Quorum-quenching activity of the AHL-lactonase from <i>Bacillus licheniformis</i> DAHB1 inhibits vibrio biofilm formation in vitro and reduces shrimp intestinal colonisation and mortality

Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture

Conformation of phylogenetic relationship of penaeidae shrimp based on Morphometric and Molecular investigations

Understanding of accurate phylogenetic relationship among Penaeidae shrimp is important for academic and fisheries industry. The Morphometric and Randomly Amplified Polymorphic DNA (RAPD) analysis was used to make the phylogenetic relationsip among 13 Penaeidae shrimp. For morphometric analysis forty variables and total lengths of shrimp were measured for each species, and removed the effect of size variation. The size normalized values obtained was subjected to UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) cluster analysis. For RAPD analysis, the four primers showed reliable differentiation between species, and used correlation coefficient between the DNA banding patterns of 13 Penaeidae species to construct UPGMA dendrogram. Phylogenetic relationship from morphometric and molecular analysis for Penaeidae species found to be congruent. We concluded that as the results from morphometry investigations concur with molecular one, phylogenetic relationship obtained for the studied Penaeidae are considered to be reliable.Понимание точных филогенетических отношений у креветок Penaeidae важно как с общенаучной точки зрения, так и для рыбной промышленности. RAPD анализ был использован для установления филогенетических связей 13 видов креветок Penaeidae. Для морфометрических анализов измерены 40 переменных и общих длин креветок для каждого вида и устранен эффект вариабельности размера. Показатели нормализованного размера обработаны с помощью кластерного анализа UPGMA (Unweighted Pair-Group Method with Arithmetic Mean). При RAPD анализе четыре праймера показали достоверные различия между видами. Коэффициенты корреляции между паттернами ДНК использованы для построения UPGMA дендрограмм. Филогенетические связи, построенные на основе морфометрических и молекулярных анализов, совпали, что позволило сделать вывод об их достоверности

In silico analysis of lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene from the haemocytes of Indian white shrimp Fenneropenaeus indicus

Lipopolysaccharide and β-1,3-glucan binding protein (LGBP) gene are involved in the pattern recognition mechanism of invertebrates, it induces the cell and humoral mediated immune responses like encapsulation, phagocytosis, nodule formation, clotting, synthesis of antimicrobial peptides and activation of the prophenoloxidase (proPO) system. The current study focuses to model the three-dimensional structure of novel immune related gene LGBP from the Indian white shrimp Fenneropeneaus indicus (F.indicus) by in silico homology modeling and its motif prediction. Fenneropeneaus indicus lipopolysaccharide and β-1,3-glucan binding protein (Fein-LGBP) consists of glycosylated regions which come under the glucanase family. Two conserved putative integrin-binding motif (cell adhesion sites), bacterial glucanase motif (GM) and two polysaccharide recognition motifs for the polysaccharide binding motif (PsBM) and β- glucan recognition motif (β-GRM) were conserved in the novel sequences of Fein-LGBP. Prediction of motifs, patterns, disulfide bridges and secondary structure were performed for functional characterization of the Fein-LGBP. Three dimensional structure of the Fein-LGBP was generated by Modeller9V8, Swiss Model and validated using NIH server. Results revealed that the modelled structure of Fein-LGBP was 75.7% of residues in allowed region. Theoretical model of Fein- LGBP facilitates to the discovery of new synthetic immune related peptides, agonists that could be useful to  understand the mechanism of LGBP involvement in the prophenoloxidase activating system of crustaceans. The tertiary structure prediction of the immune related gene Fein- LGBP will assist to explore more knowledge in immune system of crustaceans

Conformation of Phylogenetic Relationship of Penaeidae Shrimp Based on Morphometric and Molecular Investigations

Understanding of accurate phylogenetic relationship among Penaeidae shrimp is important for academic and fisheries industry. The Morphometric and Randomly Amplified Polymorphic DNA (RAPD) analysis was used to make the phylogenetic relationsip among 13 Penaeidae shrimp. For morphometric anal� ysis forty variables and total lengths of shrimp were measured for each species, and removed the effect of size variation. The size normalized values obtained was subjected to UPGMA (Unweighted Pair�Group Method with Arithmetic Mean) cluster analysis. For RAPD analysis, the four primers showed reliable differentiation between species, and used correlation coefficient between the DNA banding patterns of 13 Penaeidae species to construct UPGMA dendrogram. Phylogenetic relationship from morphometric and molecular analysis for Penaeidae species found to be congruent. We concluded that as the results from morphometry investigations concur with molecular one, phylogenetic relationship obtained for the studied Penaeidae are considered to be reliable

N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection

Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish

Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tigershrimp,Penaeus semisulcatusby gel fermentation chromatography using blue Sepharose matrix. The twopurified prophenoloxidase macromolecules are of about 76 and 75 kDa determined through SDS-PAGEand named asPenaeus semisulcatusprophenoloxidase I (PSproPO I) andPenaeus semisulcatusproph-enoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier TransformInfrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography(HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre againsthuman erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activityagainst yeastSaccharomyces cerevisiaeand encapsulation activity against Sepharose CL 6B beadscompared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I andPSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogenassociated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containingPSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria.The above results concluded that no significant differences were found between the purified PSproPO Iand PSproPO II immune indices and functions. This study might provide a sensitive platform to under-stand more about the critical roles of PSproPO I and PSproPO II in crustacean immune syste

Magnetic nanoparticles are highly toxic to chloroquine-resistant Plasmodium falciparum, dengue virus (DEN-2), and their mosquito vectors

SURESH KUMAR SUBBIAH (A04156) / / / Kadarkarai Murugan,Jiang Wei,Mohamad Saleh Alsalhi,Marcello Nicoletti,Manickam Paulpandi,Christina Mary Samidoss,Devakumar Dinesh,Balamurugan Chandramohan,Chellasamy Paneerselvam,Jayapal Subramaniam,Chithravel Vadiva

Bio-Fabrication of Human Amniotic Membrane Zinc Oxide Nanoparticles and the Wet/Dry HAM Dressing Membrane for Wound Healing

Publication history: Accepted - 25 June 2021; Published online - 28 July 2021.The preparation of unique wet and dry wound dressing products derived from unprocessed human amniotic membrane (UP-HAM) is described. The UP-HAM was decellularized, and the constituent proteins were cross-linked and stabilized before being trimmed and packed in sterile Nucril-coated laminated aluminium foil pouches with isopropyl alcohol to manufacture processed wet human amniotic membrane (PWHAM). The dry type of PD-HAM was prepared by decellularizing the membrane, UV irradiating it, lyophilizing/freeze-drying it, sterilizing it, and storing it at room temperature. The UP-HAM consists of a translucent yellowish mass of flexible membranes with an average thickness of 42 µm. PW-HAM wound dressings that had been processed, decellularized, and dehydrated had a thinner average thickness of 30 µm and lacked nuclear-cellular structures. Following successful decellularization, discrete bundle of fibrous components in the stromal spongy layers, microvilli and reticular ridges were still evident on the surface of the processed HAM, possibly representing the location of the cells that had been removed by the decellularization process. Both wet and dry HAM wound dressings are durable, portable, have a shelf life of 3–5 years, and are available all year. A slice of HAM dressing costs 1.0 US/cm2 . Automation and large-scale HAM membrane preparation, as well as storage and transportation of the dressings, can all help to establish advanced technologies, improve the efficiency of membrane production, and reduce costs. Successful treatment of wounds to the cornea of the eye was achieved with the application of the HAM wound dressings. The HAM protein analysis revealed 360 µg proteins per gram of tissue, divided into three main fractions with MWs of 100 kDa, 70 kDa, and 14 kDa, as well as seven minor proteins, with the 14 kDa protein displaying antibacterial properties against human pathogenic bacteria. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 July 2021 | Volume 9 | Article 695710 fbioe-09-695710 July 22, 2021 Time: 16:39 # 2 Ramasamy et al. HAMP-ZnO Nanoparticles HAM Wound Dressing Wet and dry wound dressings were produced. HAM proteins were purified and analysed. The zinc oxide nanoparticles (HAMP-ZnO NP) made from HAM proteins were characterised and tested for their antibacterial activity. Wounds to the cornea of the eye healed easily when treated with HAM wound dressings. Fresh human Amniotic membrane, Serological screening, selection of disease-free HAM, reome stromal layer, preparation of HAM. UNPROCESSED HAM Cuboidal epithelial cells, basement membrane, compact layer, stromal and spongy layers containing scatted fibroblast cells are visible in hsitological analysis. The flow chart depicts the methods for processing, and preparation of wet (PWHAM) and dry (PD-HAM) wound healing dressings. HAM proteins, Nanoparticle synthesis (HAMP-ZnO NP) and analysis. Antibacterial analysis show Inhibition of growth and biofilm formation of pathogenic bacteria . Processed HAM lacked a nuclear-cellular epithelium, but it did have a distinct fibrous elements in basement membrane, stromal and spongy layers. Processed PW-HAM (Light &SEM) showed smooth epithelial surface topography with microvilli,. HAM dressing, wet/dry, packed, labelled, sterilised and processed. They are durable, portable, have long shelf life . A slice of HAM dressing costs US 1.0 / cm² . The wound dressings are ready to be applied. The dermal wounds and conjunctival surface can be successfully repaired using processed HAM wound dressings GRAPHICAL ABSTRACT | Flow chart depicting the methods, preparing, and characterizing, by histological, and scanning electron microscopy, of wet (PW-HAM) and dry (PD-HAM)of wound healing dressing, and preparation of nanoparticles (HAMP ZnO NP); and application of HAM wound dressing. A wide range of antibacterial activity was observed after treatment with 75 µg/ml zinc oxide nanoparticles derived from human amniotic membrane proteins (HAMP-ZnO NP), including dose-dependent biofilm inhibition and inhibition of Gram-positive (S. aureus, S. mutans, E. faecalis, and L. fusiformis) and Gram-negative bacteria (S. sonnei, P. aeruginosa, P. vulgaris, and C. freundii).PR has acknowledged Sree Balaji Medical College and Hospital for providing the article processing charges of the journal, and moral and technical support. The support of Cologenesis Health Care Pvt. Ltd. for a study on “Human amniotic membrane for ocular and dermal applications” is sincerely appreciated

Green-synthesized CdS nano-pesticides: toxicity on young instars of malaria vectors and impact on enzymatic activities of the non-target mud crab Scylla serrata

Currently, nano-formulated mosquito larvicides have been widely proposed to control young instars of malaria vector populations. However, the fate of nanoparticles in the aquatic environment is scarcely known, with special reference to the impact of nanoparticles on enzymatic activity of non-target aquatic invertebrates. In this study, we synthesized CdS nanoparticles using a green protocol relying on the cheap extract of Valoniopsis pachynema algae. CdS nanoparticles showed high toxicity on young instars of the malaria vectors Anopheles stephensiand A. sundaicus. The antimalarial activity of the nano-synthesized product against chloroquine-resistant (CQ-r) Plasmodium falciparumparasites was investigated. From a non-target perspective, we focused on the impact of this novel nano-pesticide on antioxidant enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities of the mud crab Scylla serrata. The characterization of nanomaterials was carried out by UV–vis and FTIR spectroscopy, as well as SEM and XRD analyses. In mosquitocidal assays, LC50 of V. pachynema-synthesized CdS nanoparticles on A. stephensi ranged from 16.856 (larva I), to 30.301 μg/ml (pupa), while for An. sundaicus they ranged from 13.584 to 22.496 μg/ml. The antiplasmodial activity of V. pachynema extract and CdS nanoparticles was evaluated against CQ-r and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of V. pachynema extract was 58.1 μg/ml (CQ-s) and 71.46 μg/ml (CQ-r), while nano-CdS IC50 was 76.14 μg/ml (CQ-s) and 89.21 μg/ml (CQ-r). In enzymatic assays, S. serrata crabs were exposed to sub-lethal concentrations, i.e. 4, 6 and 8 μg/ml of CdS nanoparticles, assessing changes in GST and AChE activity after 16 days. We observed significantly higher activity of GST, if compared to the control, during the whole experiment period. In addition, a single treatment with CdS nanoparticles led to a significant decrease in AChE activity over time. The toxicity of CdS nanoparticles and Cd ions in aqueous solution was also assessed in mud crabs, showing higher toxicity of aqueous Cd ions if compared to nano-CdS. Overall, our results underlined the efficacy of green-synthesized CdS nanoparticles in malaria vector control, outlining also significant impacts on the enzymatic activity of non-target aquatic organisms, with special reference to mud crabs