Destabilizing Effect of Organo Ru(II) Salts on the Intermolecular Parallel CGG Repeat DNA Quadruplex Associated with Neurodegenerative/Neuromuscular Diseases.

The cationic organo ruthenium(II) salts ([Ru(p-cymene)(ipit)(Cl)](Cl) (RuS), 1-isopropyl-3-(pyridin-2-yl)-imidazol-2-thione (ipit) and [Ru(p-cymene)(ipis)(Cl)](Cl) (RuSe), 1-isopropyl-3-(pyridin-2-yl)-imidazol-2-selenone (ipis)) are isolated, and their binding efficacy with d(CGG)15 quadruplex is investigated. Circular dichroism (CD) wavelength scan titration experiments of RuS and RuSe compounds with the intermolecular parallel quadruplex formed by d(CGG)15 (associated with neurodegenerative/neuromuscular/neuronal intranuclear inclusion disorders like FXTAS, OPMD, OPDM types 1-4, and OPML as well as FXPOI) and with the control d(CGG)15·d(CCG)15 duplex indicate their specificity toward the former. Electrophoretic mobility shift titration experiments also confirm the binding of the ligands with d(CGG)15. CD thermal denaturation experiments indicate that both RuS and RuSe destabilize the quadruplex, specifically at 10 mM concentration of the ligands. This is further confirmed by 1D 1H NMR experiments. Such a destabilizing effect of these ligands on the d(CGG)15 quadruplex indicates that RuS and RuSe chalcogen complexes can act as a template for the design of novel molecules for the diagnostics and/or therapeutics of CGG repeat expansion-associated diseases.

Innate immune function of serine/threonine-protein kinase from Macrobrachium rosenbergii in response to host-pathogen interactions.

The occurrences of multiple drug-resistant strains have been relentlessly increasing in recent years. The aquaculture industry has encountered major disease outbreaks and crucially affected by this situation. The usage of non-specific chemicals and antibiotics expedites the stimulation of resistant strains. Triggering the natural defense mechanism would provide an effective and safest way of protecting the host system. Hence, we have investigated the innate immune function of serine/threonine-protein kinase (STPK) in Macrobrachium rosenbergii (Mr). The in-silico protein analysis resulted in the identification of cationic antimicrobial peptide, MrSL-19, with interesting properties from STPK of M. rosenbergii. Antimicrobial assay, FACS and SEM analysis demonstrated that the peptide potentially inhibits Staphylococcus aureus by interacting with its membrane. The toxic study on MrSL-19 demonstrated that the peptide is not toxic against HEK293 cells as well as human erythrocytes. This investigation showed the significant innate immune property of an efficient cationic antimicrobial peptide, MrSL-19 of STPK from M. rosenbergii.

Characteristics of Molecularly Engineered Anticancer Drug Conjugated Organic Nanomicelles for Site-Selective Cancer Cell Rupture and Growth Inhibition of Tumor Spheroids.

Site-selective uptake and specific biodistribution of chemotherapeutic drugs are essential prerequisites for targeted cancer therapy. Especially, antibody and peptide conjugated drugs have been attempted as localized therapeutic agents. However, the characteristics of drug conjugated nanosystems are less explored, which are limited with their toxicity, low therapeutic efficacy, complicated synthesis, and high costs. Herein, we report a biocompatible (about 95%) molecularly engineered anticancer drug conjugated nanomicelles (∼200 nm in size) for site-selective CD44 overexpressed cancer cell rupture and tumor growth inhibition. Microscopic analysis demonstrates the distinct visualization of organic-organic interfaces (∼5 nm), which are corroborated with spectroscopic measurements confirmed the conjugation of niclosamide drug with hyaluronic acid (NIC-HA). Uniformly distributed hemocompatible (about 99%) organic nanomicelles exhibit the cellular membrane and cytoplasmic targeting with significant cellular rupture (IC50 of 4 µM for MDA MB 231 cells) indicating their inherent targeting ability for cancer cells and cancer stem cells. An inclusive in vitro and in vivo analysis for targeted antitumor activity (HT1080 tumor xenograft model) of NIC-HA nanoconjugates (∼24.6% loading) exhibited promising cancer cell death and tumor growth inhibition (60%, p < 0.05) due to STAT-3 signaling pathway inhibition and induction of apoptosis in CD44-positive triple negative breast cancer cells.

Comprehensive Evaluation of Degradable and Cost-Effective Plasmonic Nanoshells for Localized Photothermolysis of Cancer Cells.

Integrating the concept of biodegradation and light-triggered localized therapy in a functional nanoformulation is the current approach in onco-nanomedicine. Morphology control with an enhanced photothermal response, minimal toxicity, and X-ray attenuation of polymer-based nanoparticles is a critical concern for image-guided photothermal therapy. Herein, we describe the simple design of cost-effective and degradable polycaprolactone-based plasmonic nanoshells for the integrated photothermolysis as well as localized imaging of cancer cells. The gold-deposited polycaprolactone-based plasmonic nanoshells (AuPCL NS) are synthesized in a scalable and facile way under ambient conditions. The synthesized nanoshells are monodisperse, fairly stable, and highly inert even at five times (250 µg/mL) the therapeutic concentration in a week-long test. AuPCL NS are capable of delivering standalone photothermal therapy for the complete ablation of cancer cells without using any anticancerous drugs and causing toxicity. It delivers the same therapeutic efficacy to different cancer cell lines, irrespective of their chemorefractory status and also works as a potential computed tomography contrast agent for the integrated imaging-directed photothermal cancer therapy. High biocompatibility, degradability, and promising photothermal efficacy of AuPCL NS are attractive aspects of this report that could open new horizons of localized plasmonic photothermal therapy for healthcare applications.

Enhanced EPR directed and Imaging guided Photothermal Therapy using Vitamin E Modified Toco-Photoxil.

Herein we report synthesis, characterization and preclinical applications of a novel hybrid nanomaterial Toco-Photoxil developed using vitamin E modified gold coated poly (lactic-co-glycolic acid) nanoshells incorporating Pgp inhibitor d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) as a highly inert and disintegrable photothermal therapy (PTT) agent. Toco-Photoxil is highly biocompatible, physiologically stable PTT material with an average diameter of 130 nm that shows good passive accumulation (2.3% ID) in solid tumors when delivered systemically. In comparison to its surface modified counterparts such as IR780-Toco-Photoxil, FA-Toco-Photoxil or FA-IR780-Toco-Photoxil accumulation are merely ~0.3% ID, ~0.025% ID and ~0.005% ID in folate receptor (FR) negative and positive tumor model. Further, Toco-Photoxil variants are prepared by tuning the material absorbance either at 750 nm (narrow) or 915 nm (broad) to study optimal therapeutic efficacy in terms of peak broadness and nanomaterial's concentration. Our findings suggest that Toco-Photoxil tuned at 750 nm absorbance is more efficient (P = 0.0097) in preclinical setting. Toco-Photoxil shows complete passiveness in critical biocompatibility test and reasonable body clearance. High tumor specific accumulation from systemic circulation, strong photothermal conversion and a very safe material property in body physiology makes Toco-Photoxil a superior and powerful PTT agent, which may pave its way for fast track clinical trial in future.

Bactericidal and fungistatic activity of peptide derived from GH18 domain of prawn chitinase 3 and its immunological functions during biological stress.

Chitinases play a vital role during the pathogenic invasion and immunosuppression in various organisms including invertebrates and vertebrates. In this study, we have investigated the participation of MrChit-3 (Macrobrachium rosenbergii Chitinase-3) during host-pathogenic interaction in freshwater prawn, M. rosenbergii. Quantitative real-time PCR analysis showed that the expression of MrChit-3 was up-regulated during bacterial, viral and laminarin challenge. Moreover, to understand the antimicrobial role of the GH18 domain, a putative membrane-targeting antimicrobial peptide (MrVG) was identified from the GH18 domain region of the protein and it was chemically synthesized. Physico-chemical features of the GH18 derived antimicrobial peptide (AMP) was assessed by various in silico tools and the antimicrobial property of the peptide was confirmed from in vitro studies. The membrane targeting mechanism of the peptide was determined by flow cytometry (FACS) and scanning electron microscope (SEM) analysis. Interestingly, the peptide was able to inhibit the growth of a chitinolytic fungal pathogen, Aspergillus niger, which was isolated from the shells of M. rosenbergii. The toxicity studies such as hemolysis activity on human blood erythrocytes and cell viability assay with primary kidney cells, HEK293 of MrVG revealed that the peptide was not involved in inducing any toxicity.

A mini review on immune role of chemokines and its receptors in snakehead murrel Channa striatus.

Chemokines are ubiquitous cytokine molecules involved in migration of cells during inflammation and normal physiological processes. Though the study on chemokines in mammalian species like humans have been extensively studied, characterization of chemokines in teleost fishes is still in the early stage. The present review provides an overview of chemokines and its receptors in a teleost fish, Channa striatus. C. striatus is an air breathing freshwater carnivore, which has enormous economic importance. This species is affected by an oomycete fungus, Aphanomyces invadans and a Gram negative bacteria Aeromonas hydrophila is known to cause secondary infection. These pathogens impose immune changes in the host organism, which in turn mounts several immune responses. Of these, the role of cytokines in the immune response is immense, due to their involvement in several activities of inflammation such as cell trafficking to the site of inflammation and antigen presentation. Given that importance, chemokines in fishes do have significant role in the immunological and other physiological functions of the organism, hence there is a need to understand the characteristics, activities and performace of these small molecules in details.

Gene expression and in silico analysis of snakehead murrel interleukin 8 and antimicrobial activity of C-terminal derived peptide WS12.

Chemokines have been known for their wide range of functions including chemoattractant property in humans and other vertebrate organisms. They act as a bridge between innate and adaptive immune system. In the present study, we have identified a CXC chemokine from the cDNA library of C. striatus; on the basis of orthology study, it was found highly identical to interleukin 8 (IL8). The bioinformatics analysis of the chemokine revealed the presence of a typical γ-core domain and a CXC motif at the N-terminal region of the molecule. Based on the amphipathic nature at the C terminal helical region of CstIL8 and their antimicrobial propensity observed during bioinformatics analysis, a short peptide namely WS12 comprising 12 amino acid residues was predicted and synthesized to determine its antimicrobial activity. The peptide WS12 was active against Bacillus cereus, a Gram positive bacterium. Scanning electron microscopy (SEM) results showed bleb-like formation on the surface of the bacteria after the treatment of WS12. Additionally, WS12 did not exhibit any cytotoxic activity against the fish leukocytes. Further, the gene expression studies also revealed that CstIL8 was expressed significantly in liver of Channa striatus (Cst) at basal level. The immune challenge studies with pathogens and immune-stimulants revealed an increase in the mRNA levels at different time points post-challenge. Hence, it is possible to conclude that WS12 was a potent antimicrobial agent and it was significantly expressed during the pathogen stress.

Pellino-1 derived cationic antimicrobial prawn peptide: Bactericidal activity, toxicity and mode of action.

The antimicrobial peptides (AMPs) are multifunctional molecules which represent significant roles in the innate immune system. These molecules have been well known for decades because of their role as natural antibiotics in both invertebrates and vertebrates. The development of multiple drug resistance against conventional antibiotics brought a greater focus on AMPs in recent years. The cationic peptides, in particular, proven as host defense peptides and are considered as effectors of innate immunity. Among the various innate immune molecules, functions of pellino-1 (Peli-1) have been recently studied for its remarkable role in specific immune functions. In our study, we have identified Peli-1 from the cDNA library of freshwater prawn Macrobrachium rosenbergii (Mr) and analyzed its features using various in-silico methods. Real time PCR analysis showed an induced expression of MrPeli-1 during white spot syndrome virus (WSSV), bacteria (Vibrio harveyi) and lipopolysaccharide (LPS) from Escherichia coli challenge. Also, a cationic AMP named MrDN was derived from MrPeli-1 protein sequence and its activity was confirmed against various pathogenic bacteria. The mode of action of MrDN was determined to be its membrane permeabilization ability against Bacillus cereus ATCC 2106 as well as its DNA binding ability. Further, scanning electron microscopic (SEM) images showed the membrane disruption and leakage of cellular components of B. cereus cells induced by MrDN. The toxicity of MrDN against normal cells (HEK293 cells) was demonstrated by MTT and hemolysis assays. Overall, the results demonstrated the innate immune function of MrPeli-1 with a potential cationic AMP in prawn.

In-silico analysis and mRNA modulation of detoxification enzymes GST delta and kappa against various biotic and abiotic oxidative stressors.

This study reports the comprehensive comparative information of two different detoxification enzymes such as glutathione S-transferases (GSTs) delta and kappa from freshwater giant prawn Macrobrachium rosenbergii (designated as MrGSTD and MrGSTK) by investigating their in-silico characters and mRNA modulation against various biotic and abiotic oxidative stressors. The physico-chemical properties of these cDNA and their polypeptide structure were analyzed using various bioinformatics program. The analysis indicated the variation in size of the polypeptides, presence or absence of domains and motifs and structure. Homology and phylogenetic analysis revealed that MrGSTD shared maximum identity (83%) with crustaceans GST delta, whereas MrGSTK fell in arthropods GST kappa. It is interesting to note that MrGSTD and MrGSTK shared only 21% identity; it indicated their structural difference. Structural analysis indicated that MrGSTD to be canonical dimer like shape and MrGSTK appeared to be butterfly dimer like shape, in spite of four ß-sheets being conserved in both GSTs. Tissue specific gene expression analysis showed that both MrGSTD and MrGSTK are highly expressed in immune organs such as haemocyte and hepatopancreas, respectively. To understand the role of mRNA modulation of MrGSTD and MrGSTK, the prawns were inducted with oxidative stressors such as bacteria (Vibrio harveyi), virus [white spot syndrome virus (WSSV)] and heavy metal, cadmium (Cd). The analysis revealed an interesting fact that both MrGSTD and MrGSTK showed higher (P < 0.05) up-regulation at 48 h post-challenge, except MrGSTD stressed with bacteria, where it showed up-regulation at 24 h post-challenge. Overall, the results suggested that GSTs are diverse in their structure and possibly conferring their potential involvement in immune protection in crustaceans. However, further study is necessary to focus their functional differences at proteomic level.

Multifunctional murrel caspase 1, 2, 3, 8 and 9: Conservation, uniqueness and their pathogen-induced expression pattern.

Caspases are evolutionarily conserved proteases which play fundamental role in apoptosis. Invasion of pathogen triggers the activation of caspases-mediated pro-inflammatory and pro-apoptotic pathways, where multifunctional caspases are involved. In striped murrel Channa striatus, epizootic ulcerative syndrome (EUS) causes endemics resulting in huge economic loss. Aphanomyces invadans, an oomycete is the primary causative agent of EUS which further induces secondary bacterial infections especially Aeromonas hydrophila. In order to get insights into the caspase gene family in C. striatus during EUS infection, we performed various physicochemical and structural analyses on the cDNA and protein sequences of five different murrel caspases namely CsCasp 1, 2, 3, 8 and 9. Sequence analysis of murrel caspase proteins showed that in spite of the conserved CASC domain, each caspase embraces some unique features which made them functionally different. Tissue distribution analysis showed that all the murrel caspases are highly expressed in one of the immune organs such as liver, kidney, spleen and blood cells. Further, to understand the role of caspase during EUS infection, modulation in expression of each caspase gene was analysed after inducing fungal and bacterial infection in C. striatus. Pathogen-induced gene expression pattern revealed an interesting fact that the expression of all the caspase genes reached a maximum level at 24 h post-infection (p.i) in case of bacteria, whereas it was 48 h in fungus. However, the initiation of elevated expression differed between each caspase based on their role such as pro-inflammatory, initiator and executioner caspase. Overall, the results suggested that the caspases in murrel are diverse in their structure and function. Here, we discuss the similarities and differences of five different murrel caspases.

Molecular importance of prawn large heat shock proteins 60, 70 and 90.

Considering the importance of heat shock proteins (HSPs) in the innate immune system of prawn, a comparative molecular approach was proposed to study the crustacean large HSPs 60, 70 and 90. Three different large HSPs were identified from freshwater prawn Macrobrachium rosenbergii (Mr) cDNA library during screening. The structural and functional characteristic features of HSPs were studied using various bioinformatics tools. Also, their gene expression and mRNA regulation upon various pathogenic infections was studied by relative quantification using 2(-ΔΔCT) method. MrHSP60 contains a long chaperonin 60 domain at 46-547 which carries a chaperonin 60 signature motif between 427 and 438, whereas MrHSP70 contains a long HSP70 domain at 21-624 and MrHSP90 carries a HSP90 domain at 188-719. The two dimensional analysis showed that MrHSP60 contains more amino acids (52%) in helices, whereas MrHSP70 (40.6%) and MrHSP90 (51.8%) carried more residues in coils. Gene expression results showed significant (P < 0.05) expression of MrHSP60, 70 and 90 in haemocyte, gill and hepatopancreas, respectively. Further, the expression level was up-regulated upon bacterial (Aeromonas hydrophilla and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodo virus (MrNV)] infections during various time periods. The gene expression results exhibited the potential involvement of these three HSPs in the immune system of prawn. The study indicated the potentiality of these molecules, thereby protecting cells against pathogens as well as severe cellular and environmental stresses in crustaceans.