Extraction and characterization of exosomes from the exhaled breath condensate and sputum of lung cancer patients and vulnerable tobacco consumers-potential noninvasive diagnostic biomarker source.

Noninvasive sample sources of exosomes, such as exhaled breath and sputum, which are in close proximity to the tumor microenvironment and may contain biomarkers indicative of lung cancer, are far more permissive than invasive sample sources for biomarker screening. Standardized exosome extraction and characterization approaches for low-volume noninvasive samples are critically needed. We isolated and characterized exhaled breath condensate (EBC) and sputum exosomes from healthy nonsmokers (n= 30), tobacco smokers (n= 30), and lung cancer patients (n= 40) and correlated the findings with invasive sample sources. EBC samples were collected by using commercially available R-Tubes. To collect sputum samples the participants were directed to take deep breaths, hold their breath, and cough in a collection container. Dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy were used to evaluate the exosome morphology. Protein isolation, western blotting, exosome quantification via EXOCET, and Fourier transform infrared spectroscopy were performed for molecular characterization. Exosomes were successfully isolated from EBC and sputum samples, and their yields were adequate and sufficiently pure for subsequent downstream processing and characterization. The exosomes were confirmed based on their size, shape, and surface marker expression. Remarkably, cancer exosomes were the largest in size not only in the plasma subgroups, but also in the EBC (p < 0.05) and sputum (p= 0.0036) subgroups, according to our findings. A significant difference in exosome concentrations were observed between the control sub-groups (p < 0.05). Our research confirmed that exosomes can be extracted from noninvasive sources, such as EBC and sputum, to investigate lung cancer diagnostic biomarkers for research, clinical, and early detection in smokers.

Enhancing Proliferation of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) through hTERT Expression while Preserving Stemness and Multipotency.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) hold promise in regenerative medicine owing to their multipotent capabilities resembling mesenchymal stem cells (MSCs). Despite their potential, SHED have not been extensively investigated because their limited lifespan and unavailability of cell-lines pose challenges for therapeutic applications. This study investigated the effect of ectopic human telomerase reverse transcriptase (hTERT) expression on SHEDs' proliferation while preserving stemness and genomic integrity. METHODS: Deciduous teeth were collected from children aged 6-10 years. After isolation and characterization, the SHED were transduced with pBabe-puro-hTERT retrovirus to establish SHED cell-line, which was evaluated and compared with pBabe-puro (mock control) for stemness, multipotency and growth attributes through flow cytometry, trilineage differentiation, and growth kinetics. We also estimated hTERT gene expression, genomic integrity, and validated cell-line through STR analysis. RESULTS: Following hTERT transduction, SHED displayed elevated hTERT gene expression while retaining fibroblast-like morphology and mesenchymal stem cell markers. Moreover, after hTERT transduction cellular shape remained same along with increased replicative lifespan and proliferation potential. SHED-hTERT cells exhibited multi-potency and maintained stemness, as evidenced by surface marker expression and multilineage differentiation. Furthermore, genomic integrity was not affected by hTERT integration, as confirmed by STR analysis and CDKN2A gene assessment. CONCLUSION: Ectopic hTERT expression in SHED successfully prolonged their replicative lifespan and improved their ability to proliferate and migrate, while preserving their stemness, multipotency and genomic integrity, suggesting minimal carcinogenic risk. Establishment of SHED cell-line holds potential in regenerative medicine applications, especially in cell-based drugs and tissue engineering experiments.

Exploring salivary exosomes as early predictors of oral cancer in susceptible tobacco consumers: noninvasive diagnostic and prognostic applications.

BACKGROUND: Salivary exosome analysis provides a noninvasive and comprehensive approach with potential applications in oral cancer diagnosis and prognosis. The early detection of oral cancer has remained a critical concern in enhancing the quality of life, especially for individuals who consume tobacco and are at greater risk of developing the disease. The current study investigates the potential of salivary exosomes in screening smokers for early signs and transformations of oral cancer. METHODS: Morphological characterization of salivary exosomes among three study groups (non-smokers as control, smokers as high-risk tobacco consumers, and Oral cancer) (n = 120) was carried out through dynamic light scattering, and nanoparticle tracking analysis. For molecular characterization, EXOCET and Fourier transform infrared spectroscopy methods were utilized. The expression of the exosomal surface protein CD63 was evaluated using Western blotting. RESULTS: Salivary exosomes exhibit noticeable differences in size between control group and tobacco consumers. The differentiation extended beyond exosome size and included variations in concentration and bio-molecular composition, as determined by FTIR screening. Tobacco consumers and oral cancer groups showed significantly larger and more concentrated exosomes compared to the healthy group. CONCLUSION: Our study provides strong evidence that the properties of salivary exosomes can serve as reliable noninvasive biomarkers for distinguishing tobacco consumers from non-smokers and oral cancer patients. Our results underscore the potential of exosome-based diagnostics in early oral cancer detection for high-risk individuals. The larger size and higher concentration of exosomes in tobacco consumers indicate early changes in cell secretions associated with the transformation from healthy to abnormal cells.

Ameliorative potential of stem cells from human exfoliated deciduous teeth (SHED) in preclinical studies: A meta-analysis.

The preclinical and clinical role of mesenchymal stem cells from various adult sources is extensively investigated and established in regenerative medicine. However, the comprehensive exploration of the therapeutic potential of Stem cells from human exfoliated deciduous teeth (SHED) is inadequate. Therefore, we performed a systematic meta-analysis of preclinical animal model studies in several diseases to provide insight into SHED's efficacy and therapeutic potential. Two blinded and independent investigators searched the available online databases and scrutinized the included studies. Meta-analysis was performed to evaluate the pooled effect estimate of intervention of SHED by Review Manager 5.4.1. To investigate the therapeutic efficacy of SHED intervention, we also analyzed the test of heterogeneity (I2), overall effect (Z), sensitivity, and publication bias. Among the 2156 scrutinized studies, 40 were included and evaluated as per inclusion and exclusion criteria. The intervention of SHED and its derivatives in several diseases depicted statistically significant therapeutic effects in periodontitis, pulpitis, spinal cord injury, parkinson's disease, alzheimer's disease, focal cerebral ischemia, peripheral nerve injury, and retinal pigmentosa. SHED also improved levels of alanine aminotransferase, aspartate aminotransferase, and bilirubin in liver fibrosis . In autoimmune diseases also, values were significant. SHED also showed a statistically significant reduction of wound healing area and new bone formation in bone defects. The pooled effect estimates of included preclinical studies demonstrated a statistically significant therapeutic effect of SHED in numerous diseases. Based on our data, it is suggested that the potential of SHED may be implemented in clinical trials after conducting a few more preclinical studies.

Exosomics in oral cancer diagnosis, prognosis, and therapeutics - An emergent and imperative non-invasive natural nanoparticle-based approach.

Exosomes- the natural nanoparticles belonging to heterogeneous vesicles are released via nearly all sorts of cells, including tumour cells, to oprate intercellular communication. Selective packaging of exosomes amid nucleic acids, phospholipids, and proteins makes them ideal for intercellular communications occurring among different cells. The existence of exosomes has been validated in various biofluids, including saliva. Being non-invasive and in direct contact with oral malignant cells, saliva establishes itself as a preeminent source of early cancer biomarkers. In context, the role and providence of both recipient and donor secreting cells are persuaded through exosomal cargo.Several studies have emphasized the influence of exosomal contents in different stages of cancer development, reconciling interactions between tumour cells and their surrounding niche. More explicitly, a transformation of exosomal contents such as nucleic acids, lipids, and proteins can endorse tumour progression and help ascertain a secluded pre-metastatic niche crammed with substances that errand cancer cell proliferation,angiogenesis, metastasis, and drug resistance. The blooming field of exosomes has directed the evolution of high-end isolation and characterization techniques along with the development of an entirely new field- exosomics that comprises complete analysis of exosomal cargo in various physiological conditions, including oral cancer. Researchers have discovered multiple pathways involved in exosome biogenesis to understand numerous events associated with cancer progression. Tissue-specific packaging of exosomes makes them a novel source of prognostic and diagnostic biomarkers and potential therapeutic targets. The extent of the current review confers the contemporary perception of the versatile task of exosomes, especially salivary exosomes, as potential biomarkers in the progression and diagnosis as well as therapeutics of oral cancers and their potential employment in clinical applications.

A Phytochemicals Approach Towards the Treatment of Cervical Cancer Using Polyphenols and Flavonoids.

AIM: Despite enormous progress in cancer biology, oncologists are still struggling to retrieve the methods and drugs to cure cancer which remains a global threat to humans. Plant-derived natural compounds, also known as phytochemicals, carry therapeutic potential and could be taken as dietary supplements, which are a radical way to resist as well as cure cancer. The present study reveals the anti-cancerous potential of a few phytochemical constituents under in vitro conditions and their mode of action on cervical cancer. SiHa was treated with phytochemicals viz. Quercetin dihydrate, Gallic Acid, and Naringin with varying concentrations to assess their cytotoxicity potential by various methods and also to elucidate their IC50 values. METHODS: All three compounds reduced the cell number and viability, along with alterations in cancer cell morphology. The diagnosed IC50 values of the compounds were 160µM, 200µM, and 1500µM for Quercetin dihydrate, Gallic Acid, and Naringin, respectively. DNA fragmentation assay and gene expression analysis were also used to assess apoptosis and anti-proliferative activity of compounds. RESULTS: We found fragmented DNA in treated cells as assessed by gel electrophoresis assay. These phytochemicals elicit an apoptotic response in SiHa cells by significantly up-regulating the gene expression level of p53 and p21 (p-value <0.005). CONCLUSION: Considering the anti-cancer and anti-proliferative potential of Quercetin dihydrate, Gallic Acid, and Naringin on the cervical cancer cell line, these phytochemicals could be used as an alternative or concurrent cancer therapeutic approach. However, further in-depth elucidation of their mode of action, safety, and efficacy should be explored.

Bioactive compounds and their libraries: An insight into prospective phytotherapeutics approach for oral mucocutaneous cancers.

Oral mucocutaneous cancers (OMCs) are cancers that affect both the oral mucosa and perioral cutaneous structures. Common OMCs are squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and malignant melanoma (MM). Anatomical similarities and conventions which categorizes these lesions blur the magnitude of OMCs in diverse populations. The burden of OMC is high in the sub-Saharan Africa and Indian subcontinents, and the cost of management is prohibitive in the resource-limited, developing world. Hence, there is a pressing demand for the use of cost-effective in silico approaches to identify diagnostic tools and treatment targets for diseases with high burdens in these regions. Due to their ubiquitousness and accessibility, the use of therapeutic efficacy of plant bioactive compounds in the management of OMC is both appropriate and plausible. Furthermore, screening known mechanistic disease targets with well annotated plant bioactive compound libraries is poised to improve the routine management of OMCs provided that the requisite access to database resources are available and accessible. Using natural products minimizes the side effects and morbidities associated with conventional therapies. The development of innovative treatments approaches would tremendously benefit the African and Indian populace and reduce the mortalities associated with OMCs in the developing world. Hence, we discuss herein, the potential benefits, opportunities and challenges of using bioactive compound libraries in the management of OMCs.

Mesenchymal stem cell immunomodulation and regeneration therapeutics as an ameliorative approach for COVID-19 pandemics.

The severe acute respiratory syndrome-novel coronavirus mediated COVID-19 has been recently declared a pandemic by the World Health Organization. The primary target of the SARS-CoV-2 virus is the human lungs governed by the ACE-2 receptor of epithelial type II cells/endothelial cells, which promote modulation of the immune response of host cells through generating cytokine storm, inflammation, severe pneumonia symptoms, and secondary complications such as acute respiratory distress syndrome. Although numerous antiviral and anti-parasitic drugs are under clinical trials to combat this pandemic, to date, neither a specific treatment nor any successful vaccine has been established, urging researchers to identify any potential candidate for combating the disease. Mesenchymal stem cells own self-renewal, differentiation, homing, immunomodulation and remains unaffected by the coronavirus on the virtue of the absence of ACE-2 receptors, indicating that MSC's could be used an ameliorative approach for COVID-19. MSCs have shown to combat the disease via various pathways such as repairing the lung epithelial and endothelial cells, reducing hyperimmune response, maintaining the renin-angiotensin system. Although MSCs-based treatment approaches for COVID-19 is still under consideration with limited data, many human clinical trials of MSC's has been initiated to explore their potential for COVID 19 treatment. The current review summarizes and emphasizes on how MSC's modulate the immune response, can repair the lungs from the impact of the virus, and various aspects of MSC's as a remedial source for COVID-19, to provide better insight for biomedical researchers and for those who are fascinated by stem cells as a therapeutic approach.

Hematopoietic Stem Cells Culture, Expansion and Differentiation: An Insight into Variable and Available Media.

Owing to differentiation and self-renewal capacity, hematopoietic stem cells clasp potentiality to engender all blood cell types, leading to their immense competence to play a diverse role in therapeutic applications. Although these stem cells are the most investigated and exploited until now, further research is still essential to comprehend their nature, fate, and potential. Enhanced usage of hematopoietic stem cells in research and therapeutics intensified the requirement of expansion and differentiation of hematopoietic stem cells under in vitro conditions. Since these cells remain in senescence for a prolonged period before isolation, selection of appropriate growth medium along with supplements and culture conditions are crucial to initiate their cell division and to designate their destiny. The precise equilibrium between self-renewal and differentiation of stem cells sustained by exclusive medium along with special growth or differentiation factors is accountable for generating diverse cell lineages. Maintenance of hematopoietic stem and progenitor cell lines along with the advancement of research work generate an inexorable demand for production and commercialization of specialized stem cell culture media, with or without serum along with specific growth factors and supplements. Media commercialization for precise stem cell types, culturing and differentiation is a cost-effective developing field. Here in this review, we are assembling various types of hematopoietic stem cell self-renewal, expansion and differentiation media along with supplements and culture conditions, either developed and used by various scientists or are available commercially.