Interactive effect of carbendazim and imidacloprid on buffalo bone marrow derived mesenchymal stem cells: oxidative stress, cytotoxicity and genotoxicity.

The effect of a combination of two pesticides, carbendazim (CBZ) and imidacloprid (IMI), was investigated on mesenchymal stem cells derived from the bone marrow of buffalo (bMSCs). The bMSCs were exposed to the CBZ (2.25 µM, 4.49 µM, and 8.98 µM) and IMI (0.81 mM, 1.61 mM, and 3.22 mM) alone as well as in combinations. The bMSCs were found to be positive for the stem cell markers, AP, CD73, and OCT4. The bMSCs showed a significant reduction (p ≤ 0.05) in cell viability, and status of anti-oxidants while a significant increase (p ≤ 0.05) in the level of LDH, ALP, and CK-MB in CBZ and IMI-treated groups. A significant increase (p ≤ 0.05) was noticed in LPO, O2─ radical, total ROS, loss of ΔΨm, apoptotic index, and DNA damage in CBZ and IMI-treated groups. A low-dose combination group showed an elevated effect compared to the groups treated with the single pesticide. The interaction index was calculated for CBZ-IMI combined treatment groups on various parameters that showed the majority of antagonist effects. Present findings confirmed that CBZ and IMI-induced cytotoxicity in bMSCs was mediated via ROS production, altered ΔΨm and LPO along with depressed antioxidant status which was responsible for cell apoptosis and cell damage. This study suggested that CBZ and IMI had a dose-dependent toxic effect when the pesticides were used alone, while, co-exposure to both the pesticides simultaneously had an antagonist or non-additive effect on buffalo bMSCs at lower dose combinations and they induced a potentiating effect at high-dose combination.

Identification of miR-34 regulatory networks in settings of disease and antimiR-therapy: Implications for treating cardiac pathology and other diseases.

Expression of the miR-34 family (miR-34a, -34b, -34c) is elevated in settings of heart disease, and inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy. Under chronic cardiac disease settings, targeting the entire miR-34 family is more effective than targeting miR-34a alone. The identification of transcription factor (TF)-miRNA regulatory networks has added complexity to understanding the therapeutic potential of miRNA-based therapies. Here, we sought to determine whether antimiR-34 targets secondary miRNAs via TFs which could contribute to antimiR-34-mediated protection. Using miRNA-Seq we identified differentially regulated miRNAs in hearts from mice with cardiac pathology due to transverse aortic constriction (TAC), and focused on miRNAs which were also regulated by antimiR-34. Two clusters of stress-responsive miRNAs were classified as "pathological" and "cardioprotective," respectively. Using ChIPBase we identified 45 TF binding sites on the promoters of "pathological" and "cardioprotective" miRNAs, and 5 represented direct targets of miR-34, with the capacity to regulate other miRNAs. Knockdown studies in a cardiomyoblast cell line demonstrated that expression of 2 "pathological" miRNAs (let-7e, miR-31) was regulated by one of the identified TFs. Furthermore, by qPCR we confirmed that expression of let-7e and miR-31 was lower in hearts from antimiR-34 treated TAC mice; this may explain why targeting the entire miR-34 family is more effective than targeting miR-34a alone. Finally, we showed that Acsl4 (a common target of miR-34, let-7e and miR-31) was increased in hearts from TAC antimiR-34 treated mice. In summary, antimiR-34 regulates the expression of other miRNAs and this has implications for drug development.

Sex differences in response to miRNA-34a therapy in mouse models of cardiac disease: identification of sex-, disease- and treatment-regulated miRNAs.

KEY POINTS: MicroRNA (miRNA)-based therapies are in development for numerous diseases, including heart disease. Currently, very limited basic information is available on the regulation of specific miRNAs in male and female hearts in settings of disease. The identification of sex-specific miRNA signatures has implications for translation into the clinic and suggests the need for customised therapy. In the present study, we found that a miRNA-based treatment inhibiting miRNA-34a (miR-34a) was more effective in females in a setting of moderate dilated cardiomyopathy than in males. Furthermore, the treatment showed little benefit for either sex in a setting of more severe dilated cardiomyopathy associated with atrial fibrillation. The results highlight the importance of understanding the effect of miRNA-based therapies in cardiac disease settings in males and females. ABSTRACT: MicroRNA (miRNA)-34a (miR-34a) is elevated in the diseased heart in mice and humans. Previous studies have shown that inhibiting miR-34a in male mice in settings of pathological cardiac hypertrophy or ischaemia protects the heart against progression to heart failure. Whether inhibition of miR-34a protects the female heart is unknown. Furthermore, the therapeutic potential of silencing miR-34a in settings of dilated cardiomyopathy (DCM) and atrial fibrillation (AF) has not been assessed previously. In the present study, we examined the effect of silencing miR-34a in males and females in (1) a model of moderate DCM and (2) a model of severe DCM with AF. The cardiac disease models were administered with a locked nucleic acid-modified oligonucleotide (LNA-antimiR-34a) at 6-7 weeks of age when the models display cardiac dysfunction and conduction abnormalities. Cardiac function and morphology were measured 6 weeks after treatment. In the present study, we show that inhibition of miR-34a provides more protection in the DCM model in females than males. Disease prevention in LNA-antimiR-34a treated DCM female mice was characterized by attenuated heart enlargement and lung congestion, lower expression of cardiac stress genes (B-type natriuretic peptide, collagen gene expression), less cardiac fibrosis and better cardiac function. There was no evidence of significant protection in the severe DCM and AF model in either sex. Sex- and treatment-dependent regulation of miRNAs was also identified in the diseased heart, and may explain the differential response of males and females. These studies highlight the importance of examining the impact of miRNA-based drugs in both sexes and under different disease conditions.

Encapsulation of Bacteriophage in Liposome Accentuates Its Entry in to Macrophage and Shields It from Neutralizing Antibodies.

Phage therapy has been a centre of attraction for biomedical scientists to treat infections caused by drug resistant strains. However, ability of phage to act only on extracellular bacteria and probability of interference by anti-phage antibodies in vivo is considered as a important limitation of bacteriophage therapy. To overcome these hurdles, liposome were used as delivery vehicle for phage in this study. Anti-phage antibodies were raised in mice and pooled serum was evaluated for its ability to neutralize free and liposome entrapped phage. Further, ability of phage and liposome-entrapped phage to enter mouse peritoneal macrophages and kill intracellular Klebsiella pneumoniae was compared. Also, an attempt to compare the efficacy of free phage and liposome entrapped phage, alone or in conjunction with amikacin in eradicating mature biofilm was made. The entrapment of phage in liposome provided 100% protection to phage from neutralizing antibody. On the contrary un-entrapped phage got neutralized within 3 h of its interaction with antibody. Compared to the inability of free phage to enter macrophages, the liposome were able to deliver entrapped phage inside macrophages and cause 94.6% killing of intracellular K. pneumoniae. Liposome entrapped phage showed synergistic activity along with amikacin to eradicate mature biofilm of K. pneumoniae. Our study reinforces the growing interest in using phage therapy as a means of targeting multidrug resistant bacterial infections as liposome entrapment of phage makes them highly effective in vitro as well as in vivo by overcoming the majority of the hurdles related to clinical use of phage.

Studies on Enhancement of Anti-microbial Activity of Pristine MWCNTs Against Pathogens.

Carbon nanotubes (CNTs), owing to their inherently unique properties in the domain of biomedical sciences including drug delivery, offer an exciting platform to the researchers. Of late, their applications have also been successfully established. Recently, single-walled CNTs (SWCNTs) have been explored for antibacterial efficacy, but naïve multi-walled CNTs (MWCNTs) still remained unearthed. The present studies endeavor the investigation of the potential of various non-ionic surfactants in solubility enhancement of MWCNTs and their subsequent antibacterial efficacy against Escherichia coli and Staphylococcus aureus. Polysorbates offer more solubility to MWCNTs vis-à-vis the phospholipids. However, the antibacterial effect was found to be less influenced by solubility but significantly determined by the type of surfactant. Transmission electron photomicrographs confirmed significant adhesion of MWCNTs to the bacterial walls only in the presence of unsaturated phospholipids and this was expressed in the form of lowest minimum inhibitory concentration (MIC) values of MWCNTs dispersed with the same. The findings are unique as MWCNTs were found to be active against both Gram-negative and Gram-positive bacteria to a similar extent, though somewhat milder than SWCNTs. However, when dispersed with unsaturated phospholipids, the former offer almost comparable antibacterial effects to that of the latter. The study opens a new research domain to further explore the antibacterial effects of non-functionalized and relatively safer MWCNTs, accentuating the importance of biocomponents like unsaturated phospholipids in this purview.

Bacteriophage-loaded nanostructured lipid carrier: improved pharmacokinetics mediates effective resolution of Klebsiella pneumoniae-induced lobar pneumonia.

This study examined the therapeutic and prophylactic potential of bacteriophages in a mouse model of Klebsiella pneumoniae lobar pneumonia. Phages were administered intraperitoneally. Liposome-entrapped phages (LP) were effective in treating infection, even when therapy was delayed by 3 days after the induction of pneumonia. In contrast, nonliposomal phages provided protection when administered 24 hours after infection. Administration of nonliposomal phages 6 hours prior to intranasal bacterial challenge resulted in complete protection, compared with LP, which was effective even when administered 48 hours prior to infection. Increased reduction and a greater increment in the levels of proinflammatory and antiinflammatory cytokines, respectively, in homogenates of lung from LP-treated mice were suggestive of increased efficacy of LP in the treatment of pneumonia. This is the first study to assess liposomes as a delivery vehicle for phage, and the results confirm the superiority of LP for both therapeutic and prophylactic applications.

Alterations in Hematological Profile of Experimentally Induced Subchronic Thiacloprid Toxicosis in Gallus domesticus.

OBJECTIVES: Thiacloprid, a novel neonicotinoid insecticide is chiefly used as a crop protectant therefore it is likely to cause indirect exposure to poultry through contaminated feed and water because this species is occasionally supplied with feed that is, declared unfit for human consumption. The current study was performed to explore the nonlethal toxic effects of thiacloprid in Gallus domesticus on hematological parameters. MATERIALS AND METHODS: Fifty-two birds were randomly divided into nine groups. Groups I to IV of four birds each were kept as healthy control. The Groups V, VI, VII, VIII, IX, and X contained six birds each and were administered thiacloprid at 1 mg/kg/day for 15, 30, 45, 60, 75, and 90 days, respectively. RESULTS: Thiacloprid caused variable changes in the hematological parameters. There was a significant decline in the packed cell volume (PCV), hemoglobin (Hb) concentration, and total erythrocyte count (TEC). The PCV declined to the extent of 23.33 ± 0.76% on day 90 from the 0 day value of 29.75 ± 1.26% of experiment. The Hb concentration decreased from 9.93 ± 0.57 g/dl (0 day) to 7.52 ± 0.62 g/dl (90 days). The TEC declined from the 0 day value of 2.41 ± 0.08 × 10(6)/mm(3) to 90 days value of 2.08 ± 0.05 × 10(6)/mm(3). The total leukocyte count on 0 day was 12.50 ± 0.76 × 10(3)/mm(3) and it showed a significant increase from day 45 (17.80 ± 2.67 × 10(3)/mm(3)) to day 90 (21.33 ± 1.48 × 10(3)/mm(3)) of thiacloprid treatment. There was a significant rise in value of erythrocyte sedimentation rate to 19.25 ± 1.22 mm/24 h on day 90 of treatment from the 14.42 ± 1.09 mm/24 h on 0 day. The long-term oral administration of thiacloprid produced no significant alterations in the values of erythrocytic indices. CONCLUSIONS: The repeated oral toxicity on thiacloprid in present investigation suggested that it has an adverse effect on health of birds and is moderately risk insecticide in G. domesticus.

Artificial Klebsiella pneumoniae biofilm model mimicking in vivo system: altered morphological characteristics and antibiotic resistance.

The purpose of this study was to develop a biofilm model of Klebsiella pneumoniae B5055, mimicking in vivo biofilm system so as to determine susceptibility of different phases of biofilm to antibiotics by three-dimensional analysis. Artificial mature biofilm of K. pneumoniae was made on black, polycarbonate membranes. Biofilm structure was visualized by scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM). Viable count method, CLSM and SEM analysis confirmed that mature, uniform and viable biofilms can be formed on the polycarbonate membranes by this method. The three-dimensional heterogeneity of biofilm was confirmed on the basis of results of CLSM, which is an important characteristics of in vivo biofilm system. Staining with the LIVE/DEAD BacLight viability kit and acridine orange suggested that the center of biofilm had more inactive cells compared with actively dividing cells on the periphery. Amikacin at a concentration of 40 µg ml⁻¹ was effective against younger biofilm whereas ineffective against older biofilm that showed sparsely populated dead cells using the BacLight viability staining kit. Role of altered morphological characteristics toward increased antibiotic susceptibility was also studied for different phases of K. pneumoniae biofilm by CLSM and light microscopy. Thickness of biofilm increased from 0.093 to 0.231 mm with time. So, both heterogeneity and thickness of the biofilm are likely to influence the ineffectiveness of amikacin in older biofilm. The present model holds considerable clinical relevance and may be useful for evaluating the efficacy of antimicrobial agent on bacterial biofilms in vitro.

Nanocolloidal carriers of isotretinoin: antimicrobial activity against Propionibacterium acnes and dermatokinetic modeling.

Acne, a common skin disease in teenagers, is caused by Propionibacterium acnes (P. acnes). Isotretinoin (ITR) is though reported to have immense antiacne potential, yet there are hardly any reports vouching its antimicrobial activity. The present study, therefore, was undertaken to study the antimicrobial activity of ITR and evaluate the effect of its encasement in nanocarriers on its minimum inhibitory concentration (MIC). The nanocarriers were also evaluated for the skin transport characteristics. MICs of pure drug and entrapped drug in nanolipid carriers (ITR-NLCs) and in solid lipid nanoparticles (ITR-SLNs) were determined by broth dilution method against clindamycin phosphate as the reference antibiotic. It was observed that ITR possessed marked antimicrobial activity against anaerobic pathogen, P. acnes. Nanocarriers loaded with ITR, that is, SLNs and NLCs, enhanced the antimicrobial activity even at lower concentrations vis-à-vis the drug alone and improved drug transport potential vis-à-vis the commercial gel. The unique findings could be the result of effective adhesion of ITR-loaded nanocarriers to the bacterial membranes and release of drug directly to the target. Besides establishing ITR as an antimicrobial agent against acne-causing bacteria, the current work ratifies immense potential of nanocolloidal carriers like SLNs and NLCs to treat acne in a more efficient manner.

Susceptibility of different phases of biofilm of Klebsiella pneumoniae to three different antibiotics.

The existence of majority of bacteria in biofilm mode makes it difficult to eradicate them as antibiotics at much higher concentrations than the MICs are required to destroy these bacteria. This study investigated the effect of different classes of antibiotics on different phases of biofilm formed by Klebsiella pneumoniae. The organism was grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibility to different classes of antibiotics was assessed. The results showed that planktonic organisms were susceptible to ciprofloxacin, amikacin and piperacillin, and their MBC values were same or eight times higher than their corresponding MICs. MBC of ciprofloxacin and amikacin was found to be four and eight times higher for monolayer than planktonic cells. On the other hand, MBC of piperacillin was >1024 µg ml(-1). K. pneumoniae in a biofilm growth mode was more resistant to antibiotics than all other modes. The effect of amikacin and ciprofloxacin on young and older biofilms, at the highest achievable serum concentrations, was also examined. It was observed that amikacin at a concentration of 40 µg ml(-1) was able to eradicate the young biofilms; however, with increase in the age of the biofilm, it became completely ineffective. Calcofluor staining suggested increased production of exopolysaccharide in older biofilm compared with younger biofim that might be responsible for the increased resistance of older biofilm of K. pneumoniae to antibiotics.