Removal of endotoxin from antibody preparations for clinical use

Cell Biochemistry and Biophysics - Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is...     

Passage number of cancer cell lines: Importance,intricacies, and way-forward

Cancer cell lines play a crucial role as invaluable models in cancer research, facilitating the examination of cancer progression as well as the advancement of diagnostics and treatments. While they may not perfectly replicate the original tumor, they generally exhibit similar characteristics. Low-passage cancer cell lines are generally preferred due to their closer resemblance to the original tumor, as long-term culturing can alter the genetic and molecular profiles of a cell line thereby highlighting the importance of monitoring the passage number (PN). Variations in proliferation, migration, gene expression, and drug sensitivity can be linked to PN differences. PN can also influence DNA methylation levels, metabolic profiles, and the expression of genes/or proteins in cancer cell lines. When conducting research on cancer cell lines, it is crucial for researchers to carefully select the appropriate PN to maintain consistency and reliability of results. Moreover, to ensure dependability and replicability, scientists ought to actively track the growth, migration, and gene/or protein profiles of cancer cell lines at specific PNs. This approach enables the identification of the most suitable range of PNs for experiments, guaranteeing consistent and precise results. Additionally, such efforts serve to minimize disparities and uphold the integrity of research. In this review, we have laid out recommendations for laboratories to overcome these PN discrepancies when working with cancer cell lines.     

Treatment of periodontal disease using xanthan based chlorhexidine gel

People of all ages are suffering from periodontal disease. It causes indirect damage in the oral cavity. It is of interest to evaluate the efficacy of xanthan-based chlorhexidine gel (Xan-CHX) in patients with mild-severe chronic periodontitis. Five patients with 60 sites were divided in two groups. Group A (treated with SRP) and group B (treated with Chlosite i.e., SRP + CHL). The recorded clinical parameters were Plaque index (PI), Gingival index (GI), Bleeding index (BI), and Clinical attachment Level (CAL) with sub gingival plaque subjected to microbial analysis. Significant reduction was observed in both groups. However, group B (treated with Chlosite i.e., SRP + CHL) showed statistically significant improvement on above mentioned parameters as compared to group A. Data suggest that in the treatment of periodontal disease (viz. PI, GI, BI and CAL) combination of SRP and Chlosite showed added benefits over only SRP.     

Acarbose improved survival for Apc+/Min mice

Acarbose blocks the digestion of complex carbohydrates, and the NIA Intervention Testing Program (ITP) found that it improved survival when fed to mice. Yet, we do not know if lifespan extension was caused by its effect on metabolism with regard to the soma or cancer suppression. Cancer caused death for ~80% of ITP mice. The ITP found rapamycin, an inhibitor to the pro‐growth mTORC1 (mechanistic target of rapamycin complex 1) pathway, improved survival and it suppressed tumors in Apc^+/Min mice providing a plausible rationale to ask if acarbose had a similar effect. Apc^+/Min is a mouse model prone to intestinal polyposis and a mimic of familial adenomatous polyposis in people. Polyp‐associated anemia contributed to their death. To address this knowledge gap, we fed two doses of acarbose to Apc^+/Min mice. Acarbose improved median survival at both doses. A cross‐sectional analysis was performed next. At both doses, ACA fed mice exhibited reduced intestinal crypt depth, weight loss despite increased food consumption and reduced postprandial blood glucose and plasma insulin, indicative of improved insulin sensitivity. Dose‐independent and dose‐dependent compensatory liver responses were observed for AMPK and mTORC1 activities, respectively. Only mice fed the high dose diet exhibited reductions in tumor number with higher hematocrits. Because low‐dose acarbose improved lifespan but failed to reduced tumors, its effects seem to be independent of cancer. These data implicate the importance of improved carbohydrate metabolism on survival.     

Poly ethylene glycol mediated in vitro screening and physico-biochemical changes induced in mango callus due to moisture stress

Plant Cell, Tissue and Organ Culture (PCTOC) - A method for in vitro screening and selection of drought (moisture stress) tolerant mango calli was developed. Poly ethylene glycol (PEG) (Molecular...     

Survivability of parthenogenetic embryos following in vivo transfer in naturally synchronized Capra hircus

The present study was conducted to see the in vivo developmental potency of caprine parthenogenetic embryos generated in a modified way. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and then activated by 7% ethanol followed by 2 mM 6-dimethyl amino purine (6-DMAP) and embryo development was recorded. Early stage parthenogenetic embryos (two to four cells) were surgically transferred in recipients (10). The pregnancy diagnosis was done by nonreturn to oestrus, ultrasonography (USG), and progesterone estimation. The levels of progesterone were above normal values (1 ng/ml) of pregnancy and fall below the level of pregnancy just before retuned to oestrus. Progesterone profile revealed that out of ten recipients (G1–G10), four goats (G1, G2, G3, and G5) returned to oestrus after 43?±?7.29 (Mean?±?SE) d of embryo transfer and six goats (G4, G6, G7, G8, G9, and G10) did not return to cycle even after 70 d of embryo transfer. In three recipients (G4, G5, and G6), the USG on day 40 revealed that there was fluid filled uterine body with solid fetus-like structure. These might be dead fetus and had started resorption. The progesterone profile also corroborated the assumption of pregnancy in these animals. Authors believe that this may be the first report on in vivo diploid parthenogenetic embryo development in caprine species.     

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.     

Curcumin Suppresses Gelatinase B Mediated Norepinephrine Induced Stress in H9c2 Cardiomyocytes

Background

Extracellular matrix (ECM) remodeling facilitates biomechanical signals in response to abnormal physiological conditions. This process is witnessed as one of the major effects of the stress imposed by catecholamines, such as epinephrine and norepinephrine (NE), on cardiac muscle cells. Matrix metalloproteinases (MMPs) are the key proteases involved in degradation of the ECM in heart.

Objectives

The present study focuses on studying the effect of curcumin on Gelatinase B (MMP-9), an ECM remodeling regulatory enzyme, in NE-induced cardiac stress. Curcumin, a bioactive polyphenol found in the spice turmeric, has been studied for its multi-fold beneficial properties. This study focuses on investigating the role of curcumin as a cardio-protectant.

Methods

H9c2 cardiomyocytes were subjected to NE and curcumin treatments to study the response in stress conditions. Effect on total collagen content was studied using Picrosirus red staining. Gelatinase B activity was assessed through Gel-Diffusion Assay and Zymographic techniques. RT-PCR, Western Blotting and Immunocytochemistry were performed to study effect on expression of gelatinase B. Further, the effect of curcumin on the localization of NF-κB, known to regulate gelatinase B, was also examined.

Results

Curcumin suppressed the increase in the total collagen content under hypertrophic stress and was found to inhibit the in-gel and in-situ gelatinolytic activity of gelatinase B. Moreover, it was found to suppress the mRNA and protein expression of gelatinase B.

Conclusions

The study provides an evidence for an overall inhibitory effect of curcumin on Gelatinase B in NE-induced hypertrophic stress in H9c2 cardiomyocytes which may contribute in the prevention of ECM remodeling.