phage failed to provide significant decontamination. To ensure broad applicability, phage cocktails were also loaded into microneedles to demonstrate polymicrobial decontamination against other common food contaminants including Salmonella. This platform can also be adapted to extend food shelf-life by targeting spoilage-inducing bacteria.

bioengineering↗

bioRxiv2025

Species synonyms depict changing but taxon-independent taxonomic praxis

What constitutes a single species is ultimately arbitrary, resulting in idiosyncrasies in taxonomic praxis determining species counts. One consequence of this arbitrariness is taxonomic synonymization, wherein variation determined not unique enough to constitute distinct species gets merged into one. Synonymization is non-random and subject to taxon and trait-specific biases. We explore these trends using a comprehensive dataset of the worlds marine species for all large phyla and Animalia. We match all accepted species-names from the present day with their synonyms and calculate metrics of synonymization including the time lag between the years of descriptions of a species and its newest synonym. We find that although species description trends across phyla show no consistent, discernible trends, the fraction of names that gets synonymised remains initially constant across phyla, with a subsequent gradual decrease. Most accepted species described around the early 20th century possess synonyms created in the same year, indicating synonyms arising due to new name combinations, suggesting a distinct period of taxonomic praxis. This period was followed by a period of most species descriptions lacking synonyms on moving towards the present. Across taxa, we find the mean lag to be strongly negatively correlated to time, with time not being a limiting factor determining this trend. We also find the number of synonyms per accepted name to also be strongly negatively correlated to time in a taxon-independent manner. Our study considers when the diversity characterising species in the form of once-unique species-names becomes known to science. Our results suggest that across taxa, species described more recently encompass less biological variability, indicating a gradual change in how morphologically delineated species which comprise the vast majority of species described across time are described.

ecology↗

bioRxiv2025

Strain-level and phenotypic stability contrasts with plasmid and phage variability in water kefir communities

Microbial communities can change in response to top-down factors, such as phages, and bottom-up factors, such as nutrient availability. Previous studies have successfully investigated bacterial species-level dynamics, but diversity and interactions beyond the species-level is usually lacking. Traditional fermented foods, such as water kefir, provide ideal systems to study ecological and evolutionary dynamics beyond the species-level, as they are simple and trackable systems that are cultivated in non-sterile, nutrient-rich environments which foster microbial growth and invasion. Despite the central role of only a few lactic acid bacteria for fermentation, little is known about the genomic diversity and dynamics of these community members over time. Within the framework of a graduate course, 35 students propagated water kefir across several generations under different nutrient conditions and in different households to study microbial responses over time. We found that water kefir communities were generally stable at the species-level, with only rare bacterial species replaced over long timescales (more than 2 years). While we observed little strain-level diversity with few strain replacements over long timescales, closely related strains exhibited variation in accessory gene content, often encoded on plasmids, particularly those involved in ecologically meaningful functions such as sugar utilization pathways and phage defense systems. We hypothesise that these genomic variations could reflect the adaptations of strains to different sugars and phages. Consistent with this, we observed a diverse array of phages, many likely originating from the unique household environments. By documenting the genomic landscape of microbial species, strains, plasmids, and phages, this study advances our understanding of the diversity and dynamics of microbial communities in fermented foods. Furthermore, our course material is publicly available and offers a blueprint for bridging the gap between teaching and research, inspiring the next generation of scientists to unravel the complexities of microbial ecosystems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/640646v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@56db6borg.highwire.dtl.DTLVardef@5f6dc6org.highwire.dtl.DTLVardef@11fff14org.highwire.dtl.DTLVardef@1a37a9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Evaluation of distal facial nerve branches contribution to facial nerve paralysis in rodents

Introduction/AimsFacial nerve paralysis is a complex and devastating condition. Translational research of facial paralysis recovery remains largely limited to animal studies, for which there are many potential models employed. When studying facial nerve regeneration in rodents, it is important to understand the converging contributions of the motor supply into the whisker pad. A consensus surgical approach and animal model has yet to be defined. Of particular interest for movement of the nose and whiskers are the buccal and marginal mandibular nerves. This study aims to evaluate how these distal nerve branches contribute to facial nerve paralysis and identify key morphological changes at the neuromuscular junctions (NMJs) in the whisker pad of rodents. MethodsAdult rats underwent isolated transection of the buccal branch of the facial nerve, both the buccal and marginal mandibular branches of the facial nerve, or control sham surgery. ResultsHistological, electrophysiological, and behavior assessments confirmed that the transection of the buccal branch alone did not cease whisker movement in rats, but when combined with a transection of the marginal mandibular branch, it resulted in full paralysis of the whisker and nose movement. DiscussionThese results are indicative of the distinct roles of these nerves branches in facial paralysis repair following a transection injury. Further, our results suggest additional targets for facial nerve repair treatments.

neuroscience↗

bioRxiv2025

Transgenic A53T mice have astrocytic α-synuclein aggregates in dopamine and striatal regions

AimsParkinsons disease is considered biologically a neuronal -synuclein disease, largely ignoring the more widespread -synuclein deposition that occurs in astrocytes, with the aim of this study to identify whether astrocytes accumulate small -synuclein aggregates before or after neurons. MethodsFixed serial midbrain and striatal sections from M83 A53T transgenic mouse model of Parkinsons disease and wild-type controls were histologically processed for multiplex labelling of -synuclein and astrocytic markers and astrocyte quantitation performed on digital images using QuPath software. ResultsThe density of astrocytes within the substantia nigra pars compacta was approximately 30% greater compared with other sampled regions (P<0.005). Small aggregates of -synuclein were observed in astrocytic processes, including in wild-type mice where a quarter of all astrocytes had an obvious -synuclein aggregate. Compared to wild-type, A53T transgenic astrocytes had significantly enlarged somas (P<0.001) with more processes (P<0.001) consistent with a reactive phenotype. The A53T transgenic mice had more than double the numbers of astrocytes (P<0.001) and 2.5 times more astrocytes with -synuclein aggregates compared to wild-type mice (P<0.001). ConclusionsThese data suggest that small -synuclein aggregates are normally cleared by astrocytes and that the substantia nigra pars compacta requires more astrocytic support for this function than other midbrain dopaminergic regions or the striatum. This adds another vulnerability factor to those already known for the substantia nigra with early deficits in clearance of small -synuclein aggregates by astrocytes associated with an increased astrocytic reactivity in the A53T transgenic mouse model. Key PointsO_LIThe substantia nigra pars compacta contains a higher density of astrocytes than the ventral tegmental area or striatum, indicating a greater reliance on astrocytic function and a greater vulnerability to astrocyte dysfunction C_LIO_LISmall aggregates of -synuclein were observed in wild-type midbrain and striatal astrocytes, indicating normal clearance of -synuclein by these astrocytes C_LIO_LIMidbrain and striatal astrocytes from A53T transgenic astrocytes have more than double the number of astrocytes and more astrocytes containing -synuclein aggregates which have a reactive morphological phenotype. C_LI

neuroscience↗

bioRxiv2024

Cell Morphology accurately predicts the nuclear shape of adherent cells

Cells are internally tensed, or prestressed, largely by actomyosin contractility. We hypothesized that nuclear shape is quantitatively predictable from cell shape since prestress couples them both. We trained machine learning models on a publicly available image database of the WTC-11 cell line and predicted shape modes of the nucleus with high accuracy. We develop a U-Net architecture-based model, Cell2Nuc, that predicted nuclear voxels from the cell membrane with accuracies between 74%-87%. To investigate prestress, we cultured and imaged HeLa cells after inhibiting actomyosin contractility. The Cell2Nuc model retrained on the HeLa cells predicted nuclear voxels with slightly lower accuracy. Statistical analysis revealed changes in nuclear size and chromatin organization upon prestress inhibition. Similar trends were seen in images taken from NIH3T3 cells. Thus, cell shape encodes features of nuclear shape, their coupling is partly due to actomyosin contractility, whose abrogation leads to changes in chromatin organization of mechanosensitive origin.

cell biology↗

bioRxiv2024

A Genetic Toggle Switch in Plants

In synthetic biology, genetic components are assembled to make transcriptional units, and transcriptional units are assembled into circuits to perform specific and predictable functions of a genetic device. Genetic devices have been described in bacteria, mammalian cell cultures and small organoids, yet development of programmable genetic circuits for devices in plants has lagged. Programmable genetic devices require defining the components quantitative functions. Because plants have long life spans, studies often use transient analysis to define quantitative functions while verification in stably engineered plants is often neglected and largely unknown. This raises a question if unique attributes of plants such as environmental sensitivity, developmental plasticity, or alternation of generations, adversely impacts predictability of plant genetic circuits and devices. Alternatively, it is also possible that genetic elements to produce predictable genetic devices for plants require rigorous characterization with detailed mathematical modeling. Here we use plant genetic elements with quantitatively characterized transfer functions and developed in silico models to guide their assembly into a genetic device: a toggle switch or a mutually inhibitory gene-regulatory device. Our approach allows computational selection of plant genetic components and iterative refinement of the circuit if the desired genetic functions are not initially achieved. We show that our computationally selected genetic circuit functions as predicted in stably engineered plants including through tissue and organ differentiation. Developing abilities to produce predictable and programmable plant genetic devices opens the prospect of predictably engineering plants unique abilities in sustainable human and environmental systems.

synthetic biology↗

bioRxiv2024

DNA-templated spatially controlled proteolysis targeting chimeras for CyclinD1-CDK4/6 complex protein degradation

Constraining proximity-based drugs, such as proteolysis-targeting chimeras (PROTACs), into its bioactive conformation can significantly impact their selectivity and potency. However, traditional methods for achieving this often involve complex and time-consuming synthetic procedures. Here, we introduced an alternative approach by demonstrating DNA-templated spatially controlled PROTACs (DTACs), which leverage the programmability of nucleic-acid based self-assembly for efficient synthesis, providing precise control over inhibitors spacing and orientation. The resulting constructs revealed distance- and orientation-dependent selectivity and degradation potency for the CyclinD1-CDK4/6 protein complex in cancer cells. Notably, an optimal construct DTAC-V1 demonstrated the unprecedented synchronous degradation of entire CyclinD1-CDK4/6 complex. This resulted in the effective cell cycle arrest in G1 phase, and further therapeutic studies showed its potent anti-tumor effects compared to inhibitors alone. These findings present a novel framework for PROTACs design, offering critical insights that may inform the development of other proximity-induced therapeutic modalities.

synthetic biology↗

bioRxiv2024

Symbiont loss and gain, rather than co-diversification shapes honeybee gut microbiota diversity and function

Studying gut microbiota evolution across animals is crucial for understanding symbiotic interactions but is hampered by the lack of high-resolution genomic data. Honeybees, with their specialized gut microbiota and well-known ecology, offer an ideal system to study this evolution. Using shotgun metagenomics on 200 worker bees from five honeybee species, we recover thousands of metagenome-assembled genomes and identify several novel bacterial species. While microbial communities were mostly host-specific, we found both specialists and generalists, even among closely related bacterial species, with notable variation between honeybee hosts. Some bacterial generalists emerged host-specific only at the strain level, suggesting recent host switches. While we found some signal of co-diversification between hosts and symbionts, this was not more than expected by chance and was much less pronounced than what has been observed for gut bacteria of hominids and small mammals. Instead, symbiont gains, losses, and replacements emerged as the predominant process for honeybees. This highly dynamic evolution of the specialized honey bee gut microbiota has led to taxonomic and functional differences across hosts, such as the ability to degrade pollen-derived pectin. Our results provide new insights into the evolutionary processes govern gut microbiota diversity across closely related hosts and uncover the functional potential of the previously underexplored gut microbiota of these important pollinators.

microbiology↗

bioRxiv2024

Exact distributions of threshold crossing times of proteins under post-transcriptional regulation by small RNAs

The timings of several cellular events like cell lysis, cell division, or pore formation in endosomes are regulated by the time taken for the relevant proteins to cross a threshold in number or concentration. Since protein synthesis is stochastic, the threshold crossing time is a first passage problem. The exact distributions of these first passage processes have been obtained recently for unregulated and auto-regulated genes. Many proteins are however regulated by post-transcriptional regulation, controlled by small non-coding RNAs (sRNAs). Certain mathematical models of gene expression with post-transcriptional sRNA regulation have been recently exactly mapped to models without sRNA regulation. Utilizing this mapping and the exact distributions, we calculate exact results on fluctuations (full distribution, all cumulants, and characteristic times) of protein threshold crossing times in the presence of sRNA regulation. We derive two interesting predictions from these exact results. We show that the size of the fluctuation of the threshold crossing times have a non-monotonic U-shaped behavior as a function of the rates of binding and unbinding of the sRNA-mRNA complex. Thus there are optimal parameters that minimize noise. Furthermore, the fluctuations in models with sRNA regulation may be higher or lower compared to the model without regulation, depending on the mean protein burst size.

biophysics↗

bioRxiv2024

Novel tools to quantify total, phospho-Ser129 and aggregated alpha-synuclein in the mouse brain

Assays for quantifying aggregated and phosphorylated (S129) human -synuclein protein are widely used to evaluate pathological burden in patients suffering from synucleinopathy disorders. Many of these assays, however, do not cross-react with mouse -synuclein or exhibit poor sensitivity for this target, which is problematic considering the preponderance of mouse models at the forefront of pre-clinical -synuclein research. In this project, we addressed this unmet need by reformulating two existing AlphaLISA(R) SureFire(R) UltraTM total and pS129 -synuclein assay kits to yield robust and ultrasensitive (LLoQ [≤]0.5pg/mL) quantification of mouse and human wild-type and pS129 -synuclein protein. We then employed these assays, together with the BioLegend -synuclein aggregate ELISA, to assess the relationship between -synuclein S129 phosphorylation and aggregation in different mouse brain tissue preparations. Overall, we highlight the compatibility of these new immunoassays with rodent models and demonstrate their potential to advance knowledge surrounding -synuclein phosphorylation and aggregation in synucleinopathies.

neuroscience↗

bioRxiv2024

Widespread variation in molecular interactions and regulatory properties among transcription factor isoforms

Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.

systems biology↗

bioRxiv2023

High yield, low magnesium flexizyme reactions in a water-ice eutectic phase

Flexizymes enable the stoichiometric acylation of tRNAs with a variety of compounds, enabling the in vitro translation of peptides with both non-natural backbones and side chains. However, flexizyme reactions have several drawbacks, including single-turnover kinetics, high Mg(II) carryover inhibiting in vitro translation, and rapid product hydrolysis. Here we present flexizyme reactions utilizing an ice-eutectic phase, with high yields, 30X lower Mg(II), and long-term product stability. The eutectic flexizyme reactions increase the ease of use and flexibility of flexizyme aminoacylation, and increase the in vitro protein production.

synthetic biology↗