Circulating small extracellular vesicles in Alzheimer's disease: a case-control study of neuro-inflammation and synaptic dysfunction.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aß plaques and neurofibrillary tangles. Chronic inflammation and synaptic dysfunction lead to disease progression and cognitive decline. Small extracellular vesicles (sEVs) are implicated in AD progression by facilitating the spread of pathological proteins and inflammatory cytokines. This study investigates synaptic dysfunction and neuroinflammation protein markers in plasma-derived sEVs (PsEVs), their association with Amyloid-ß and tau pathologies, and their correlation with AD progression. METHODS: A total of 90 [AD = 35, mild cognitive impairment (MCI) = 25, and healthy age-matched controls (AMC) = 30] participants were recruited. PsEVs were isolated using a chemical precipitation method, and their morphology was characterized by transmission electron microscopy. Using nanoparticle tracking analysis, the size and concentration of PsEVs were determined. Antibody-based validation of PsEVs was done using CD63, CD81, TSG101, and L1CAM antibodies. Synaptic dysfunction and neuroinflammation were evaluated with synaptophysin, TNF-α, IL-1ß, and GFAP antibodies. AD-specific markers, amyloid-ß (1-42), and p-Tau were examined within PsEVs using Western blot and ELISA. RESULTS: Our findings reveal higher concentrations of PsEVs in AD and MCI compared to AMC (p < 0.0001). Amyloid-ß (1-42) expression within PsEVs is significantly elevated in MCI and AD compared to AMC. We could also differentiate between the amyloid-ß (1-42) expression in AD and MCI. Similarly, PsEVs-derived p-Tau exhibited elevated expression in MCI compared with AMC, which is further increased in AD. Synaptophysin exhibited downregulated expression in PsEVs from MCI to AD (p = 0.047) compared to AMC, whereas IL-1ß, TNF-α, and GFAP showed increased expression in MCI and AD compared to AMC. The correlation between the neuropsychological tests and PsEVs-derived proteins (which included markers for synaptic integrity, neuroinflammation, and disease pathology) was also performed in our study. The increased number of PsEVs correlates with disease pathological markers, synaptic dysfunction, and neuroinflammation. CONCLUSIONS: Elevated PsEVs, upregulated amyloid-ß (1-42), and p-Tau expression show high diagnostic accuracy in AD. The downregulated synaptophysin expression and upregulated neuroinflammatory markers in AD and MCI patients suggest potential synaptic degeneration and neuroinflammation. These findings support the potential of PsEV-associated biomarkers for AD diagnosis and highlight synaptic dysfunction and neuroinflammation in disease progression.

Deciphering pancreatic neuroendocrine tumors: Unveiling through circulating small extracellular vesicles.

The survival rate over a five-year period for rare pancreatic neuroendocrine tumors (PanNET) is notably lower compared to other neuroendocrine tumors due to late-stage detection, which is a consequence of the absence of suitable diagnostic markers; therefore, there exists a critical need for an early-stage biomarker-specific to PanNETs. This study introduces a novel approach, investigating the impact of small extracellular vesicles (sEV) in PanNET growth and metastasis. As proof of concept, this study shows a correlation between sEV concentration in controls and PanNET. Notably, higher sEV concentrations were observed in PanNETs than in controls (p < 0.0001) with a sensitivity of 100%. Further, apparent differences were observed in the sEV concentrations between controls and grades 1 PanNET (p = 0.005). The expression of sEV markers was confirmed using CD63, TSG101, CD9, Flotillin-1, and GAD65 antibodies. Additionally, the expression of cancer marker BIRC2/cIAP1 (p = 0.002) and autophagy marker Beclin-1 (p = 0.02) were observed in plasma-derived sEVs and PanNET tissue. This study represents the first to indicate the increased secretion of sEV in PanNET patients' blood plasma, proposing potential function of sEV as a new biomarker for early-stage PanNET detection.

C1QA and COMP: plasma-based biomarkers for early diagnosis of pancreatic neuroendocrine tumors.

Pancreatic Neuroendocrine tumors (PanNET) are challenging to diagnose and often detected at advanced stages due to a lack of specific and sensitive biomarkers. This study utilized proteomics as a valuable approach for cancer biomarker discovery; therefore, mass spectrometry-based proteomic profiling was conducted on plasma samples from 12 subjects (3 controls; 5 Grade I, 4 Grade II PanNET patients) to identify potential proteins capable of effectively distinguishing PanNET from healthy controls. Data are available via ProteomeXchange with the identifier PXD045045. 13.2% of proteins were uniquely identified in PanNET, while 60% were commonly expressed in PanNET and controls. 17 proteins exhibiting significant differential expression between PanNET and controls were identified with downstream analysis. Further, 5 proteins (C1QA, COMP, HSP90B1, ITGA2B, and FN1) were selected by pathway analysis and were validated using Western blot analysis. Significant downregulation of C1QA (p = 0.001: within groups, 0.03: control vs. grade I, 0.0013: grade I vs. grade II) and COMP (p = 0.011: within groups, 0.019: control vs grade I) were observed in PanNET Grade I & II than in controls. Subsequently, ELISA on 38 samples revealed significant downregulation of C1QA and COMP with increasing disease severity. This study shows the potential of C1QA and COMP in the early detection of PanNET, highlighting their role in the search for early-stage (Grade-I and Grade-II) diagnostic markers and therapeutic targets for PanNET.

Circulating plasma miR-23b-3p as a biomarker target for idiopathic Parkinson's disease: comparison with small extracellular vesicle miRNA.

Background: Parkinson's disease (PD) is an increasingly common neurodegenerative condition, which causes movement dysfunction and a broad range of non-motor symptoms. There is no molecular or biochemical diagnosis test for PD. The miRNAs are a class of small non-coding RNAs and are extensively studied owing to their altered expression in pathological states and facile harvesting and analysis techniques. Methods: A total of 48 samples (16 each of PD, aged-matched, and young controls) were recruited. The small extracellular vesicles (sEVs) were isolated and validated using Western blot, transmission electron microscope, and nanoparticle tracking analysis. Small RNA isolation, library preparation, and small RNA sequencing followed by differential expression and targeted prediction of miRNA were performed. The real-time PCR was performed with the targeted miRNA on PD, age-matched, and young healthy control of plasma and plasma-derived sEVs to demonstrate their potential as a diagnostic biomarker. Results: In RNA sequencing, we identified 14.89% upregulated (fold change 1.11 to 11.04, p < 0.05) and 16.54% downregulated (fold change -1.04 to -7.28, p < 0.05) miRNAs in PD and controls. Four differentially expressed miRNAs (miR-23b-3p, miR-29a-3p, miR-19b-3p, and miR-150-3p) were selected. The expression of miR-23b-3p was "upregulated" (p = 0.002) in plasma, whereas "downregulated" (p = 0.0284) in plasma-derived sEVs in PD than age-matched controls. The ROC analysis of miR-23b-3p revealed better AUC values in plasma (AUC = 0.8086, p = 0.0029) and plasma-derived sEVs (AUC = 0.7278, p = 0.0483) of PD and age-matched controls. Conclusion: We observed an opposite expression profile of miR-23b-3p in PD and age-matched healthy control in plasma and plasma-derived sEV fractions, where the expression of miR-23b-3p is increased in PD plasma while decreased in plasma-derived sEV fractions. We further observed the different miR-23b-3p expression profiles in young and age-matched healthy control.

Fluorescence-tagged salivary small extracellular vesicles as a nanotool in early diagnosis of Parkinson's disease.

BACKGROUND: Parkinson's disease is generally asymptomatic at earlier stages. At an early stage, there is an extensive progression in the neuropathological hallmarks, although, at this stage, diagnosis is not possible with currently available diagnostic methods. Therefore, the pressing need is for susceptibility risk biomarkers that can aid in better diagnosis and therapeutics as well can objectively serve to measure the endpoint of disease progression. The role of small extracellular vesicles (sEV) in the progression of neurodegenerative diseases could be potent in playing a revolutionary role in biomarker discovery. METHODS: In our study, the salivary sEV were efficiently isolated by chemical precipitation combined with ultrafiltration from subjects (PD = 70, healthy controls = 26, and prodromal PD = 08), followed by antibody-based validation with CD63, CD9, GAPDH, Flotillin-1, and L1CAM. Morphological characterization of the isolated sEV through transmission electron microscopy. The quantification of sEV was achieved by fluorescence (lipid-binding dye-labeled) nanoparticle tracking analysis and antibody-based (CD63 Alexa fluor 488 tagged sEV) nanoparticle tracking analysis. The total alpha-synuclein (α-synTotal) in salivary sEVs cargo was quantified by ELISA. The disease severity staging confirmation for n = 18 clinically diagnosed Parkinson's disease patients was done by 99mTc-TRODAT-single-photon emission computed tomography. RESULTS: We observed a significant increase in total sEVs concentration in PD patients than in the healthy control (HC), where fluorescence lipid-binding dye-tagged sEV were observed to be higher in PD (p = 0.0001) than in the HC using NTA with a sensitivity of 94.34%. In the prodromal PD cases, the fluorescence lipid-binding dye-tagged sEV concentration was found to be higher (p = 0.008) than in HC. This result was validated through anti-CD63 tagged sEV (p = 0.0006) with similar sensitivity of 94.12%. We further validated our findings with the ELISA based on α-synTotal concentration in sEV, where it was observed to be higher in PD (p = 0.004) with a sensitivity of 88.24%. The caudate binding ratios in 99mTc-TRODAT-SPECT represent a positive correlation with sEV concentration (r = 0.8117 with p = 0.0112). CONCLUSIONS: In this study, for the first time, we have found that the fluorescence-tagged sEV has the potential to screen the progression of disease with clinically acceptable sensitivity and can be a potent early detection method for PD.

A tale of exosomes and their implication in cancer.

Cancer is a cause of high deaths worldwide and also a huge burden for the health system. Cancer cells have unique properties such as a high rate of proliferation, self-renewal, metastasis, and treatment resistance, therefore, the development of novel diagnoses of cancers is a tedious task. Exosomes are secreted by virtually all cell types and have the ability to carry a multitude of biomolecules crucial for intercellular communication, hence, contributing a crucial part in the onset and spread of cancer. These exosomal components can be utilized in the development of markers for diagnostic and prognostic purposes for various cancers. This review emphasized primarily the following topics: exosomes structure and functions, isolation and characterization strategies of exosomes, the role of exosomal contents in cancer with a focus in particular on noncoding RNA and protein, exosomes, and the cancer microenvironment interactions, cancer stem cells, and tumor diagnosis and prognosis based on exosomes.

Osteogenic markers in peri-implant crevicular fluid in immediate and delayed-loaded dental implants: A randomized controlled trial.

INTRODUCTION: The study evaluates the levels of matrix metalloprotease-8 (MMP-8), and Cathepsin-K (CatK) in peri-implant crevicular fluid (PICF) among patients with immediate loaded (IL) and delayed-loaded (DL) implants at different time points to know the inflammation and osteogenic status. METHODS: The study population consisted of two groups (n = 25, each group) with a mean age of 28.7 ± 3.5 years, and PICF was collected. MMP-8 and CatK levels were quantified through ELISA. RESULTS: We observed the concentrations of inflammatory markers (MMP-8 and CatK) at three time points in the IL and DL groups. The mean concentration of MMP-8 in the IL group was 9468 ± 1230 pg/mL, 5547 ± 1088 pg/mL, and 7248 ± 1396 pg/mL at 2 weeks, 3 months, and 12 months, respectively; while in the DL group was 10 816 ± 779.7 pg/mL, 9531 ± 1245 pg/mL, and 9132 ± 1265 pg/mL at 2 weeks, 3 and 12 months, respectively. The mean concentration of Cat-K in the IL group was observed at 422.1 ± 36.46 pg/mL, 242.9 ± 25.87 pg/mL, and 469 ± 75.38 pg/mL at 2 weeks, 3, and 12 months, whereas in the DL group was 654.6 ± 152.9 pg/mL, 314.7 ± 28.29 pg/mL, and 539.8 ± 115.1 pg/mL at 2 weeks, 3 months and 12 months, respectively. CONCLUSION: In this study, the levels of CatK and MMP-8 levels decline at 12 months in both groups, and the IL group shows lower values compared to the DL group; however, no significant changes were observed after analyses were adjusted for multiple comparisons (p > 0.025). Therefore, there is not much difference observed in the inflammation process between immediate and delayed loading. (Clinical trial identifier: CTRI/2017/09/009668).

BCL-2 (-938C>A), BAX (-248G>A), and HER2 Ile655Val Polymorphisms and Breast Cancer Risk in Indian Population.

Breast cancer is the most common carcinoma in women worldwide. The present case-control study was aimed to examine the association of BCL-2 (-938C> A), BAX (-248G > A), and HER2 (I655V i.e. A > G) polymorphisms with breast cancer risk in Indian population. This study enrolled 117 breast cancer cases and 104 controls. BCL-2 (-938C > A), BAX (-248G > A), and HER2 Ile655Val polymorphisms were screened by PCR-RFLP method. There was no significance difference in the allelic and genotype frequency of the BCL-2 (-938C > A) and BAX (-248G > A) polymorphisms between cases and controls. In relation to HER2 Ile655Val polymorphism, the statistical analysis of observed genotypic frequencies showed significant association (p-0.0059). Compared to Ile/Ile (A/A) genotype, frequency of Ile/Val (A/G) genotype was significantly higher among cases than in control group and observed to increase the breast cancer risk (OR, 2.43; 95%CI, 1.32-4.46; p-0.004). The frequency of Val (G) allele was significantly higher in cases as compared to controls (6.83% vs 2.88%, resp.). Compared to Ile (A) allele, significant increase in the risk of breast cancer was observed with Val (G) allele (OR, 2.21; 95% CI, 1.35-3.63; p-0.0016). We observed significant association between HER2 Ile655Val polymorphism and breast cancer risk under the dominant (OR = 2.52; 95% CI: 1.41-4.51; p-0.001) and codominant (OR, 2.24; 95% CI: 1.23-4.09; p-0.008) model. In our study, BCL-2 (-938C > A) and BAX (-248G > A) polymorphism were not found to be associated with breast cancer risk. This present study for the first time shows significant association of HER2 Ile655Val polymorphism with risk of breast cancer in Indian population. Therefore, we suggest that each population need to evaluate its own genetic profile for breast cancer risk that may be helpful for better understanding the racial and geographic differences reported for breast cancer incidence and mortality.

Autophagy Paradox of Cancer: Role, Regulation, and Duality.

Autophagy, a catabolic process, degrades damaged and defective cellular materials through lysosomes, thus working as a recycling mechanism of the cell. It is an evolutionarily conserved and highly regulated process that plays an important role in maintaining cellular homeostasis. Autophagy is constitutively active at the basal level; however, it gets enhanced to meet cellular needs in various stress conditions. The process involves various autophagy-related genes that ultimately lead to the degradation of targeted cytosolic substrates. Many factors modulate both upstream and downstream autophagy pathways like nutritional status, energy level, growth factors, hypoxic conditions, and localization of p53. Any problem in executing autophagy can lead to various pathological conditions including neurodegeneration, aging, and cancer. In cancer, autophagy plays a contradictory role; it inhibits the formation of tumors, whereas, during advanced stages, autophagy promotes tumor progression. Besides, autophagy protects the tumor from various therapies by providing recycled nutrition and energy to the tumor cells. Autophagy is stimulated by tumor suppressor proteins, whereas it gets inhibited by oncogenes. Due to its dynamic and dual role in the pathogenesis of cancer, autophagy provides promising opportunities in developing novel and effective cancer therapies along with managing chemoresistant cancers. In this article, we summarize different strategies that can modulate autophagy in cancer to overcome the major obstacle, i.e., resistance developed in cancer to anticancer therapies.

The Evolving Landscape of Exosomes in Neurodegenerative Diseases: Exosomes Characteristics and a Promising Role in Early Diagnosis.

Neurodegenerative diseases (ND) remains to be one of the biggest burdens on healthcare systems and serves as a leading cause of disability and death. Alzheimer's disease (AD) is among the most common of such disorders, followed by Parkinson's disease (PD). The basic molecular details of disease initiation and pathology are still under research. Only recently, the role of exosomes has been linked to the initiation and progression of these neurodegenerative diseases. Exosomes are small bilipid layer enclosed extracellular vesicles, which were once considered as a cellular waste and functionless. These nano-vesicles of 30-150 nm in diameter carry specific proteins, lipids, functional mRNAs, and high amounts of non-coding RNAs (miRNAs, lncRNAs, and circRNAs). As the exosomes content is known to vary as per their originating and recipient cells, these vesicles can be utilized as a diagnostic biomarker for early disease detection. Here we review exosomes, their biogenesis, composition, and role in neurodegenerative diseases. We have also provided details for their characterization through an array of available techniques. Their updated role in neurodegenerative disease pathology is also discussed. Finally, we have shed light on a novel field of salivary exosomes as a potential candidate for early diagnosis in neurodegenerative diseases and compared the biomarkers of salivary exosomes with other blood/cerebrospinal fluid (CSF) based exosomes within these neurological ailments.

A novel approach to correlate the salivary exosomes and their protein cargo in the progression of cognitive impairment into Alzheimer's disease.

BACKGROUND: Cognition is the ability of a person to think, remember, and interconnect ideas from various dimensions to strive for solutions. Cognitive defects accompany all forms of dementia and the decline in cognition is a most feared aspect. Mild cognitive impairment is considered as a transitional phase and the progressive loss in cognition can finally lead to Alzheimer's disease. NEW METHOD: In this study, we demonstrated a novel method based on nanoparticle tracking analysis (NTA) technique to directly correlate salivary exosomes concentration with the progression of cognitive impairment (CI) in Alzheimer's disease (AD).This could open up the possibility for an early and cost-effective screening of Alzheimer's disease. RESULTS: Using our novel method, the total salivary exosomes concentration was measured by NTA technique, followed by validation of key exosomal cargo proteins through an automated western blot analyzer. We observed significant differences in salivary exosomes concentration among the groups of cognitively impaired and Alzheimer's disease patients (p = 0.0023) compared to the healthy control cohort. The method was validated through CD63 (exosomes surface marker) fluorescent antibody based quantification, which yielded a similar outcome (p = 0.0286). We further corroborated our findings with the expression level of oligomeric amyloid-beta, phosphorylated-tau protein from salivary exosomes. The Aß oligomer/fibril abundance (p = 0.0291), phospho-tau (p = 0.0325) and Aß protein abundance (p = 0.0198) was significantly higher in Alzheimer's and cognitively impaired patients in comparison to the healthy controls. COMPARISON WITH EXISTING METHOD(S): There are few molecular biomarkers available to differentiate between various stages of cognitive impairment. Moreover, the current methodologies utilizing the few biomarkers available are either invasive or expensive; also, for a patient with mild cognitive complains, it is impractical to use these as a screening tool. CONCLUSION: Our initial results indicate that the salivary exosomes concentration based on the nano-tracking technique has the potential to be used as a cost-effective screening method for early disease detection.

Frequency of I655V SNP of HER-2/neu in colorectal cancer: a study from India.

The present study was conducted to determine the prognostic significance of I655V SNP (rs1136201) is a genetic one in HER-2 oncoprotein in cases of colorectal cancer (CRC). We conducted a case-control study analysing 83 subjects (naïve primary CRC cases) who underwent CRC biopsy/colectomy and included 57 healthy control subjects. Analysis of HER-2 polymorphism was done by PCR-RFLP technique. The mean age was found to be 55.9 years; median age was 56 years and mode age was 54 years with a range of 43 (30-73). Males constitute 63 (75.9%) and females constitute 20 (24.1%) of patient population. According to gradewise distribution, 12 (14.45%) patients were of Grade I, 53 (63.85%) of Grade II, and 18 (21.68%) were of Grade III. We found out that out of 83 patients, 52 (62.65%) were of homozygous wild type (A/A; Ile/Ile); 27 (32.53%) were of heterozygous type (A/G; Ile/Val) and 4 (4.81%) were of homozygous mutant type (G/G; Val/Val). Allelic frequency of Ile (A) was found out to be 0.79 and that of Val (G) is 0.21 and were not significantly different from the healthy control population. Fischer's exact p value obtained was 0.86.