Computational exploration of novel antimicrobial modalities targeting fucose-binding lectins and ribosomes in Mycobacterium smegmatis using tRNA-encoded peptides.

tRNA-Encoded Peptides (tREPs), encoded by small open reading frames (smORFs) within tRNA genes, have recently emerged as a new class of functional peptides exhibiting antiparasitic activity. The discovery of tREPs has led to a re-evaluation of the role of tRNAs in biology and has expanded our understanding of the genetic code. This presents an immense, unexplored potential in the realm of tRNA-peptide interactions, paving the way for groundbreaking discoveries and innovative applications in various biological functions. This study explores the antimicrobial potential of tREPs against protein targets by employing a computational method that uses verified data sources and highly recognized predictive algorithms to provide a sorted list of likely antimicrobial peptides, which were then filtered for toxicity, cell permeability, allergenicity and half-life. These peptides were then docked with screened protein targets and computationally validated using molecular dynamics (MD) simulations for 150 ns and the binding free energy was estimated. The peptides Pep2 (VVLWRKPRVRKTG) and Pep6 (HRLRLRRRKPWW) exhibited good binding affinities of -110.5 +/- 2.5 and -129.0 +/- 3.9, respectively, with RMSD values of 0.4 and 0.25 nm against the fucose-binding lectin (7NEF) and the 30S ribosome of Mycobacterium smegmatis (5O5J) protein targets. The 7NEF-Pep2 and 5O5J-Pep6 complexes indicated higher negative binding free energies of -52.55 kcal/mol and -55.52 kcal/mol respectively, as calculated by Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA). Thus, the tREPs derived peptides designed as a part of this study, provide novel approaches for potential anti-bacterial therapeutic modalities.Communicated by Ramaswamy H. Sarma.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Independent component analysis reveals 49 independently modulated gene sets within the global transcriptional regulatory architecture of multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii causes severe infections in humans, resists multiple antibiotics, and survives in stressful environmental conditions due to modulations of its complex transcriptional regulatory network (TRN). Unfortunately, our global understanding of the TRN in this emerging opportunistic pathogen is limited. Here, we apply independent component analysis, an unsupervised machine learning method, to a compendium of 139 RNA-seq data sets of three multidrug-resistant A. baumannii international clonal complex I strains (AB5075, AYE, and AB0057). This analysis allows us to define 49 independently modulated gene sets, which we call iModulons. Analysis of the identified A. baumannii iModulons reveals validating parallels to previously defined biological operons/regulons and provides a framework for defining unknown regulons. By utilizing the iModulons, we uncover potential mechanisms for a RpoS-independent general stress response, define global stress-virulence trade-offs, and identify conditions that may induce plasmid-borne multidrug resistance. The iModulons provide a model of the TRN that emphasizes the importance of transcriptional regulation of virulence phenotypes in A. baumannii. Furthermore, they suggest the possibility of future interventions to guide gene expression toward diminished pathogenic potential.IMPORTANCEThe rise in hospital outbreaks of multidrug-resistant Acinetobacter baumannii infections underscores the urgent need for alternatives to traditional broad-spectrum antibiotic therapies. The success of A. baumannii as a significant nosocomial pathogen is largely attributed to its ability to resist antibiotics and survive environmental stressors. However, there is limited literature available on the global, complex regulatory circuitry that shapes these phenotypes. Computational tools that can assist in the elucidation of A. baumannii's transcriptional regulatory network architecture can provide much-needed context for a comprehensive understanding of pathogenesis and virulence, as well as for the development of targeted therapies that modulate these pathways.

Comparative molecular profiling of multidrug-resistant Pseudomonas aeruginosa identifies novel mutations in regional clinical isolates from South India.

Objectives: We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. Methods: We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Results: Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (blaNDM-1) within our sample. Conclusions: This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates impacts quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous bacterial virulence phenotypes. Most work on Pf phages has focused on strain Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf strains infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, virulence factor production, and virulence. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates, but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.

Patient, family member, and health care provider perspective on barriers and facilitators to diabetic retinopathy screening in Thailand: A qualitative study.

OBJECTIVES: Diabetic retinopathy (DR) can cause significant visual impairment which can be largely avoided by early detection through proper screening and treatment. People with DR face a number of challenges from early detection to treatment. The aim of this study was to investigate factors that influence DR screening in Thailand and to identify barriers to follow-up compliance from patient, family member, and health care provider (HCP) perspectives. METHODS: A total of 15 focus group discussions (FGDs) were held, each with five to twelve participants. There were three distinct stakeholders: diabetic patients (n = 47) presenting to a diabetic retinopathy clinic in Thailand, their family members (n = 41), and health care providers (n = 34). All focus group conversations were transcribed verbatim. Thematic analysis was used to examine textual material. RESULTS: Different themes emerged from the FGD on knowledge about diabetes, self-care behaviors of diabetes mellitus (DM), awareness about DR, barriers to DR screening, and the suggested solutions to address those barriers. Data showed lower knowledge and awareness about diabetes and DR in both patients and family members. Long waiting times, financial issues, and lack of a person to accompany appointments were identified as the major deterrents for attending DR screening. Family support for patients was found to vary widely, with some patients reporting to have received adequate support while others reported having received minimal support. Even though insurance covered the cost of attending diabetes/DR screening program, some patients did not show up for their appointments. CONCLUSION: Patients need to be well-informed about the asymptomatic nature of diabetes and DR. Communication at the patient level and shared decision-making with HCPs are essential. Family members and non-physician clinicians (such as diabetes nurses, diabetes educators, physician assistants) who work in the field of diabetes play a vital role in encouraging patients to attend diabetes and DR follow-ups visits regularly.

Compliance with follow-up in patients with diabetic macular edema: Eye care center vs. diabetes care center.

Purpose: The study was conducted to compare the compliance to intravitreal injection treatment and follow-up in patients with center-involving diabetic macular edema (CI-DME) and treatment outcomes between a tertiary eye care facility and a tertiary diabetes care center. Methods: A retrospective review was conducted on treatment naïve DME patients who had received intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections in 2019. Participants were people with type 2 diabetes who were under regular care at the eye care center or the diabetes care center in Chennai. The outcome measures were noted at months 1, 2, 3, 6, and 12. Results: A review of 136 patients treated for CI-DME (72 from the eye care center and 64 from a diabetes care center) was carried out. The severity of diabetic retinopathy (DR) was similar in both centers. There was no statistically significant (P > 0.05) difference in the choice of initial intravitreal drug in the two centers. At 12-month follow-up, only 29.16% came for a follow-up in the eye center vs. 76.56% in a diabetes care center (P = 0.000). The multivariate logistic regression showed increasing age was associated with non-compliance in both the groups (eye care center: odds ratio [OR] 0.91; 95% confidence interval [CI] 0.82-1.21; P = 0.044) and diabetes care center (OR 1.15; 95% CI 1.02-1.29; P = 0.020). Conclusion: The follow-up rate between eye care and diabetic care center with DME showed a significant disparity. By providing comprehensive diabetes care for all complications under one roof, compliance with follow-up can be improved in people with DME.

'I don`t need an eye check-up'. A qualitative study using a behavioural model to understand treatment-seeking behaviour of patients with sight threatening diabetic retinopathy (STDR) in India.

Diabetic Retinopathy (DR) affects about 27% of patients with diabetes globally. According to the World Health Organization (WHO), DR is responsible for37 million cases of blindness worldwide. The SMART India study (October 2020-August 2021) documented the prevalence of diabetes, and DR in people40 years and above across ten Indian states and one Union Territory by conducting community screening. About 90% of people with sight threatening diabetic retinopathy (STDR) were referred from this screening study to eye hospitals for management, but failed to attend. This qualitative study, a component of the SMART India study, explored perceptions of referred patients regarding their susceptibility to eye related problems in diabetes and the benefits/barriers to seeking care. Perceived barriers from the viewpoint of ophthalmologists were also explored. Guided by the Health Beliefs Model (HBM), 20 semi structured interviews were carried out with consenting patients diagnosed with STDR. They included nine patients who had sought care recruited from eight eye hospitals across different states in India and eleven patients who did not seek care. Eleven ophthalmologists also participated. Four themes of analysis based on the HBM were, understanding of DR and its treatment, perceptions about susceptibility and severity, perceived barriers, perceived benefits and cues to action. Findings revealed poor understanding of the effects of diabetes on the eye contributing to low risk perception. Prohibitive costs of treatment, difficulties in accessing care services and poor social support were major barriers to seeking care. Ophthalmologists acknowledged that the absence of symptoms and the slow progressive nature of the disease deluded patients into thinking that they were fine. The study attests to the need for greater health literacy around diabetes, DR and STDR; for making treatment more affordable and accessible and for the development of effective patient education and communication strategies towards increasing compliance.

Man vs Microbes - The Race of the Century.

The complexity of the antimicrobial resistance (AMR) crisis and its global impact on healthcare invokes an urgent need to understand the underlying forces and to conceive and implement innovative solutions. Beyond focusing on a traditional pathogen-centric approach to antibiotic discovery yielding diminishing returns, future therapeutic interventions can expand to focus more comprehensively on host-pathogen interactions. In this manner, increasing the resiliency of our innate immune system or attenuating the virulence mechanisms of the pathogens can be explored to improve therapeutic outcomes. Key pathogen survival strategies such as tolerance, persistence, aggregation, and biofilm formation can be considered and interrupted to sensitize pathogens for more efficient immune clearance. Understanding the evolution and emergence of so-called 'super clones' that drive AMR spread with rapid clonotyping assays may guide more precise antibiotic regimens. Innovative alternatives to classical antibiotics such as bacteriophage therapy, novel engineered peptide antibiotics, ionophores, nanomedicines, and repurposing drugs from other domains of medicine to boost innate immunity are beginning to be successfully implemented to combat AMR. Policy changes supporting shorter durations of antibiotic treatment, greater antibiotic stewardship, and increased surveillance measures can enhance patient safety and enable implementation of the next generation of targeted prevention and control programmes at a global level.

In silico based multi-epitope vaccine design against norovirus.

Norovirus (NoV) belongs to the Calciviridae family that causes diarrhoea, vomiting, and stomach pain in people who have acute gastroenteritis (AGE). Identifying multi-epitope dependent vaccines for single stranded positive sense viruses such as NoV has been a long due. Although efforts have been in place to look into the candidate epitopes, understanding molecular mimicry and finding new epitopes for inducing immune responses against the T/B-cells which play an important role for the cell-mediated and humoral immunity was not dealt with in great detail. The current study focuses on identifying new epitopes from various databases that were filtered for antigenicity, allergenicity, and toxicity. The adjuvant ß-defensin along with different linkers were used for vaccine construction. Further, the binding relationship between the vaccine construct and toll-like immune receptor (TLR3) complex was determined using a molecular docking analysis, followed by molecular dynamics simulation of 100 ns. The vaccine candidate developed expresses good solubility with a score of 0.530, Z-score of -4.39 and molecular docking score of -140.4 ± 12.1. The MD trajectories reveal that there is a stability between TLR3 and the developed vaccine candidate with an average of 0.91 nm RMSD value and also the system highest occupancy H-bond formed between GLU127 of TLR3 and TYR10 of vaccine candidate (61.55%). Four more H-bonds exist with an occupancy of more than 32% between TLR3 and the vaccine candidates which makes it stable. Thus, the multi-epitope based vaccine developed in the present study forms the basis for further experimental investigations to develop a potentially good vaccine against NoV.Communicated by Ramaswamy H. Sarma.

Mitigation of quorum sensing mediated virulence factors of Pseudomonas aeruginosa: the role of Meldrum's acid activated furan.

The rapid emergence of drug resistant pathogens is a major threat which has warranted the development of alternative strategies to combat infectious diseases. In this work, we have tested the anti-virulent activity of Meldrum's acid activated furan (MAF) and 1,3-dimethyl barbituric acid activated furan (BAF) against Chromobacterium violaceum and Pseudomonas aeruginosa. It was found that MAF significantly reduced the violacein production and biofilm formation of C. violaceum at sub-inhibitory concentrations. The quorum sensing (QS) regulated virulence factors of P. aeruginosa including biofilm formation, motility, pigment production, and elastase activity were also found to be reduced considerably at sub-inhibitory concentrations of MAF. Additionally, MAF downregulated the expression of genes in the QS circuitry of P. aeruginosa, demonstrating the potential of MAF in lowering the pathogenicity of P. aeruginosa. In silico studies demonstrated the potential of MAF to compete with the signaling molecules of C. violaceum and P. aeruginosa for the QS receptor interaction. In vivo studies using Caenorhabditis elegans demonstrated the anti-pathogenicity of MAF by enhancing the survival of P. aeruginosa-infected C. elegans. These results suggest that activated furan compounds could be potential inhibitors of QS-mediated virulence factors in C. violaceum and P. aeruginosa, encouraging their use in combating multidrug-resistant pathogens.

Increased Innate Immune Susceptibility in Hyperpigmented Bacteriophage-Resistant Mutants of Pseudomonas aeruginosa.

Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa. Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation. During in vitro phage treatment of a P. aeruginosa-type strain, we observed the emergence of phage-resistant mutants with brown pigmentation that was indicative of pyomelanin. As increased pyomelanin (due to hmgA gene mutation) was recently associated with enhanced resistance to hydrogen peroxide and persistence in experimental lung infection, we questioned if therapeutic phage applications could inadvertently select for hypervirulent populations. Pyomelanogenic phage-resistant mutants of P. aeruginosa PAO1 were selected for upon treatment with three distinct phages. Phage-resistant pyomelanogenic mutants did not possess increased survival of pyomelanogenic ΔhmgA in hydrogen peroxide. At the genomic level, large (~300 kb) deletions in the phage-resistant mutants resulted in the loss of ≥227 genes, many of which had roles in survival, virulence, and antibiotic resistance. Phage-resistant pyomelanogenic mutants were hypersusceptible to cationic peptides LL-37 and colistin and were more easily cleared in human whole blood, serum, and a murine infection model. Our findings suggest that hyperpigmented phage-resistant mutants that may arise during phage therapy are markedly less virulent than their predecessors due to large genomic deletions. Thus, their existence does not present a contraindication to using anti-pseudomonal phage therapy, especially considering that these mutants develop drug susceptibility to the familiar FDA-approved antibiotic, colistin.

Systematic understanding of anti-tumor mechanisms of Tamarixetin through network and experimental analyses.

Tamarixetin, a flavonoid derived from Quercetin, was shown to possess anti-cancer properties in various types of cancer. However, the mechanism of action of this compound is not well understood. Observations from reverse docking and network pharmacology analysis, were validated by cell based studies to analyse the chemotherapeutic potential and elucidate the molecular mechanism of action of Tamarixetin in breast cancer. In silico analysis using reverse docking and PPI analysis clearly indicated that out of 35 proteins targeted by Tamarixetin, the top 3 hub genes, namely, AKT1, ESR1 and HSP90AA1, were upregulated in breast tumor tissues and more importantly showed strong negative correlation to breast cancer patient survival. Furthermore, the KEGG pathway analysis showed enrichment of target proteins of Tamarixetin in 33 pathways which are mainly involved in neoplastic signalling. In vitro cell-based studies demonstrated that Tamarixetin could inhibit cell proliferation, induce ROS and reduce mitochondrial membrane potential, leading to cell death. Tamarixetin induced cell cycle arrest at G2/M phase and inhibited the migration as well as the invasion of breast cancer cells. Taken together, the combination of in silico and in vitro approaches used in the present study clearly provides evidence for the chemotherapeutic potential of Tamarixetin in breast cancer.

Mathematical Modeling of Severe Acute Respiratory Syndrome Coronavirus 2 Infection Network with Cytokine Storm, Oxidative Stress, Thrombosis, Insulin Resistance, and Nitric Oxide Pathways.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a systemic disease affecting not only the lungs but also multiple organ systems. Clinical studies implicate that SARS-CoV-2 infection causes imbalance of cellular homeostasis and immune response that trigger cytokine storm, oxidative stress, thrombosis, and insulin resistance. Mathematical modeling can offer in-depth understanding of the SARS-CoV-2 infection and illuminate how subcellular mechanisms and feedback loops underpin disease progression and multiorgan failure. We report here a mathematical model of SARS-CoV-2 infection pathway network with cytokine storm, oxidative stress, thrombosis, insulin resistance, and nitric oxide (NO) pathways. The biochemical systems theory model shows autocrine loops with positive feedback enabling excessive immune response, cytokines, transcription factors, and interferons, which can imbalance homeostasis of the system. The simulations suggest that changes in immune response led to uncontrolled release of cytokines and chemokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNFα), and affect insulin, coagulation, and NO signaling pathways. Increased production of NETs (neutrophil extracellular traps), thrombin, PAI-1 (plasminogen activator inhibitor-1), and other procoagulant factors led to thrombosis. By analyzing complex biochemical reactions, this model forecasts the key intermediates, potential biomarkers, and risk factors at different stages of COVID-19. These insights can be useful for drug discovery and development, as well as precision treatment of multiorgan implications of COVID-19 as seen in systems medicine.

A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates.

Multidrug-resistant community-acquired infections caused by the opportunistic human pathogen Pseudomonas aeruginosa are increasingly reported in India and other locations globally. Since this organism is ubiquitous in the environment, samples such as sewage and wastewater are rich reservoirs of P. aeruginosa bacteriophages. In this study, we report the isolation and characterization of a novel P. aeruginosa N4-like lytic bacteriophage, vB_Pae_AM.P2 (AM.P2), from wastewater in Kerala, India. AM.P2 is a double-stranded DNA podovirus that efficiently lyses the model strain, PAO1, at a multiplicity of infection as low as 0.1 phage per bacterium and resistance frequency of 6.59 × 10-4 Synergy in bactericidal activity was observed between AM.P2 and subinhibitory concentrations of the antibiotic ciprofloxacin. Genome sequencing of AM.P2 revealed features similar to those of the N4-like P. aeruginosa phages LUZ7 and KPP21. As judged by two independent assay methods, spot tests and growth inhibition, AM.P2 successfully inhibited the growth of almost 30% of strains from a contemporary collection of multidrug-resistant P. aeruginosa clinical isolates from South India. Thus, AM.P2 may represent an intriguing candidate for inclusion in bacteriophage cocktails developed for various applications, including water decontamination and clinical bacteriophage therapy.IMPORTANCE In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen P. aeruginosa in sewage and water bodies in India is well documented. The isolation and characterization of bacteriophages that could target emerging P. aeruginosa strains, representing possible epicenters for community-acquired infections, could serve as a useful alternative tool for various applications, such as phage therapy and environmental treatment. Continuing to supplement the repertoire of broad-spectrum bacteriophages is an essential tool in confronting this problem.

Nuclear factor-κB plays an important role in Tamarixetin-mediated inhibition of matrix metalloproteinase-9 expression.

Flavonoids possess a broad spectrum of pharmacological properties, including anti-cancer, anti-oxidant and immunomodulatory activities. The current study explored the potential of some less-studied flavonoids in inhibiting Matrix Metalloproteinase-9 (MMP-9), a prominent biomarker, upregulated in a variety of cancers and known to promote migration and invasion of cancer cells. Amongst these, Tamarixetin, a naturally occurring flavonoid derivative of Quercetin, demonstrated significant dose-dependent inhibition of MMP-9 expression. Furthermore, a substantial inhibition of migration, invasion and clonogenic potential of HT1080 cells was also observed in the presence of Tamarixetin, which further suggests its role as a potential anti-cancer agent. It is noteworthy that Tamarixetin inhibits nuclear translocation as well the activity of nuclear factor kappa B (NFκB), both of which are functions essential for the activation of MMP-9 in promoting tumorigenesis. Additionally, the endogenous regulators of MMP-9 that tightly control its activity were also modulated by Tamarixetin, as evident from the 1.9 fold increase in the expression of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), with a concomitant 2.2 fold decrease in Matrix Metalloproteinase-14 (MMP-14) expression. The results obtained were further corroborated in three dimensional (3D) tumor models, which showed significant inhibition of MMP-9 activity as well as reduced invasive potential in the presence of Tamarixetin. Taken together, our observations demonstrate for the first time, the anti-invasive potential of Tamarixetin in cancer cells, indicating its possible use as a template for novel therapeutic applications.

Patient and provider perspectives on barriers to screening for diabetic retinopathy: an exploratory study from southern India.

OBJECTIVE: Diabetic retinopathy is one of the leading causes of visual impairment after cataract and uncorrected refractive error. It has major public health implications globally, especially in countries such as India where the prevalence of diabetes is high. With timely screening and intervention, the disease progression to blindness can be prevented, but several barriers exist. As compliance to diabetic retinopathy screening in people with diabetes is very poor in India, this study was conducted to explore understanding of and barriers to diabetic retinopathy screening from the perspectives of patients and healthcare providers. METHODS: Using qualitative methods, 15 consenting adult patients with diabetes were selected purposively from those attending a large tertiary care private eye hospital in southern India. Eight semistructured interviews were carried out with healthcare providers working in large private hospitals. All interviews were audiotaped, transcribed verbatim and analysed using the framework analytical approach. RESULTS: Four themes that best explained the data were recognising and living with diabetes, care-seeking practices, awareness about diabetic retinopathy and barriers to diabetic retinopathy screening. Findings showed that patients were aware of diabetes but understanding of diabetic retinopathy and its complications was poor. Absence of symptoms, difficulties in doctor-patient interactions and tedious nature of follow-up care were some major deterrents to care seeking reported by patients. Difficulties in communicating information about diabetic retinopathy to less literate patients, heavy work pressure and silent progression of the disease were major barriers to patients coming for follow-up care as reported by healthcare providers. CONCLUSIONS: Enhancing patient understanding through friendly doctor-patient interactions will promote trust in the doctor. The use of an integrated treatment approach including education by counsellors, setting up of patient support groups, telescreening approaches and use of conversation maps may prove more effective in the long run.

RECK and TIMP-2 mediate inhibition of MMP-2 and MMP-9 by Annona muricata.

Up-regulation of MMP-2 and MMP-9 plays a significant role in promoting cancer progression by degrading the components of the extracellular matrix, thereby enhancing the migration of tumor cells. Although the antiproliferative and apoptotic effect of Annona muricata is well established, its effect on MMP-2 and MMP-9, a major target in several types of cancers, has not been studied. Powdered samples of various parts of A. muricata like fruit, stem, seed, and twig extracted using aqueous methanol showed significant dose-dependent inhibition of MMP-2 and MMP-9 in a highly metastatic fibrosarcoma cell line, HT1080. Additionally, these extracts also up-regulated the expression of several endogenous inhibitors of MMP-2 and MMP-9 like REversion-inducing Cysteine-rich protein with Kazal motifs (RECK) and Tissue Inhibitor of Metalloproteinase- 2 (TIMP-2). Furthermore, primary cells developed from tumor tissues obtained from patients not exposed to chemotherapy, also exhibited similar results. Remarkably, the inhibition of MMP-2 and MMP-9 observed was tumor specific, with the A. muricata fruit extract showing only 2% inhibition in cells obtained from normal tissues, when compared to 60% inhibition observed in cells obtained from tumor samples. The present study elucidates a novel mechanism by which A. muricata extracts selectively exhibit their anti-cancer activity in tumor cells by down-regulating MMP-2 and MMP-9 that are important biomarkers in cancer.