Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

VapC12 ribonuclease toxin modulates host immune response during Mycobacterium tuberculosis infection.

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with ΔvapC12 induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with ΔvapC12 succumbed early to the disease. Finally, we reported that the above phenotype of ΔvapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the ΔvapC12 infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host.

An integrated deep-learning model for smart waste classification.

Efficient waste management is essential for human well-being and environmental health, as neglecting proper disposal practices can lead to financial losses and the depletion of natural resources. Given the rapid urbanization and population growth, developing an automated, innovative waste classification model becomes imperative. To address this need, our paper introduces a novel and robust solution - a smart waste classification model that leverages a hybrid deep learning model (Optimized DenseNet-121 + SVM) to categorize waste items using the TrashNet datasets. Our proposed approach uses the advanced deep learning model DenseNet-121, optimized for superior performance, to extract meaningful features from an expanded TrashNet dataset. These features are subsequently fed into a support vector machine (SVM) for precise classification. Employing data augmentation techniques further enhances classification accuracy while mitigating the risk of overfitting, especially when working with limited TrashNet data. The results of our experimental evaluation of this hybrid deep learning model are highly promising, with an impressive accuracy rate of 99.84%. This accuracy surpasses similar existing models, affirming the efficacy and potential of our approach to revolutionizing waste classification for a sustainable and cleaner future.

LncRNA-mediated orchestrations of alternative splicing in the landscape of breast cancer.

Alternative splicing (AS) is a fundamental post-transcriptional process in eukaryotes, enabling a single gene to generate diverse mRNA transcripts, thereby enhancing protein variability. This process involves the excision of introns and the joining of exons in pre-mRNA(s) to form mature mRNA. The resulting mature mRNAs exhibit various combinations of exons, contributing to functional diversity. Dysregulation of AS can substantially modulate protein functions, impacting the onset and progression of numerous diseases, including cancer. Non-coding RNAs (ncRNAs) are distinct from protein-coding RNAs and consist of short and long types. Long non-coding RNAs (lncRNAs) play an important role in regulating several cellular processes, particularly alternative splicing, according to new research. This review provides insight into the latest discoveries concerning how lncRNAs influence alternative splicing within the realm of breast cancer. Additionally, it explores potential therapeutic strategies focused on targeting lncRNAs.

Rv0495c regulates redox homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has evolved sophisticated surveillance mechanisms to neutralize the ROS-induces toxicity which otherwise would degrade a variety of biological molecules including proteins, nucleic acids and lipids. In the present study, we find that Mtb lacking the Rv0495c gene (ΔRv0495c) is presented with a highly oxidized cytosolic environment. The superoxide-induced lipid peroxidation resulted in altered colony morphology and loss of membrane integrity in ΔRv0495c. As a consequence, ΔRv0495c demonstrated enhanced susceptibility when exposed to various host-induced stress conditions. Further, as expected, we observed a mutant-specific increase in the abundance of transcripts that encode proteins involved in antioxidant defence. Surprisingly, despite showing a growth defect phenotype in macrophages, the absence of the Rv0495c enhanced the pathogenicity and augmented the ability of the Mtb to grow inside the host. Additionally, our study revealed that Rv0495c-mediated immunomodulation by the pathogen helps create a favorable niche for long-term survival of Mtb inside the host. In summary, the current study underscores the fact that the truce in the war between the host and the pathogen favours long-term disease persistence in tuberculosis. We believe targeting Rv0495c could potentially be explored as a strategy to potentiate the current anti-TB regimen.

Biocompatible Human Placental Extracellular Matrix Derived Hydrogels.

Solubilizing extracellular matrix (ECM) materials and transforming them into hydrogels has expanded their potential applications both in vitro and in vivo. In this study, hydrogels are prepared by decellularization of human placental tissue using detergent and enzymes and by the subsequent creation of a homogenized acellular placental tissue powder (P-ECM). A perfusion-based decellularization approach is employed using detergent and enzymes. The P-ECM with and without gamma irradiation is then utilized to prepare P-ECM hydrogels. Physical and biological evaluations are conducted to assess the suitability of the P-ECM hydrogels for biocompatibility. The decellularized tissue has significantly reduced cellular content and retains the major ECM proteins. Increasing the concentration of P-ECM leads to improved mechanical properties of the P-ECM hydrogels. The biocompatibility of the P-ECM hydrogel is demonstrated through cell proliferation and viability assays. Notably, gamma-sterilized P-ECM does not support the formation of a stable hydrogel. Nonetheless, the use of HCl during the digestion process effectively decreases spore growth and bacterial bioburden. The study demonstrates that P-ECM hydrogels exhibit physical and biological attributes conducive to soft tissue reconstruction. These hydrogels establish a favorable microenvironment for cell growth and the need for investigating innovative sterilization methods.

Small-molecule inhibitors of kinases in breast cancer therapy: recent advances, opportunities, and challenges.

Breast cancer is the most common malignancy in women worldwide and despite significant advancements in detection, treatment, and management of cancer, it is still the leading cause of malignancy related deaths in women. Understanding the fundamental biology of breast cancer and creating fresh diagnostic and therapeutic strategies have gained renewed focus in recent studies. In the onset and spread of breast cancer, a group of enzymes known as kinases are extremely important. Small-molecule kinase inhibitors have become a promising class of medications for the treatment of breast cancer owing to their capacity to specifically target kinases involved in the growth and progression of cancer. The creation of targeted treatments that block these kinases and the signalling pathways that they activate has completely changed how breast cancer is treated. Many of these targeted treatments have been approved for the treatment of breast cancer as clinical trials have demonstrated their great efficacy. CDK4/6 inhibitors, like palbociclib, abemaciclib, and ribociclib, EGFR inhibitors such as gefitinib and erlotinib and HER2-targeting small-molecule kinases like neratinib and tucatinib are some examples that have shown potential in treating breast cancer. Yet, there are still difficulties in the development of targeted medicines for breast cancer, such as figuring out which patient subgroups may benefit from these therapies and dealing with drug resistance problems. Notwithstanding these difficulties, kinase-targeted treatments for breast cancer still have a lot of potential. The development of tailored medicines will continue to be fuelled by the identification of novel targets and biomarkers for breast cancer as a result of advancements in genomic and proteomic technology.

Omicron sub-lineage BA.5 infection results in attenuated pathology in hACE2 transgenic mice.

A recently emerged sub-lineage of Omicron, BA.5, together with BA.4, caused a fifth wave of coronavirus disease (COVID-19) in South Africa and subsequently emerged as a predominant strain globally due to its high transmissibility. The lethality of BA.5 infection has not been studied in an acute hACE2 transgenic (hACE2.Tg) mouse model. Here, we investigated tissue-tropism and immuno-pathology induced by BA.5 infection in hACE2.Tg mice. Our data show that intranasal infection of BA.5 in hACE2.Tg mice resulted in attenuated pulmonary infection and pathology with diminished COVID-19-induced clinical and pathological manifestations. BA.5, similar to Omicron (B.1.1.529), infection led to attenuated production of inflammatory cytokines, anti-viral response and effector T cell response as compared to the ancestral strain of SARS-CoV-2, Wuhan-Hu-1. We show that mice recovered from B.1.1.529 infection showed robust protection against BA.5 infection associated with reduced lung viral load and pathology. Together, our data provide insights as to why BA.5 infection escapes previous SARS-CoV-2 exposure induced-T cell immunity but may result in milder immuno-pathology and alleviated chances of re-infectivity in Omicron-recovered individuals.

PTEN regulates expression of its pseudogene in glioblastoma cells in DNA methylation-dependent manner.

Glioblastoma (GBM) is the most aggressive and frequent type of primary brain cancer in adult patients. One of the key molecular features associated with GBM pathogenesis is the dysfunction of PTEN oncosuppressor. In addition to PTEN gene, humans and several primates possess processed PTEN pseudogene (PTENP1) that gives rise to long non-coding RNA lncPTENP1-S. Regulation and functions of PTEN and PTENP1 are highly interconnected, however, the exact molecular mechanism of how these two genes affect each other remains unclear. Here, we analyzed the methylation level of the CpG islands (CpGIs) in the promoter regions of PTEN and PTENP1 in patient-derived GBM neurospheres. We found that increased PTEN methylation corelates with decreased PTEN mRNA level. Unexpectedly, we showed the opposite trend for PTENP1. Using targeted methylation and demethylation of PTENP1 CpGI, we demonstrated that DNA methylation increases lncPTENP1-S expression in the presence of wild type PTEN protein but decreases lncPTENP1-S expression if PTEN protein is absent. Further experiments revealed that PTEN protein binds to PTENP1 promoter region and inhibits lncPTENP1-S expression if its CpGI is demethylated. Interestingly, we did not detect any effect of lncPTENP1-S on the level of PTEN mRNA, indicating that in GBM cells PTENP1 is a downstream target of PTEN rather than its upstream regulator. Finally, we studied the functions of lncPTENP1-S and demonstrated that it plays a pro-oncogenic role in GBM cells by upregulating the expression of cancer stem cell markers and decreasing cell adhesion.

The emerging regulatory roles of non-coding RNAs associated with glucose metabolism in breast cancer.

Altered energy metabolism is one of the hallmarks of tumorigenesis and essential for fulfilling the high demand for metabolic energy in a tumor through accelerating glycolysis and reprogramming the glycolysis metabolism through the Warburg effect. The dysregulated glucose metabolic pathways are coordinated not only by proteins coding genes but also by non-coding RNAs (ncRNAs) during the initiation and cancer progression. The ncRNAs are responsible for regulating numerous cellular processes under developmental and pathological conditions. Recent studies have shown that various ncRNAs such as microRNAs, circular RNAs, and long noncoding RNAs are extensively involved in rewriting glucose metabolism in human cancers. In this review, we demonstrated the role of ncRNAs in the progression of breast cancer with a focus on outlining the aberrant expression of glucose metabolic pathways. Moreover, we have discussed the existing and probable future applications of ncRNAs to regulate energy pathways along with their importance in the prognosis, diagnosis, and future therapeutics for human breast carcinoma.

Transcription factor mce3R modulates antibiotics and disease persistence in Mycobacteriumtuberculosis.

Transcription factors (TFs) of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis, regulate a network of pathways that help prolong the survival of Mtb inside the host. In this study, we have characterized a transcription repressor gene (mce3R) from the TetR family, that encodes for Mce3R protein in Mtb. We demonstrated that the mce3R gene is dispensable for the growth of Mtb on cholesterol. Gene expression analysis suggests that the transcription of genes belonging to the mce3R regulon is independent of the carbon source. We found that, in comparison to the wild type, the mce3R deleted strain (Δmce3R) generated more intracellular ROS and demonstrated reduced susceptibility to oxidative stress. Total lipid analysis suggests that mce3R regulon encoded proteins modulate the biosynthesis of cell wall lipids in Mtb. Interestingly, the absence of Mce3R increased the frequency of generation of antibiotic persisters in Mtb and imparted in-vivo growth advantage phenotype in guinea pigs. In conclusion, genes belonging to the mce3R regulon modulate the frequency of generation of persisters in Mtb. Hence, targeting mce3R regulon encoded proteins could potentiate the current regimen by eliminating persisters during Mtb infection.

Assessment of anti-arthritic activity of lipid matrix encased berberine in rheumatic animal model.

The purpose of this study was to evaluate the drug delivery and therapeutic potential of berberine (Br) loaded nanoformulation in rheumatoid arthritis (RA)-induced animal model. The Br-loaded NLCs (nanostructured lipid carriers) were prepared employing melt-emulsification process, and optimised through Box-Behnken design. The prepared NLCs were assessed for in-vitro and in-vivo evaluations. The optimised NLCs exhibited a mean diameter of 180.2 ± 0.31 nm with 88.32 ± 2.43% entrapment efficiency. An enhanced anti-arthritic activity with reduced arthritic scores to 0.66 ± 0.51, reduction in ankle diameter to 5.80 ± 0.27 mm, decline in paw withdrawal timing, and improvements in walking behaviour were observed in the Br-NLCs treated group. The radiographic images revealed a reduction in bone and cartilage deformation. The Br-NLCs showed promising results in the management of RA disease, can be developed as an efficient delivery system at commercial levels, and may be explored for clinical application after suitable experiments in the future.

miRNA therapeutics in precision oncology: a natural premium to nurture.

The dynamic spectrum of microRNA (miRNA) has grown significantly over the years with its identification and exploration in cancer therapeutics and is currently identified as an important resource for innovative strategies due to its functional behavior for gene regulation and modulation of complex biological networks. The progression of cancer is the consequence of uncontrolled, nonsynchronous procedural faults in the biological system. Diversified and variable cellular response of cancerous cells has always raised challenges in effective cancer therapy. miRNAs, a class of non-coding RNAs (ncRNAs), are the natural genetic gift, responsible to preserve the homeostasis of cell to nurture. The unprecedented significance of endogenous miRNAs has exhibited promising therapeutic potential in cancer therapeutics. Currently, miRNA mimic miR-34, and an antimiR aimed against miR-122 has entered the clinical trials for cancer treatments. This review, highlights the recent breakthroughs, challenges, clinical trials, and advanced delivery vehicles in the administration of miRNA therapies for precision oncology.

Synthesis of a Zirconium Complex of an N,O-type p-tert-Butylcalix[4]arene and Its Application in Some Multicomponent Reactions.

The synthesis and characterization of a new octahedral Zr(IV) complex of oxygen-depleted N,O-type calixarene ligand comprising two distal-functionalized pyrazole rings have been reported. The cone shape and structure of the prepared complex were confirmed univocally by single-crystal X-ray diffraction and NMR studies. The Zr metal lies at 2.091 Å from the plane of the calixarene ring. This complex has been utilized as an efficient catalyst for the synthesis of Biginelli adducts, bis(indolyl)methanes, and coumarins. This complex (Cl2Zr-calixarene) showed superior activity for these multicomponent reactions in comparison to the corresponding Ti(IV) and Zn(II) analogues. Ferrocene-appended bis(indolyl)methane, prepared using this catalyst, was also evaluated for its anticancer activity against the A-172 cell line.

Overexpression of laminin-5 gamma-2 promotes tumorigenesis of pancreatic ductal adenocarcinoma through EGFR/ERK1/2/AKT/mTOR cascade.

Pancreatic ductal adenocarcinoma (PDAC) is correlated with poor outcomes because of limited therapeutic options. Laminin-5 gamma-2 (LAMC2) plays a critical role in key biological processes. However, the detailed molecular mechanism and potential roles of LAMC2 in PDAC stay unexplored. The present study examines the essential role and molecular mechanisms of LAMC2 in the tumorigenesis of PDAC. Here, we identified that LAMC2 is significantly upregulated in microarray cohorts and TCGA RNA sequencing data of PDAC patients compared to non-cancerous/normal tissues. Patients with higher transcript levels of LAMC2 were correlated with clinical stages; dismal overall, as well as, disease-free survival. Additionally, we confirmed significant upregulation of LAMC2 in a panel of PDAC cell lines and PDAC tumor specimens in contrast to normal pancreatic tissues and cells. Inhibition of LAMC2 significantly decreased cell growth, clonogenic ability, migration and invasion of PDAC cells, and tumor growth in the PDAC xenograft model. Mechanistically, silencing of LAMC2 suppressed expression of ZEB1, SNAIL, N-cadherin (CDH2), vimentin (VIM), and induced E-cadherin (CDH1) expression leading to a reversal of mesenchymal to an epithelial phenotype. Interestingly, co-immunoprecipitation experiments demonstrated LAMC2 interaction with epidermal growth factor receptor (EGFR). Further, stable knockdown of LAMC2 inhibited phosphorylation of EGFR, ERK1/2, AKT, mTOR, and P70S6 kinase signaling cascade in PDAC cells. Altogether, our findings suggest that silencing of LAMC2 inhibited PDAC tumorigenesis and metastasis through repression of epithelial-mesenchymal transition and modulation of EGFR/ERK1/2/AKT/mTOR axis and could be a potential diagnostic, prognostic, and therapeutic target for PDAC.

Functions of long non-coding RNA ROR in patient-derived glioblastoma cells.

Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer in adult patients. A variety of long non-coding RNAs play an important role in the pathogenesis of GBM, however the molecular functions of most of them still remain elusive. Here, we investigated linc-RoR (long intergenic non-protein coding RNA, regulator of reprogramming) using GBM neurospheres obtained from 12 different patients. We demonstrated that the highest level of this transcript is detected in cells with increased EGFR expression. According to our data, linc-RoR knockdown decreases cell proliferation, increases sensitivity to DNA damage, and downregulates the level of cancer stem cell (CSC) markers. On the other hand, linc-RoR overexpression promote cell growth and increases the proportion of CSCs. Analysis of RNA sequencing data revealed that linc-RoR affects expression of genes involved in the regulation of mitosis. In agreement with this observation, we have showen that the highest level of linc-RoR is detected in the G2/M phase of the cell cycle, when linc-RoR is localized on the chromosomes of dividing cells. Based on our results, we can propose that linc-RoR performs pro-oncogenic functions in human gliobalstoma cells, which may be associated with the regulation of mitotic progression and GBM stemness.

Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, demonstrates immense plasticity with which it adapts to a highly dynamic and hostile host environment. This is facilitated by a web of signalling pathways constantly modulated by a multitude of proteins that regulate the flow of genetic information inside the pathogen. Transcription factors (TFs) belongs to one such family of proteins that modulate the signalling by regulating the abundance of proteins at the transcript level. In the current study, we have characterized the putative transcriptional regulatory protein encoded by the Rv1719 gene of Mycobacterium tuberculosis. This TF belongs to the IclR family of proteins with orthologs found in both bacterial and archaeal species. We cloned the Rv1719 gene into the pET28a expression vector and performed heterologous expression of the recombinant protein with E coli as the host. Further, optimization of the purification protocol by affinity chromatography and characterization of proteins for their functional viability has been demonstrated using various biochemical and/or biophysical approaches. Scale-up of purification yielded approximately 30 mg of ~ 28 kDa protein per litre of culture. In-silico protein domain analysis of Rv1719 protein predicted the presence of the helix-turn-helix (HTH) domain suggesting its ability to bind DNA sequence and modulate transcription; a hallmark of a transcriptional regulatory protein. Further, by performing electrophoretic mobility shift assay (EMSA) we demonstrated that the protein binds to a specific DNA fragment harboring the probable binding site of one of the predicted promoters.

Golden Syrian hamster as a model to study cardiovascular complications associated with SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the Golden Syrian hamster causes lung pathology that resembles human coronavirus disease (COVID-19). However, extrapulmonary pathologies associated with SARS-CoV-2 infection and post-COVID sequelae remain to be understood. Here, we show, using a hamster model, that the early phase of SARS-CoV-2 infection leads to an acute inflammatory response and lung pathologies, while the late phase of infection causes cardiovascular complications (CVCs) characterized by ventricular wall thickening associated with increased ventricular mass/body mass ratio and interstitial coronary fibrosis. Molecular profiling further substantiated our findings of CVC as SARS-CoV-2-infected hamsters showed elevated levels of serum cardiac troponin I, cholesterol, low-density lipoprotein, and long-chain fatty acid triglycerides. Serum metabolomics profiling of SARS-CoV-2-infected hamsters identified N-acetylneuraminate, a functional metabolite found to be associated with CVC, as a metabolic marker was found to be common between SARS-CoV-2-infected hamsters and COVID-19 patients. Together, we propose hamsters as a suitable animal model to study post-COVID sequelae associated with CVC, which could be extended to therapeutic interventions.