Comparative transcriptome profile of embryos at different developmental stages derived from somatic cell nuclear transfer (SCNT) and in-vitro fertilization (IVF) in riverine buffalo (Bubalus bubalis).

Somatic cell nuclear transfer (SCNT) is a very important reproductive technology with many diverse applications, such as fast multiplication of elite animals, the production of transgenic animals and embryonic stem (ES) cells. However, low cloning efficiency, a low live birth rate and the abnormally high incidence of abnormalities in the offspring born are attributed to incomplete or aberrant nuclear reprogramming. In SCNT embryos, the aberrant expression pattern of the genes throughout embryonic development is responsible for the incomplete nuclear reprogramming. The present study was carried out to identify the differential gene expression (DEGs) profile and molecular pathways of the SCNT and IVF embryos at different developmental stages (2 cell, 8 cell and blastocyst stages). In the present study, 1164 (2 cell), 1004 (8 cell) and 530 (blastocyst stage) DEGs were identified in the SCNT embryos as compared to IVF embryos. In addition, several genes such as ZEB1, GDF1, HSF5, PDE3B, VIM, TNNC, HSD3B1, TAGLN, ITGA4 and AGMAT were affecting the development of SCNT embryos as compared to IVF embryos. Further, Gene Ontology (GO) and molecular pathways analysis suggested, SCNT embryos exhibit variations compared to their IVF counterparts and affected the development of embryos throughout the different developmental stages. Apart from this, q-PCR analysis of the GDF1, TMEM114, and IGSF22 genes were utilized to validate the RNA-seq data. These findings contribute valuable insights about the different genes and molecular pathways underlying SCNT embryo development and offer crucial information for improving SCNT efficiency.

Diabetes, Diabetic Retinopathy, and Inflammatory Disorders.

This review summarizes the impact of systemic and ocular inflammatory disorders on diabetes mellitus (DM) and diabetic retinopathy (DR). Local inflammation is a key pathology in diabetic retinopathy (DR) and is also an evolving target for clinical therapy. The legacy effects of local inflammation at the intracellular level make DR a persistent self-driven vicious process. Ocular inflammation is accompanied as well as incited by systemic inflammation due to diabetes mellitus (DM) itself. Over the years, a multitude of studies have evaluated the impact of systemic inflammatory disorders (SIDs, like rheumatoid arthritis, lupus, psoriasis, etc.) and anti-inflammatory drugs prescribed for managing them on manifestations of DM. Recent studies have indicated increased insulin resistance to be a result of chronic inflammation, and the anti-inflammatory drugs to have a protective effect towards DM. Very few studies have evaluated the impact of SIDs on DR. Furthermore, the evidence from these studies is conflicting, and while local anti-inflammatory therapy has shown a lot of clinical potential for use in DR, the results of systemic anti-inflammatory therapies have been inconsistent. The impact of local ocular inflammation due to uveitis on DR is a crucial aspect that has not been evaluated well at present. Initial pre-clinical studies and small-sized clinical reports have shown a strong and positive relationship between the presence of uveitis and the severity of DR as well as its progression, while larger cross-sectional patient surveys have refuted the same. The long term impact of ocular inflammation due to uveitis on DR needs to be studied while adjusting for confounders.

Exploring the involvement of the alternative complement pathway in non-infectious uveitis pathogenesis.

Purpose: Non-infectious uveitis is a complex disease characterized by intraocular inflammation of the uveal area and the leading cause of vision impairment and blindness in young people globally. However, what triggers inflammation and contributes to its recurrence remains unclear. The complement system has been linked to various immunological and inflammatory conditions. In the present study, we have systematically evaluated the role of the alternative complement pathway in the pathogenesis of non-infectious uveitis. Methodology: Quantitative PCR was done in the peripheral leukocytes to study the expression of genes and regulatory miRNA in both anterior and posterior uveitis (n=28 in each category). Multiplex ELISA was performed to measure alternative pathway complement components, such as C3b, factor B, and CFH, and aqueous humor of infectious and non-infectious uveitis patients and non-inflammatory controls (n=10 each). Western blotting was done to validate the ELISA findings in a subset of patients and controls. Results: Downregulation of C3 and CFH mRNA in the peripheral blood was shown by quantitative PCR in the group of anterior uveiits (AU), while the opposite result was found in the group of posterior uveitis (PU). ELISA levels of C3b and CFH proteins were significantly higher in aqueous humor of infectious and non-infectious uveitis (*p = 0.03 and **p = 0.0007 respectively) as compared to the control group. Western blotting further validated (VitH) the activation of the complement cascade in the aqueous (AH) and vitreous humor of patients with non-infectious uveitis, with an increased level of C3b (n=6) and CFH (n=4) in aqueous humor. C3b level was significantly increased while CFH was reduced relative to controls in the vitreous humor (VitH) of posterior uveitis patients compared to controls (n=27 in each category). A C3b to CFH ratio was computed to assess the regulation of complement activation and this index was several folds higher in both anterior and posterior uveitis (n=10 each). The expression of miRNA-hsa-miR-146a and miRNA-hsa-miR-155-5p that regulates CFH was downregulated and nicely correlated with the increased complement proteins in both anterior and posterior uveitis (n=10 each). Conclusion: Our results demonstrate a clear role of CFH and the activation of the alternative complement pathway in the pathogenesis of non-infectious uveitis; however, its therapeutic potential warrants further investigations.

Breast Cancer Detection on Histopathological Images Using a Composite Dilated Backbone Network.

Breast cancer is a lethal illness that has a high mortality rate. In treatment, the accuracy of diagnosis is crucial. Machine learning and deep learning may be beneficial to doctors. The proposed backbone network is critical for the present performance of CNN-based detectors. Integrating dilated convolution, ResNet, and Alexnet increases detection performance. The composite dilated backbone network (CDBN) is an innovative method for integrating many identical backbones into a single robust backbone. Hence, CDBN uses the lead backbone feature maps to identify objects. It feeds high-level output features from previous backbones into the next backbone in a stepwise way. We show that most contemporary detectors can easily include CDBN to improve performance achieved mAP improvements ranging from 1.5 to 3.0 percent on the breast cancer histopathological image classification (BreakHis) dataset. Experiments have also shown that instance segmentation may be improved. In the BreakHis dataset, CDBN enhances the baseline detector cascade mask R-CNN (mAP = 53.3). The proposed CDBN detector does not need pretraining. It creates high-level traits by combining low-level elements. This network is made up of several identical backbones that are linked together. The composite dilated backbone considers the linked backbones CDBN.

An In Vitro Study Comparing the Antimicrobial Efficacy of 0.2% Chitosan, 3% Sodium Hypochlorite, 2% Chlorhexidine against Enterococcus faecalis, Alone and in Conjunction with Diode Laser.

The aim and objective of this study was to compare the antimicrobial efficacy of 0.2% chitosan, 3% sodium hypochlorite, 2% chlorhexidine against Enterococcus faecalis, alone and in conjunction with diode laser. Materials and methods: The root canals of 72 extracted intact human single-rooted teeth with single canals were prepared, and E. faecalis was incubated in the root canals for 7 days. The teeth were then randomly divided into the following four experimental groups: group I: Saline, group II: 0.2% Chitosan, group III: 3% Sodium hypochlorite, and group IV: 2% Chlorhexidine. These groups were further subdivided into three groups: (1) 10 mL irrigant only, (2) 10 mL irrigant, dried and irradiation with diode laser, (3) Diode laser was used for activation of irrigant solution. Samples were obtained from subgroups in each group and checked for turbidity. The effect of each irrigant was evaluated by counting the number of colony-forming units observed on inoculation with samples taken from the irrigated canal on bile esculin azide agar. The data thus obtained was recorded and put to statistical analysis. Results: Significant reductions were noted in E. faecalis colony counts in all groups (p < 0.05). The greatest reduction in colony count (0%) was noted in group IV followed by group II. Also, samples disinfected with diode laser after root canal irrigation showed less number of colony-forming units per mL as compared to the samples irrigated with root canal solutions alone or diode laser alone. Conclusion: Chitosan has the capability for use as an accessory for disinfection of the root canal system. The application of an 810-nm diode laser by itself did not have the adequate antimicrobial activity to be used as an adjunct in root canal therapy. Irradiation with diode laser ought to be used in conjunction with the irrigant to gain maximum antibacterial effect against E. faecalis. How to cite this article: Goel P, Galhotra V, Makkar S, et al. An In-vitro Study Comparing the Antimicrobial Efficacy of 0.2% Chitosan, 3% Sodium Hypochlorite, 2% Chlorhexidine against Enterococcus faecalis, Alone and in Conjunction with Diode Laser. Int J Clin Pediatr Dent 2022;15(1):109-114.

Talon's cusp in a primary maxillary central incisor: A report of a rare odontogenic anomaly.

Talon's cusp, a type of dens evaginatus (DE) in a primary tooth, is a rare odontogenic anomaly which is reported sparingly in the literature. We report this case describing the presence of a talon's cusp on the right primary maxillary central incisor in a 2-year-old boy precipitating discomfort, owing to occlusal interference. The treatment plan involved pulpectomy and reduction of the lingula DE extension under general anesthesia.

Global MicroRNA Expression Profiling of Buffalo (Bubalus bubalis) Embryos at Different Developmental Stages Produced by Somatic Cell Nuclear Transfer and In-Vitro Fertilization Using RNA Sequencing.

Despite the success of cloning technology in the production of offspring across several species, its application on a wide scale is severely limited by the very low offspring rate obtained with cloned embryos. The expression profile of microRNAs (miRNAs) in cloned embryos throughout embryonic development is reported to deviate from regular patterns. The present study is aimed at determining the dynamics of the global expression of miRNA profile in cloned and in-vitro fertilization (IVF) pre-implantation embryos at different developmental stages, i.e., the two-cell, eight-cell, and blastocyst stages, using next-generation sequencing. The results of this study suggest that there is a profound difference in global miRNA profile between cloned and IVF embryos. These differences are manifested throughout the course of embryonic development. The cloned embryos differ from their IVF counterparts in enriched Gene Ontology (GO) terms of biological process, molecular function, cellular component, and protein class categories in terms of the targets of differentially expressed miRNAs. The major pathways related to embryonic development, such as the Wnt signaling pathway, the apoptosis signaling pathway, the FGF signaling pathway, the p53 pathway, etc., were found to be affected in cloned relative to IVF embryos. Overall, these data reveal the distinct miRNA profile of cloned relative to IVF embryos, suggesting that the molecules or pathways affected may play an important role in cloned embryo development.

Global DNA methylation profiles of buffalo (Bubalus bubalis) preimplantation embryos produced by handmade cloning and in vitro fertilization.

Somatic cell nuclear transfer technique (SCNT) has proved to be an outstanding method of multiplication of elite animals but accompanied with low efficiency and live birth rate of cloned animals. Epigenetic alterations of DNA has been one of the culprits behind this issue. Cloned embryos are found to deviate slightly from regular pattern of demethylation and re-methylation at the time of nuclear reprogramming and embryonic development when compared with embryos produced by in vitro fertilization (IVF). Thus, the present study was aimed at evaluating global DNA methylation profiles of cloned embryos at 2-cell, 8-cell and blastocyst stages and compare it with corresponding stages of embryos produced by IVF by using MeDIP-Sequencing on Illumina-based platform. We found out that cloned embryos exhibited significantly different DNA methylation pattern as compared to IVF embryos with respect to distribution of differentially methylated regions in different components of genome, CpG islands distribution and methylation status, gene ontological profiles and pathways affected throughout the developmental stages. The data generated from MeDIP-Seq was validated at blastocyst stage cloned and IVF embryos by bisulfite-sequencing PCR on five randomly selected gene regions.

Recent advancement of piperidine moiety in treatment of cancer- A review.

Piperidine is an important pharmacophore, a privileged scaffold and an excellent heterocyclic system in the field of drug discovery which provides numerous opportunities in studying/exploring this moiety as an anticancer agent by acting on various receptors of utmost importance. Cancer is an uncontrolled division of cells that results in the formation of tumour which have the potential to migrate to other parts of the body (metastasis) finally becoming a major threat to human population. Since piperidine displayed great potential in this area it is being further probed to get novel entities for the treatment of cancer. This review throws light on recent biological expansions of piperidine along with structure activity relationships to deliver association between various synthesized newer/novel derivatives and receptor sites.

Assessment of C-reactive Proteins, Cytokines, and Plasma Protein Levels in Hypertensive Patients with Apical Periodontitis.

INTRODUCTION: Chronic apical periodontitis (CAP) manifests mostly as periapical radiolucency. Various inflammatory mediators play a significant role in the pathogenesis of apical periodontitis. In acute inflammatory conditions, C-reactive proteins (CRP) and fibrinogen show a rise in their concentrations. In chronic diseases with high inflammatory components, an increased prevalence of hypertension has been observed. Hence, we assessed the association of CAP and plasma levels of various inflammatory markers (CRP, interleukin 6 [IL-6], and fibrinogen) in severely hypertensive patients. MATERIALS AND METHODS: This study was conducted in the conservative wing of the institute and included assessment of 250 hypertensive patients with apical periodontitis. With the help of periapical radiographs and clinical examination, the assessment of following parameters was done: Amount of teeth present, visible plaque index, periodontal pocket probing depth, clinical attachment level, bleeding on probing, presence/absence of carious lesions, which included assessment of caries in crown portion, in the root portion, and residual tooth roots (RR), presence of CAP from each patient; 8 mm of venous blood was collected in the morning for the assessment of plasma levels of IL-6, CRP, and fibrinogen levels. Immediate collection and processing of the samples were done in the hospital laboratory. All the results were analyzed by Statistical Package for the Social Sciences software. RESULTS: Out of 250, 155 patients were females. Mean plasma levels of CRP observed in our study were 0.8 mg/dL. Mean plasma levels of IL-6 and fibrinogen were found to be 3.3 and 337.1 mg/dL respectively. A significant correlation was observed while comparing mean body mass index (BMI), RR, and CAP in hypertensive patients. While comparing the mean plasma IL-6 levels, mean BMI, and CAP in the patients, significant results were obtained. Significant correlation was observed while comparing the mean BMI and CAP in hypertensive patients. CONCLUSION: Systemic levels of CRP, IL-6, and fibrinogen levels are influenced by the presence of CAP in hypertensive patients. CLINICAL SIGNIFICANCE: In hypertensive patients, CAP alters the systemic levels of various inflammatory markers.

Assessment of the Incidence of Posttreatment Endodontic Flare-ups in Patients undergoing Single-sitting Root Canal Therapies: A Clinical Study.

INTRODUCTION: Endodontic therapy is one of the commonly used procedures for treating the teeth affected by various pathologies. One of the major problems for endodontists despite the advancements in the root canal procedures is the posttreatment endodontic flare-ups. Much debate exists regarding the completion of endodontic therapy in a single sitting or multiple sittings. Hence, we assessed the incidence of endodontic flare-ups in patients undergoing single-sitting root canal therapies. MATERIALS AND METHODS: The present study included 200 patients who underwent single-sitting endodontic therapy. Clinical details and conditions of each and every tooth of every patient were recorded before and after the completion of endodontic therapy. Irrigation during the root canal procedures was done by 2.5% NaOCl solution in most of the cases while others were irrigated with various combinations of ethylenediaminetetraacetic acid (EDTA) and cycloheximide (CHX) solutions. Follow-up records and readings of the patents were noted and were subjected to statistical analysis. RESULTS: Four groups were formed which divided the patients equally on the basis of their age. Out of 50 patients in the age group of 21 to 30 years, only 4 showed posttreatment endodontic flare-ups, while no endodontic flare-up was recorded in patients with age group of 31 to 50 years. Only two male and four females showed flare-ups postoperatively. A nonsignificant correlation was obtained when flare-up cases were compared on the basis of type of irrigation solution used during canal preparation. CONCLUSION: Single-sitting endodontic therapy appears to be a successful procedure with good prognosis and minimal posttreatment flare-up results, even in patients with periapical pathologies. CLINICAL SIGNIFICANCE: Single-sitting root canal procedures can be successfully carried in patients with vital or nonvital pulp tissues and also in patients with periapical lesions.

An infectious HHV-6B isolate from a healthy adult with chromosomally integrated virus and a reporter based relative viral titer assay.

Human herpesvirus 6B (HHV-6B) primary infections occur in early childhood and establish a life-long latency in the most healthy adults. HHV-6B was detectable in the peripheral blood mononuclear cells (PBMC) and granulocytes by serial genomic DNA dilution PCR till 10 pg of template DNA, in a healthy adult. Epstein Barr virus (EBV) mediated transformation of the PBMC resulted in establishment of a B-cell line. Southern hybridization with the PBMC as well as the cell line DNA showed distinct signals for high copy viral genomes and Gardella gel analysis indicated chromosomal integration of the HHV-6B. Integration site analysis in the PBMC and the cell line indicated an atypical viral integration in non-telomeric region of chromosome 12. Cell free culture medium of the cell line could infect different mononuclear cell lines, naïve or mitogen stimulated PBMC and was found to impart productive infection in a recipient T cell line. An HIV-1 LTR driven luciferase based reporter cell line was made and a single step assay was developed for estimating HHV-6B relative concentration in the culture supernatants. This study thus reports a new infectious HHV-6B isolate with uncommon integration site, spontaneous production from a cell line and also development of a simple relative HHV-6B titer assay.