Shrimp Cells Research Articles

Formulation and evaluation of Phaseolus lunatus seed coat mediated silver nanoparticles mouthwash: A comprehensive study on biomedical properties and toxicological assessment

Silver nanoparticles have promising therapeutic potential in the field of dentistry, as newly emerging oral therapeutics, in the form of mouthwashes based on silver nanoparticles, demonstrate significant potential for enhancing oral management thus the present investigation aims to formulate silver nanoparticles-based mouthwash from an aqueous extract of Phaseolus lunatus seed coat (PLSC) and evaluate its biomedical properties. The green synthesized AgNPs in the mouthwash were characterized using UV–visible spectroscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and x-ray diffraction (XRD). The formulated mouthwash was assessed for its anti-microbial activity using the agar well diffusion technique and time-kill curve assay. Its anti-inflammatory and anti-oxidant activities are assessed through egg albumin assay and hydrogen peroxide assays. The Cytotoxic effect of formulated mouthwash was assessed through a brine shrimp lethality assay and MTT assay over the human osteoblast cell line (MG-63). Furthermore, the study also assessed the toxicity effect of formulated mouthwash through zebrafish embryos. The results suggest that the green synthesized AgNPs were spherical and had an average size of 42 nm and the formulated mouthwash exhibited significant anti-microbial activity, anti-inflammatory, and antioxidant properties, making them a better candidate for oral health therapeutics. The Zebrafish embryo toxicology studies of mouthwash revealed a consistent lack of abnormalities and a good viability and hatchability rate and the cytotoxic effect shows less toxicity over brine shrimp and human osteoblast cells. In conclusion, PLSC-AgNPs mouthwash is a potential oral therapeutic option with minimal toxicity for better oral management.

Killing of xenogenous and virally infected homogenous target cells by shrimp lymphocyte-like haemocytes

Haemocytes play a crucial role in the invertebrate's immune system. In our lab, five subpopulations of shrimp haemocytes were identified in the past: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. In the latter two subpopulations, their characteristics such as having small cytoplasmic rims and not adhering to plastic cell-culture plates are very similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity like natural killer cells, with the ability to recognize and kill target cells. In our study, K562 cells and Sf9 cells were used as xenogenous target cells to detect the cytotoxic activity of the shrimp non-adherent lymphocyte-like haemocytes. Non-adherent haemocytes were collected and mixed with K562 cells and Sf9 cells at a 5:1 ratio and the binding activity was examined under a microscope. The binding rate of non-adherent haemocytes to K562 cells and Sf9 cells reached 6.6 % and 2.4 % after 240 min of culture, respectively. Then, the killing activity of non-adherent haemocytes was detected by an EMA staining (fluorescence microscopy), which showed 3.75 % dead K562 cells and 1.025 % dead Sf9 cells, and by Sytox® blue staining (flow cytometry), which showed 4.97 % of dead K562 cells. Next, a killing assay was developed to visualize the killing activity of shrimp non-adherent haemocytes. Non-adherent haemocytes were pre-labeled in blue (CellTracker blue) and K562/Sf9 cells in green (CFSE); dead cells were differentially stained red with ethidium bromide. The cytotoxic activity increased and reached a level of 2.59 % in K562 cells and 0.925 % in Sf9 cells at 120 min after co-culture. Furthermore, in the co-cultures of non-adherent haemocytes with K562 cells and Sf9 cells, upregulation of the gene and protein expression of the cytotoxic molecules torso-like protein and granzyme B was observed by RT-qPCR at 240 min and western blotting at 180 min. Additionally, non-adherent haemocytes were co-cultured with WSSV-inoculated shrimp ovary and lymphoid organ cells to detect the cytotoxicity to homogenous target cells. The binding activity started at 60 min in both the ovary and lymphoid organ cultures and reached at 240 min 50.62 % and 40.7 %, respectively. The killing activity was detected by EMA staining and the percentage of dead ovary and lymphoid organ cells increased respectively from 10.84 % to 6.89 % at 0 min to 13.09 % and 8.37 % at 240 min. In conclusion, we demonstrated the existence of cytotoxic activity of shrimp lymphocyte-like haemocytes against xenogenous cells from mammals and insects and against WSSV-infected homogenous shrimp cells.

Development and Evaluation of a Shrimp Virus (IHHNV)-Mediated Gene Transfer and Expression System for Shrimps.

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Synthesis of alga-coated copper oxide nanoparticles with potential applications in shrimp farming

Copper (Cu) is a crucial element that plays a vital role in facilitating proper biological activities in living organisms. In this study, copper oxide nanoparticles (CuO NPs) were synthesized using a straightforward precipitation chemical method from a copper nitrate precursor at a temperature of 85 °C. Subsequently, these NPs were coated with the aqueous extract of Sargassum angustifolium algae. The size, morphology, and coating of the NPs were analyzed through various methods, revealing dimensions of approximately 50 nm, a multidimensional shaped structure, and successful algae coating. The antibacterial activity of both coated and uncoated CuO NPs against Vibrio harveyi, a significant pathogen in Litopenaeus vannamei, was investigated. Results indicated that the minimum inhibitory concentration (MIC) for uncoated CuO NPs was 1000 μg/mL, whereas for coated CuO NPs, it was 500 μg/mL. Moreover, the antioxidant activity of the synthesized NPs was assessed. Interestingly, uncoated CuO NPs exhibited superior antioxidant activity (IC50 ≥ 16 μg/mL). The study also explored the cytotoxicity of different concentrations (10–100 μg/mL) of both coated and uncoated CuO NPs. Following 48 h of incubation, cell viability assays on shrimp hemocytes and human lymphocytes were conducted. The findings indicated that CuO NPs coated with alga extract at a concentration of 10 μg/mL increased shrimp hemocyte viability. In contrast, uncoated CuO NPs at a concentration of 25 μg/mL and higher, as well as CuO NPs at a concentration of 50 μg/mL and higher, led to a decrease in shrimp hemocyte survival. Notably, this study represents the first quantitative assessment of the toxicity of CuO NPs on shrimp cells, allowing for a comparative analysis with human cells.

Identification of active shrimp U6 promoters for efficient CRISPR/Cas9 gene editing in penaeid shrimps based on a VP28-pseudotyped baculovirus expression system

The activity of species-specific U6 promoter is crucial for efficient expression of sgRNA in target cells when CRISPR/Cas9 is delivered in plasmid, thus the use of CRISPR/Cas9 technology was markedly limited in shrimps due to the lack of endogenous U6 promoter. To overcome this, in this study four candidate U6 promoters (LvU6 A-D) had been identified and cloned from the genome of penaeid shrimp (Litopenaeus vannamei), and then their activities to drive the expression of shRNA in insect Sf9 cells and shrimp primary hemolymph cells were successively analyzed using insect baculovirus expression system. It was found that all the isolated four LvU6 promoters contained all the characteristic elements (DSE, PSE and TATA box) of RNA polymerase III U6 promoter. Both a dual plasmids delivery system (Bacmid-PH-LvU6-shEGFP-P2-gBFP and Bacmid-PH-EGFP) and an all-in-one plasmid delivery system (Bacmid-PH-EGFP-LvU6-shEGFP-P2-gBFP) were constructed for RNAi analysis of reporter gene EGFP which are not pseudo-typed for the infection of Sf9 cells but pseudo-typed for the infection of shrimp cells by co-transfection with pFastBac1-VP28 (an envelope protein of shrimp virus). The obtained results showed that only LvU6 A, B and D could successfully drive the expression of shEGFP in both of the tested cells, and the order of interference efficiency was LvU6 B > LvU6 D > LvU6 A. Furthermore, the RNAi efficiency of LvU6 B was up to 27% (P < 0.05) when the dual plasmids were co-transfected at a mass ratio of 1: 0.1 in Sf9 cells, which was higher than that of the all-in-one plasmid (15%, P < 0.05). In shrimp cells, no noticeable fluorescence could be observed when dual plasmids delivery system was used; in contrast, the efficiency could reach 19% when all-in-one delivery system was used (P < 0.05). However, when LvU6 B was used in the shrimp CRISPR/Cas9 gene editing system with a co-expressed VP28 gene in the baculovirus vector, there were no mutations detected in the target gene (Bax) of both in vitro cultured shrimp cells and adult shrimps. Surprisingly, after optimizing the system by using a truncated LvU6 B promoter and introducing double sgRNAs, the insertion, deletion or replacement of target sites occurred in both shrimp cells and adult tissues (spermatophore, Oka and intestine), and the mutation rates increased up to 22% and 46%, respectively. This work has provided one active shrimp U6 promoter for efficient gene editing in shrimps and shrimp cells.

Synthesis, characterization, biomolecular interaction, cytotoxicity, and computational studies of quinoxaline-based platinum(II) complexes

The combination of ninhydrin and o-phenylenediamine, followed by their reaction with a phenylhydrazine derivative produces quinoxaline-based ligands (L1 – L6). These ligands react with K2PtCl4 to form Pt(II) complexes (I – VI). The ligands and Pt(II) chelate were characterized through various spectroscopic and analytical techniques, like 1H NMR, 13C NMR, C, H, N-elemental analysis, IR spectroscopy, and mass spectrometry. DFT calculations were used to optimizethe structures of metal complexes. To investigate the binding mode of complexes to CT-DNA/BSA, absorption titration, viscosity measurements, and molecular docking analysis were employed. The results indicated that the DNA binding mechanism involved intercalation. The antibacterial efficacy of the compounds was evaluated against five strains of bacteria. The lower minimum inhibitory concentration (MIC) of complexes suggests that they are more potent than quinoxaline-based ligands. The synthesized compounds were evaluated for cytotoxicity using brine shrimp. The LC50 values of ligands and complexes ranged from 7.64 to 11.45 μg/mL and 5.27 to 7.02 μg/mL, respectively. The capacity of the molecule to suppress cell proliferation using the MCF-7 cancer cell was tested, and the IC50 value was discovered to be comparable to that of astandard drug. Thus in this work, complexes demonstrated intercalation as the binding mode with CT-DNA/BSA and exhibited enhanced antibacterial efficacy compared to the ligands. The compounds also showed promising cytotoxicity against brine shrimp and MCF-7 cancer cells, with comparable IC50 values to a standard drug, indicating their potential as therapeutic agents.

Histopathological condition of Litopenaeus vannamei infected with Infectious Myonecrosis Virus (IMNV) on different days of culture collected from Aceh, Sidoarjo, Probolinggo and Lampung Indonesia

Histopathological detection is one of many methods that has been used on shrimp diseases detection. A study on histopathology of Litopenaeus vannamei infected by infectious Myonecrosis Virus (IMNV) has been conducted in Sidoarjo, East Java. The purpose of this study was to give a clear view of the histopathological condition of shrimp infected by IMNV which is necessary to examine the destructive level of the disease within the shrimp cells and hopefully can contribute to adding more information about the disease. About 16 samples of shrimp from different days of cultured that shown an IMNV infection clinically sign has been examined on their muscle tissue, lymphoid organ and hepatopancreas condition. The result has shown that shrimp infected by IMNV has a necrosis and haemocyte infiltration on muscle, spheroids on lymphoid organ and shown congestions and haemocyte infiltrations on hepatopancreas. There were significant differences (P<0.05, Kruskal-Wallis) on muscle and lymphoid organ condition between shrimp before and after 2 months cultured. But there was no significant difference in shrimp hepatopancreas condition.

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

Improvement and applications of adeno-associated virus 2 (AAV-2) system for efficient gene transfer and expression in penaeid shrimp cells

Adeno-associated virus 2 (AAV-2)-mediated expression system is a well-developed and widely used gene transfer and expression tool in mammalian cells, but no attempt has been done to examine its infection and expression efficiency in shrimp cells. In this study, using eGFP as a reporter gene, we found that unmodified AAV-2 could infect primarily cultured shrimp hemolymph cells, but with an extremely low infection efficiency of up to 0.0067%. However, all the improvements of AAV-2 expression system tried in this study including the insertion of shrimp virus (IHHNV)-sourced P2 promoter into the AAV-2 vector (i.e. pAAV-CMV-P2-eGFP) and the inclusion of shrimp virus (IHHNV)-sourced nucleocapsid protein （IHCP） into or association of shrimp virus (WSSV)-sourced tegument protein (VP26) with the capsid of the improved AAV-2 s, respectively, could significantly increase the infection and expression efficiencies of this expression system in the primarily cultured shrimp hemolymph cells in comparison with wild-type AAV-2. Moreover, from the viewpoint of improving the infection and expression efficiency, the insertion of P2 promoter did best, followed by the association of WSSV tegument protein of VP26, and the last was the inclusion of IHHNV nucleocapsid protein of IHCP. In conclusion, an improved AAV-2 expression system which consists of four expression plasmids of pAAV-CMV-P2-eGFP, pAAV-RC, pHelper and pcDNA3.1-VP26 and one packaging cell line of HEK293T has been successfully developed in this study. This improved AAV-2 expression system will provide us a useful tool for efficient gene transfer and expression in shrimp cells.

Affecting mechanism of Chlorella sorokiniana meal replacing fish meal on growth and immunity of Litopenaeus vannamei based on transcriptome analysis

The purpose of this study was to analyze the affecting mechanism of Chlorella sorokiniana meal replacing fish meal on the growth performance and immune function of Pacific white shrimp, Litopenaeus vannamei by transcriptomics analysis. In a control diet containing 560 g % /kg fish meal, chlorella was used to replace 0 %, 20 %, 40 %, 60 %, 80 % and 100 % of dietary fish meal to form six iso-nitrogenous and iso-lipidic diets (C0, C20, C40, C60, C80, C100). The six diets were fed to white shrimp (1.37 ± 0.02 g) for 8 weeks. Compared to the control group, the final body weight (FBW), specific growth rate (SGR) and feed efficiency (FE) of the C20 group were significantly improved (P < 0.05). There was no significant difference in growth performance between the C40 group and the C0 group (P > 0.05). When the replacement level of fish meal by chlorella was ≥ 60 %, the FBW, SGR and FE of shrimp were significantly decreased (P < 0.05). In the analysis of the hepatopancreatic transcriptome, the amount of differentially expressed genes (DEGs) in C20, C40, C60, C80 and C100 groups were 322 (176 up and 146 down), 482 (246 up and 236 down), 804 (487 up and 317 down), 1080 (684 up and 396 down) and 1262 (827 up and 435 down), respectively, when compared to the C0 group. GO Term and KEGG pathway enrichment analysis of DEGs showed that DEGs between the C20 and C0 groups were significantly enriched in chitin binding and inositol metabolism processes. Several immune-related signaling pathways were also enriched, including the C-type lectin receptor pathway, complement and coagulation cascades, the B-cell receptor pathway, and most of these genes were up-regulated in the C20 group. Compared to the C0 group, DEGs in C40, C60, C80 and C100 groups were significantly enriched in lysosomal, protein digestion, and absorption pathways. In addition, in the C80 and C100 groups, the DEGs were also significantly enriched in lipid catabolism process. The high replacement of fish meal with chlorella meal affected the absorption and utilization of nutrients by regulating the signaling pathways related to protein digestion and absorption, and aggravated lipid catabolism of the body, thereby affecting the growth of shrimp. The high replacement of fish meal might also affect the homeostasis of shrimp cells and reduce immunity through the lysosomal signaling pathway. In summary, the low replacement of fish meal (20 %) with chlorella meal significantly improved the growth performance of Litopenaeus vannamei, which might be realized through activating the C-type lectin receptor signaling pathway to regulate immune function of the body, but the high substitution of fish meal (≥ 60 %) reduced the growth performance and immunity by affecting the lysosomal signaling pathway.

Assessment of the characterization antimicrobial and green synthesis of magnesium oxide nanoparticles using Psydium guajava - in-vitro analysi

Aim: To assess the characterization, antimicrobial activity of green synthesised magnesium oxide nanoparticles using Psydium guajava leaves. Materials And Methods: Psydium guajava leaves were dried and ground, of which 2 g of the powder was used for the study.the powder was then dissolved in 100ml of distilled water. 1 mg of magnesium chloride salt was dissolved in 20 ml of distilled water. Both the solutions were mixed and subjected to centrifuge. After the preparation of the nanoparticle, it was subjected for further investigation like SEM analysis, EDAX, UV-VIs, cytotoxicity and anti microbial tests. The cytotoxicity was investigated using a brine shrimps bioassay.The number of surviving naupli cells were manually counted under dissection microscope. The antibacterial activity was examined against four bacterial strains- E.coli, E.fecalis,S.aureus,P.aurogenosa by agar well diffusion method .The inoculated plates were incubated overnight (for about 24 hours at 37 degree celsius) and the inhibition zone around the nanoparticle solution was calculated. The antimicrobial tests were carried against standard antibiotic and the results were tabulated. Results: SEM analysis showed a particle size of 70 nm and the UV -Visible spectroscopy showed a peak at 320nm confirming the presence of magnesium oxide nanoparticles that is prepared using Psydium guajava. Cytotoxicty tests revealed that 83% of the brine shrimp cells survived .The anti microbial tests revealed that the nanoparticle was effective against two micro-organisms -S.aureus and E.fecalis and their zone of inhibition was nearly equal to that of standard antibiotic against which the organisms were incubated. Conclusion: Psydium guajava synthesized magnesium nanoparticles were found to have good cytotoxicity and acceptable anti-microbial activity especially against two most commonly harbouring micro-organism(S.aureus and E.faecalis) .Hence they can be extended for use in soft tissue healing.

Viral Capsid Change upon Encapsulation of Double-Stranded DNA into an Infectious Hypodermal and Hematopoietic Necrosis Virus-like Particle.

In this study, we aimed to encapsulate the sizable double-stranded DNA (dsDNA, 3.9 kbp) into a small-sized infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP; T = 1) and compared the changes in capsid structure between dsDNA-filled VLP and empty VLP. Based on our encapsulation protocol, IHHNV-VLP was able to load dsDNA at an efficiency of 30-40% (w/w) into its cavity. Structural analysis revealed two subclasses of IHHNV-VLP, so-called empty and dsDNA-filled VLPs. The three-dimensional (3D) structure of the empty VLP produced in E. coli was similar to that of the empty IHHNV-VLP produced in Sf9 insect cells. The size of the dsDNA-filled VLP was slightly bigger (50 Å) than its empty VLP counterpart; however, the capsid structure was drastically altered. The capsid was about 1.5-fold thicker due to the thickening of the capsid interior, presumably from DNA-capsid interaction evident from capsid protrusions or nodules on the interior surface. In addition, the morphological changes of the capsid exterior were particularly observed in the vicinity of the five-fold axes, where the counter-clockwise twisting of the "tripod" structure at the vertex of the five-fold channel was evident, resulting in a widening of the channel's opening. Whether these capsid changes are similar to virion capsid maturation in the host cells remains to be investigated. Nevertheless, the ability of IHHNV-VLP to encapsulate the sizable dsDNA has opened up the opportunity to package a dsDNA vector that can insert exogenous genes and target susceptible shrimp cells in order to halt viral infection.

Single-Cell Sequencing Reveals Types of Hepatopancreatic Cells and Haemocytes in Black Tiger Shrimp (Penaeus monodon) and Their Molecular Responses to Ammonia Stress.

The cell types and developmental trajectories of shrimp cells based on the transcriptional level have not been established, and gene expression profile and function at the single-cell level is unclear. We aimed to use scRNA-seq to construct a single-cell resolution transcriptional map of hepatopancreas and haemocytes in shrimp to analyse the molecular mechanisms of the immune response to ammonia nitrogen stress. In the present study, seven cell clusters were successfully identified in each of the two tissues (haemocytes, Hem1-7; hepatopancreas, Hep1-7) based on specifically-expressed marker genes. The developmental starting points of haemocytes and hepatopancreatic cells were Hem2 and Hep1, respectively. We propose that Hem2 has oligopotent potential as the initiation site for haemocyte development and that Hem4 and Hem5, located at the end of development, are the most mature immune cell types in haemocytes. Hep5 and Hep6 were the developing terminal cells of hepatopancreas. The antioxidant system and proPO system of shrimp were activated under ammonia nitrogen stress. A large number of DEGs were involved in oxidative stress, detoxification metabolism, and immune defence. In particular, important response genes such as AMPs, proPO, and GST were not only marker genes for identifying cell groups but also played an important role in shrimp cell differentiation and functional plasticity. By successfully applying 10× Genomics based scRNA-seq to the study of shrimp, the single-cell transcriptional profiles of hepatopancreatic cells and haemocytes of shrimp innate immune responses under ammonia stress were constructed for the first time. This atlas of invertebrate hepatopancreatic cells and haemocytes at single-cell resolution identifies molecular events that underpin shrimp innate immune system responses to stress.

Investigations on chitin and coconut fiber reinforcements on mechanical and moisture absorption properties of corn starch bioplastics

Plastic garbage stacking up in landfills poses a severe environmental danger. Bioplastic manufactured from several biodegradable materials is one approach for reducing such problems. In this work, corn starch bioplastic (BP) was prepared by gelatinization. The BP was reinforced with 10 wt% chitin as wells as 10 wt% coconut fibers to obtain chitin reinforced bioplastic (CH-BP) and coconut fiber reinforced bioplastic (CF-BP). The chitin was extracted from shrimp cells. SEM and XRD characterization of chitin extracted were conducted to study the fiber formation and structure. The effect of plasticizer glycerol on the tensile strength, elongation, and moisture absorption, were compared for BP, CH-BP and CF-BP. The increase in glycerol content increased the elongation, flexibility and moisture absorption for all the three types of bioplastics, however, the tensile strength decreased. The tensile strength of CF-BP was slightly more at various glycerol concentration, whereas, the elongation percentage and moisture absorption was low compared to CH-BP and BP. The CF-BP can be employed for applications that requires more strength, the CH-BP in applications that require flexibility and BP where high moisture absorption is necessary. Nevertheless, the concentration of fillers and glycerol should be optimized for various applications.

Inhibitory Effect of Chitosan and Phosphate Cross-linked Chitosan against Cucumber Mosaic Virus and Pepper Mild Mottle Virus.

Cucumber mosaic virus (CMV) and Pepper mild mottle virus (PMMoV) causes severe economic loss in crop productivity of both agriculture and horticulture crops in Korea. The previous surveys showed that naturally available biopolymer material – chitosan (CS), which is from shrimp cells, reduced CMV accumulation on pepper. To improve the antiviral activity of CS, it was synthesized to form phosphate cross-linked chitosan (PCS) and compared with the original CS. Initially, the activity of CS and PCS (0.01%, 0.05%, and 0.1% concentration) compound against PMMoV infection and replication was tested using a half-leaf assay on Nicotiana glutinosa leaves. The total number of local lesions represented on a leaf of N. glutinosa were counted and analyzed with phosphate buffer treated leaves as a negative control. The leaves treated with a 0.1% concentration of CS or PCS compounds exhibited an inhibition effect by 40–75% compared with the control leaves. The same treatment significantly reduced about 40% CMV accumulation measured by double antibody sandwich enzyme-linked immunosorbent assay and increased the relative expression levels of the NPR1, PR-1, cysteine protease inhibitor gene, LOX, PAL, SRC2, CRF3 and ERF4 genes analyzed by quantitative reverse transcriptase-polymerase chain reaction, in chili pepper plants.

Effect of UV Irradiation (A and C) on Casuarina equisetifolia-Mediated Biosynthesis and Characterization of Antimicrobial and Anticancer Activity of Biocompatible Zinc Oxide Nanoparticles.

The green synthesis of nanoparticles has emerged as a simple, safe, sustainable, reliable and eco-friendly protocol. Among different types of NPs, green-synthesized zinc oxide NPs (ZnONPs) show various promising biological uses due to their interesting magnetic, electrical, optical and chemical characteristics. Keeping in view the dependence of the therapeutic efficacy of NPs on their physico-chemical characteristics, the green synthesis of ZnONPs using Casuarina equisetifolia leaf extract under UV-A and UV-C light was carried out in this study. UV-irradiation helped to control the size and morphology of ZnONPs by exciting the electrons in the photoactive compounds of plant extracts to enhance the bio-reduction of ZnO into ZnONPs. C. equisetifolia leaf extract was found enriched with phenolic (2.47 ± 0.12 mg GAE/g DW) and flavonoid content (0.88 ± 0.28 mg QE/g DW) contributing to its 74.33% free-radical scavenging activity. FTIR spectra showed the involvement of polyphenols in the bio-reduction, stabilization and capping of ZnONPs. Moreover, SEM-EDX and XRD analyses showed great potential of UV-C light in yielding smaller (34–39 nm) oval-shaped ZnONPs, whereas UV-A irradiation resulted in the formation of fairly spherical 67–71 nm ZnONPs and control ZnONPs were of mixed shape and even larger size (84–89 nm). Green-synthesized ZnONPs, notably CE-UV-C-ZnONPs, showed promising anti-bacterial activities against Bacillus subtilis, Pseudomonas fluorescens and Pseudomonas aeruginosa. Moreover, ZnONPs also enhanced ROS production which led to a significant loss of mitochondrial membrane potential and activated caspase-3 gene expression and caspase-3/7 activity in human hepatocellular carcinoma (HepG2) cells. CE-UV-C-ZnONP treatment reduced HepG2 cell viability to as low as 36.97% owing to their unique shape and smaller size. Lastly, ZnONPs were found to be highly biocompatible towards brine shrimp and human red blood cells suggesting their bio-safe nature. This research study sheds light on the plausible role of UV radiation in the green synthesis of ZnONPs with reasonable control over their size and morphology, thus improving their biological efficacy.

Field-usable aptamer‑gold nanoparticles-based colorimetric sensor for rapid detection of white spot syndrome virus in shrimp

White spot syndrome virus (WSSV) is one of the most threatening viral pathogens of shrimp worldwide. VP28, the major capsid protein of WSSV, is reported to play an essential role in the interaction with the host cells. Usually, the diagnostic test of WSSV is performed by one-step PCR, which is neither field-usable nor rapid enough. Therefore, the development of early diagnostic tests is imperative for the management of disease and to prevent huge economic losses. In this work, a rapid colorimetric aptasensor with high selectivity and sensitivity was introduced. First, a new ssDNA aptamer for the detection of WSSV was designed from a random pool of aptamer sequences based on the docking score and bioconjugate free energy, and then, evaluated experimentally. The designed aptamer was used to prepare an aptasensor conjugate gold nanoparticles (AuNPs) as a recognition element that increased the resistance of AuNPs to salt-induced aggregation. Therefore, the AuNPs with spherical morphology and average particle size of about 20 nm were synthesized for this reason. The results showed that the aptamer didn't attach to the other pathogens (i.e., IHHN and Vibrio harveyi) and healthy shrimp cells, and remained stable. Interestingly, the gray-blue color of aptasensor was seen only by the presence of WSSV, indicating the aptasensor assay showed good specificity to WSSV. The limit of detection (LOD) of the aptasensor was 104 copies of WSSV that could be detected only utilizing naked eyes. It was concluded that the designed aptamer had excellent potential for the detection of WSSV as a rapid visual colorimetric assay in aquaculture shrimp farmers.

Molecular modelling approaches predicted 1,2,3-triazolyl ester of ketorolac (15K) to be a novel allosteric modulator of the oncogenic kinase PAK1

P21-activated kinases (PAKs) are serine/threonine protein kinase which have six different isoforms (PAK1–6). Of those, PAK1 is overexpressed in many cancers and considered to be a major chemotherapeutic target. Most of the developed PAK1 inhibitor drugs work as pan-PAK inhibitors and show undesirable toxicity due to having untargeted kinase inhibition activities. Selective PAK1 inhibitors are therefore highly desired and oncogenic drug hunters are trying to develop allosteric PAK1 inhibitors. We previously synthesized 1,2,3-triazolyl ester of ketorolac (15K) through click chemistry technique, which exhibits significant anti-cancer effects via inhibiting PAK1. Based on the selective anticancer effects of 15K against PAK1-dependent cancer cells, we hypothesize that it may act as an allosteric PAK1 inhibitor. In this study, computational analysis was done with 15K to explore its quantum chemical and thermodynamic properties, molecular interactions and binding stability with PAK1, physicochemical properties, ADMET, bioactivities, and druglikeness features. Molecular docking analysis demonstrates 15K as a potent allosteric ligand that strongly binds to a novel allosteric site of PAK1 (binding energy ranges – 8.6 to – 9.2 kcal/mol) and does not target other PAK isoforms; even 15K shows better interactions than another synthesized PAK1 inhibitor. Molecular dynamics simulation clearly supports the stable binding properties of 15K with PAK1 crystal. Density functional theory-based calculations reveal that it can be an active drug with high softness and moderate polarity, and ADMET predictions categorize it as a non-toxic drug as evidenced by in vitro studies with brine shrimp and fibroblast cells. Structure–activity relationship clarifies the role of ester bond and triazol moiety of 15K in establishing novel allosteric interactions. Our results summarize that 15K selectively inhibits PAK1 as an allosteric inhibitor and in turn shows anticancer effects without toxicity.

A Novel Approach of Transducing Recombinant Baculovirus into Primary Lymphoid Cells of Penaeus monodon for Developing Continuous Cell Line.

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5mM sodium butyrate to the culture medium.

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding.

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded β-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.