A comparative assessment of antibacterial properties of neem oil coated sutures: An in vitro study.

Background: When it comes to surgical site infection (SSI), it has been shown that two-thirds of wound infections starts around the incision line and the number is even greater in the presence of sutures. Therefore, a number of compounds have been used to coat the suture materials which tend to decrease the SSI some of which include ciprofloxacin, Aloe vera, and chlorhexidine. Objective: The objective of this study was to assess the antibacterial properties of neem oil-coated sutures in anaerobic culture media when compared to triclosan-coated sutures and uncoated sutures. Materials and Methods: The phosphate-buffered saline solution containing the plaque samples of the patients was transferred evenly onto the sterile agar media. Equal segments of 100% neem oil coated (Group 1), 50% neem oil coated (Group 2), triclosan-coated (Group 3), and uncoated (Group 4) sutures were placed on the agar plate. These were then incubated at 37°C for 24 h. The inhibition zone was calculated in mm by measuring of the zone of inhibition (ZOI) in terms of length, breadth, and area. Results: The results were calculated following the incubation. The mean length for 100% neem oil-coated suture was 2.61 mm ± 0.2 and for 50% neem oil-coated suture was 2.49 mm ± 0.24. The mean breadth for 100% neem oil-coated suture was 1.5 mm ± 0.41 and for 50% neem oil-coated suture the mean was 0.95 mm ± 0.58. The mean area for 100% neem oil-coated suture was 77 mm ± 32.9 and for 50% neem oil-coated suture was 16.8 mm ± 11.14. Triclosan and uncoated sutures did not show any ZOI. Conclusion: The 100% neem oil-coated sutures had the largest length, breadth, and area of ZOI in the anaerobic culture media, followed by 50% neem oil-coated sutures. Triclosan-coated and uncoated sutures did not show any ZOI. Therefore, neem oil-coated suture can be used in the overall healing and prevention of postoperative discomfort after oral surgical procedures.

A simplified protocol to induce hypoxia in a standard incubator: A focus on retinal cells.

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl2), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl2 concentrations used typically range from 100 to 600 µM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl2. In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells.

In vitro and in vivo evaluation of dual Clofazimine and Verapamil loaded PLGA nanoparticles.

Combination therapy may counter the risk caused by efflux pumps mediated resistance developed by mycobacteria with a concomitant increase of the bactericidal effect of anti-TB drugs. In the present study, combination of two drugs in a nanoformulation was prepared. Clofazimine targets type 2 NADH dehydrogenase of the electron transport chain, and Verapamil inhibits various mycobacterial efflux pumps. The nanotechnology approach was adopted to overcome limitations associated with administration of free form of drugs by using poly (D, L-lactic-co-glycolic acid) as a polymer. Nanoparticles were prepared by oil/water single emulsion solvent evaporation procedure and characterized by various techniques. The results thus highlighted that developed nanoparticles were spherical with nano range size (200-450 nm). Fourier transform infrared spectroscopy revealed successful encapsulation of drugs in developed nanoformulations. Drugs in combination showed higher encapsulation efficiency and percentage drug loading capacity as compared to individual drug nanoformulations. Also, reduced toxicity of nanoformulation was observed in hemolysis assay as compared to free drugs. Ex-vivo analysis demonstrated efficient uptake of rhodamine encapsulated nanoparticles by THP-1 cells, while in-vivo results revealed sustained drug release of nanoformulation as compared to free drugs in combination. Therefore, we were able to achieve development of a single nanoformulation encapsulating Clofazimine and Verapamil in combination. Based on these findings, future studies can be designed to explore the potential of co-encapsulated Clofazimine and Verapamil nanoparticles in management of tuberculosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01062-8.

Defective quality control autophagy in Hyperhomocysteinemia promotes ER stress and consequent neuronal apoptosis through proteotoxicity.

Homocysteine (Hcy), produced physiologically in all cells, is an intermediate metabolite of methionine and cysteine metabolism. Hyperhomocysteinemia (HHcy) resulting from an in-born error of metabolism that leads to accumulation of high levels of Hcy, is associated with vascular damage, neurodegeneration and cognitive decline. Using a HHcy model in neuronal cells, primary cortical neurons and transgenic zebrafish, we demonstrate diminished autophagy and Hcy-induced neurotoxicity associated with mitochondrial dysfunction, fragmentation and apoptosis. We find this mitochondrial dysfunction is due to Hcy-induced proteotoxicity leading to ER stress. We show this sustained proteotoxicity originates from the perturbation of upstream autophagic pathways through an aberrant activation of mTOR and that protetoxic stress act as a feedforward cues to aggravate a sustained ER stress that culminate to mitochondrial apoptosis in HHcy model systems. Using chemical chaperones to mitigate sustained ER stress, Hcy-induced proteotoxicity and consequent neurotoxicity were rescued. We also rescue neuronal lethality by activation of autophagy and thereby reducing proteotoxicity and ER stress. Our findings pave the way to devise new strategies for the treatment of neural and cognitive pathologies reported in HHcy, by either activation of upstream autophagy or by suppression of downstream ER stress. Video Abstract.

Proteomic-based identification of APCS as candidate protein for diagnosis of patients exhibiting anti-tubercular drug induced liver injury.

Traditional markers evaluate anti-tubercular drug-induced liver injury (AT-DILI). However, these markers have certain limitations and studies are in progress to characterize AT-DILI at an early stage. In the present study, 40 patients were categorized and equally distributed into healthy controls, newly diagnosed tuberculosis (TB), TB without hepatotoxicity and TB with hepatotoxicity groups based on their conventional liver function tests. Relative protein quantification was performed on depleted pooled serum samples of each representative group by LC-MS/MS, and validation of shortlisted protein was done by ELISA. Levels of all analysed biochemical parameters showed a statistical increment in the hepatotoxicity group compared to the other three groups, representing AT-DILI. Comparative proteomic analysis between TB with hepatotoxicity versus TB without hepatotoxicity groups highlighted 24 significant differentially expressed proteins, including PROS1, KNG1, CFH, LCAT, APCS and ADIPOQ. Identified proteins were involved in complement activation, triglyceride-rich lipoprotein particle remodelling and pathways comprising complement, coagulation cascades and cholesterol metabolism. Based on functional relevance, the serum amyloid P component (APCS) was shortlisted for validation, and it showed a similar trend as observed in the discovery phase with 100% sensitivity and 87% specificity; however, findings need exploration in larger cohorts.

The lupus susceptibility allele DRB1*03:01 encodes a disease-driving epitope.

The HLA-DRB1*03:01 allele is a major genetic risk factor in systemic lupus erythematosus (SLE), but the mechanistic basis of the association is unclear. Here we show that in the presence of interferon gamma (IFN-γ), a short DRB1*03:01-encoded allelic epitope activates a characteristic lupus transcriptome in mouse and human macrophages. It also triggers a cascade of SLE-associated cellular aberrations, including endoplasmic reticulum stress, unfolded protein response, mitochondrial dysfunction, necroptotic cell death, and production of pro-inflammatory cytokines. Parenteral administration of IFN-γ to naïve DRB1*03:01 transgenic mice causes increased serum levels of anti-double stranded DNA antibodies, glomerular immune complex deposition and histopathological renal changes that resemble human lupus nephritis. This study provides evidence for a noncanonical, antigen presentation-independent mechanism of HLA-disease association in SLE and could lay new foundations for our understanding of key molecular mechanisms that trigger and propagate this devastating autoimmune disease.

Noncoding RNAs as novel immunotherapeutic tools against cancer.

Immunotherapy is implemented as an important treatment strategy in various malignancies. In cancer, immunotherapy is employed for successful killing of tumor cells with high specificity and greater efficacy, with minimum side effects. Despite various available strategies, cellular immunotherapy including innate (NK cells, macrophages, dendritic cells) and adaptive (B cells and T cells) immune cells plays a critical role in tumor microenvironment. Since past few years, many drugs targeting immune checkpoint proteins including CTLA-4 and PD-1/PD-L1 have been investigated as immunotherapy approach against cancer but complete effectiveness still remains a question, as diverse mechanisms involved in tumorigenesis may result in the development of cancer cell resistance. Number of evidences have highlighted the significant role of non-coding RNAs (ncRNAs) in regulating multiple stages of cancer initiation, progression & immunity. ncRNAs comprises 98% human transcriptome and are basically considered as dark genome. Among ncRNAs, miRNAs and lncRNAs have been extensively studied in regulating diverse processes of cancer tumorigenesis. Upregulation of oncogenic and downregulation of tumor suppressive miRNAs/lncRNAs has been reported to facilitate the cancer progression and invasiveness. This chapter summarizes how an interplay between ncRNAs and immune cells in cancer pathogenesis can be therapeutically targeted to improve current treatment regimen. Strategies should be employed to improve the efficacy and reduce off-target effects of ncRNA based immunotherapy. Henceforth, combination of ncRNAs and available immunotherapy can be argued to enhance the efficacy of existing immunotherapeutic approaches against cancer to improve patient's survival.

Proteomic profile of 4-PBA treated human neuronal cells during ER stress.

Perturbations affecting the homoeostasis of endoplasmic reticulum (ER) activate an adaptive signaling known as the unfolded protein response or UPR. Many studies have reported the association between neurological disorders and ER stress. Decreasing ER stress may therefore aid in therapeutic control of neuronal diseases. Sodium 4-phenylbutyrate (4-PBA), a small molecule, has been shown to alleviate ER stress and various neurological diseases, but the mechanistic basis of its action is not well understood. Using an iTRAQ based LC-MS technique we have delineated the effect of 4-PBA on the proteome of human neuroblastoma cells (SK-N-SH) during Tunicamycin-induced ER stress. The proteomic profile of 4-PBA-treated cells revealed that 4-PBA does not alter the cellular proteome to adapt towards ER stress. However, it can alleviate both the toxicity and proteomic alterations, induced by an ER stress inducer. Hence, the therapeutic effect of 4-PBA is primarily due to its ability to resolve ER stress rather than its ability to alter the expression of proteins required for maintaining ER proteostasis. Thus, we posit here that 4-PBA acts as an authentic chemical chaperone by aiding protein folding in the ER.

Systems approaches to unraveling plant metabolism: identifying biosynthetic genes of secondary metabolic pathways.

The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants.

Evaluation of oral health awareness in parents of preschool children.

OBJECTIVES: Little data are available on the initiative shown by the parents for dental health care of their preschool children in India. This study was conducted to evaluate the status of oral health awareness in parents of preschool children. MATERIALS AND METHODS: A total of 230 preschool children were included in the study and their parents were analyzed for their child dental awareness by holding free dental checkups and interactive meetings with the help of their respective schools. RESULTS AND CONCLUSION: Results revealed that there is a low initiation of the parents when oral health care of small children is concerned; however, an active collective effort of the school and dental team can make awareness program effective.