Differential Expression of SRY-Related HMG-Box Transcription Factor 2, Oligodendrocyte Lineage Transcription Factor 2, and Zinc Finger E-Box Binding Homeobox 1 in Serum-Derived Extracellular Vesicles: Implications for Mithramycin Sensitivity and Targeted Therapy in High-Grade Glioma.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and is often resistant to traditional therapies. Evidence suggests that glioma stem cells (GSCs) contribute to this resistance. Mithramycin (Mit-A) targets GSCs and exhibits antitumor activity in GBM by affecting transcriptional targets such as SRY-related HMG-box transcription factor 2 (SOX2), oligodendrocyte lineage transcription factor 2 (OLIG2), and zinc finger E-box binding homeobox 1 (ZEB1). However, its clinical use has been limited by toxicity. This study explored the diagnostic potential of serum extracellular vesicles (EVs) to identify Mit-A responders. Serum EVs were isolated from 70 glioma patients, and targeted gene expression was analyzed using qRT-PCR. Using chemosensitivity assay, we identified 8 Mit-A responders and 17 nonresponders among 25 glioma patients. The M-score showed a significant correlation (p = 0.045) with isocitrate dehydrogenase 1 mutation but not other clinical variables. The genes SOX2 (p = 0.005), OLIG2 (p = 0.003), and ZEB1 (p = 0.0281) were found to be upregulated in the responder EVs. SOX2 had the highest diagnostic potential (AUC = 0.875), followed by OLIG2 (AUC = 0.772) and ZEB1 (AUC = 0.632).The combined gene panel showed significant diagnostic efficacy (AUC = 0.956) through logistic regression analysis. The gene panel was further validated in the serum EVs of 45 glioma patients. These findings highlight the potential of Mit-A as a targeted therapy for high-grade glioma based on differential gene expression in serum EVs. The gene panel could serve as a diagnostic tool to predict Mit-A sensitivity, offering a promising approach for personalized treatment strategies and emphasizing the role of GSCs in therapeutic resistance.

Polyethylene glycol-based isolation of urinary extracellular vesicles, an easily adoptable protocol.

Urine is a highly advantageous biological specimen for biomarker research and is a non-invasive source. Most of the urinary biomarkers are non-specific, volatile and need extensive validation before clinical adoption. Extracellular vesicles are secreted by almost all cells and are involved in homoeostasis, intercellular communication, and cellular processes in healthy and pathophysiological states. Urinary extracellular vesicles (UEVs) are released from the urogenital system and mirror the molecular processes of physiological and pathological states of their source cells. Therefore, UEVs serve as a valuable source of biomarkers for the non-invasive diagnosis of various pathologies. They hold a promising source of multiplex biomarkers suitable for prognosis, diagnosis, and therapy monitoring. UEVs are easily accessible, non-invasive, and suited for longitudinal sampling. Although various techniques are available for isolating UEVs, there is yet to be a consensus on a standard and ideal protocol. We have optimized an efficient, reliable, and easily adoptable polyethylene glycol (PEG) based UEV isolation technique following MISEV guidelines. The method is suitable for various downstream applications of UEVs. This could be a cost-effective, consistent, and accessible procedure for many clinical labs and is most suited for longitudinal analysis. Adopting the protocol will pave the way for establishing UEVs as the ideal biomarker source. •Urine can be collected non-invasively and repeatedly, hence a very useful specimen for biomarker discovery. Urinary EVs (UEVs), derived from urine, offer a stable diagnostic tool, but standardised isolation and analysis approaches are warranted.•To have enough UEVs for any study, large volumes of urine sample are necessary, which limits different isolation methods by cost, yield, and time.•The protocol developed could help researchers by offering a cost-effective and dependable UEV isolation method and may lay the foundation for UEVs adoption in clinical space.

Exploring urinary extracellular vesicles for organ transplant monitoring: A comprehensive study for detection of allograft dysfunction using immune-specific markers.

BACKGROUND: Allograft dysfunction (AGD) is a common complication following solid organ transplantation (SOT). This study leverages the potential of urinary extracellular vesicles (UEVs) for the non-invasive detection of AGD. AIM: We aimed to assess the diagnostic value of T-cell and B-cell markers characteristic of T-cell-mediated and antibody-mediated rejection in UEV-mRNA using renal transplantation as a model. MATERIALS AND METHODS: UEVs were isolated from 123 participants, spanning healthy controls, functional transplant recipients, and biopsy-proven AGD patients. T-cell and B-cell marker mRNA expressions were evaluated using RT-qPCR. RESULTS: We observed significant differences in marker expression between healthy controls and AGD patients. ROC analysis revealed an AUC of 0.80 for T-cell markers, 0.98 for B-cell markers, and 0.94 for combined markers. T-cell markers achieved 81.3 % sensitivity, 80 % specificity, and 80.4 % efficiency. A triad of T-cell markers (PRF1, OX40, and CD3e) increased sensitivity to 87.5 % and efficiency to 82.1 %. B-cell markers (CD20, CXCL3, CD46, and CF3) delivered 100 % sensitivity and 97.5 % specificity. The combined gene signature of T-cell and B-cell markers offered 93.8 % sensitivity and 95 % specificity. CONCLUSION: Our findings underscore the diagnostic potential of UEV-derived mRNA markers for T-cells and B-cells in AGD, suggesting a promising non-invasive strategy for monitoring graft health.

Identifying stable reference genes in polyethene glycol precipitated urinary extracellular vesicles for RT-qPCR-based gene expression studies in renal graft dysfunction patients.

BACKGROUND: Urinary extracellular vesicles (UEVs) hold RNA in their cargo and are potential sources of biomarkers for gene expression studies. The most used technique for gene-expression studies is quantitative polymerase chain reaction (qPCR). It is critical to use stable reference genes (RGs) as internal controls for normalising gene expression data, which aren't currently available for UEVs. METHODS: UEVs were precipitated from urine of graft dysfunction patients and healthy controls by Polyethylene glycol, Mn6000 (PEG6K). Vesicular characterisation confirmed the presence of UEVs. Gene expression levels of five commonly used RGs, i.e., Beta-2-Microglobulin (B2M), ribosomal-protein-L13a (RPL13A), Peptidylprolyl-Isomerase-A (PPIA), hydroxymethylbilane synthase (HMBS), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were quantified, and their stability was established through the RefFinder. The stability of identified RGs was validated by quantification of Perforin and granzyme B, signature molecules of renal graft dysfunction. RESULTS: Urine precipitated with 12% 6 K PEG yielded round and double-membraned UEVs of size ranging from 30 to 100 nm, as confirmed through transmission electron microscopy. Nanoparticle tracking analysis (59 ± 22 nm) and Dynamic-light-scattering (78 ± 56.5 nm) confirmed their size profile. Semi-quantitative Exocheck antibody array demonstrated the presence of EV protein markers in UEV. Using the comparative ΔCт method and RefFinder analysis, B2M (1.6) and RPL13A (1.8) genes emerged as the most stable reference genes. Validation of target gene expression in renal graft dysfunction patients confirmed the efficiency of B2M and RPL13A through significant upregulation compared to other RGs. CONCLUSIONS: Our study identified and validated B2M and RPL13A as optimal RGs for mRNA quantification studies in the UEVs of patients with renal graft dysfunction.

Nano-Messengers of the Heart: Promising Theranostic Candidates for Cardiovascular Maladies.

Till date, cardiovascular diseases remain a leading cause of morbidity and mortality across the globe. Several commonly used treatment methods are unable to offer safety from future complications and longevity to the patients. Therefore, better and more effective treatment measures are needed. A potential cutting-edge technology comprises stem cell-derived exosomes. These nanobodies secreted by cells are intended to transfer molecular cargo to other cells for the establishment of intercellular communication and homeostasis. They carry DNA, RNA, lipids, and proteins; many of these molecules are of diagnostic and therapeutic potential. Several stem cell exosomal derivatives have been found to mimic the cardioprotective attributes of their parent stem cells, thus holding the potential to act analogous to stem cell therapies. Their translational value remains high as they have minimal immunogenicity, toxicity, and teratogenicity. The current review highlights the potential of various stem cell exosomes in cardiac repair, emphasizing the recent advancements made in the development of cell-free therapeutics, particularly as biomarkers and as carriers of therapeutic molecules. With the use of genetic engineering and biomimetics, the field of exosome research for heart treatment is expected to solve various theranostic requirements in the field paving its way to the clinics.

Diagnostic Values of Laboratory Biomarkers in Predicting a Severe Course of COVID-19 on Hospital Admission.

Objective: We intend to identify differences in the clinicodemographic and laboratory findings of COVID-19 patients to predict disease severity and outcome on admission. Methods: This single-centred retrospective study retrieved laboratory and clinical data from 350 COVID-19 patients on admission, represented as frequency tables. A multivariate regression model was used to assess the statistically significant association between the explanatory variables and COVID-19 infection outcomes, where adjusted odds ratio (AOR), p value, and 95% CI were used for testing significance. Results: Among the 350 COVID-19 patients studied, there was a significant increase in the WBC count, neutrophils, aggregate index of systemic inflammation (AISI), neutrophil-to-lymphocyte ratio (dNLR), neutrophil-to-lymphocyte and platelet ratio (NLPR), monocyte-to-lymphocyte ratio (MLR), systemic immune-inflammation index (SII), systemic inflammation response index (SIRI), D-dimer, interleukin-6 (IL-6), ferritin, lactate dehydrogenase (LDH), prothrombin time (PT), glucose, urea, urea nitrogen, creatinine, alanine phosphatase (ALP), and aspartate aminotransferase (AST) and a significant decrease in lymphocytes, eosinophils, total protein, albumin, prealbumin serum, and albumin/globulin (A/G) ratio in the severe group when compared with the mild and moderate groups. However, after adjusting their age, gender, and comorbidities, WBC count (adjusted odds ratio (AOR) = 6.888, 95% CI = 1.590-29.839, p = 0.010), neutrophils (AOR = 5.912, 95% CI = 2.131-16.402, p = 0.001), and urea (AOR = 4.843, 95% CI = 1.988-11.755, p = 0.001) were strongly associated with disease severity. Interpretation and Conclusion. On admission, WBC count, neutrophils, and urea, with their cut of values, can identify at-risk COVID-19 patients who could develop severe COVID-19.

NIR triggered liposome gold nanoparticles entrapping curcumin as in situ adjuvant for photothermal treatment of skin cancer.

We report the synthesis of a biodegradable liposome gold nanoparticles for curcumin (Au-Lipos Cur NPs) delivery. This entrapped curcumin served as an in situ adjuvant for photothermal therapy. Curcumin was loaded in Au-Lipos NPs with an encapsulation efficiency of ∼70%. The gold coating enabled the NPs to specifically absorb NIR light (780nm) by virtue of Surface Plasmon Resonance (SPR) and this light energy was converted to heat. The generated heat destabilized the liposomal core enhancing the release of encapsulated curcumin. Photothermal transduction efficacy of Au-Lipos NPs (loaded with curcumin) showed a significant temperature rise upon laser irradiation causing irreversible cellular damage. In vitro photothermal effect and intracellular uptake was evaluated in B16 F10 (melanoma) cell line. Au-Lipos Cur NPs showed significantly enhanced uptake when compared with free curcumin. Enhancement in cancer cell cytotoxicity was observed in Au-Lipos Cur NPs treated group upon laser irradiation owing to curcumin. Our findings indicate that curcumin could serve as a potential in situ adjuvant for photothermal therapy of melanoma.