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Ghosh, K.

Publications and source records attributed to Ghosh, K..

At least 37 records · Page 2Linked to original sources

Non-enzymatic error correction in self-replicators without extraneous energy supply

Accurate propagation of sequence information in nucleic acids is central to the evolutionary dynamics of self-replicating systems. Modern biological systems achieve high fidelity using enzymes that actively correct errors through energy-driven mechanisms. However, such complex machinery was absent under prebiotic conditions. Here, we present a theoretical model of error correction in self-replicating heteropolymers that requires neither enzymes nor an extraneous energy supply. The model relies solely on the free-energy gradient driving strand growth and requires asymmetric cooperativity -- a kinetic asymmetry known to promote unidirectional elongation. Despite its simplicity, we demonstrate that this minimal model facilitates kinetic discrimination between correct and incorrect base pair incorporations, and for specific set of parameters, reproduces the error ratio of [~] 10-4, experimentally observed in passive base selection processes. It replicates key features observed in DNA error correction, including stalling, fraying, next-nucleotide effects, and the speed-accuracy trade-off. Our results provide plausible answers for longstanding questions, such as the energy source for the enhanced base selectivity of passive DNA polymerases and the role of thermodynamics and kinetics of phosphodiester bond formation in error correction. We show that catalysis of the phosphodiester bond plays a central role in error correction, even without explicit enzymatic structural discrimination. This observation points to a plausible pathway for accurate oligomer synthesis under prebiotic conditions, driven solely by the thermodynamic gradient favoring strand elongation. More broadly, the model highlights how persistent molecular order can emerge from non-equilibrium dynamics - a central requirement for emergence of life.

biochemistry↗

Lysyl oxidase promotes actin-dependent neutrophil activation and cytotoxicity in diabetes: Implications for diabetic retinopathy

Activated neutrophils contribute to retinal endothelial cell (EC) death and capillary degeneration associated with early diabetic retinopathy (DR). However, the factors and mechanisms driving neutrophil activation and cytotoxicity in diabetes remain insufficiently understood. Here we show that lysyl oxidase (LOX), a collagen crosslinking and matrix stiffening enzyme that increases retinal EC susceptibility to activated neutrophils, simultaneously activates neutrophils in its alternate soluble form. Specifically, treatment of diabetic mice with LOX inhibitor {beta}-aminopropionitrile (BAPN) prevented the diabetes-induced increase in neutrophil activation (extracellular release of neutrophil elastase and superoxide) and cytotoxicity towards co-cultured mouse retinal ECs. Mouse neutrophils and differentiated (neutrophil-like) human HL-60 cells treated with recombinant LOX alone exhibited similar activation and cytotoxicity. Mechanistically, this LOX-induced neutrophil activation was associated with biphasic F-actin remodeling, with the initial and rapid (<15 min) F-actin depolymerization followed by a significant increase in F-actin polymerization and polarization. Preventing the initial F-actin depolymerization blocked LOX-induced neutrophil activation and cytotoxicity towards co-cultured retinal ECs. Finally, we show that this biphasic F-actin remodeling is essential for LOX-induced membrane clustering of azurophilic granule marker CD63 and NADPH organizer p47 that are associated with extracellular release of neutrophil elastase and superoxide, respectively. By revealing a causal and previously unrecognized link between LOX and actin-dependent neutrophil activation in diabetes, these findings provide fresh mechanistic insights into the proinflammatory role of LOX in early DR that goes beyond its canonical matrix-stiffening effects.

pathology↗

Eukaryotes evade information storage-replication rate trade-off with endosymbiont assistance leading to larger genomes

Genome length varies widely among organisms, from compact genomes of prokaryotes to vast and complex genomes of eukaryotes. In this study, we theoretically identify the evolutionary pressures that may have driven this divergence in genome length. We use a parameter-free model to study genome length evolution under selection pressure to minimize replication time and maximize information storage capacity. We show that prokaryotes tend to reduce genome length, constrained by a single replication origin, while eukaryotes expand their genomes by incorporating multiple replication origins. We propose a connection between genome length and cellular energetics, suggesting that endosymbiotic organelles, mitochondria and chloroplasts, evolutionarily regulate the number of replication origins, thereby influencing genome length in eukaryotes. We show that the above two selection pressures also lead to strict equalization of the number of purines and their corresponding base-pairing pyrimidines within a single DNA strand, known as Chagraffs second parity rule, a hitherto unexplained observation in genomes of nearly all known species. This arises from the symmetrization of replichore length, another observation that has been shown to hold across species, which our model reproduces. The model also reproduces other experimentally observed phenomena, such as a general preference for deletions over insertions, and elongation and high variance of genome lengths under reduced selection pressure for replication rate, termed the C-value paradox. We highlight the possibility of regulation of the firing of latent replication origins in response to cues from the extracellular environment leading to the regulation of cell cycle rates in multicellular eukaryotes. Significance StatementUnderstanding the forces shaping genome architecture is a long-standing challenge in evolutionary biology. Our study demonstrates that the balance between replication speed and information storage, constrained by cellular en-ergetics, drives the divergence in genome lengths between prokaryotes and eukaryotes. By quantifying selection pressure as the ratio of replication time to genomic information content, we show that this pressure enforces adaptive constraints, giving rise to features such as symmetric replichores and Chargaffs second parity rule. These insights not only help us resolve an enduring evolutionary puzzle, but also offer a unified framework linking genome organization, cellular specialization, and even potential mechanisms underlying carcinogenesis.

evolutionary biology↗

EHE cell cultures: a platform for mechanistic and therapeutic investigation

Epithelioid hemangioendothelioma (EHE) is a difficult to treat vascular sarcoma defined by TAZ- CAMTA1 or YAP-TFE3 fusion proteins. Human cell lines needed to further understand the pathogenesis of EHE have been lacking. Herein, we describe a method to generate EHE extended primary cell cultures. An integrated multi -omic and functional approach was used to characterize these cultures. The cell cultures, relatively homogenous by single cell RNA-Seq, demonstrated established characteristics of EHE including increased proliferation, anchorage independent growth, as well as the overall gene expression profile and secondary genetic alterations seen in EHE. Whole genome sequencing (WGS) identified links to epigenetic modifying complexes, metabolic processes, and pointed to the importance of the extracellular matrix (ECM) in these tumors. Bulk RNA-Seq demonstrated upregulation of pathways including PI3K-Akt signaling, ECM/ECM receptor interaction, and the Hippo signaling pathway. Development of these extended primary cell cultures allowed for single-cell profiling which demonstrated different cell compartments within the cultures. Furthermore, the cultures served as a therapeutic platform to test the efficacy of TEAD inhibitors in vitro. Overall, the development of EHE primary cell cultures will aid in the mechanistic understanding of this sarcoma and serve as a model system to test new therapeutic approaches.

cancer biology↗

PI3K regulates TAZ/YAP and mTORC1 axes that can be synergistically targeted

Abstract/SummaryO_ST_ABSPurposeC_ST_ABSSarcomas are a heterogeneous group of cancers with few shared therapeutic targets. PI3K signaling is activated in various subsets of sarcomas, representing a shared oncogenic signaling pathway. Oncogenic PI3K signaling has been challenging to target therapeutically. An integrated view of PI3K and Hippo pathway signaling is examined to determine if this could be leveraged therapeutically. Experimental designA tissue microarray containing sarcomas of various histological types was evaluated for PTEN loss and correlated with levels of activated TAZ and YAP. PI3K and Hippo pathways were dissected in sarcoma cell lines. The role of TAZ and YAP were evaluated in a PI3K-driven mouse model. The efficacy of mTORC1 inhibition and TEAD inhibition were evaluated in sarcoma cell lines and in vivo. ResultsPI3K signaling is frequently activated in sarcomas due to PTEN loss (in 30-60%), representing a common therapeutic target. TAZ and YAP are transcriptional co-activators regulated by PI3K and drive a transcriptome necessary for tumor growth in a PI3K-driven sarcoma mouse model. Combination therapy using IK-930 (TEAD inhibitor) and everolimus (mTORC1 inhibitor) synergistically diminished proliferation and anchorage independent growth of PI3K-activated sarcoma cell lines at low, physiologically achievable doses. Furthermore, this combination therapy showed a synergistic effect in vivo, reducing tumor proliferation and size. ConclusionsTAZ and YAP are transcriptional co-activators downstream of PI3K signaling, a pathway that has lacked a well-defined oncogenic transcription factor. This PI3K-TAZ/YAP axis exists in parallel to the known PI3K-Akt-mTORC1 axis allowing for synergistic combination therapy targeting the TAZ/YAP-TEAD interaction and mTORC1 in sarcomas.

cancer biology↗

Modulation of cellular ageing regulates Integrated Stress Response signalling to control blood cell homeostasis

Organismal ageing is associated with a decline in cellular and molecular functions. One of the hallmarks of ageing is stem cell exhaustion which occurs due to dynamic changes in the stem cell - niche microenvironment. Ageing affects stem cell potency impacting self-renewal and differentiation trajectories. Blood cell homeostasis in the hematopoietic system is tightly regulated by a fine balance between stemness and differentiation. Here, we employ the Drosophila hematopoietic organ, the larval lymph gland (LG) to investigate the impact of modulating cellular ageing on organ homeostasis. LG consists of a Posterior Signalling Center (PSC) that acts as a stem cell niche and maintains the prohemocytes housed in the Medullary Zone (MZ). Cortical Zone (CZ) consists of differentiated hemocytes namely plasmatocytes, crystal cells and lamellocytes. Intricate signalling networks active in the PSC and MZ orchestrate and regulate homeostasis. In this study, we characterize the effect of genetic perturbation of the molecular circuitry of ageing locally and systemically to investigate its effect on LG blood cell homeostasis. Genetic modulation of cellular ageing displays traditional hallmarks of ageing validating our model in the LG. Our results indicate that induction of accelerated ageing both locally and systemically leads to a reduction in stem cell niche size, DNA damage accumulation and increased progenitor differentiation whereas decelerated ageing shows an opposite trend. We show that the Integrated Stress Response (ISR) pathway is activated upon inducing accelerated ageing in the LG hemocytes possibly to recoup back to homeostatic conditions. Furthermore, LGs of ISR pathway mutants or upon perturbation of ISR pathway components in prohemocytes show increased blood cell differentiation indicating disruption of homeostasis. Genetic epistasis analysis shows that ectopic over-expression of ISR pathway genes in an accelerated ageing scenario over and above the existing levels can rescue the defects in blood cell homeostasis. Overall, our study explores how modulation of cellular aging locally or systemically can impact tissue homeostasis. Our research paves way to understand the cellular mechanisms underlying an aged versus young stem cell-niche microenvironment and how its abrogation may lead to onset of disease.

developmental biology↗

G-quadruplexes catalyze protein folding by reshaping the energetic landscape

Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing G-quadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. As short nucleic acids can catalyze protein folding without the input of ATP, the ability of the cell to fold proteins is far higher than previously anticipated. Significance StatementHow folding of proteins occurs en masse in the cell is still a daunting unsolved problem. Many proteins have complicated and difficult folding trajectories, with in vitro folding times that are physiologically untenable. The acceleration of protein folding to physiologically relevant timescales is a biologically essential function thought to be accomplished by a small set of ATP-dependent chaperonins. In this work, we surprisingly show that small nucleic acid sequences containing G-quadruplexes can catalyze protein folding and reshape protein folding energy landscapes. As a result, the capacity for accelerating protein folding in the cells is far higher than previously suggested, potentially explaining the accommodation of large number of proteins with physiologically unreasonable folding times.

biophysics↗

Ellagic acid: a potential inhibitor of enhancer of zeste homolog-2 and protein arginine methyltransferase-5

Dysregulation of epigenetic processes, characterized by aberrant DNA methylation patterns and histone modifications, is a hallmark of cancer, driving its initiation, progression, and metastasis by silencing tumor suppressor genes or activating oncogenes. Perturbations in histone modifications such as H3K27me3 by EZH2 and H4R3me2s by PRMT5 play significant roles in these epigenetic alterations, disrupting normal gene expression and facilitating oncogene activation while suppressing tumor suppressor genes. Consequently, inhibitors targeting enzymes involved in DNA methylation, histone modification, or chromatin remodeling, such as PRMTs and PRC complexes, are promising anti-cancer agents, with several undergoing pre-clinical and clinical trials. Our screening of a phytochemical library revealed ellagic acid as an effective inhibitor of both EZH2 and PRMT5. Ellagic acid interacts strongly with the EZH2 and PRMT5:MEP50 complex, binding to their active sites through {pi}-cation interactions and hydrogen bonds. Surface Plasmon Resonance study confirmed potent binding affinities of ellagic acid, with KD values of 3.28E-06 and 6.54E-05 for EZH2 and PRMT5:MEP50 respectively. In-vitro assays validated inhibitory effects on EZH2 and PRMT5:MEP50 by reducing the levels of their catalytic products H3K27me3 and H4R3me2s respectively and induction of autophagy and apoptosis. Further In-vivo studies using mouse xenografts further demonstrated significant tumor size reductions upon oral administration of ellagic acid, with decreased expression of the proliferative marker ki67 and histone repressive marks. Taken together we showed that inhibition of EZH2 and PRMT5:MEP50 by ellagic acid could be used to develop breast cancer therapeutic drug.

cancer biology↗

PATHOGEN GROWTH AND VIRULENCE DYNAMICS DRIVE THE HOST EVOLUTION AGAINST COINFECTIONS

Coinfections, or the simultaneous infection of hosts by multiple pathogens, are widespread in nature with significant negative impacts on global health. Can hosts evolve against such coinfections as effectively as they would against individual pathogens? Also, what roles do individual pathogens play during such evolution? Here, we combined theoretical models and experiments with Tribolium castaneum populations evolving against two coinfecting bacterial pathogens, with contrasting growth and virulence dynamics, to reveal that fast-growing pathogens inflicting rapid mortality surges (i.e., fast-acting) restrict adaptive success against coinfections. While hosts rapidly evolved better survival against slow-growing bacteria causing long-lasting infections, evolution against coinfection was significantly delayed and resembled slow adaptation against fast-acting pathogens. Moreover, limited scopes of immunomodulation against fast-acting pathogens during coinfections can drive the observed adaptive patterns. Overall, we provide new insights into how adaptive dynamics and mechanistic bases against coinfections are critically regulated by individual pathogens growth and virulence dynamics.

evolutionary biology↗

Stiffness Measurement of Retinal Capillaries and Subendothelial Matrix using Atomic Force Microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced increase in retinal capillary stiffness plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. Retinal capillary stiffening results from overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and retinal subendothelial matrix using atomic force microscopy.

pathology↗

Nerve injury downregulates δ-opioid and cannabinoid CB1 receptor genes through REST in primary sensory neurons

The transcription repressor REST in the dorsal root ganglion (DRG) is upregulated by peripheral nerve injury and promotes the development of chronic pain. However, the genes targeted by REST in neuropathic pain development remain unclear. The expression levels of 4 opioid receptor (Oprm1, Oprd1, Oprl1, Oprk1) and the cannabinoid CB1 receptor (Cnr1) genes in the DRG regulate nociception. In this study, we determined the role of REST in the control of their expression in the DRG induced by spared nerve injury (SNI) in both male and female mice. Transcriptomic analyses of male mouse DRGs followed by quantitative reverse transcription polymerase chain reaction analyses of both male and female mouse DRGs showed that SNI upregulated expression of Rest and downregulated mRNA levels of all 4 opioid receptor and Cnr1 genes, but Oprm1 was upregulated in female mice. Analysis of publicly available bioinformatic data suggested that REST binds to the promoter regions of Oprm1 and Cnr1. Chromatin immunoprecipitation analyses indicated differing levels of REST at these promoters in male and female mice. Full-length Rest conditional knockout in primary sensory neurons reduced SNI-induced pain hypersensitivity and rescued the SNI-induced reduction in the expression of Oprd1 and Cnr1 in the DRG in both male and female mice. Our results suggest that nerve injury represses the transcription of Oprd1 and Cnr1 via REST in primary sensory neurons and that REST is a potential therapeutic target for neuropathic pain.

neuroscience↗

Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer both in vitro and in vivo

Histone methyltransferases are selectively catalyzing the methylation of lysine or arginine residues of target histone and non-histone proteins, classified as lysine methyltransferases and arginine methyltransferases. The EZH2 and PRMT5 catalyze trimethylation of H3 at K27 and symmetric dimethylation of H4 at R3 respectively. These histone repressive marks have been considered as hallmarks in cancer. Both PRMT5 and EZH2 over expressed in several cancers and have been considered as important target of drug development. As a result, many synthetic molecules as inhibitors of both PRMT5 and EZH2 are at different level of preclinical and clinical phases. Cancer atlas data analysis revealed that both PRMT5 and EZH2 had shown more than 90% amplification in breast cancer alone. We screened an array of phytocompounds towards the inhibition of PRMT5 and EZH2 using in silico, in vitro assays. Among them Epigallocatechin-3-gallate (EGCG) has interacted with human PRMT5: MEP50 and EZH2 efficiently. The EGCG interacted within the SAM binding site, with a {pi}-cation interaction at Lys 333 and H-bonds with Tyr324, Tyr334, Gly365, Leu437, and Glu444. Surface plasmon resonance analysis revealed that EGCG has strong binding affinity in nanomolar concentrations with both PRMT5-MEP50 than EZH2. Further in vitro methylation and cell-based assays proved the inhibitory potential of EGCG by reducing the catalytic products of PRMT5 and EZH2 i.e., H4R3me2s & H3K27me3 respectively and showed that it induced autophagy and apoptosis. Furthermore in vivo, mouse xenografts studies demonstrated that oral dosage, reduced tumor size significantly with reduction in proliferation marker Ki67 and these histone repressive marks. Finally conclude that inhibition of PRMT5 and EZH2 by EGCG potentially can be used to develop combined therapeutic approaches.

cancer biology↗

Experimental evolution of Pseudomonas aeruginosa to colistin in spatially confined microdroplets identifies evolutionary trajectories consistent with adaptation in microaerobic lung environments

Antibiotic resistance is a continuing global health crisis. Identifying the evolutionary trajectories leading to increased antimicrobial resistance can be critical to the discovery of biomarkers for clinical diagnostics and new targets for drug discovery. While the combination of patient data and in vitro experimental evolution has been remarkably successful in extending our understanding of antimicrobial resistance, it can be difficult for in vitro methods to recapitulate the spatial structure and consequent microenvironments that characterize in vivo infection. Notably, in cystic fibrosis (CF) patients, changes to either the PmrA/PmrB or PhoP/PhoQ two-component systems have been identified as critical drivers for high levels of colistin and polymyxin resistance. When using microfluidic emulsions to provide spatially structured, low-competition environments, we found that adaptive mutations to phoQ were more successful than pmrB in increasing colistin resistance. Conversely, mutations to pmrB were readily identified using well-mixed unstructured cultures. We found that oxygen concentration gradients within the microdroplet emulsions favored adaptive changes to the PhoP/PhoQ pathway consistent with microaerobic conditions that can be found in the lungs of CF patients. We also observed mutations linked to hallmark adaptations to the CF lung environment, such as loss of motility (fleQ, fliC, fleS, flg, flh, and fleQ) and loss of O antigen biosynthesis (wbpL). Mutation to wbpL, in addition to causing loss of O antigen, was additionally shown to confer moderately increased colistin resistance. Taken together, our data suggest that distinct evolutionary trajectories to colistin resistance may be shaped by the microaerobic partitioning and spatial separation imposed within the CF lung. ImportanceAntibiotic resistance remains one of the great challenges confronting public health in the world today. Individuals with compromised immune systems or underlying health conditions are often at an increased for bacterial infections. Patients with Cystic Fibrosis (CF) produce thick mucus that clogs airways and provides a very favorable environment for infection by bacteria that further decrease lung function and, ultimately, mortality. CF patients are often infected by bacteria such as Pseudomonas aeruginosa early in life and experience a series of chronic infections that, over time, become increasingly difficult to treat due to increased antibiotic resistance. Colistin is a major antibiotic used to treat CF patients. Clinical and laboratory studies have identified PmrA/PmrB and PhoP/PhoQ as responsible for increased resistance to colistin. Both have been identified in CF patient lungs, but why, in some cases, is it one and not the other? In this study, we show that distinct evolutionary trajectories to colistin resistance may be favored by the microaerobic partitioning found within the damaged CF lung.

microbiology↗

Mechanical regulation of retinal vascular inflammation and degeneration in diabetic retinopathy

Vascular inflammation is known to cause degeneration of retinal vessels in early diabetic retinopathy (DR). Past studies investigating these diabetes-induced vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in DR are also mechanically regulated by retinal vascular stiffening that is caused by overexpression of collagen-crosslinking enzyme lysyl oxidase (LOX) in retinal vessels. Treatment of diabetic mice with LOX inhibitor BAPN prevented the increase in retinal vascular stiffness, vascular ICAM-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal vascular degeneration and the diabetes-induced loss of contrast sensitivity in these mice. Finally, we show that increasing substrate stiffness alone increases the adhesiveness and neutrophil elastase-induced death of cultured retinal endothelial cells. By uncovering a link between LOX-dependent vascular stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer unique insights into DR pathogenesis that has important translational potential.

pathology↗

Lysyl oxidase-dependent subendothelial matrix stiffening promotes RAGE-mediated retinal endothelial activation in diabetes

Endothelial cell (EC) activation is a crucial determinant of retinal vascular inflammation associated with diabetic retinopathy (DR), a major microvascular complication of diabetes. We previously showed that, similar to abnormal biochemical factors, aberrant mechanical cues in the form of lysyl oxidase (LOX)-dependent subendothelial matrix stiffening also contribute significantly to retinal EC activation in diabetes. Yet, how LOX is itself regulated and precisely how it mechanically controls retinal EC activation in diabetes is poorly understood. Here we show that high glucose-induced LOX upregulation in human retinal ECs (HRECs) is mediated by proinflammatory RAGE (receptor for advanced glycation end products/AGEs). HRECs treated with methylglyoxal (MGO), an active precursor to the AGE MG-H1, exhibited LOX upregulation that was blocked by a RAGE inhibitor, thus confirming the ability of RAGE to promote LOX expression. Crucially, as a downstream effector of RAGE, LOX was found to mediate both the proinflammatory and matrix remodeling effects of MGO/RAGE, primarily through its ability to crosslink/stiffen matrix. Finally, using decellularized HREC-derived matrices and a mouse model of diabetes, we demonstrate that LOX-dependent matrix stiffening feeds back to enhance RAGE, thereby achieving its autoregulation and proinflammatory effects. These fresh insights into the regulation and proinflammatory role of LOX-dependent mechanical cues may help identify new therapeutic targets to block AGE/RAGE signaling in DR.

pathology↗

Cell Stiffening Contributes to Complement-mediated Injury of Choroidal Endothelial Cells in Early AMD

Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet, no therapies exist for [~]85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigment epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of small GTPases Rac and Rho because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential.

pathology↗

Effect of Leaf Aqueous Extract of Manilkara hexandra on Digestive and Glucose Metabolic Enzymes, and Serum Glucose Level in Labeo rohita Fingerlings

Manilkara hexandra (Roxb.) Dubard (common name Khirni, family: Sapotaceae) is an evergreen traditionally used medicinal plant. The present study aimed to see the effects of leaf aqueous extract of M. hexandra (LAEMH) on digestive and glucose metabolic enzyme action and serum glucose level in rohu, Labeo rohita, fingerlings. Experimental fish were fed a basal diet (Group I), and diets supplemented with LAEMH at 300 mg (Group II) and 600 mg (Group III) kg-1body weight. A significant reduction in serum glucose was noticed in the treated groups when compared with the control group. The reduced amylase activity was noticed in the treated fingerlings in a dose-dependent manner. However, lipase and protease activities didnt differ significantly among the experimental groups. Reduced serum glucose level might be correlated with the decline in the activity of digestive amylase in fish. Group II (3.35{+/-}0.19 U) and Group III (3.49{+/-}0.13 U) were recorded with reduced glucose-6-phosphatase activities than the control group (4.4{+/-}0.39 U). Moreover, the study revealed a decline in fructose-1,6-phosphatase activities in the treated groups in comparison to the control group. The decline in the activities of the metabolic enzymes might be associated with the non-availability of glucose owing to reduced activity of the digestive amylase in the treated groups. In conclusion, the present study established the hypoglycaemic effect of the leaf aqueous extract of M. hexandra in a fish model.

pharmacology and toxicology↗

A toxin-antitoxin system associated transcription factor of Caulobacter crescentus can influence cell cycle-regulated gene expression during the SOS response

Toxin-antitoxin (TA) systems are widespread in bacterial chromosomes but their functions remain enigmatic. Although many are transcriptionally upregulated by stress conditions, it is unclear what role they play in cellular responses to stress and to what extent the role of a given TA system homologue varies between different bacterial species. In this work we investigate the role of the DNA damage-inducible TA system HigBA of Caulobacter crescentus in the SOS response and discover that in addition to the toxin HigB affecting cell cycle gene expression through inhibition of the master regulator CtrA, HigBA possesses a transcription factor third component, HigC, which both auto-regulates the TA system and acts independently of it. Through HigC, the system exerts downstream effects on antibiotic (ciprofloxacin) resistance and cell cycle gene expression. HigB and HigC had inverse effects on cell cycle gene regulation, with HigB reducing and HigC increasing the expression of CtrA-dependent promoters. Neither HigBA nor HigC had any effect on formation of persister cells in response to ciprofloxacin. Rather, their role in the SOS response appears to be as transcriptional and post-transcriptional regulators of cell cycle-dependent gene expression, transmitting the status of the SOS response as a regulatory input into the cell cycle control network via CtrA. ImportanceAlmost all bacteria respond to DNA damage by upregulating a set of genes that helps them to repair and recover from the damage, known as the SOS response. The set of genes induced during the SOS response varies between species, but frequently includes toxin-antitoxin systems. However, it is unknown what the consequence of inducing these systems is, and whether they provide any benefit to the cells. We show here that the DNA damage-induced TA system HigBA of the asymmetrically dividing bacterium Caulobacter crescentus affects the cell cycle regulation of this bacterium. HigBA also has a transcription factor encoded immediately downstream of it, named here HigC, which controls expression of the TA system and potentially other genes as well. Therefore, this work identifies a new role for TA systems in the DNA damage response, distinct from non-specific stress tolerance mechanisms which had been proposed previously.

microbiology↗