Antibacterial Efficacy and Mechanisms of Action of a Novel Beta-Defensin from Snakehead Murrel, Channa striata.

Beta-defensins, identified from fishes, constitute a crucial category of antimicrobial peptides important in combating bacterial fish pathogens. The present investigation centers on the molecular and functional characterization of CsDef, a 63-amino acid beta-defensin antimicrobial peptide derived from snakehead murrel (Channa striata). The physicochemical attributes of CsDef align with the distinctive characteristics observed in AMPs. CsDef was recombinantly produced, and the recombinant peptide, rCsDef, exhibited notable antibacterial efficacy against bacterial fish pathogens with an MIC of 16 µM for V. proteolyticus. A. hydrophila exhibited 91% inhibition, E. tarda 92%, and V. harveyi 53% at 32 µM of rCsDef. The rCsDef exhibited a multifaceted mechanism of action against bacteria, i.e., through membrane depolarization, membrane permeabilization, and generation of ROS. The rCsDef was non-hemolytic to hRBCs and non-cytotoxic to normal mammalian cell line CHO-K1. However, it exhibited anticancer properties in MCF-7. rCsDef demonstrated notable stability with respect to pH, temperature, salt, metal ions, and proteases. These findings suggest it is a potential candidate molecule for prospective applications in aquaculture.

Antibacterial activity and modes of action of a novel hepcidin isoform from the shrimp scad, Alepes djedaba (Forsskål, 1775).

Hepcidin, initially identified in human blood ultrafiltrate as cysteine rich Liver Expressed Antimicrobial Peptide (LEAP-1), is a core molecular conduit between iron trafficking and immune response. Though a great share of studies has been focused on the iron regulatory function of hepcidins, investigations on the antimicrobial aspects are relatively less. The present study is aimed at identification of hepcidin from a teleost fish, Alepes djedaba followed by its recombinant expression, testing antibacterial property, stability and evaluation of cytotoxicity. Modes of action on bacterial pathogens were also examined. A novel hepcidin isoform, Ad-Hep belonging to the HAMP1 (Hepcidin antimicrobial peptide 1) group of hepcidins was identified from the shrimp scad, Alepes djedaba. Ad-Hep with 2.9 kDa size was found to be a cysteine rich, cationic peptide (+4) with antiparallel beta sheet conformation, a furin cleavage site (RXXR) and 'ATCUN' motif. It was heterologously expressed in E. coli Rosettagami B(DE3)PLysS cells and the recombinant peptide, rAd-Hep was found to have significant antibacterial activity, especially against Edwardsiella tarda, Vibrio parahaemolyticus and Escherichia coli. Membrane depolarization followed by membrane permeabilization and Reactive Oxygen Species (ROS) production were found to be the modes of action of rAd-Hep on bacterial cells. Ad-Hep was found to be non-haemolytic to hRBC and non-cytotoxic in mammalian cell line. Stability of the peptide at varying temperature, pH and metal salts qualify them for applications in vivo. With significant bactericidal activity coupled with direct killing mechanisms, the rAd-Hep can be a promising drug candidate for therapeutic applications in medicine and fish culture systems.

A Minor Groove Binder with Significant Cytotoxicity on Human Lung Cancer Cells: The Potential of Hesperetin Functionalised Silver Nanoparticles.

Natural drug functionalised silver (Ag) nanoparticles (NPs) have gained significant interest in pharmacology related applications due to their therapeutic efficiency. We have synthesised silver nanoparticle using hesperetin as a reducing and capping agent. This work aims to discuss the relevance of the hesperetin functionalised silver nanoparticles (H-AgNPs) in the field of nano-medicine. The article primarily investigates the anticancer activity of H-AgNPs and then their interactions with calf thymus DNA (ctDNA) through spectroscopic and thermodynamic techniques. The green synthesised H-AgNPs are stable, spherical in shape and size of 10 ± 3 nm average diameter. The complex formation of H-AgNPs with ctDNA was established by UV-Visible absorption, fluorescent dye displacement assay, isothermal calorimetry and viscosity measurements. The binding constants obtained from these experiments were consistently in the order of 104 Mol-1. The melting temperature analysis and FTIR measurements confirmed that the structural alterations of ctDNA by the presence of H-AgNPs are minimal. All the thermodynamic variables and the endothermic binding nature were acquired from ITC experiments. All these experimental outcomes reveal the formation of H-AgNPs-ctDNA complex, and the results consistently verify the minor groove binding mode of H-AgNPs. The binding constant and limit of detection of 1.8 µM found from the interaction studies imply the DNA detection efficiency of H-AgNPs. The cytotoxicity of H-AgNPs against A549 and L929 cell lines were determined by in vitro MTT cell viability assay and lactate dehydrogenase (LDH) assay. The cell viability and LDH enzyme release are confirmed that the H-AgNPs has high anticancer activity. Moreover, the calculated LD50 value for H-AgNPs against lung cancer cells is 118.49 µl/ml, which is a low value comparing with the value for fibroblast cells (269.35 µl/ml). In short, the results of in vitro cytotoxicity assays revealed that the synthesised nanoparticles can be considered in applications related to cancer treatments. Also, we have found that, H-AgNPs is a minor groove binder, and having high DNA detection efficiency.

Exploring the cytotoxicity on human lung cancer cells and DNA binding stratagem of camptothecin functionalised silver nanoparticles through multi-spectroscopic, and calorimetric approach.

The influence of nanoparticles inside the human body and their interactions with biological macromolecules need to be explored/studied prior to specific applications. The objective of this study is to find the potential of camptothecin functionalised silver nanoparticles (CMT-AgNPs) in biomedical applications. This article primarily investigates the binding stratagem of CMT-AgNPs with calf thymus DNA (ctDNA) through a series of spectroscopic and calorimetric methods and then analyses the anticancer activity and cytotoxicity of CMT-AgNPs. The nanoparticles were synthesized using a simple one pot method and characterized using UV-Visible, fourier transform infrared (FTIR) spectroscopy, X-ray diffraction and high-resolution transmission electron microscopy (HRTEM). The average size of CMT-AgNPs is 10 ± 2 nm. A group of experimental techniques such as UV-Visible spectrophotometry, fluorescence dye displacement assay, circular dichroism (CD) and viscosity analysis unravelled the typical groove binding mode of CMT-AgNPs with ctDNA. The CD measurement evidenced the minor conformational alterations of double helical structure of ctDNA in the presence of CMT-AgNPs. The information deduced from the isothermal titration calorimetry (ITC) experiment is that the binding was exothermic and spontaneous in nature. Moreover, all the thermodynamic binding parameters were extracted from the ITC data. The binding constants obtained from UV absorption experiments, fluorescence dye displacement studies and ITC were consistently in the order of 104 Mol-1. All these results validated the formation of CMT-AgNPs-ctDNA complex and the results unambiguously confirm the typical groove binding mode of CMT-AgNPs. An exhaustive in vitro MTT assay by CMT-AgNPs and CMT against A549, HT29, HeLa and L929 cell lines revealed the capability of CMT-AgNPs as a potential anticancer agent.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression.

BACKGROUND: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. RESULTS: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four ß sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. CONCLUSION: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Characterisation of a novel crustin isoform from mud crab, Scylla serrata (Forsskål, 1775) and its functional analysis in silico.

A 336-base pair (bp) sized mRNA sequence encoding 111 amino acid size crustin isoform (MC-crustin) was obtained from the gill sample of the green mud crab, Scylla serrata. MC-crustin possessed an N-terminal signal peptide region comprising of 21 amino acid residues, followed by a 90 amino acid mature peptide region having a molecular weight of 10.164 kDa, charge + 4.25 and theoretical pI of 8.27. Sequence alignment and phylogenetic tree analyses revealed the peptide to be a Type I crustin, with four conserved cysteine residues forming the cysteine rich region, followed by WAP domain. MC-crustin was cationic with cysteine/proline rich structure and was predicted with antimicrobial, anti-inflammatory, anti-angiogenic and anti-hypertensive property making it a potential molecule for possible therapeutic applications.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization.

BACKGROUND: Increase of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. RESULTS: Open reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. CONCLUSION: Functional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

Molecular characterization of a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger (Ruppel, 1836).

BACKGROUND: The concern regarding a post-antibiotic era with increasing drug resistance by pathogens imposes the need to discover alternatives for existing antibiotics. Antimicrobial peptides (AMPs) with their versatile therapeutic properties are a group of promising molecules with curative potentials. These evolutionarily conserved molecules play important roles in the innate immune system of several organisms. The ß-defensins are a group of cysteine rich cationic antimicrobial peptides that play an important role in the innate immune system by their antimicrobial activity against the invading pathogens. The present study deals with a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger. Total RNA was isolated from the gills, cDNA was synthesized and the ß-defensin isoform obtained by polymerase chain reaction was cloned and subjected to structural and functional characterization in silico. RESULTS: A ß-defensin isoform could be detected from the gill mRNA of red-toothed trigger fish, Odonus niger. The cDNA encoded a 63 amino acid peptide, ß-defensin, with a 20 amino acid signal sequence followed by 43 amino acid cationic mature peptide (On-Def) having a molecular weight of 5.214 kDa and theoretical pI of 8.89. On-Def possessed six highly conserved cysteine residues forming disulfide bonds between C1-C5, C2-C4, and C3-C6, typical of ß-defensins. An anionic pro-region was observed prior to the ß-defensin domain within the mature peptide. Clustal alignment and phylogenetic analyses revealed On-Def as a group 2 ß-defensin. Furthermore, it shared some structural similarities and functional motifs with ß-defensins from other organisms. On-Def was predicted to be non-hemolytic with anti-bacterial, anti-viral, anti-fungal, anti-cancer, and immunomodulatory potential. CONCLUSION: On-Def is the first report of a ß-defensin from the red-toothed trigger fish, Odonus niger. The antimicrobial profile showed the potential for further studies as a suitable candidate for antimicrobial peptide therapeutics.

Epidemiology and transmission dynamics of COVID-19 in two Indian states.

Although most cases of coronavirus disease 2019 (COVID-19) have occurred in low-resource countries, little is known about the epidemiology of the disease in such contexts. Data from the Indian states of Tamil Nadu and Andhra Pradesh provide a detailed view into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission pathways and mortality in a high-incidence setting. Reported cases and deaths have been concentrated in younger cohorts than would be expected from observations in higher-income countries, even after accounting for demographic differences across settings. Among 575,071 individuals exposed to 84,965 confirmed cases, infection probabilities ranged from 4.7 to 10.7% for low-risk and high-risk contact types, respectively. Same-age contacts were associated with the greatest infection risk. Case fatality ratios spanned 0.05% at ages of 5 to 17 years to 16.6% at ages of 85 years or more. Primary data from low-resource countries are urgently needed to guide control measures.

A histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himantura walga: Molecular and functional characterisation.

Antimicrobial peptides (AMPs) are biologically dynamic molecules produced by all type of organisms as a fundamental component of their innate immune system. The present study deals with the identification of a histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himantura walga. A 243 base pair fragment encoding 81 amino acid residues amplified from complementary DNA was identified as Hipposin and termed as Hw-Hip. Homologous analysis showed that Hw-Hip belongs to the Histone H2A superfamily and shares sequence identity with other histone-derived AMPs from fishes. Phylogenetic analysis of Hw-Hip displayed clustering with the fish H2A histones. Secondary structure analysis showed the presence of three α-helices and four random coils with a prominent proline hinge. The physicochemical properties of Hw-Hip are in agreement with the properties of antimicrobial peptides. A 39-mer active peptide sequence was released by proteolytic cleavage in silico. Functional characterisation of active peptide in silico revealed antibacterial, anticancer and antibiofilm activities making Hw-Hip a promising candidate for further exploration.

Foreign Body in Left Main Bronchus.

Tracheobronchial foreign body (TFB) aspiration is rare in adults, although incidence rates increases with advancing age. We report a case of foreign body in left main bronchus in an adult female who had no risk factor. She was successfully treated with removal of betel nuts by bronchoscopy. Unusual presentation and high index of suspicion can help in proper management.

A clinical study of the relationship between diabetes mellitus and periodontal disease.

UNLABELLED: The relationship between diabetes mellitus and periodontal disease is not clear, even though studied intensively. From the available data, it seemed reasonable to believe that diabetics were more susceptible to periodontal disease than non.diabetics. AIM: The present study was to clinically evaluate the relationship of diabetes mellitus with periodontal disease along with various parameters. MATERIALS AND METHODS: Fifteen hundred patients with diabetes mellitus were examined. A thorough oral examination was carried out and relevant history was recorded for all the patients. RESULTS: Results indicated that the prevalence of periodontal disease in diabetic patients was 86.8%. CONCLUSION: It can be concluded that poorer the glycemic control, and longer the duration of diabetes, the greater will be the prevalence and severity of periodontal disease.

Children with medically unexplained pain symptoms: categorization and effective management.

CONTEXT: Medically unexplained pain symptoms are common in children, and their incidence is on the rise. There is often a lack of clearly articulated pathophysiology in these patients. There is need to improve understanding about varied causes and presentations of these patients which would generate further insight in management of these patients. Documentation and detailed assessment of such children in Indian setting is not seen in literature. MATERIALS AND METHODS: A series of 17 cases, 10 boys and 7 girls referred from pediatrics department is discussed, so as to categorize them in three different subgroups for management. RESULT AND DISCUSSION: Although there were often no overt anxiety or depressive features, some psychosocial stress which was mostly unnoticed by the child, the parents and the doctor, preceded such a pain. It was often an academic stress, familial separation or parental psychiatric illness. They were at times not able to verbalize their distress, which was revealed with the help of Children's Apperception Test (C.A.T.). They mainly had anxieties about loss of love or disapproval by parents and also fear of harm or injury. They used defence mechanisms like denial, reaction formation and repression, which were ineffective in handling the overwhelming anxiety. Most of these children had either above average or borderline intelligence. Somatic expression of emotional needs and fears in these children was managed effectively by supportive therapy and antidepressant drugs.

Avian reovirus induces an inhibitory effect on lymphoproliferation in chickens.

The cellular immune responses of chickens inoculated with the vaccine strain S-1133 and/or a field isolate VA-1 of avian reovirus (ARV) were studied. Both strains of virus caused inhibition of the phytohaemagglutinin (PHA)-induced lymphoproliferative response of peripheral blood mononuclear cells (PBMC) and splenic mononuclear cells (SMC) during the initial stage from day 4 up to day 10 post-inoculation (PI), with a later return to the normal value. The inhibition in the PHA-induced lymphoproliferation of SMC could be partially overcome by depletion of adherent cells. The supernatant of the PHA-stimulated SMC culture was also checked in vitro for the presence of suppressive factor(s) produced in response to ARV infection. The culture supernatant from chickens at day 5 PI caused significant inhibition of the PHA-induced lymphoproliferation of control birds, suggesting the presence of suppressive factor(s). ARV infection also significantly inhibited IL-2 production on day 5. There was a significant increase in nitric oxide production by the splenic mononuclear cells of chickens inoculated with either strain of ARV.