Biology subjects

Agarwal, R.

Publications and source records attributed to Agarwal, R..

46 records · Page 3Linked to original sources

bioRxiv2021

Rapamycin microparticles induce autophagy, prevent senescence and are effective in treatment of Osteoarthritis

Trauma to the knee joint is associated with significant cartilage degeneration and erosion of subchondral bone, which eventually leads to osteoarthritis (OA), resulting in substantial morbidity and healthcare burden. With no disease-modifying drugs in clinics, the current standard of care focuses on symptomatic relief and viscosupplementation. Modulation of autophagy and targeting senescence pathways are emerging as potential treatment strategies. Rapamycin has shown promise in OA disease amelioration by autophagy upregulation, yet its clinical use is hindered by difficulties in achieving therapeutic concentrations, necessitating multiple weekly injections. Here, we have synthesized rapamycin - loaded poly (lactic-co-glycolic acid) microparticles (RMPs) that induced autophagy, prevented senescence and sustained sulphated glycosaminoglycans(sGAG) production in primary human articular chondrocytes from OA patients. RMPs were potent, nontoxic, and exhibited high retention time (up to 35 days) in mice joints. Intra-articular delivery of RMPs effectively mitigated cartilage damage and inflammation in surgery-induced OA when administered as a prophylactic or therapeutic regimen. Together, our studies demonstrate the feasibility of using RMPs as a potential clinically translatable therapy to prevent and treat post-traumatic osteoarthritis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/453073v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@29863org.highwire.dtl.DTLVardef@628da3org.highwire.dtl.DTLVardef@40ec6eorg.highwire.dtl.DTLVardef@626355_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Pseudomonas can prevent the parasitic fungus, while keeping the crop fungus unaffected, in the gardens of Odontotermes obesus.

Insects that farm monocultures of fungi are canonical examples of nutritional symbiosis as well as independent evolution of agriculture in non-human animals. But just like in human agriculture, these fungal crops face constant threat of invasion by weeds which, if unchecked, takes over the crop fungus. In fungus-growing termites, the crop fungus (Termitomyces) faces such challenges from the parasitic fungus Pseudoxylaria. The mechanism by which Pseudoxylaria is suppressed is not known. However, evidence suggests that some bacterial secondary symbionts can serve as defensive mutualists by preventing the growth of Pseudoxylaria. However, such secondary symbionts must possess the dual, yet contrasting, capabilities of suppressing the weedy fungus while keeping the growth of the crop fungus unaffected. This study describes the isolation, identification and culture-dependent estimation of the roles of several such putative defensive mutualists from the colonies of the wide-spread fungus-growing termite from India, Odontotermes obesus. From the 38 bacterial cultures tested, a strain of Pseudomonas showed significantly greater suppression of the weedy fungus than the crop fungus. Moreover, a 16S rRNA pan-microbiome survey, using the Nanopore platform, revealed Pseudomonas to be a part of the core microbiota of Odontotermes obesus. A meta-analysis of microbiota composition across different species of Odontotermes also confirms the wide-spread prevalence of Pseudomonas within this termite. These evidence indicate that Pseudomonas could be playing the role of defensive mutualist within Odontotermes.

evolutionary biology↗

bioRxiv2021

Iron acquisition across outer membrane: The role of major outer membrane protein Slr1908 under iron supplemented conditions in Synechocystis 6803

The Ton-B dependent outer membrane (OM) transporters are responsible for active iron (Fe) import in Synechocystis sp. strain PCC 6803 (S. 6803 or WT) under Fe depletion. However, the mechanism of Fe acquisition under Fe supplemented conditions remains uncharacterised. In the present study, functional role of OMP Slr1908 in S. 6803 was addressed by insertional mutagenesis. The{Delta} slr1908 cells exhibited slower growth in the first week in comparison to the WT and displayed an absorption and 77K fluorescence spectrum typical of Fe deficiency. Indeed, the mutant had [~] 80% less Fe as confirmed by atomic absorption spectroscopy and 55Fe-radiotracer uptake. The iron deficiency was paralleled with low Mn content. The mutant had low SOD content as well as activity, less cytochromes, less chlorophyll content, less Fv/ Fm, lower ETRII and high oxidative stress in comparison to the WT at the end of first week. Interestingly, the mutant showed transcriptional upregulation of iron stress induced protein isiA and isiB signifying intracellular Fe deficiency. Upregulation of OMP Slr0042 was also observed at RNA and protein level. The results indicate that Slr1908 is a major Fe uptake OMP in S. 6803 the deletion of which leads to initial slow growth that gets partially offset by induction of other Fe importing OMPs.

microbiology↗

bioRxiv2020

The molecular logic of synaptic wiring at the single cell level

The correct wiring of neuronal circuits is one of the most complex processes in development, since axons form highly specific connections out of a vast number of possibilities. Circuit structure is genetically determined in vertebrates and invertebrates, but the mechanism guiding each axon to precisely innervate a unique pre-specified target cell is poorly understood. Here, we used single cell genomics, imaging and genetics to show that single-cell specific connections between motoneurons and target muscles are specified through a combinatorial code of immunoglobulin domain proteins. These programs are orchestrated by a homeodomain transcription factor code that specifies cellular identities down to the level of biologically unique cells. Using spatial mapping, we show that this code follows spatial patterns already observed at early embryonic stages, while acting at a much later stage to specify motor circuits. Taken together, our data suggest that a relatively simple homeo-immunoglobulin-code determines neuronal circuit structure.

developmental biology↗

bioRxiv2020

Diverse viral proteases activate the NLRP1 inflammasome

The NLRP1 inflammasome is a multiprotein complex that is a potent activator of inflammation. Mouse NLRP1B can be activated through proteolytic cleavage by the bacterial Lethal Toxin (LeTx) protease, resulting in degradation of the N-terminal domains of NLRP1B and liberation of the bioactive C-terminal domain, which includes the caspase activation and recruitment domain (CARD). However, a natural pathogen-derived effector that can activate human NLRP1 remains unknown. Here, we use an evolutionary model to identify several proteases from diverse picornaviruses that cleave human NLRP1 within a rapidly evolving region of the protein, leading to host-specific and virus-specific activation of the NLRP1 inflammasome. Our work demonstrates that NLRP1 acts as a "tripwire" to recognize the enzymatic function of a wide range of viral proteases, and suggests that host mimicry of viral polyprotein cleavage sites can be an evolutionary strategy to activate a robust inflammatory immune response.

microbiology↗

bioRxiv2020

Antimycobacterial potential of Mycobacteriophage under pathophysiological conditions

Antibiotic resistance continues to be a major global health risk with an increase in multi-drug resistant infections seen across nearly all bacterial diseases. Mycobacterial infections such as Tuberculosis (TB) and Non-Tuberculosis infections have seen a significant increase in the incidence of multi-drug resistant and extensively drug-resistant infections. With this increase in drug-resistant Mycobacteria, mycobacteriophage therapy offers a promising alternative. However, a comprehensive study on the infection dynamics of mycobacteriophage against their host bacteria and the evolution of bacteriophage (phage) resistance in the bacteria remains elusive. We aim to study the infection dynamics of a phage cocktail against Mycobacteria under various pathophysiological conditions such as low pH, low growth rate and hypoxia. We show that mycobacteriophages are effective against M. smegmatis under various conditions and the phage cocktail prevents emergence of resistance for long durations. Although the phages are able to amplify after infection, the initial multiplicity of infection plays an important role in reducing the bacterial growth and prolonging efficacy. Mycobacteriophages are effective against antibiotic-resistant strains of Mycobacterium and show synergy with antibiotics such as rifampicin and isoniazid. Finally, we also show that mycobacteriophages are efficient against M. tuberculosis both under lag and log phase for several weeks. These findings have important implications for developing phage therapy for Mycobacterium.

microbiology↗

bioRxiv2020

Rapamycin-PLGA microspheres induce autophagy and prevent senescence in chondrocytes and exhibit long in vivo residence

Osteoarthritis (OA) is a joint disease that results in progressive destruction of articular cartilage and the adjoining subchondral bone. The current treatment is focused on symptomatic relief due to the absence of disease-modifying drugs. The primary cells of the cartilage, chondrocytes, have limited regenerative capacity and when they undergo stress due to trauma or with aging, they senesce or become apoptotic. Autophagy, a cellular homeostasis mechanism has a protective role in OA during stress but gets downregulated in OA. Rapamycin, a potent immunomodulator, has shown promise in OA treatment by autophagy activation and is known to prevent senescence. However, its clinical translation for OA is hampered due to systemic toxicity as high and frequent doses are required. Hence, there is a need to develop suitable delivery carriers that can result in sustained and controlled release of the drug in the joint. In this study, we have fabricated rapamycin encapsulated poly (lactic-co-glycolic acid) (PLGA) based carriers that induced autophagy and prevented cellular senescence in human chondrocytes. The microparticle (MP) delivery system showed sustained release of drug for several weeks. Rapamycin-microparticles protected in-vitro cartilage mimics from degradation, allowing sustained production of sGAG, and demonstrated a prolonged senescence preventive effect in vitro under oxidative and genomic stress conditions. These microparticles also exhibited a long residence time of more than 19 days in the joint after intra-articular injections in murine knee joints. Such particulate systems are a promising candidate for intra-articular delivery of rapamycin for treatment of osteoarthritis. Statement of SignificanceCurrent OA treatment is symptomatic and does not change the disease progression. Many drugs have failed as they are rapidly cleared and are unable to maintain therapeutic concentration in the OA joint. Direct joint administration of drugs using sustained-release systems offer several advantages, which includes increased bioavailability, fewer off-target effects, and lower total drug cost. We have engineered a suitable drug carrier which provides a tunable drug release pattern. This study provides evidence that PLGA encapsulated rapamycin remained potent and prevented OA like changes in chondrocytes under genomic and oxidative stress. The particle formulation also had a longer residence time in the knee joint of mice which can be translated in clinics for intra-articular therapeutic injections for increased patient compliance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/027136v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c5901borg.highwire.dtl.DTLVardef@177fa3eorg.highwire.dtl.DTLVardef@1ce2a55org.highwire.dtl.DTLVardef@3d5d6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Structure based virtual screening identifies novel competitive inhibitors for the sialoglycan binding protein Hsa

Infective endocarditis (IE) is a cardiovascular disease often caused by bacteria of the viridans group of streptococci, which includes Streptococcus gordonii and Streptococcus sanguinis. Previous research has found that a serine-rich repeat (SRR) proteins on the S. gordonii bacterial surface play a critical role in pathogenesis by facilitating bacterial attachment to sialyated glycans displayed on human platelets. Despite its important role in disease progression, there are currently no anti-adhesive drugs available on the market. Here, we performed structure-based virtual screening using an ensemble docking approach followed by consensus scoring to identify novel inhibitors against the sialoglycan binding domain of the SRR adhesin protein Hsa from the S. gordonii strain DL1. In silico cross screening against the glycan binding domains of closely related SRR proteins from five other S. gordonii or S. sanguinis strains was also performed to further reduce false positives. Using our in silico screening strategy we successfully predicted nine compounds which were able to displace the native ligand (sialyl-T antigen) in an in vitro assay and bind competitively to adhesin protein Hsa ([~]20% hit rate).

microbiology↗

bioRxiv2020

Mesophilic enzyme function at high temperature: moleculardynamics of hyperthermophilic and mesophilicpyrophosphatases

The mesophilic inorganic pyrophosphatase from Escherichia coli (EcPPase) retains function at 353 K, the physiological temperature of hyperthermophilic Thermoccoccus thioreducens, whereas, the homolog protein from the hyperthermophilic organism (TtPPase) cannot function at room temperature. To explain this asymmetric behavior, we examined structural and dynamical properties of the two proteins using molecular dynamics simulations. The global flexibility of TtPPase is significantly higher than its mesophilic homolog at all tested temperature/pressure conditions. However, at 353 K, EcPPase reduces its solvent-exposed surface area and increases subunit compaction while maintaining flexibility in its catalytic pocket. In contrast, TtPPase lacks this adaptability and has increased rigidity and reduced protein:water interactions in its catalytic pocket at room temperature, providing a plausible explanation for its inactivity near room temperature.

biophysics↗

bioRxiv2019

Selectivity and engineering of the sialoglycan-binding spectrum in Siglec-like adhesins

The Siglec-like Serine-Rich Repeat (SRR) adhesins mediate bacterial attachment to mammalian hosts via sialoglycan receptors. Here, we combine structural, computational, biochemical, and phylogenetic approaches to elucidate the determinants of the sialoglycan-binding spectrum across the family of Siglec-like SRR adhesins. We further identified mutable positions that disproportionately affect sialoglycan selectivity, as demonstrated by increases in binding to alternative ligands of 2- to 3-orders of magnitude. Biologically, these studies highlight how bacteria nimbly modulate the receptor interaction during coevolution of host and pathogen. These studies additionally created binding proteins specific for sialyl-T antigen or 6S-sialyl LewisX that can recognize glycosylation of human plasma proteins. The engineered binding proteins can facilitate the characterization of normal cellular glycan modifications or may be used as diagnostic tools in disease states with altered glycosylation.\n\nSignificanceThe ability of bacteria to bind selectively to host receptors underlies both commensalism and pathogenesis. Here, we identify the molecular basis for receptor selectivity in streptococci that bind to sialoglycan receptors. This revealed how to convert these adhesins into selective probes that measure triand tetrasacharides within the context of larger glycosylations. These probes that can be used in a laboratory with no specialized equipment and can be used to address biological questions relating to sialoglycan-dependent signaling and adhesion.

biochemistry↗