Biology subjects

Singh, G.

Publications and source records attributed to Singh, G..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

MK2 Expression Promotes Non-Small Cell Lung Cancer Cell Death and Predicts Survival

Non-small cell lung cancers demonstrate intrinsic resistance to cell death even in response to chemotherapy. Previous work suggested that defective nuclear translocation of active caspase 3 may play a role in resistance to cell death. Separately, our group has identified that mitogen activated protein kinase activated protein kinase 2 (MK2) is required for nuclear translocation of active caspase 3 in the execution of apoptosis. This study demonstrates a relatively low expression of MK2 in non-small cell lung carcinoma cell lines compared to small cell carcinoma cell lines. Further, overexpression of MK2 in non-small cell lung carcinoma cell lines results in increased caspase 3 activity and caspase 3 mediated cell death. Higher MK2 transcript levels were observed in patients with earlier-stage non-small cell lung cancer. Higher expression of MK2 is associated with better survival in patients with early stage non-small cell lung cancer across two independent clinical datasets. Using data sets spanning multiple cancer types, we observed improved survival with higher MK2 expression was unique to lung adenocarcinoma. Mechanistically, MK2 promotes nuclear translocation of caspase 3 leading to PARP1 cleavage and execution of cell death. While MK2 can directly phosphorylate caspase 3, neither phosphorylation status of caspase 3 nor the kinase activity of MK2 impacts caspase 3 activation, nuclear translocation and execution of cell death. Rather, a non-kinase function of MK2, specifically trafficking via its nuclear localization sequence, is required for caspase 3 mediated cell death. In summary this study highlights the importance of a non-enzymatic function of MK2 in the execution of apoptosis, which may be leveraged in the adjunctive treatment of NSCLC or other conditions where regulation of apoptosis is crucial.

cancer biology↗

bioRxiv2021

Population Dynamics of Hybrid State during Adaptive Therapy in Cancer

Drug resistance emerges due to drug-induced phenotypic switching of drug-sensitive to drug-resistant subpopulations in cancer during therapy. Existing models indicate the competitive advantage of sensitive over resistant population to regulate tumor and reducing the treatment cost with increased time to progression of tumor ultimately benefiting the patient in a clinical setting. Here, we present a Lotka Volterra (LV) based population dynamics (PD) model of the drug-sensitive, drug-resistant, and transient drug-hybrid state along with phenotypic switching during adaptive therapy based on a simple cancer biomarker (CB) to decide the adaptive therapy dosage to regulate cancer. We identified that the strength of intra competition along with phenotypic switching parameters is crucial to mediate the effectiveness of adaptive therapy and also investigated the significance of the initial fraction of subpopulations on AT. We hypothesize and predict the dynamics of drug-induced transient hybrid state playing a key role in the cancer cells undergoing metastasis.

cancer biology↗

bioRxiv2021

Unravelling consensus genomic regions associated with quality traits in wheat (Triticum aestivum L.) using meta-analysis of quantitative trait loci

A meta-analysis of quantitative trait loci (QTLs) associated with following six major quality traits (i) arabinoxylan, (ii) dough rheology properties, (iii) nutritional traits, (iv) polyphenol content, (v) processing quality traits, and (vi) sedimentation volume was conducted in wheat. For this purpose, as many as 2458 QTLs were collected from the 50 mapping studies published during 2013-20. Of the total QTLs, 1126 QTLs were projected on to the consensus map saturated with 2,50,077 markers resulting into the identification of 110 meta-QTLs (MQTLs) with average confidence interval (CI) of 5.6 cM. These MQTLs had 18.84 times reduced CI compared to CI of initial QTLs. Fifty-one (51) MQTLs were also verified with the marker-trait associations (MTAs) detected in earlier genome-wide association studies (GWAS). Physical region occupied by a single MQTL ranged from 0.12 to 749.71 Mb with an average of 130.25 Mb. Candidate gene mining allowed the identification of 2533 unique gene models from the MQTL regions. In-silico expression analysis discovered 439 differentially expressed gene models with >2 transcripts per million (TPM) expression in grains and related tissues which also included 44 high-confidence candidate genes known to be involved in the various cellular and biochemical processes related to quality traits. Further, nine functionally characterized wheat genes associated with grain protein content, high molecular weight glutenin and starch synthase enzymes were also found to be co-localized with some of the MQTLs. In addition, synteny analysis between wheat and rice MQTL regions identified 23 wheat MQTLs syntenic to 16 rice MQTLs. Furthermore, 64 wheat orthologues of 30 known rice genes were detected in 44 MQTL regions. These genes encoded proteins mainly belonging to the following families: starch synthase, glycosyl transferase, aldehyde dehydrogenase, SWEET sugar transporter, alpha amylase, glycoside hydrolase, glycogen debranching enzyme, protein kinase, peptidase, legumain and seed storage protein enzyme. Main ConclusionMeta-QTL analysis in wheat for major quality traits identified 110 MQTLs with reduced confidence interval. Candidate gene mining and expression analysis discovered differentially expressed genes involve in quality traits.

genetics↗

bioRxiv2021

Ab initio modelling of an essential mammalian protein: Transcription Termination Factor 1 (TTF1)

Transcription Termination Factor 1 (TTF1) is an essential mammalian protein that regulates cellular transcription, replication fork arrest, DNA damage repair, chromatin remodelling etc. TTF1 interacts with numerous cellular proteins to regulate various cellular phenomena, and plays a crucial role in maintaining normal cellular physiology, dysregulation of which has been reported towards cancerous transformation of the cells. However, despite its key role in cellular physiology, the complete structure of human TTF1 has not been elucidated to date, either experimentally or computationally. Hence, understanding the structure of human TTF1 becomes highly important for studying its functions and interactions with other cellular factors. Therefore, the aim of this study was to construct the complete structure of human TTF1 protein, using molecular modelling approaches. Owing to the lack of suitable homologues in the PDB, the complete structure of human TTF1 was constructed using ab initio modelling. The structural stability was determined using molecular dynamics (MD) simulations in explicit solvent, and trajectory analyses. The representative structure of human TTF1 was obtained by trajectory clustering, and the central residues were determined by centrality analyses of the residue interaction network of TTF1. Two residue clusters, in the oligomerisation domain and C-terminal domain, were determined to be central to the structural stability of human TTF1. To the best of our knowledge, this study is the first to report the complete structure of human TTF1, and the results obtained herein will provide structural insights for future research in cancer biology and related studies. Author SummaryThe transcription termination factor 1 (TTF1) is an essential multifunctional mammalian protein which plays important role in regulating important cellular process like transcription, replication, DNA damage repair, chromatin remodelling etc. and its dysregulation leads to various cancers. Despite its being such an important factor, the complete structure of human TTF1 has not been determined to date, either using experimental techniques or computationally. Therefore, the aim of this study was to construct the complete structure of human TTF1 using computational modelling. In this study the complete structure of human TTF1 was constructed by ab initio modelling using iTasser. The stability of this model was determined by 200 ns molecular dynamics (MD) simulations. The representative conformation of human TTF1 was further determined by clustering the simulation trajectory and the residues that are central to the stability of this structure were identified. The results demonstrate the presence of two residue clusters in human TTF1, one in the oligomerisation domain and other in the C-terminal domain, which were found to be crucial for the structural stability of this protein. Hence, the results of this study will aid future studies in this field towards engineering this important protein for further biochemistry and cell biology research.

bioinformatics↗

bioRxiv2021

Prioritizing Management for Cumulative Impacts

Determining where environmental management is best applied, either through regulating single sectors of human activities or across sectors, is complicated by interactions between human impacts and the environment. In this article, we show how an explicit representation of human-environment interactions can help, via "impact networks" including activities (e.g. shipping), stressors (e.g. ship strikes), species (e.g. humpback whales) or ecosystem services (e.g. marine recreation). Impact networks can enable the identification of "leverage nodes", which, if present, can direct managers to the activities and stressors crucial for reducing risk to important ecosystem components. Exploring an impact network for a coastal ecosystem in British Columbia, Canada, we seek to identify these leverage nodes using a new approach employing Bayesian Belief Networks of risks to ecosystems. In so doing, we address three key questions: (1) Do leverage nodes exist? (2) Do management plans for species correctly identify leverage nodes? (3) Does the management of leverage nodes for certain species realize benefits for other species and ecosystem services? We show that there are several leverage nodes across all species investigated, and show that preconceptions about the regulation of risk to species can misidentify leverage nodes, potentially leading to ineffective management. Notably, we show that managing fisheries does not reduce overall risk to herring whereas managing diverse cumulative impacts including nutrient runoff, oil spills, and marine debris can reduce risk to herring, additional species, and related ecosystem services. Thus, by targeting leverage nodes, managers can efficiently mitigate risks for whole communities, ecosystems, and ecosystem services.

ecology↗

bioRxiv2021

Beneficial effects of two Ayurvedic formulations, Saraswata Ghrita and Kalyanaka Ghrita on survival and on toxic aggregates in Drosophila models of Huntington's and Alzheimer's disease

In order to understand the health promotive, rejuvenative and disease preventive approach of the Ayurvedic system of medicine in the light of current principles, we examined two Rasayana formulations, viz., Kalayanaka Ghrita (KG) and Saraswata Ghrita (SG) for their effects in Alzheimers (AD) and Huntingtons (HD) neurodegenerative disease models of Drosophila. Initial experiments involving feeding of wild type flies on food supplemented with 0.05%, 0.25% and 0.5% (w/v) KG or SG revealed 0.05% to be without any adverse effect while higher concentrations caused dose-dependent reduction in pupation frequency and adult life span in wild type flies. Rearing GMR-GAL4>127Q (HD model) and ey-GAL4>A{beta}42 (AD model) larvae and adults on 0.05% or 0.25% SG or KG supplemented food enhanced the otherwise significantly reduced larval lethality and enhanced their median life span, with the 0.25% SG or KG concentrations being less effective than the 0.05%. In parallel with the better larval survival and enhanced adult life span, feeding the HD and AD model larvae on either of the Ghrita supplemented food (0.05% and 0.25%) substantially reduced the polyQ aggregates or amyloid plaques, respectively, in the larval eye discs. The present first in vivo organismic model study results have clinical implications for the increasing burden of age-associated dementia and neurodegenerative diseases like AD and HD in human populations.

neuroscience↗

bioRxiv2021

Mechanistic Model of Replication Fork Progression in Yeast

Replication fork progression complex plays an essential role during DNA replication. It travels along with the DNA with a particular speed called replication fork speed. Faithful duplication of the genome requires strict control over replication fork speed. Both acceleration and pausing mechanisms of the replication fork complex are regulated at the molecular level. Based on the experimental evidence, DNA replicates faster in normal cells than cancer cells, whereas cancer cells duplicate themselves more quickly than normal cells. Then in principle, accelerating the replication fork complex in cancer cells beyond a specific threshold speed limit can cause DNA damage and plausibly kill them. A modular mathematical model is proposed to explain the dynamics of replication fork control during DNA replication using the underlying molecular mechanisms in yeast which can extend to the mammalian system in the future.

systems biology↗

bioRxiv2021

Mechanistic Model of Telomere Length Homeostasisin Yeast

Cells maintain homeostatic telomere length, and this homeostatic disruption leads to various types of diseases. Presently, it is not clear how telomeres achieve homeostasis. One of the prevailing hypotheses is a protein-counting model with a built-in sensor mechanism that counts proteins that directly regulate the telomeric length. However, it does not explain telomere length regulation at the mechanistic level. Here, we present a mathematical model based on the underlying molecular mechanisms of length regulation needed to establish telomere length homeostasis in yeast. We perform both deterministic and stochastic simulations to validate the models with the experimental data of Teixeira et al., rate-balance plot, and phase plane analysis to understand the nature of dynamics exhibited by the models. For global analysis, we constructed bifurcation diagrams. The model explains the role of negative and positive feedback loops and a delay between telomerase and telomere-bound proteins, leading to oscillations in telomere length. We map these in-silico results to Teixeiras proposition of telomeres making a transition between extendible and non-extendible states.

systems biology↗

bioRxiv2021

Depside and depsidone synthesis in lichenized fungi comes into focus through a genome-wide comparison of the olivetoric and physodic acid chemotype of Pseudevernia furfuracea

Primary biosynthetic enzymes involved in the synthesis of lichen polyphenolic compounds depsides and depsidones are Non-Reducing Polyketide Synthases (NR-PKSs), and cytochrome P450s (CytP450). However, for most depsides and depsidones the corresponding PKSs are unknown. Additionally, in non-lichenized fungi specific fatty acyl synthases (FASs) provide starters to the PKSs. Yet, the presence of such FASs in lichenized fungi remains to be investigated. Here we implement comparative genomics and metatranscriptomics to identify the most likely PKS and FASs for the synthesis of olivetoric and physodic acid, the primary depside and depsidone defining the two chemotypes of the lichen Pseudevernia furfuracea. We propose that the gene cluster PF33-1_006185, found in both chemotypes, is the most likely candidate for olivetoric and physodic acid biosynthesis. This is the first study to identify the gene cluster and the FAS likely responsible for physodic and olivetoric acid biosynthesis in a lichenized fungus. Our findings suggest that gene regulation and other epigenetic factors determine whether the mycobiont produces the depside or the depsidone, providing the first direct indication that chemotype diversity in lichens can arise through regulatory and not only through genetic diversity. Combining these results and existing literature, we propose a detailed scheme for depside/depsidone synthesis.

genomics↗

bioRxiv2021

Generation of tumors in wing imaginal discs of Drosophila melanogaster third instar larvae to study temporal and spatial patterns of expression of major heat shock proteins

Cancer cells generally overexpress heat shock proteins (Hsps), the major components of cellular stress response, to overcome and survive the diverse stresses. However, the specific roles of Hsps in initiation and establishment of cancers remain unclear. Using loss of Lgl-mediated epithelial tumorigenesis in Drosophila, we induced tumorigenic somatic clones of different genetic backgrounds to examine the temporal and spatial expression and roles of major heat shock proteins in tumor growth. The constitutively expressed Hsp83, Hsc70 (heat shock cognate), Hsp60 and Hsp27 show elevated levels in all cells of the tumorigenic clone since early stages, which persists till their transformation. However, the stress-inducible Hsp70 is expressd only in a few cells at later stage of established tumorous clones that show high F-actin aggregation. Intriguingly, levels of heat shock factor (HSF), the master regulator of Hsps, remain unaltered in these tumorous cells and its down-regulation does not affect tumorigenic growth of lgl- clones overexpressing Yorkie, although down-regulation of Hsp83 prevents their survival and growth. Interestingly, overexpression of HSF or Hsp83 in lgl- cells makes them competitively successfully in establishing tumorous clones. These results show that the major constitutively expressed Hsps, but not the stress-inducible Hsp70, are involved in early as well as late stages of epithelial tumors and their elevated expression in lgl- clones co-overexpressing Yorkie is independent of HSF.

cancer biology↗

bioRxiv2021

Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains

Although mRNA vaccines prevent COVID-19, variants jeopardize their efficacy as immunity wanes. Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike) or modified (mRNA-1273.351, designed for B.1.351 spike) preclinical Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Immunization with high or low dose formulations of mRNA vaccines induced neutralizing antibodies in serum against ancestral SARS-CoV-2 and several variants, although levels were lower particularly against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. Nonetheless, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, and serve as a possible model for waning immunity, showed breakthrough lung infection and pneumonia with B.1.617.2. Thus, as levels of immunity induced by mRNA vaccines decline, breakthrough infection and disease likely will occur with some SARS-CoV-2 variants, suggesting a need for additional booster regimens.

microbiology↗

bioRxiv2021

Mammalian UPF3A and UPF3B activate NMD independently of their EJC binding

Nonsense-mediated mRNA decay (NMD) is governed by the three conserved factors - UPF1, UPF2 and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, with its paralog UPF3A suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here we characterize the UPF3B-dependent and -independent NMD in human cell lines knocked-out of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, EJC-mediated NMD can operate in UPF3B-dependent and -independent manner. While UPF3A is almost completely dispensable for NMD in wild-type cells, it strongly activates EJC-mediated NMD in cells lacking UPF3B. Surprisingly, this major NMD branch can operate in UPF3-independent manner questioning the idea that UPF3 is needed to bridge UPF proteins to the EJC during NMD. Complementation studies in UPF3 knockout cells further show that EJC-binding domain of UPF3 paralogs is not essential for NMD. Instead, the conserved mid domain of UPF3B, previously shown to engage with ribosome release factors, is required for its full NMD activity. Altogether, UPF3 plays a more active role in NMD than simply being a bridge between the EJC and the UPF complex.

molecular biology↗

bioRxiv2021

TOR represses stress responses through global regulation of H3K27 trimethylation in plants

Target of Rapamycin (TOR) functions as a central sensory hub to link a wide range of external stimuli to gene expression. However, the mechanisms underlying stimulus-specific transcriptional reprogramming by TOR remains elusive. Our in silico analysis in Arabidopsis demonstrates that TOR-repressed genes are associated with either bistable or silent chromatin states. Both states regulated by TOR signaling pathway are associated with high level of H3K27me3 deposited by CURLY LEAF (CLF) in specific context with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Combinations of epigenetic modifications H3K4me3 and H3K27me3 implicate bistable feature which alternates between on and off state allowing rapid transcriptional changes upon external stimuli. Chromatin remodeler SWI2/SNF2 ATPase BRAHMA (BRM) activates TOR-repressed genes only at bistable chromatin domains to rapidly induce biotic stress responses. Here we demonstrated both in silico and in vivo that TOR represses transcriptional stress responses through global maintenance of H3K27me3.

plant biology↗

bioRxiv2020

Can crop phenology and plant height be channelized to improvise wheat productivity in diverse production environments?

Non-grain parameters like height, flowering and maturity should also be tried to break yield plateau in wheat. This study explores such possibilities by analysing performance of released and pre-released varieties evaluated in ten diverse production environments of India during the period 2000-2020. Regression analysis supports relevance of such non-grain determinants in grain yield under every environment but magnitude of impact can vary. Collective contribution of non-grain parameters can be high in a production environment where growth condition is most favourable for wheat growth and every factor is important in such situations. They contribute less in the environments engrossed with abiotic stress and merely one or two factors can be earmarked for selection. Besides yield, this selection strategy can also enhance grain weight in certain environments. At a time when selection for grain attributes is not providing further push; it would be worth trying to explore these non-grain field indicators as selection strategy for further advancement in productivity and grain weight of bread wheat.

developmental biology↗

bioRxiv2020

A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD up, two-RBD down state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFN{gamma}+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).

immunology↗

bioRxiv2020

Integrating deep learning with microfluidics for biophysical classification of sickle red blood cells

Sickle cell disease (SCD), a group of inherited blood disorders with significant morbidity and early mortality, affects a sizeable global demographic largely of African and Indian descent. It is manifested in a mutated form of hemoglobin that distorts the red blood cells into a characteristic sickle shape with altered biophysical properties. Sickle red blood cells (sRBCs) show heightened adhesive interactions with inflamed endothelium, triggering obstruction of blood vessels and painful vaso-occlusive crisis events. Numerous studies have reported microfluidic-assay-based disease monitoring tools which rely on quantifying adhesion characteristics of adhered sRBCs from high resolution channel images. The current workflow for analyzing images from these assays relies on manual cell counting and detailed morphological characterization by a specially trained worker, which is time and labor intensive. Moreover manual counts by different individuals are prone to artifacts due to user bias. We present here a standardized and reproducible image analysis workflow designed to tackle these issues, using a two part deep neural network architecture that works in tandem for automatic, fast and reliable segmentation and classification into subtypes of adhered cell images. Our training utilized an exhaustive data set of images generated by the SCD BioChip, a microfluidic assay which injects clinical whole blood samples into protein-functionalized microchannels, mimicking physiological conditions in the microvasculature. The automated image analysis performs robustly in comparison to human classification: accuracies were similar to or better than those of the trained personnel, while the overall analysis time was improved by two orders of magnitude.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Cross talk between tumor stroma and cancer cells plays a critical role in progressive enrichment of cancer stem cell phenotype in primary breast tumors

In order to delineate the underlying molecular mechanisms responsible for intra tumoral enrichment of BCSCs in aggressive breast tumors, firstly we evaluated the frequency and characteristics of breast cancer stem cells (BCSCs) within the tumor mass as well as in pathologically normal adjacent tissues in primary breast carcinomas of various clinical and histological grades. Then, we evaluated the expression profiles of various genes in non-cancer stem cells from these tumors to delineate the role played by cellular niche in de novo origin and/or expansion of intra-tumoral cancer stem cells. The study included primary tumor and adjacent normal breast tissue specimens from chemotherapy-naive breast carcinoma patients. The BCSCs, identified as Lin-CD44+CD24- and aldehyde dehydrogenase 1 A1 positive were enumerated. The frequency of intra-tumoral BCSCs was correlated with various clinicopathological parameters of breast cancer. The flow-cytometrically sorted stromal cells and cancer cells from treatment naive primary breast tumors were processed for gene expression profiling using a custom designed PCR array of genes known to facilitate cancer cell proliferation and disease progression. The frequency of BCSCs within the tumor mass as well as in the adjacent normal tissue correlated significantly with histopathological and molecular grades of tumors indicating a direct relationship of BCSC with aggressive behavior of breast cancer. A significantly higher number of BCSCs was also detected in metastatic LN group as compared to non-metastatic LN. Further, a significantly increased expression of the genes associated with growth factors, cytokines & matricellular proteins in tumors with high BCSCs content (> 5%; Hi-BCSCs tumors) as compared to Lo-BCSC tumors (with <5% intratumoral BCSC content) suggested the possible contribution of stromal cells and cancer cells in intra-tumoral expansion of CSCs. Similarly, a significant up-regulation of genes associated with hypoxia and angiogenesis in Hi-BCSCs tumors further supported the role of hypoxic environment. The expression levels of genes associated with epithelial to mesenchymal transition also followed a similar pattern. On the other hand, downregulated SNAI1 gene (generally upregulated in onset of EMT) in stromal cells of Hi-BCSCs tumors suggests a post EMT environment in Hi-BCSCs tumors. The findings suggest that the molecular crosstalk between the non-BCSC niche cells and the cancer stem cells within the breast cancer microenvironment directly contribute to formation of biologically conducive conditions for expansion of cancer stem cells.

cancer biology↗

bioRxiv2020

CYCLODEXTRIN DERIVATIVE ENHANCES THE OPHTHALMIC DELIVERY OF POORLY SOLUBLE AZITHROMYCIN.

Azithromycin (AZM), a macrolide antibiotic used for the treatment of Chlamydial conjunctivitis, is less effective for the treatment of this disease due to its poor bioavailability (38%). Various alternatives have been developed for improving the physico-chemical properties (i.e., solubility) of the AZM without much success. To overcome the problems associated with AZM, an inclusion complex employing a modified cyclodextrin i.e., sulfobutylether-{beta}-cyclodextrin (SBE-{beta}-CD) was prepared and characterized by phase solubility studies, pXRD and FTIR techniques. The results portrayed the formation of the inclusion complex of AZM with sulfobutylether {beta}-cyclodextrin (SBE-{beta}-CD) in 1:2 molar stoichiometric ratios. This inclusion complex was later incorporated into a polymer matrix to prepare an in situ gel. Various combinations of carbopol 934P and hydroxypropyl methylcellulose (HPMC K4M) polymers were used and evaluated by rheological and in vitro drug release studies. The optimized formulation (F4), containing carbopol 934P 0.2% (w/v) and HPMC K4M 0.2% (w/v), was evaluated for clarity, pH, gelling capacity, drug content, rheological properties, in vitro drug release pattern, ocular irritation test and antimicrobial efficacy. Finally, owing to the improved antimicrobial efficacy and increased residence time, AZM:SBE-{beta}-CD in situ gel was found to be a promising formulation for the efficient treatment of bacterial ocular disease.

microbiology↗