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Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

Fluorescent magnetic iron oxide nanoparticles have been used to label cells for imaging as well as for therapeutic purposes. The purpose of this study was to modify the approach to develop a nanoprobe for cell selection and imaging with a direct therapeutic translational focus. The approach involves physical coincubation and adsorption of superparamagnetic iron oxide nanoparticle-polyethylene glycol (SPION-PEG) complexes with a monoclonal antibody (mAb) or a set of antibodies. Flow cytometry, confocal laser scanning microscopy, transmission electron microscopy, iron staining, and magnetic resonance imaging were used to assess cell viability, function, and labeling efficiency. This process has been validated by selecting adipose tissue-derived cardiac progenitor cells from the stromal vascular fraction using signal regulatory protein alpha (SIRPA)/kinase domain receptor (KDR) mAbs. These markers were chosen because of their sustained expression during cardiomyocyte differentiation. Sorting of cells positive for SIRPA and KDR allowed the enrichment of cardiac progenitors with 90% troponin-I positivity in differentiation cultures. SPION labeled cardiac progenitor cells (1×10(5) cells) was mixed with gel and used for 3T magnetic resonance imaging at a concentration, as low as 12.5 μg of iron. The toxicity assays, at cellular and molecular levels, did not show any detrimental effects of SPION. Our study has the potential to achieve moderate to high specific cell selection for the dual purpose of imaging and therapy.

Background Invasive ductal carcinoma (IDC) is the most common form of breast cancer which accounts for 85% of all breast cancer diagnoses. Non-invasive and early stages have a better prognosis than late-stage invasive cancer that has spread to lymph nodes. The involvement of microRNAs (miRNAs) in the initiation and progression of breast cancer holds great promise for the development of molecular tools for early diagnosis and prognosis. Therefore, developing a cost effective, quick and robust early detection protocol using miRNAs for breast cancer diagnosis is an imminent need that could strengthen the health care system to tackle this disease around the world. Methods We have analyzed putative miRNAs signatures in 100 breast cancer samples using two independent high fidelity array systems. Unique and common miRNA signatures from both array systems were validated using stringent double-blind individual TaqMan assays and their expression pattern was confirmed with tissue microarrays and northern analysis. In silico analysis were carried out to find miRNA targets and were validated with q-PCR and immunoblotting. In addition, functional validation using antibody arrays was also carried out to confirm the oncotargets and their networking in different pathways. Similar profiling was carried out in Brca2/p53 double knock out mice models using rodent miRNA microarrays that revealed common signatures with human arrays which could be used for future in vivo functional validation. Results Expression profile revealed 85% downregulated and 15% upregulated microRNAs in the patient samples of IDC. Among them, 439 miRNAs were associated with breast cancer, out of which 107 miRNAs qualified to be potential biomarkers for the stratification of different types, grades and stages of IDC after stringent validation. Functional validation of their putative targets revealed extensive miRNA network in different oncogenic pathways thus contributing to epithelial-mesenchymal transition (EMT) and cellular plasticity. Conclusion This study revealed potential biomarkers for the robust classification as well as rapid, cost effective and early detection of IDC of breast cancer. It not only confirmed the role of these miRNAs in cancer development but also revealed the oncogenic pathways involved in different progressive grades and stages thus suggesting a role in EMT and cellular plasticity during breast tumorigenesis per se and IDC in particular. Thus, our findings have provided newer insights into the miRNA signatures for the classification and early detection of IDC.

The prognosis for glioma patients remains grim despite aggressive treatment approaches. Current molecular profiles have limitations in predicting glioma recurrence, highlighting the need for new and improved prognostic biomarkers. We investigated whether the growth kinetics of patient-derived glioma cultures (PDGCs) can offer valuable prognostic insights to predict tumor recurrence. Additionally, we examined the expression of glial-mesenchymal transition (GMT) markers in PDGCs to assess their potential as additional prognostic biomarkers. 130 patients diagnosed with primary glioma via MRI scans were prospectively enrolled. Surgical tumor tissues were collected from all participants and used to establish patient-derived glioma cultures (PDGCs). The growth kinetics and colony-forming ability of the respective PDGCs were calculated to derive proliferation index (PI) for each patient. Progression-free survival (PFS) and overall survival (OS) served as the primary outcome measures. We established short-term glioma cultures in 98 clinical samples, regardless of the CNS WHO tumor grade, IDH1/2 mutation and 19/19q codeletion status and maintained active cell proliferation for at least 10–12 passages. However, we observed two distinct growth kinetic patterns among PDGCs. Based on their proliferation index (PI), we categorized patients into either high proliferation index (HPI) or low proliferation index (LPI) group. Furthermore, we noted a differential expression profile of GMT markers between HPI and LPI patients. The proliferation index (PI) exhibited a significant correlation with progression-free survival (PFS), while the expression of GMT marker vimentin was associated with overall survival (OS). The PDGC-derived Proliferation Index (PI) can serve as a predictive tool for tumor recurrence, independent of clinical or tumor-related factors. Moreover, reduced vimentin expression is a positive indicator for glioma patients' overall survival status. [Display omitted] •PDGCs unveil real-time glioma cell kinetics, offering insights into tumor recurrence potential.•PDGCs were established regardless of tumor grade and other molecular characteristics of original tumor.•Distinct growth kinetics within tumors with similar histopathology reveal glioma heterogeneity.•Glial-mesenchymal transition is evident in glioma and is associated with a highly proliferative phenotype of PDGCs.•Low PDGC proliferation and reduced vimentin predict favorable glioma prognosis, enhancing survival outcomes.

Avian influenza has raised many apprehension in the recent years because of its potential transmitability to humans. With the increasing emergence of drug-resistant avian influenza strains, development of potential vaccines are imperative to manage this disease. Two structural antigens, haemagglutinin and neuraminidase, have been the target candidates for the development of subunit vaccine against influenza. In an effort to develop a faster and economically beneficial vaccine, the neuraminidase gene of a highly pathogenic avian influenza isolate was cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant neuraminidase (rNA) antigen was purified, and its bioactivity was analysed. The rNA was found to be functional, as determined by the neuraminidase assay. Four groups of mice were immunized with different concentrations of purified rNA antigen, which were adjuvanted with aluminium hydroxide. The immune response against rNA was analysed by enzyme-linked immunosorbent assay (ELISA) and neuraminidase inhibition assay. The mice groups immunized with 25 μg and 10 μg of antigen had a significant immune response against rNA. This method can be utilized for faster and cost-effective development of vaccines for a circulating and newer strain of avian influenza, and would aid in combating the disease in a pandemic situation, in which production time matters greatly.

Aqueous, methanol, ethyl acetate, and chloroform extracts of the root, stem, and leaf of Raphanus sativus were studied for antibacterial activity against food-borne and resistant pathogens. All extracts except the aqueous extracts had significant broad-spectrum inhibitory activity. The ethyl acetate extract of the root had the potent antibacterial activity, with a minimum inhibitory concentration (MIC) of 0.016-0.064 mg/ml and a minimum bactericidal concentration (MBC) of 0.016-0.512 mg/ml against health-damaging bacteria. This was followed by the ethyl acetate extracts of the leaf and stem with MICs of 0.064-0.256 and 0.128-0.256 mg/ml, respectively and MBCs of 0.128-2.05 and 0.256-2.05 mg/ml, respectively. The ethyl acetate extracts of the different parts of R. sativus retained their antibacterial activity after heat treatment at 100°C for 30 min, and their antibacterial activity was enhanced when pH was maintained in the acidic range. Hence this study, for the first time, demonstrated that the root, stem, and leaf of R. sativus had significant bactericidal effects against human pathogenic bacteria, justifying their traditional use as anti-infective agents in herbal medicines.

Raphanus sativus , a common cruciferous vegetable has been attributed to possess a number of pharmacological and therapeutic properties. This present study evaluated the protective effect of different parts of R. sativus such as root, stem and leaf obtained with a variety of extraction solvents against cell death and oxidative DNA damage induced by hydrogen peroxide (H 2 O 2 ) in normal human lymphocytes. R. sativus extracts as such showed no cytotoxicity and genotoxicity to the lymphocytes at the tested concentrations. Of the different extracts, hexane extract of root and methanolic extract of stem and leaf showed significant protective effect against oxidative damage induced by 200 μM H 2 O 2 in a dose dependent manner, as compared to cells exposed only to H 2 O 2 . Our results suggest that the protective effect afforded by R. sativus extract could be related to the presence of isothiocyanates and polyphenolics, as they possess significant capacity to remove reactive species by virtue of their ability to scavenge free radicals and induce antioxidant enzyme system in the cells.

Raphanus sativus, a common cruciferous vegetable has been attributed to possess a number of pharmacological and therapeutic properties. This present study evaluated the protective effect of different parts of R. sativus such as root, stem and leaf obtained with a variety of extraction solvents against cell death and oxidative DNA damage induced by hydrogen peroxide (H sub(2)O sub(2)) in normal human lymphocytes. R. sativus extracts as such showed no cytotoxicity and genotoxicity to the lymphocytes at the tested concentrations. Of the different extracts, hexane extract of root and methanolic extract of stem and leaf showed significant protective effect against oxidative damage induced by 200kM H sub(2)O sub(2) in a dose dependent manner, as compared to cells exposed only to H sub(2)O sub(2). Our results suggest that the protective effect afforded by R. sativus extract could be related to the presence of isothiocyanates and polyphenolics, as they possess significant capacity to remove reactive species by virtue of their ability to scavenge free radicals and induce antioxidant enzyme system in the cells.

Introduction: Cancer is the second leading cause of mortality in India, with conventional chemotherapy often limited by drug resistance, off-target toxicity, and metastasis promotion. These treatments indiscriminately attack rapidly dividing cells, including healthy tissues and immune cells, leading to severe side effects and weakened antitumor immunity. Hence, alternative strategies are needed to selectively target cancer cells while minimizing collateral damage. Methods: Mesenchymal stem cells (MSCs) were isolated, cultured up to passage 4 (P4), and characterized via flow cytometry and immunocytochemistry for stemness markers. Their differentiation potential was validated using adipogenic and chondrogenic lineage assays. The MSC-derived secretome, collected under hypoxic conditions, was analyzed for its cytotoxic effects on cancer cells using MTT, TMRM staining, and scratch assays. NanoLC-MS/MS profiling and STRING-DB analysis identified key proteins involved in apoptosis, tumor suppression, and DNA damage response. Results: P2 secretomes exhibited the highest protein concentration and strongest cytotoxic effects. UCSC-derived secretomes showed superior anti-cancer activity, significantly reducing cancer cell viability, mitochondrial membrane potential, and migration. Dose-dependent studies revealed up to 95% cancer cell death with UCSC secretome, while ADSC secretome achieved 80%. In contrast, HEK293-derived secretomes had minimal effects. LC-MS profiling identified tumor-suppressive and apoptosis-inducing proteins, with unique protein signatures distinguishing UCSC and ADSC secretomes. STRING analysis mapped key regulatory pathways in tumor inhibition. Conclusion: MSC-derived secretomes, particularly from UCSCs, effectively target aggressive breast cancer cells while sparing normal cells, offering a promising alternative to conventional therapies. Future research should optimize secretome formulations, identify key bioactive components, and validate their efficacy in in vivo models and clinical trials to advance this therapy toward clinical applications. Key words: Mesenchyal stem cells, umbilical cord tissue, gene network, secretomes, cytotoxicity, chemotherapeutic drugs