Diagnosis and biomarkers for ocular tuberculosis: From the present into the future.

Tuberculosis is an airborne disease caused by Mycobacterium tuberculosis (Mtb) and can manifest both pulmonary and extrapulmonary disease, including ocular tuberculosis (OTB). Accurate diagnosis and swift optimal treatment initiation for OTB is faced by many challenges combined with the lack of standardized treatment regimens this results in uncertain OTB outcomes. The purpose of this study is to summarize existing diagnostic approaches and recently discovered biomarkers that may contribute to establishing OTB diagnosis, choice of anti-tubercular therapy (ATT) regimen, and treatment monitoring. The keywords ocular tuberculosis, tuberculosis, Mycobacterium, biomarkers, molecular diagnosis, multi-omics, proteomics, genomics, transcriptomics, metabolomics, T-lymphocytes profiling were searched on PubMed and MEDLINE databases. Articles and books published with at least one of the keywords were included and screened for relevance. There was no time limit for study inclusion. More emphasis was placed on recent publications that contributed new information about the pathogenesis, diagnosis, or treatment of OTB. We excluded abstracts and articles that were not written in the English language. References cited within the identified articles were used to further supplement the search. We found 10 studies evaluating the sensitivity and specificity of interferon-gamma release assay (IGRA), and 6 studies evaluating that of tuberculin skin test (TST) in OTB patients. IGRA (Sp = 71-100%, Se = 36-100%) achieves overall better sensitivity and specificity than TST (Sp = 51.1-85.7%; Se = 70.9-98.5%). For nuclear acid amplification tests (NAAT), we found 7 studies on uniplex polymerase chain reaction (PCR) with different Mtb targets, 7 studies on DNA-based multiplex PCR, 1 study on mRNA-based multiplex PCR, 4 studies on loop-mediated isothermal amplification (LAMP) assay with different Mtb targets, 3 studies on GeneXpert assay, 1 study on GeneXpert Ultra assay and 1 study for MTBDRplus assay for OTB. Specificity is overall improved but sensitivity is highly variable for NAATs (excluding uniplex PCR, Sp = 50-100%; Se = 10.5-98%) as compared to IGRA. We also found 3 transcriptomic studies, 6 proteomic studies, 2 studies on stimulation assays, 1 study on intraocular protein analysis and 1 study on T-lymphocyte profiling in OTB patients. All except 1 study evaluated novel, previously undiscovered biomarkers. Only 1 study has been externally validated by a large independent cohort. Future theranostic marker discovery by a multi-omics approach is essential to deepen pathophysiological understanding of OTB. Combined these might result in swift, optimal and personalized treatment regimens to modulate the heterogeneous mechanisms of OTB. Eventually, these studies could improve the current cumbersome diagnosis and management of OTB.

Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities.

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

A-kinase anchor protein 12 (AKAP12) inhibits cell migration in breast cancer.

A-kinase anchor protein 12 (AKAP12) also known as Gravin and SSeCKS, is a novel potent scaffold protein for many key signaling factors, such as protein kinase C (PKC), PKA, cyclins as well as F-actin. AKAP12 expression is known to be suppressed in several human malignancies including breast, prostate, gastric and colon cancers. In this study, we evaluated the role of AKAP12 in the migration of breast cancer cells, an important cellular process in cancer progression. AKAP12 gene expression was analyzed in human breast cancer tissues using the Gene expression-based Outcome for Breast cancer Online (GOBO) database and TissueScan array, followed by relapse free survival (RFS) analysis with the Kaplan-Meier Plotter. AKAP12 protein was then analyzed in normal MCF10A breast cell line and six different breast cancer cell lines (AU565, Hs578T, MCF7, MDA-MB-231, T47D and ZR751). After which, siRNA-mediated knockdown of AKAP12 was carried out in MCF10A, MDA-MB-231 and Hs578T cells, followed by phenotypic assays. AKAP12 was observed to be reduced in breast cancer tissues as analyzed by GOBO and TissueScan array. Kaplan Meier survival analysis revealed that patients with AKAP12 gene expression had a higher RFS survival. There was also decreased AKAP12 protein expression in breast cancer cell lines compared to MCF10A normal epithelial breast cell line. Knockdown of AKAP12 in both MCF10A cells and Hs578T cells induced cell migration but did not alter cell proliferation. Moreover, siAKAP12 in aggressive MDA-MB-231 breast cancer cells led to an increase in cell migration. Immunofluorescence analysis of AKAP12 depleted MCF10A cells also revealed formation of thick stress fibers which could affect cell migration. Hence, the findings in this study suggest that AKAP12 is a potential metastasis suppressor in breast cancer.

Identification of natural peptides as a new class of antimalarial drugs by in silico approaches.

Malaria is one of the most widespread and serious parasitic diseases worldwide. Currently available antimalarial drugs have side effects, and many strains of Plasmodia have developed resistance to such drugs. The present review examines the use of annexins and of natural peptides from snake venom as a new class of anti-malarial agents, with the key property of reducing inflammation. Severe cases of malaria manifest elevated serum levels of liver enzymes, inflammation, fibrin deposition, apoptosis, and reduction in peripheral CD8+ T cells. The annexin-A1/5 proteins trigger inflammation via increased expression of diverse cytokines (tumor necrosis factor alpha, interleukin-1 beta, interleukin-10), however, by shielding microbial phospholipids they prevent injury via damage-associated molecular patterns (DAMPs). Here, we also review an in silico-based bioengineering approach that may allow for a better design, synthesis and characterization of novel peptides from snake venom as a more effective approach to treatment due to their improved antimalarial activity.

ANXA1 inhibits miRNA-196a in a negative feedback loop through NF-kB and c-Myc to reduce breast cancer proliferation.

MiRNAs are endogenous ~22 nt RNAs which play critical regulatory roles in a wide range of biological and pathological processes, which can act as oncogenes or tumor suppressor genes depending on their target genes. We have recently shown that ANXA1 inhibits the expression of miRNAs including miR196a. Here, we show that miR196a was highly expressed in ER+ MCF-7 breast cancer cells when compared to normal mammary gland cells, with expression levels negatively correlating to ANXA1. ANXA1 inhibits the biogenesis of oncogenic miR-196a by suppressing primary-miR196a indirectly through the stimulation of c-myc and NFkB expression and activity in breast cancer cells. In a negative feedback loop, miR-196a directly inhibits ANXA1 and enhances breast cancer cell proliferation in vitro. Finally, miR196a promotes breast tumor growth in vivo. This study reports a novel regulatory circuit between ANXA1, NF-kB, c-myc and miR-196a which regulates breast cancer cell proliferation and tumor growth.

A brief update on potential molecular mechanisms underlying antimicrobial and wound-healing potency of snake venom molecules.

Infectious diseases remain a significant cause of morbidity and mortality worldwide. A wide range of diverse, novel classes of natural antibiotics have been isolated from different snake species in the recent past. Snake venoms contain diverse groups of proteins with potent antibacterial activity against a wide range of human pathogens. Some snake venom molecules are pharmacologically attractive, as they possess promising broad-spectrum antibacterial activities. Furthermore, snake venom proteins (SVPs)/peptides also bind to integrins with high affinity, thereby inhibiting cell adhesion and accelerating wound healing in animal models. Thus, SVPs are a potential alternative to chemical antibiotics. The mode of action for many antibacterial peptides involves pore formation and disruption of the plasma membrane. This activity often includes modulation of nuclear factor kappa B (NF-κB) activation during skin wound healing. The NF-κB pathway negatively regulates the transforming growth factor (TGF)-ß1/Smad pathway by inducing the expression of Smad7 and eventually reducing in vivo collagen production at the wound sites. In this context, SVPs that regulate the NF-κB signaling pathway may serve as potential targets for drug development.

New frontiers for anti-biofilm drug development.

Pathogenic microbial biofilm, a consortium of microbial cells protected by a self-produced polymer matrix, is considered a worldwide challenge due to the inherent antibiotic resistance conferred by its lifestyle. Living, as it does, in a community of microbial organisms in a clinical situation, makes it responsible for severe and dangerous cases of infection. Combating this organisation of cells usually requires high antibiotic doses for a prolonged time, and these approaches often fail, contributing to infection persistence. In addition to therapeutic limitations, biofilms can be a source of infections when they grow in medical devices. The challenge imposed by biofilms has mobilised researchers in the entire world to prospect or develop alternatives to control biofilms. In this context, this review summarises the new frontiers that could be used in clinical circumstances in order to prevent or eliminate pathogenic biofilms.

Ageing and the telomere connection: An intimate relationship with inflammation.

Telomeres are the heterochromatic repeat regions at the ends of eukaryotic chromosomes, whose length is considered to be a determinant of biological ageing. Normal ageing itself is associated with telomere shortening. Here, critically short telomeres trigger senescence and eventually cell death. This shortening rate may be further increased by inflammation and oxidative stress and thus affect the ageing process. Apart from shortened or dysfunctional telomeres, cells undergoing senescence are also associated with hyperactivity of the transcription factor NF-κB and overexpression of inflammatory cytokines such as TNF-α, IL-6, and IFN-γ in circulating macrophages. Interestingly, telomerase, a reverse transcriptase that elongates telomeres, is involved in modulating NF-κB activity. Furthermore, inflammation and oxidative stress are implicated as pre-disease mechanisms for chronic diseases of ageing such as neurodegenerative diseases, cardiovascular disease, and cancer. To date, inflammation and telomere shortening have mostly been studied individually in terms of ageing and the associated disease phenotype. However, the interdependent nature of the two demands a more synergistic approach in understanding the ageing process itself and for developing new therapeutic approaches. In this review, we aim to summarize the intricate association between the various inflammatory molecules and telomeres that together contribute to the ageing process and related diseases.

Targeting transcription factor STAT3 for cancer prevention and therapy.

Signal Transducers and Activators of Transcription (STATs) comprise an important class of transcription factors that have been implicated in a wide variety of essential cellular functions related to proliferation, survival, and angiogenesis. Among various STAT members, STAT3 is frequently overexpressed in tumor cells as well as tissue samples, and regulates the expression of numerous oncogenic genes controlling the growth and metastasis of tumor cells. The current review briefly discusses the importance of STAT3 as a potential target for cancer therapy and also provides novel insights into various classes of existing pharmacological inhibitors of this transcription factor that can be potentially developed as anti-cancer drugs.

DAMPs and influenza virus infection in ageing.

Influenza A virus (IAV) is a serious global health problem worldwide due to frequent and severe outbreaks. IAV causes significant morbidity and mortality in the elderly population, due to the ineffectiveness of the vaccine and the alteration of T cell immunity with ageing. The cellular and molecular link between ageing and virus infection is unclear and it is possible that damage associated molecular patterns (DAMPs) may play a role in the raised severity and susceptibility of virus infections in the elderly. DAMPs which are released from damaged cells following activation, injury or cell death can activate the immune response through the stimulation of the inflammasome through several types of receptors found on the plasma membrane, inside endosomes after endocytosis as well as in the cytosol. In this review, the detriment in the immune system during ageing and the links between influenza virus infection and ageing will be discussed. In addition, the role of DAMPs such as HMGB1 and S100/Annexin in ageing, and the enhanced morbidity and mortality to severe influenza infection in ageing will be highlighted.

Novel phospholipase A2 inhibitors from python serum are potent peptide antibiotics.

Antimicrobial peptides (AMPs) play a vital role in defense against resistant bacteria. In this study, eight different AMPs synthesized from Python reticulatus serum protein were tested for bactericidal activity against various Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Burkholderia pseudomallei (KHW and TES strains), and Proteus vulgaris) using a disc-diffusion method (20 µg/disc). Among the tested peptides, phospholipase A2 inhibitory peptide (PIP)-18[59-76], ß-Asp65-PIP[59-67], D-Ala66-PNT.II, and D60,65E-PIP[59-67] displayed the most potent bactericidal activity against all tested pathogens in a dose-dependent manner (100-6.8 µg/ml), with a remarkable activity noted against S. aureus at 6.8 µg/ml dose within 6 h of incubation. Determination of minimum inhibitory concentrations (MICs) by a micro-broth dilution method at 100-3.125 µg/ml revealed that PIP-18[59-76], ß-Asp65-PIP[59-67] and D-Ala66-PNT.II peptides exerted a potent inhibitory effect against S. aureus and B. pseudomallei (KHW) (MICs 3.125 µg/ml), while a much less inhibitory potency (MICs 12.5 µg/ml) was noted for ß-Asp65-PIP[59-67] and D-Ala66-PNT.II peptides against B. pseudomallei (TES). Higher doses of peptides had no effect on the other two strains (i.e., Klebsiella pneumoniae and Streptococcus pneumoniae). Overall, PIP-18[59-76] possessed higher antimicrobial activity than that of chloramphenicol (CHL), ceftazidime (CF) and streptomycin (ST) (30 µg/disc). When the two most active peptides, PIP-18[59-76] and ß-Asp65-PIP[59-67], were applied topically at a 150 mg/kg dose for testing wound healing activity in a mouse model of S. aureus infection, the former accelerates faster wound healing than the latter peptide at 14 days post-treatment. The western blot data suggest that the topical application of peptides (PIP-18[59-67] and ß-Asp65-PIP[59-67]) modulates NF-kB mediated wound repair in mice with relatively little haemolytic (100-1.56 µg/ml) and cytotoxic (1000-3.125 µg/ml) effects evident on human cells in vitro.

Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become a rising threat to public health. There is an urgent need for development of promising new therapeutic agents against drug resistant bacteria like S. aureus. This report discusses purification and characterization of proteins from Indian Russell's viper snake venom. Novel 15-kDa proteins called "Viperatoxin" (VipTx-I and VipTx-II) were extracted from the whole venom and evaluated using in vitro antimicrobial experiments. The N-terminal amino acid sequence of "Viperatoxin" showed high sequence homology to daboiatoxin isolated from the same venom and also matched phospholipase A2 (PLA2) enzymes isolated from other snake venoms. In an in vitro plate assay, VipTx-II but not VipTx-I showed strong antimicrobial effects against S. aureus and Burkholderia pseudomallei (KHW & TES), Proteus vulgaris and P. mirabilis. The VipTx-II was further tested by a broth-dilution assay at 100-3.1 µg/ml concentrations. The most potent bactericidal effect was found at the lowest dilutions (MICs of 6.25 µg/ml) against B. pseudomallei, S. aureus and P. vulgaris (MICs of 12.25 µg/ml). Electron microscopic investigation revealed that the protein-induced bactericidal potency was closely associated with pore formation and membrane damage, even at the lowest concentrations (<20 µg/ml). The toxin caused a low level of cytotoxic effects as observed in human (THP-1) cells at higher concentrations. Molecular weight determinations of VipTx-II by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major, along with a few minor bands. The results indicate that VipTx-II plays a significant role in bactericidal and membrane damaging effects in vitro. Non-cytotoxic properties on human cells highlight it as a promising candidate for further evaluation of antimicrobial potential in vivo.

Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin.

Wound healing activity and mechanisms of action of an antibacterial protein from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus).

Basic phospholipase A2 was identified from the venom of the eastern diamondback rattlesnake. The Crotalus adamanteus toxin-II (CaTx-II) induced bactericidal effects (7.8 µg/ml) on Staphylococcus aureus, while on Burkholderia pseudomallei (KHW), and Enterobacter aerogenes were killed at 15.6 µg/ml. CaTx-II caused pore formation and membrane damaging effects on the bacterial cell wall. CaTx-II was not cytotoxic on lung (MRC-5), skin fibroblast (HEPK) cells and in mice. CaTx-II-treated mice showed significant wound closure and complete healing by 16 days as compared to untreated controls (**P<0.01). Histological examination revealed enhanced collagen synthesis and neovascularization after treatment with CaTx-II versus 2% Fusidic Acid ointment (FAO) treated controls. Measurement of tissue cytokines revealed that interleukin-1 beta (IL-1ß) expression in CaTx-II treated mice was significantly suppressed versus untreated controls. In contrast, cytokines involved in wound healing and cell migration i.e., monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor-basic (FGF-b), chemokine (KC), granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly enhanced in CaTx-II treated mice, but not in the controls. CaTx-II also modulated nuclear factor-kappa B (NF-κB) activation during skin wound healing. The CaTx-II protein highlights distinct snake proteins as a potential source of novel antimicrobial agents with significant therapeutic application for bacterial skin infections.

Plant-based hydrocarbon esters from Tragia involucrata possess antimicrobial and anti-inflammatory activities.

Antimicrobial and anti-inflammatory activities of hydrocarbon esters obtained from Tragia involucrata were evaluated by disk-diffusion (250 µg/ml), and broth-dilution (500-7.8 µg/ml), methods against bacteria. Among the compounds, shellsol showed the most potent activity against Burkholderia pseudomallei (KHW), Aeromonas hydrophila, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, Klebsiella pneumoniae, Proteus mirabilis, and Streptococcus pneumoniae. Interestingly, vinyl hexylether was active against food-spoilage bacteria (Bacillus cereus and Proteus vulgaris), 2, 4-methyl hexane also exerted antimicrobial activity against K. pneumoniae, S. pyogenes, B. pseudomallei, Alcaligens viscolactis, and Pseudomonas aeruginosa. 2-methylnonane and 2, 6-dimethyl heptane showed only weak activity. For example, shellsol showed bacteriostatic effect (MIC of 7.8 µg/ml) against A. hydrophila, vinyl hexylether (MIC of 15.6 µg/ml) against P. mirabilis, and 2, 4-methyl hexane (MIC of 31.25 µg/ml) on B. pseudomallei. Cytotoxic effects of compounds were assayed in human skin and monkey kidney cells (62.5-2000 µg/ml) by an XTT assay. The vinyl hexylether, 2, 4-dimethyl hexane and shellsol did not show any toxicity up to 1000 µg/ml concentrations. The 2-methylnonane and 2, 6-dimethyl heptane induced morphological changes (e.g. cell disintegration and lysis) of both cell types at a 2000 µg/ml. The vinyl hexylether, 2, 4-dimethyl hexane and shellsol were devoid of toxic effects; however, 2-methylnonane induced weight loss and severe necrosis as evidenced by histopathological and serum biochemical analysis in rats. Interestingly, shellsol showed the maximum inhibition of carrageenan-induced, paw oedema in rats. In conclusion, findings of this study clearly indicate that biologically active hydrocarbon esters, such as shellsol, vinyl hexylether, and 2, 4-dimethyl hexane isolated from T. involucrata, may effectively control the growth of certain food-borne and food-spoilage pathogens.

Emodin suppresses migration and invasion through the modulation of CXCR4 expression in an orthotopic model of human hepatocellular carcinoma.

Accumulating evidence(s) indicate that CXCL12-CXCR4 signaling cascade plays an important role in the process of invasion and metastasis that accounts for more than 80% of deaths in hepatocellular carcinoma (HCC) patients. Thus, identification of novel agents that can downregulate CXCR4 expression and its associated functions have a great potential in the treatment of metastatic HCC. In the present report, we investigated an anthraquinone derivative, emodin for its ability to affect CXCR4 expression as well as function in HCC cells. We observed that emodin downregulated the expression of CXCR4 in a dose-and time-dependent manner in HCC cells. Treatment with pharmacological proteasome and lysosomal inhibitors did not have substantial effect on emodin-induced decrease in CXCR4 expression. When investigated for the molecular mechanism(s), it was observed that the suppression of CXCR4 expression was due to downregulation of mRNA expression, inhibition of NF-κB activation, and abrogation of chromatin immunoprecipitation activity. Inhibition of CXCR4 expression by emodin further correlated with the suppression of CXCL12-induced migration and invasion in HCC cell lines. In addition, emodin treatment significantly suppressed metastasis to the lungs in an orthotopic HCC mice model and CXCR4 expression in tumor tissues. Overall, our results show that emodin exerts its anti-metastatic effect through the downregulation of CXCR4 expression and thus has the potential for the treatment of HCC.

Zinc supplementation prevents cardiomyocyte apoptosis and congenital heart defects in embryos of diabetic mice.

Oxidative stress induced by maternal diabetes plays an important role in the development of cardiac malformations. Zinc (Zn) supplementation of animals and humans has been shown to ameliorate oxidative stress induced by diabetic cardiomyopathy. However, the role of Zn in the prevention of oxidative stress induced by diabetic cardiac embryopathy remains unknown. We analyzed the preventive role of Zn in diabetic cardiac embryopathy by both in vivo and in vitro studies. In vivo study revealed a significant decrease in lipid peroxidation, superoxide ions, and oxidized glutathione and an increase in reduced glutathione, nitric oxide, and superoxide dismutase in the developing heart at embryonic days (E) 13.5 and 15.5 in the Zn-supplemented diabetic group when compared to the diabetic group. In addition, significantly down-regulated protein and mRNA expression of metallothionein (MT) in the developing heart of embryos from diabetic group was rescued by Zn supplement. Further, the nuclear microscopy results showed that trace elements such as phosphorus, calcium, and Zn levels were significantly increased (P<0.001), whereas the iron level was significantly decreased (P<0.05) in the developing heart of embryos from the Zn-supplemented diabetic group. In vitro study showed a significant increase in cellular apoptosis and the generation of reactive oxygen species (ROS) in H9c2 (rat embryonic cardiomyoblast) cells exposed to high glucose concentrations. Supplementation with Zn significantly decreased apoptosis and reduced the levels of ROS. In summary, oxidative stress induced by maternal diabetes could play a role in the development and progression of cardiac embryopathy, and Zn supplementation could be a potential therapy for diabetic cardiac embryopathy.

Carboxylate microsphere-induced cellular toxicity in human lung fibroblasts.

Carboxylate microspheres (CMs) are mainly used in industrial, biomedical and various household products. In this study, we assessed the cytotoxic effects of CMs on human MRC-5 lung fibroblasts by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Oxidative stress was determined by measurements of reactive oxygen species and antioxidant (superoxide dismutase and catalase) levels and proinflammatory cytokines quantified by enzyme-linked immunosorbent assay. Morphological changes were examined by light microscopy, confocal microscopy and transmission electron microscopy. The lung fibroblasts were exposed to increasing concentrations of CMs (0.1-1000 µmol/L) for 24 h. The results showed significant changes in cell morphology with induction of cytotoxicity and oxidative stress observed in 10-1000 µmol/L concentrations of CM-treated fibroblasts. Ultrastructural examination revealed the presence of CMs inside the cytoplasm of treated lung fibroblasts. CMs also induced elevated interleukin (IL)-1, IL-6, IL-8, IL-10 and tumor necrosis factor α levels at higher concentrations. We have demonstrated that CMs significantly reduce cell viability in a dose-dependant manner in lung fibroblasts at 0.1-1000 µmol/L doses. The findings suggest that high doses of CMs have the potential to induce cellular toxicity to the lung in vitro.

Identification of a novel Calotropis procera protein that can suppress tumor growth in breast cancer through the suppression of NF-κB pathway.

Breast cancer is the most common cancer among women. To date, improvements in hormonal and cytotoxic therapies have not yet led to a sustained remission or cure. In the present study, we investigated the in vitro and in vivo antitumor activities of a novel Calotropis procera protein (CP-P) isolated from root bark. CP-P protein inhibited the proliferation and induced apoptosis of breast cancer cells through the suppression of nuclear factor kappaB (NF-kB) activation. CP-P, when administered individually or in combination with cyclophosphamide (CYC, 0.2 mg/kg) to rats with 7, 12-dimethyl benz(a)anthracene (DMBA)-induced breast cancer decreased tumor volume significantly without affecting the body weight. To elucidate the anticancer mechanism of CP-P, antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST) and non-enzymatic antioxidant - reduced glutathione (GSH), vitamin E and C generation in the breast were analyzed by various assays. SOD, CAT, GST, GSH, vitamin E and C levels were high in combination-treated groups (CP-P+CYC) versus the CYC alone-treated groups. Also, the combination was more effective in down-regulating the expression of NF-kB-regulated gene products (cyclin D1 and Bcl-2) in breast tumor tissues. Our findings indicate that CP-P possesses significant antitumor activity comparable to a commonly used anticancer drug, cyclophosphamide, and may form the basis of a novel therapy for breast cancer.