Biology subjects

Agrawal, S.

Publications and source records attributed to Agrawal, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Presynaptic inhibition selectively suppresses leg proprioception in behaving Drosophila

Controlling arms and legs requires feedback from proprioceptive sensory neurons that detect joint position and movement. Proprioceptive feedback must be tuned for different behavioral contexts, but the underlying circuit mechanisms remain poorly understood. Using calcium imaging in behaving Drosophila, we find that the axons of position-encoding leg proprioceptors are active across behaviors, whereas the axons of movement-encoding leg proprioceptors are suppressed during walking and grooming. Using connectomics, we identify a specific class of interneurons that provide GABAergic presynaptic inhibition to the axons of movement-encoding proprioceptors. The predominant synaptic inputs to these interneurons are descending neurons, suggesting they are driven by predictions of leg movement originating in the brain. Calcium imaging from both the interneurons and their descending inputs confirmed that their activity is correlated with self-generated but not passive leg movements. Overall, our findings elucidate a neural circuit for suppressing specific proprioceptive feedback signals during self-generated movements.

neuroscience↗

bioRxiv2023

Differential effects of acute and prolonged morphine withdrawal on motivational and goal-directed control over reward-seeking behavior

Opioid addiction is a relapsing disorder marked by uncontrolled drug use and reduced interest in normally rewarding activities. The current study investigated the impact of spontaneous withdrawal from chronic morphine exposure on emotional, motivational, and cognitive processes involved in regulating the pursuit and consumption of natural food rewards in male rats. In Experiment 1, rats experiencing acute morphine withdrawal lost weight and displayed somatic signs of drug dependence. However, hedonically-driven sucrose consumption was significantly elevated, suggesting intact and potentially heightened emotional reward processing. In Experiment 2, rats undergoing acute morphine withdrawal displayed reduced motivation when performing an effortful response for palatable food reward. Subsequent reward devaluation testing revealed that acute withdrawal also disrupted their ability to exert flexible goal-directed control over their reward-seeking behavior. Specifically, morphine-withdrawn rats displayed insensitivity to reward devaluation both when relying on prior action-outcome learning and when given direct feedback about the consequences of their actions. In Experiment 3, rats tested after prolonged morphine withdrawal displayed heightened rather than diminished motivation for food rewards and retained their ability to engage in flexible goal-directed action selection. However, brief re-exposure to morphine was sufficient to impair motivation and disrupt goal-directed action selection, though in this case insensitivity to reward devaluation was only observed in the presence of morphine-paired context cues and in the absence of response-contingent feedback. We suggest that these opioid-withdrawal induced deficits in motivation and goal-directed control may contribute to addiction by interfering with the pursuit of adaptive alternatives to drug use.

neuroscience↗

bioRxiv2023

Unidirectional expression of enhancers with cell type-dependent direction of transcription

Enhancers are genomic regulatory elements that can affect expression of genes over megabases of genomic distances. As previous evidence suggested that transcription initiation at enhancers occurs in a bidirectional pattern, balanced expression of divergently transcribed capped RNAs has been used as a hallmark for enhancer detection from transcriptome data. By analyzing deep Cap Analysis Gene Expression (CAGE) data from FANTOM5, FANTOM6, and other studies, we show that enhancers are usually unidirectionally transcribed in a given cell type. However, since the preferred direction of transcription of an enhancer may switch between cell types, enhancers may appear to be bidirectionally expressed when using CAGE data aggregated over many cell types. By analyzing expression directionality of enhancers across cell types, we classified enhancers into bidirectionally expressed, unidirectionally expressed with a consistent direction of transcription, and unidirectionally expressed with a switching direction, which was the largest category. We conclude that requiring bidirectional expression during enhancer prediction from transcriptome data may lead to false negatives. Also, the preference of a given cell type for a specific direction of transcription at an enhancer suggests that enhancer RNAs are not transcriptional noise, but may be functional.

genomics↗

bioRxiv2023

Impact of reference design on estimating SARS-CoV-2 lineage abundances from wastewater sequencing data

BackgroundSequencing of SARS-CoV-2 RNA from wastewater samples has emerged as a valuable tool for detecting the presence and relative abundances of SARS-CoV-2 variants in a community. By analyzing the viral genetic material present in wastewater, public health officials can gain early insights into the spread of the virus and inform timely intervention measures. The construction of reference datasets from known SARS-CoV-2 lineages and their mutation profies has become state-of-the-art for assigning viral lineages and their relative abundances from wastewater sequencing data. However, the selection of reference sequences or mutations directly affects the predictive power. ResultsHere, we show the impact of a mutation- and sequence-based reference reconstruction for SARS-CoV-2 abundance estimation. We benchmark three data sets: 1) synthetic "spike-in" mixtures, 2) German samples from early 2021, mainly comprising Alpha, and 3) samples obtained from wastewater at an international airport in Germany from the end of 2021, including 1rst signals of Omicron. The two approaches differ in sub-lineage detection, with the marker-mutation-based method, in particular, being challenged by the increasing number of mutations and lineages. However, the estimations of both approaches depend on selecting representative references and optimized parameter settings. By performing parameter escalation experiments, we demonstrate the effects of reference size and alternative allele frequency cutoffs for abundance estimation. We show how different parameter settings can lead to different results for our test data sets, and illustrate the effects of virus lineage composition of wastewater samples and references. ConclusionsHere, we compare a mutation- and sequence-based reference construction and assignment for SARS-CoV-2 abundance estimation from wastewater samples. Our study highlights current computational challenges, focusing on the general reference design, which significantly and directly impacts abundance allocations. We illustrate advantages and disadvantages that may be relevant for further developments in the wastewater community and in the context of higher standardization.

bioinformatics↗

bioRxiv2023

Synaptic architecture of leg and wing motor control networks in Drosophila

Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles. MN activity is coordinated by complex premotor networks that allow individual muscles to contribute to many different behaviors. Here, we use connectomics to analyze the wiring logic of premotor circuits controlling the Drosophila leg and wing. We find that both premotor networks cluster into modules that link MNs innervating muscles with related functions. Within most leg motor modules, the synaptic weights of each premotor neuron are proportional to the size of their target MNs, establishing a circuit basis for hierarchical MN recruitment. In contrast, wing premotor networks lack proportional synaptic connectivity, which may allow wing steering muscles to be recruited with different relative timing. By comparing the architecture of distinct limb motor control systems within the same animal, we identify common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.

neuroscience↗

bioRxiv2023

Physiological Responses of The Leaves of a High-Altitude Plant Picrorhiza kurroa to Cold Stress

There are various plants that grow in high elevations and experience various environmental stresses, cold stress being the most prevalent one. Plants undergo biochemical, metabolic, molecular, and physiological changes under cold stress hence, they adopt various mechanisms to tolerate it. The antioxidant defense system, osmotic regulators and photosynthetic pigments in the plant provide them stress tolerance. The present study is conducted on a high-altitude plant, Picrorhiza kurroa which grows in such environmental conditions, to study the physiological parameters that provides it the coping mechanism against the cold stress. For this study, the leaves were collected from Pothivasa (2200 m asl) and Tungnath (3600 m asl) in Rudraprayag, Uttarakhand, India. The photosynthetic pigments, lipid peroxidation, antioxidant enzymes, and osmoprotectants present in the leaves were determined to visualize the impact of cold stress. It was revealed that the concentration of chlorophyll a, chlorophyll b, and carotenoids increased with increase in elevation. The activity of SOD, CAT, POD, APOX and GR were analyzed and they were observed to decrease with altitude. The MDA concentration which is an indicator of lipid peroxidation is higher in Pothivasa (2.214 mol L-1) and lower in Tungnath (1.697 mol L-1). There is a significant increase in the total protein content, total soluble sugar content and total proline content along the altitudinal gradient. Therefore, the leaves from both the sampling locations revealed the physiological changes occurred in them to adapt to the cold stress conditions.

plant biology↗

bioRxiv2022

Tools for comprehensive reconstruction and analysis of Drosophila motor circuits

Like the vertebrate spinal cord, the insect ventral nerve cord (VNC) mediates limb sensation and motor control. Here, we apply automated tools for electron microscopy volume alignment, neuron segmentation, and synapse prediction toward creating a connectome of an adult female Drosophila VNC. To interpret a connectome, it is crucial to know its relationship with the rest of the body. We therefore mapped the muscle targets of leg and wing motor neurons in the connectome by comparing their morphology to genetic driver lines, dye fills, and X-ray nano-tomography of the fly leg and wing. Knowing the outputs of the connectome allowed us to identify neural circuits that coordinate the wings and legs during escape takeoff. We provide the reconstruction of VNC circuits and motor neuron atlas, along with tools for programmatic and interactive access, as community resources to support experimental and theoretical studies of how the fly nervous system controls behavior.

neuroscience↗

bioRxiv2022

Genomic evaluation of a case of Lacticaseibacillus rhamnosus endocarditis: comparison with commercial yogurt isolates and examination of global population structure

Probiotics, including yogurt commonly containing bacteria of the Lactobacillus group, are widely used to promote microbiome health and to prevent gastrointestinal or other infections, especially in the setting of antibiotic use. Rarely, however, Lactobacillus bacteria have caused invasive or severe infections in patients. In this report, we present a patient with fatal Lacticaseibacillus bacteremia and endocarditis who frequently consumed yogurt. Whole genome sequencing identified the patients bloodstream isolate as Lacticaseibacillus (formerly Lactobacillus) rhamnosus, and sequencing of Lactobacillus isolates from selected commercial yogurt preparations revealed one isolate nearly identical to the patients isolate, differing by only one nucleotide (adenine to guanine substitution leading to a M1A amino acid change in the start codon of the nrdR gene). To place these sequences into broader context of L. rhamnosus strains, we compared them to 602 L. rhamnosus genome sequences publicly available through the National Center for Biotechnology Information repository. Both isolates belonged to a clade, identified in this report as clade YC, composed of mostly gastrointestinal isolates from healthy individuals, some of which also differed by only a single nucleotide from the patients isolate. Members of clade YC had only rarely been previously identified in invasive infections. In summary, the Lacticaseibacillus strain causing this patients infection may have originated from a commercial dairy product or from the commensal flora of his gastrointestinal tract. This study demonstrates that whole genome sequencing may be insufficient to reliably determine the source of invasive infections caused by L. rhamnosus. IMPORTANCEActive culture and probiotic foods and supplements are often used to promote gut health. Though largely considered safe for human consumption, there are rare reports of severe infections linked to bacterial species used in these products. Using whole genome sequencing, we examined a case of fatal infection with Lacticaseibacillus rhamnosus in the patients blood. By comparing the bacterial genome sequence from the patient to strains cultured from 4 commercial yogurt samples and 602 publicly available sequences of L. rhamnosus from around the world, we found that it was nearly identical to one of the yogurt strains and equally similar to other strains from both probiotics and healthy individuals gastrointestinal tracts. These findings show that even with whole genome sequencing it can be difficult to determine the source of an invasive L. rhamnosus infection, whether from recent consumption of a probiotic or dairy product versus from the gastrointestinal microbiome.

microbiology↗

bioRxiv2022

Cell-Sized Lipid Vesicles as Artificial Antigen-Presenting Cells for Antigen-Specific T Cell Activation

In this study, efficient T cell activation is demonstrated using cell-sized artificial antigen-presenting cells (aAPCs) with protein-conjugated bilayer lipid membranes that mimic biological cell membranes. The highly uniform aAPCs are generated by a facile method based on standard droplet microfluidic devices. These aAPCs are able to activate the T cells in peripheral blood mononuclear cells (PBMCs), showing a 28-fold increase in IFN{gamma} secretion, a 233-fold increase in antigen-specific CD8 T cells expansion, and a 16-fold increase of CD4 T cell expansion. The aAPCs do not require repetitive boosting or additional stimulants and can function at a relatively low aAPC-to-T cell ratio (1-to-17). The research presents strong evidence that the surface fluidity and size of the aAPCs are critical to the effective formation of immune synapses essential for T cell activation. The findings demonstrate that the microfluidic-generated aAPCs can be instrumental in investigating the physiological conditions and mechanisms for T cell activation. Finally, this method demonstrates the feasibility of customizable aAPCs for a cost-effective off-the-shelf approach to immunotherapy.

bioengineering↗

bioRxiv2022

Alternatively Spliced Landscape of PPARγ mRNA in Podocytes is Distinct from Adipose Tissue

Podocytes are highly differentiated epithelial cells, and their structural and functional integrity is compromised in a majority of glomerular and renal diseases, leading to proteinuria, chronic kidney disease, and kidney failure. Traditional agonists (e.g., pioglitazone) and selective modulators (e.g., GQ-16) of peroxisome-proliferator-activated-receptor-{gamma} (PPAR{gamma}) reduce proteinuria in animal models of glomerular disease and protect podocytes from injury via PPAR{gamma} activation. This indicates a pivotal role for PPAR{gamma} in maintaining glomerular function through preservation of podocytes distinct from its well-understood role in driving insulin sensitivity and adipogenesis. While its transcriptional role in activating adipokines and adipogenic genes is well-established in adipose tissue, liver and muscle, understanding of podocyte PPAR{gamma} signaling remains limited. We performed a comprehensive analysis of PPAR{gamma} mRNA variants due to alternative splicing, in human podocytes and compared with adipose tissue. We found that podocytes express the ubiquitous PPAR{gamma} Var 1 (encoding {gamma}1) and not Var2 (encoding {gamma}2), which is mostly restricted to adipose tissue and liver. Additionally, we detected expression at very low level of Var4, and barely detectable levels of other variants, Var3, Var11, VartORF4 and Var9, in podocytes. Furthermore, a distinct podocyte vs adipocyte PPAR-promoter-response-element containing gene expression, enrichment and pathway signature was observed, suggesting differential regulation by podocyte specific PPAR{gamma}1 variant, distinct from the adipocyte-specific {gamma}2 variant. In summary, podocytes and glomeruli express several PPAR{gamma} variants, including Var1 ({gamma}1) and excluding adipocyte-specific Var2 ({gamma}2), which may have implications in podocyte specific signaling and pathophysiology. This suggests that new selective PPAR{gamma} modulators can be potentially developed that will be able to distinguish between the two forms, {gamma}1 and {gamma}2, thus forming a basis of novel targeted therapeutic avenues.

molecular biology↗

bioRxiv2022

The high-throughput perturbation of long non-coding RNA reveals functional features in stem cells and across cell-types

Within the scope of FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem (iPS) cells, and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell type enrichment analysis identifies lncRNAs associated with pluripotency while the knockdown of LINC02595, CATG00000090305.1 and RP11-148B6.2 modulates colony formation of iPS cells. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights molecular phenotyping is more comprehensive in revealing affected pathways.

genomics↗

bioRxiv2022

Three-dimensional actuation of hummingbird wings requires diverse effects from the primary flight muscles

Hummingbirds have evolved to hover and maneuver with exceptional flight control. This is directly enabled by their musculoskeletal system that successfully exploits the agile motion of flapping wings. Here, we reveal novel principles of hummingbird wing actuation that provide insights into the evolution and robotic emulation of hummingbird flight. We develop a functional model of hummingbird musculoskeletal system, which predicts instantaneous, three-dimensional torque produced by primary (pectoralis and supracoracoideus) and combined secondary muscles. It also reveals primary muscle contractile behavior, including stress, strain, elasticity, and work. Results show that the primary muscles (i.e., the flight "engine") function as diverse effectors, as they do not simply power the stroke, but also actively deviate and pitch the wing. The secondary muscles produce controlled-tightening effects, by acting against primary muscles in deviation and pitching. The diverse effector capacity of pectoralis is associated with the evolution of a comparatively enormous bicipital crest on humerus.

biophysics↗

bioRxiv2022

Machine Learning-Based Enzyme Engineering of PETase for Improved Efficiency in Degrading Non-Biodegradable Plastic

Globally, nearly a million plastic bottles are produced every minute (1). These non-biodegradable plastic products are composed of Polyethylene terephthalate (PET). In 2016, researchers discovered PETase, an enzyme from the bacteria Ideonella sakaiensis which breaks down PET and nonbiodegradable plastic. However, PETase has low efficiency at high temperatures. In this project, we optimized the rate of PET degradation by PETase by designing new mutant enzymes which could break down PET much faster than PETase, which is currently the gold standard. We used machine learning (ML) guided directed evolution to modify the PETase enzyme to have a higher optimal temperature (Topt), which would allow the enzyme to degrade PET more efficiently. First, we trained three machine learning models to predict Topt with high performance, including Logistic Regression, Linear Regression and Random Forest. We then used Random Forest to perform ML-guided directed evolution. Our algorithm generated hundreds of mutants of PETase and screened them using Random Forest to select mutants with the highest Topt, and then used the top mutants as the enzyme being mutated. After 1000 iterations, we produced a new mutant of PETase with Topt of 71.38{degrees}C. We also produced a new mutant enzyme after 29 iterations with Topt of 61.3{degrees}C. To ensure these mutant enzymes would remain stable, we predicted their melting temperatures using an external predictor and found the 29-iteration mutant had improved thermostability over PETase.Our research is significant because using our approach and algorithm, scientists can optimize additional enzymes for improved efficiency.

synthetic biology↗

bioRxiv2021

A single cell atlas of human and mouse white adipose tissue

White adipose tissue (WAT), once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic, heterogenous, and involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control, and host defense1. High fat feeding and other metabolic stressors cause dramatic changes in adipose morphology, physiology, and cellular composition1, and alterations in adiposity are associated with insulin resistance, dyslipidemia, and type 2 diabetes (T2D)2. Here, we provide detailed cellular atlases of human and murine subcutaneous and visceral white fat at single cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells (ASPCs), vascular, and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease, and we provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. These data comprise an extensive resource for the exploration of genes, traits, and cell types in the function of WAT across species, depots, and nutritional conditions.

physiology↗

bioRxiv2021

A Transparent Ultrasound Array for Real-time Optical, Ultrasound and Photoacoustic Imaging

Objective and Impact StatementSimultaneous imaging of ultrasound and optical contrasts can help map structural, functional and molecular biomarkers inside living subjects with high spatial resolution. There is a need to develop a platform to facilitate this multimodal imaging capability to improve diagnostic sensitivity and specificity. IntroductionCurrently, combining ultrasound, photoacoustic and optical imaging modalities is challenging because con-ventional ultrasound transducer arrays are optically opaque. As a result, complex geometries are used to co-align both optical and ultrasound waves in the same field of view. MethodsOne elegant solution is to make the ultrasound transducer transparent to light. Here, we demonstrate a novel transparent ultrasound transducer (TUT) liner array fabricated using a transparent lithium niobate piezoelectric material for real-time multimodal imaging. ResultsThe TUT array consisted of 64 elements and centered at [~] 6 MHz frequency. We demonstrate a quad-mode ultrasound, Doppler ultrasound, photoacoustic and fluorescence imaging in real-time using the TUT array directly coupled to the tissue mimicking phantoms. ConclusionThe TUT array successfully showed a multimodal imaging capability, and has potential applications in diagnosing cancer, neuro and vascular diseases, including image-guided endoscopy and wearable imaging.

bioengineering↗

bioRxiv2021

Binding Affinity Estimation From Restrained Umbrella Sampling Simulations

The protein-ligand binding affinity quantifies the binding strength between a protein and its ligand. Computer modeling and simulations can be used to estimate the binding affinity or binding free energy using data- or physics-driven methods or a combination thereof. Here, we discuss a purely physics-based sampling approach based on biased molecular dynamics (MD) simulations, which in spirit is similar to the stratification strategy suggested previously by Woo and Roux. The proposed methodology uses umbrella sampling (US) simulations with additional restraints based on collective variables such as the orientation of the ligand. The novel extension of this strategy presented here uses a simplified and more general scheme that can be easily tailored for any system of interest. We estimate the binding affinity of human fibroblast growth factor 1 (hFGF1) to heparin hexasaccharide based on the available crystal structure of the complex as the initial model and four different variations of the proposed method to compare against the experimentally determined binding affinity obtained from isothermal calorimetry (ITC) experiments. Our results indicate that enhanced sampling methods that sample along the ligand-protein distance without restraining other degrees of freedom do not perform as well as those with additional restraint. In particular, restraining the orientation of the ligands plays a crucial role in reaching a reasonable estimate for binding affinity. The general framework presented here provides a flexible scheme for designing practical binding free energy estimation methods.

biophysics↗

bioRxiv2021

Selective Modulator of Nuclear Receptor PPARy with Reduced Adipogenic Potential Ameliorates Experimental Nephrotic Syndrome

BackgroundGlomerular disease, often manifesting as nephrotic syndrome (NS) with high proteinuria, can be refractory to standard treatment and is typically associated with hypoalbuminemia, hypercholesterolemia and hypercoagulopathy. We hypothesized that the nuclear receptor PPAR{gamma} can be selectively modulated using a novel partial agonist, GQ-16, to gain therapeutic advantage over traditional PPAR{gamma} agonists (e.g. thiazolidinediones) for the treatment of glomerular disease. MethodsNephropathy was induced with puromycin amino-nucleoside (PAN) in Wistar rats and treated with Pioglitazone (Pio) or GQ-16. Plasma, serum, and urine chemistries were performed, and kidneys, glomeruli, liver, and white adipose tissue (WAT) were harvested. Lipid accumulation and adipogenic gene expression were measured in adipocytes. ResultsPAN-induced proteinuria was significantly reduced with Pio to 64% of PAN-value. It was reduced robustly with GQ-16 to 81% of PAN, which was comparable to controls. While both GQ-16 and Pio restored glomerular Nphs1 and hepatic Pcsk9 expression and reduced hypercholesterolemia, GQ-16 also restored glomerular Nrf2, and reduced hypoalbuminemia and hypercoagulopathy. Furthermore, RNA-seq analysis identified both common and distinct restored glomerular genes downstream of Pio and GQ-16. Pio but not GQ-16 significantly induced aP2 (fatty acid binding protein) in adipocytes and in WAT. Pio induced more lipid accumulation than GQ-16 in differentiated adipocytes. Both, Pio and GQ-16 induced insulin sensitizing adipokines in WAT with varying degrees. ConclusionsSelective modulation of PPAR{gamma} by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria and NS associated co-morbidities, while reducing the adipogenic side-effects conferred by traditional PPAR{gamma} full agonists. Translational StatementThe authors have previously reported that type-II diabetes drugs, thiazolidinediones (PPAR{gamma} agonists), also provide beneficial effects in reducing podocyte and glomerular injury. However, these drugs are associated with adverse effects such as weight gain, and their effects on glomerular disease-associated features are largely unexplored. Their current findings demonstrate that PPAR{gamma} can be selectively modulated by its partial agonist, GQ-16, which reduces proteinuria and improves nephrotic syndrome (NS) with reduced side-effects typically conferred by thiazolidinediones. These findings not only deepen our molecular understanding of the role of PPAR{gamma} in glomerular disease and underscore the potential for partial agonists of PPAR{gamma}, such as GQ-16 as a treatment modality for NS, but also lend the possibility of its potential benefits in diabetic nephropathy.

molecular biology↗

bioRxiv2021

Stability Comparisons between Natural versus Engineered Archaeal Heat Shock Proteins

Crenarchaeal group II chaperonins (a.k.a., "heat shock" proteins, HSPs) are abundantly expressed in species of the family Sulfolobaceae. HSP and HSP{beta} expression is upregulated during thermal shock. HSPs are subunits of larger octadecameric complexes that function to protect intracellular proteins during thermal stress. Engineered HSPs have been constructed with the idea of protecting enzymes in industrial reactions. HSP{beta}-coh, a fusion protein comprised of HSP{beta} and type 1 cohesin from Clostridium thermocellum was used for proof-of-concept. Dockerin-endowed cellulolytic enzymes bind to the complex via cohesin-dockerin interactions. Enzymatic activity (i.e., hydrolysis of lignocellulose) is retained when the platform is used at high temperatures (e.g., 85-88{degrees}C). Moreover, enhancement persists on acid-pretreated substrates prompting the question: Are HSPs acid tolerant? In this study, HSP structural integrity is examined at different temperatures and pH. Far-UV circular dichroism and intrinsic fluorescence indicate HSP and HSP{beta} retain structural integrity at neutral pH over a range of temperatures (25-90{degrees}C) while HSP{beta}-coh is less tolerant to thermal stress. Structural integrity is compromised for all subunits at ultra-low pH (i.e., pH 2) with HSP showing the most susceptibility. Secondary structures of all HSPs are resilient under mildly acidic conditions (pH 4). ANS binding assays indicate a shift in tertiary structure for all subunits at ultra-low pH. Limited trypsin digestion reveals that the backbone of HSP{beta}-coh is the most flexible and HSP{beta} is the most resistant. Results suggest that HSP and HSP{beta} are more resilient than HSP{beta}-coh under thermal challenge and that there are limits to the acid tolerance of all HSPs.

biochemistry↗