Exosomal microRNAs in Parkinson's disease: insights into biomarker potential and disease pathology.

Parkinson's disease (PD) is a prevalent neurodegenerative condition primarily affecting the elderly population. Despite its high incidence in aged individuals, there are no reliable blood-based biomarkers for clinical diagnosis of PD and early screening of susceptible individuals. Recent studies have revealed the significance of exosomes in mediating cell-to-cell communications by transferring bioactive molecules, such as proteins, nucleic acids (including miRNAs), lipids, and metabolites, between cells. Due to their ability to carry diverse molecular cargo and their involvement in various physiological and pathological processes, exosomes have gained significant attention as potential disease biomarkers. Notably, exosomes have the ability to cross the blood-brain barrier, and as a result, they can be found in circulating body fluids, including cerebrospinal fluid (CSF), serum, and plasma. Therefore, the identification of PD-specific exosomes in blood samples could be a promising avenue with biomarker potential for advancing clinical diagnosis and planning therapeutic strategies. This review highlights the current understanding of exosomal miRNAs in PD pathology, emphasising their potential for clinical utility as biomarkers even though several challenges may have to be overcome to precisely utilize exosomal miRNAs as biomarkers specific to PD.

Functional assessment of DNA extraction methods from frozen human blood samples for Sanger sequencing analysis.

The quality of input DNA is crucial for obtaining significant inferences from molecular techniques like Sanger sequencing and Next Generation Sequencing experiments. Many of the extraction methods are suitable for retrieving quality DNA from fresh blood and tissue samples, regardless of the isolation principle. However, while isolating DNA from frozen blood samples, processed tissue samples or low-quality samples, careful selection of suitable extraction methods is extremely important. Moreover, there is no standard protocol recommended for genomic DNA extraction from stored blood samples, particularly those stored in a Biobank, for applications like Sanger sequencing. Consequently, we have systematically compared different commercial DNA isolation kits with a modified manual extraction method for blood samples frozen for up to three years and assessed their quality, yield and suitability for PCR, Real-Time PCR and Sanger sequencing. The manual DNA extraction method was improved by incorporating a few modifications: a lower NaCl concentration was used for precipitating DNA and excluded the use of phenol. The modified method provided the maximum DNA yield from stored blood. Although all the methods tested were suitable for recovering DNA from stored blood, the modified method described here may be preferred for large-scale applications as it provides cost-effective ways to obtain large quantities of quality DNA. Most importantly, the DNA isolated by the modified method appears to be more stable in long-term storage at -80°C.

Biomarkers predict hemorrhagic transformation and stroke severity after acute ischemic stroke.

OBJECTIVES: Hemorrhagic transformation (HT) is a complication occurring in patients with acute ischemic stroke (AIS) either spontaneously or post-thrombolysis leading to significant morbidity and mortality. We assessed circulating matrix metalloproteinase-9 (MMP-9), Claudin-5, and soluble serum stimulation-2 (sST2) in HT and stroke severity in AIS based on their temporal distribution. MATERIALS AND METHODS: We prospectively enrolled 111 AIS patients within 12 h from onset. Patient demographic, clinical, and imaging details were documented. Follow-up imaging was conducted 24-48 h after admission. Blood samples were taken at three time-points from stroke onset. HT was classified according to the European Co-operative Acute Stroke Study-III(ECASS-III). Stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS). Multiple logistic regression and receiver operating characteristic curve were conducted to determine the discriminative capacity. RESULTS: Mean age was 62.3 ± 11.7 years and median baseline NIHSS was 12[IQR 8.0-18.0]. HT was detected in 30(27%) patients. Biomarker levels at 12 h were elevated with median MMP-9 concentration of 153.9 ng/mL[IQR 110.6-309 ng/mL] indicating a trend toward significant positive correlation with HT(P = 0.05). Claudin-5 levels at 12 h was elevated but was not statistically significant (43.1 pg/mL[IQR:26.7-72.6 pg/mL] vs 59.4 pg/mL[IQR:24.5-100.8 pg/mL];P = 0.4). Multiple logistic regression indicated Claudin-5 levels at 12 h (OR 9.46;95% CI:1.97-64.6;P = 0.010) and baseline low ASPECTS score(OR 20.3;95% CI:3.46-193; P = 0.003) independently predicted HT. MMP-9 at 12 h was significantly elevated in patients with moderate to severe strokes (P = 0.04). CONCLUSIONS: Claudin-5 and low ASPECTS independently predicted HT. MMP-9 was positively correlated with baseline stroke severity.

SHARPIN: Role in Finding NEMO and in Amyloid-Beta Clearance and Degradation (ABCD) Pathway in Alzheimer's Disease?

SHANK- associated RH domain-interacting protein (SHARPIN) is a multifunctional protein associated with numerous physiological functions and many diseases. The primary role of the protein as a LUBAC-dependent component in regulating the activation of the transcription factor NF-κB accounts to its role in inflammation and antiapoptosis. Hence, an alteration of SHARPIN expression or genetic mutations or polymorphisms leads to the alteration of the above-mentioned primary physiological functions contributing to inflammation-associated diseases and cancer, respectively. However, there are complications of targeting SHARPIN as a therapeutic approach, which arises from the wide-range of LUBAC-independent functions and yet unknown roles of SHARPIN including neuronal functions. The identification of SHARPIN as a postsynaptic protein and the emerging studies indicating its role in several neurodegenerative diseases including Alzheimer's disease suggests a strong role of SHARPIN in neuronal functioning. This review summarizes the functional roles of SHARPIN in normal physiology and disease pathogenesis and strongly suggests a need for concentrating more studies on identifying the unknown neuronal functions of SHARPIN and hence its role in neurodegenerative diseases.

Diminished substrate-mediated cardiac mitochondrial respiration and elevated autophagy in adult male offspring of gestational diabetic rats.

OBJECTIVE: Heart diseases are common in offspring of gestational diabetic mother (ODM). Defects in mitochondrial metabolism and autophagy may be one of the basic reasons behind the abnormal structural and functional behaviour of offspring's heart. So the main objective of the present study was to explore the cardiac mitochondrial respiration and autophagy in male and female offspring of diabetic pregnancy at two different developmental stages of life. METHODS: High-resolution respirometry was used to measure substrate-mediated mitochondrial respiration in isolated mitochondria from ventricular tissues of offspring of streptozotocin-induced diabetic mother rats. To find the expression of proteins involved in autophagic process and oxidative stress, western blotting and densitometric analysis were done. RESULTS: Mitochondrial complex I and complex II respiration was found to be decreased in adult male offspring while it was unaltered or less affected in weaning male and female offspring. Elevated autophagy was shown by adult male, while there was no change observed in adult female. Also absence of elevated expression of oxidative stress markers was observed in all groups. CONCUSION: The present study reports altered cardiac mitochondrial respiration and autophagy in male offspring of diabetic mothers than the control ones. The study also analysed the expression of various candidates of cardiac autophagic process in male and female offspring of diabetic pregnancy at two time points of development.

Diminished substrate-mediated cardiac mitochondrial respiration and elevated autophagy in adult male offspring of gestational diabetic rats.

Heart diseases are common in the offspring of diabetic mother (ODM). Defects in mitochondrial metabolism and autophagy may, in part, be responsible for the adverse structural and functional alterations in the heart. The principal objective of this study was to investigate cardiac mitochondrial respiration and autophagy in male and female offspring of diabetic pregnancy at two different developmental stages of life, weaning and adult. Male and female offspring of rats with streptozotocin-induced gestational diabetes were used for the study and compared with offspring of control (non-diabetic) mother (OCM) rats. High-resolution respirometry was used to measure substrate-mediated respiration in mitochondria isolated from ventricular tissues of ODM and OCM. Expression of proteins associated with autophagy and oxidative stress was examined by western blot analysis. Mitochondrial complex I and complex II respiration was significantly reduced in adult male ODM while it was unaltered or less affected in weaning male, adult and weaning female ODM. Elevated autophagy was observed in adult male but not in adult female ODM. Expression of oxidative stress markers was observed to be similar in all the groups. Altered cardiac mitochondrial respiration and autophagy were observed in adult male ODM compared to OCM, while the male and female offspring at weaning stage were less affected. The results of the study show that maternal hyperglycemia affects mitochondrial respiration and autophagy in the ODM heart, which may potentially be responsible for the cardiovascular complications observed in adult life.

To be Wild or Mutant: Role of Isocitrate Dehydrogenase 1 (IDH1) and 2-Hydroxy Glutarate (2-HG) in Gliomagenesis and Treatment Outcome in Glioma.

Molecular and clinical research based on isocitrate dehydrogenase (IDH) mutations is much sought after in glioma research since a decade of its discovery in 2008. IDH enzyme normally catalyzes isocitrate to α-keto-glutarate (α-KG), but once the gene is mutated it produces an 'oncometabolite', 2-hydroxyglutarate (2-HG). 2-HG is proposed to inhibit α-KG-dependent dioxygenases and also blocks cellular differentiation. Here, we discuss the role of the IDH1 mutation in gliomagenesis. The review also focuses on the effect of 2-HG on glioma epigenetics, the cellular signaling involved in IDH1 mutant glioma cells and the therapeutic response seen in mutant IDH1(mIDH1) harboring glioma patients in comparison to the patients with wild-type IDH1. The review encompasses the debatable impacts of the mutation on immune microenvironment a propos of various mIDH1 inhibitors in practice or in trials. Recent studies revealing the relation of IDH mutation with the immune microenvironment and inflammatory status in untreated versus treated glioblastoma patients are highlighted with respect to prospective therapeutic targets. Also at the molecular level, the association of mIDH1/2-HG with the intracellular components such as mitochondria and other neighboring cells is discussed.

Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Despite the strong association between diabetes and dementia, it remains to be fully elucidated how insulin deficiency adversely affects brain functions. We show that insulin deficiency in streptozotocin-induced diabetic mice decreased mitochondrial ATP production and/or citrate synthase and cytochrome oxidase activities in the cerebrum, hypothalamus, and hippocampus. Concomitant decrease in mitochondrial fusion proteins and increased fission proteins in these brain regions likely contributed to altered mitochondrial function. Although insulin deficiency did not cause any detectable increase in reactive oxygen species (ROS) emission, inhibition of monocarboxylate transporters increased ROS emission and further reduced ATP production, indicating the causative roles of elevated ketones and lactate in counteracting oxidative stress and as a fuel source for ATP production during insulin deficiency. Moreover, in healthy mice, intranasal insulin administration increased mitochondrial ATP production, demonstrating a direct regulatory role of insulin on brain mitochondrial function. Proteomics analysis of the cerebrum showed that although insulin deficiency led to oxidative post-translational modification of several proteins that cause tau phosphorylation and neurofibrillary degeneration, insulin administration enhanced neuronal development and neurotransmission pathways. Together these results render support for the critical role of insulin to maintain brain mitochondrial homeostasis and provide mechanistic insight into the potential therapeutic benefits of intranasal insulin.-Ruegsegger, G. N., Manjunatha, S., Summer, P., Gopala, S., Zabeilski, P., Dasari, S., Vanderboom, P. M., Lanza, I. R., Klaus, K. A., Nair, K. S. Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Apolipoproteins B and A1 in Ischemic Stroke Subtypes.

INTRODUCTION: Elevated serum apolipoprotein B and the apolipoprotein B/A1 ratio have been associated with ischemic stroke and intracranial atherosclerotic disease. We sought to assess the relationship between serum levels of apolipoprotein B, apolipoprotein A1, and the apolipoprotein B/A1 ratio with ischemic stroke subtypes and large artery atherosclerosis location. MATERIALS AND METHODS: We evaluated serum apolipoprotein B and apolipoprotein A1 levels in consecutive, statin-naïve, adult ischemic stroke patients admitted to an academic medical center in southern India. We evaluated for differences in the mean serum levels of apolipoprotein B, apolipoprotein A1, and the apolipoprotein B/A1 ratio between patients with ischemic stroke attributed to intracranial atherosclerotic disease, extracranial atherosclerotic disease, small vessel disease, and cardioembolism. In secondary analysis, we assessed for differences in these serum apolipoproteins between patients with moderate-severe intracranial atherosclerotic disease and extracranial atherosclerotic disease, irrespective of ischemic stroke subtype. RESULTS: Among the 156 ischemic stroke patients enrolled in this study, there were no significant differences in serum levels of apolipoprotein B, apolipoprotein A1, and the apolipoprotein B/A1 ratio between patients with distinct ischemic stroke subtypes. No significant differences were found in serum levels of apolipoprotein B, A1 and the apolipoprotein B/A1 ratio between patients with moderate-severe intracranial atherosclerotic disease and moderate-severe extracranial atherosclerotic disease. DISCUSSION: Serum levels of apolipoprotein B and A1 did not differ between ischemic stroke subtypes. Additional studies are needed to validate our findings and to better understand the relationship between serum apolipoproteins and stroke.

In vitro neurosphere formation correlates with poor survival in glioma.

Sphere formation is an indicator of tumor aggressiveness independent of the tumor grade; however, its relation to progression-free survival (PFS) is less known. This study was designed to assess the neurosphere forming ability among low grade glioma (LGG) and high-grade glioma (HGG), its stem cell marker expression, and correlation to PFS. Tumor samples of 140 patients, including (LGG; n = 67) and (HGG; n = 73) were analyzed. We used sphere forming assay, immunofluorescence, and immunohistochemistry to characterize the tumors. Our study shows that, irrespective of the pathological sub type, both LGG and HGG formed neurospheres in vitro under conventional sphere forming conditions. However, the number of neurospheres formed from tumor tissues were significantly higher in HGG compared to LGG (P < 0.0001). Different grades of glioma were further characterized for the expression of stem cell marker proteins and lineage markers. When neurospheres were analyzed, CD133 positive cells were identified in addition to CD15 and nestin positive cells in both LGG and HGG. When these neurospheres were subjected to differentiation, cells positive for GFAP and ß-tubulin III were observed. Expression of stem cell markers and ß-tubulin III were prominent in HGG compared to LGG, whereas GFAP expression was higher in LGG than in HGG. Kaplan-Meier survival analysis demonstrated that neurosphere forming ability was significantly associated with shorter PFS (P < 0.05) in both LGG and HGG. Our results supports earlier studies that neurosphere formation may serve as a definitive indicator of stem cell population within the tumor and thus a better predictor of PFS than the tumor grades alone. © 2018 IUBMB Life, 71(1):244-253, 2019.

Apolipoprotein E Polymorphism and Oxidative Stress in Peripheral Blood-Derived Macrophage-Mediated Amyloid-Beta Phagocytosis in Alzheimer's Disease Patients.

Peripheral blood-derived macrophages isolated from Alzheimer's disease (AD) patients have earlier been reported to demonstrate ineffective phagocytosis of amyloid-beta compared to the age-matched control subjects. However, the mechanisms causing unsuccessful phagocytosis remain unclear. Oxidative stress and the presence of ApoEε4 allele has been reported to play a major role in the pathogenesis of AD, but the contribution of oxidative stress and ApoEε4 in macrophage dysfunction leading to ineffective Aß phagocytosis needs to be analyzed. Aß phagocytosis assay has been performed using FITC-labeled Aß and analyzed using flow cytometry and confocal imaging in patient samples and in THP-1 cells. Oxidative stress in patient-derived macrophages was analyzed by assessing the DNA damage using comet assay. ApoE polymorphism was analyzed using sequence-specific PCR and Hixson & Vernier Restriction isotyping protocol. In this study, we have analyzed the patterns of phagocytic inefficiency of macrophages in Indian population with a gradual decline in the phagocytic potential from mild cognitive impairment (MCI) to AD patients. Further, we have shown that the presence of ApoEε4 allele might also have a possible effect on the phagocytosis efficiency of the macrophages. Here, we demonstrate for the first time that oxidative stress could affect the amyloid-beta phagocytic potential of macrophages and hence by alleviating oxidative stress using curcumin, an anti-oxidant could enhance the amyloid-beta phagocytic efficacy of macrophages of patients with AD and MCI, although the responsiveness to curcumin might depends on the presence or absence of APOEε4 allele. Oxidative stress contributes significantly to decreased phagocytosis of Aß by macrophages. Moreover, the phagocytic inefficiency of macrophages was correlated to the presence of ApoEε4 allele. This study also found that the Aß-phagocytic potential of macrophage gets significantly enhanced in curcumin-treated patient-derived macrophages.

Interaction with the MAPT H1H1 Genotype Increases Dementia Risk in APOE ε4 Carriers in a Population of Southern India.

BACKGROUND: This study delineates the role of the interaction of apolipoprotein E (APOE) and MAPT alleles in contributing to disease risks of dementia in a southern Indian population. METHODS: A sample of 419 patients comprising Alzheimer's disease (AD; n = 156), mild cognitive impairment (MCI; n = 87), frontotemporal dementia (FTD; n = 127), vascular dementia (VD; n = 37), and dementia with Lewy bodies (DLB; n = 12) was analysed in comparison with a control group (n = 138). APOE genotyping and MAPT haplotyping were performed on all study subjects. RESULTS: Multivariate logistic regression analysis showed that variability on the APOE locus influenced the relative risk of dementia in the study population. The APOE ε4 allele increased the disease risk most significantly for AD (OR = 3.468, p < 0.0001) and MCI (OR = 2.901, p < 0.0001). The APOE ε2 allele remained protective for AD (OR = 0.205, p < 0.05). For FTD, VD, and DLB, the APOE ε4 allele was ineffectual in modulating disease risk. The MAPT H1 haplotype was not an overrepresented marker of neurodegenerative diseases. The H1H1 genotype had an additive effect in contributing to either disease risk in combination with the APOE ε4 allele or protection in combination with the APOE ε2 or ε3 allele. CONCLUSIONS: This study is a reappraisal of the strong association of APOE variability with AD in southern India when compared to other dementia groups, while the transcriptional differences between MAPT haplotypes have a limited role in Indian dementia patients.

Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice.

Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3ß phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3ß when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.

Are nitric oxide-mediated protein modifications of functional significance in diabetic heart? ye'S, -NO', wh'Y-NO't?

Protein modifications effected by nitric oxide (NO) primarily in conjunction with reactive oxygen species (ROS) include tyrosine nitration, cysteine S-nitrosylation, and glutathionylation. The physiological and pathological relevance of these three modifications is determined by the amino acids on which these modifications occur -cysteine and tyrosine, for instance, ranging from altering structural integrity/catalytic activity of proteins or by altering propensity towards protein degradation. Even though tyrosine nitration is a well-established nitroxidative stress marker, instilled as a footprint of oxygen- and nitrogen-derived oxidants, newer data suggest its wider role in embryonic heart development and substantiate the need to focus on elucidating the underlying mechanisms of reversibility and specificity of tyrosine nitration. S-nitrosylation is a covalent modification in specific cysteine residues of proteins and is suggested as one of the ways in which NO contributes to its ubiquitous signalling. Several sensitive and specific techniques including biotin switch assay and mass spectrometry based analysis make it possible to identify a large number of these modified proteins, and provide a great deal of potential S-nitrosylation sites. The number of studies that have documented nitrated proteins in diabetic heart is relatively much less compared to what has been published in the normal physiology and other cardiac pathologies. Nevertheless, elucidation of nitrated proteome of diabetic heart has revealed the presence of many mitochondrial and cytosolic proteins of functional importance. But, the existence of different models of diabetes and analyses at diverse stages of this disease have impeded scientists from gaining insights that would be essential to understand the cardiac complications during diabetes. This review summarizes NO mediated protein modifications documented in normal and abnormal heart physiology including diabetes.

'How can I halt thee?' The puzzles involved in autophagic inhibition.

The strategy for interpreting the role of autophagy on the basis of evidence obtained through autophagic inhibition sounds logical, but is beset with practical constraints. The knock down of autophagy-related (ATG) gene(s) or blockage of class III PI3-Kinase are the most common approaches for inhibiting autophagy. However, during stressful conditions, autophagy may operate in synchrony with other processes such as apoptosis; autophagy-related genes, unlike what their name implies, exert their regulation on apoptosis as well. Knocking down such genes not only blocks autophagy but also renders apoptosis defective, making the interpretation of autophagic roles unreliable. Similarly, class III PI3-Kinase aids in initiating autophagy but it is not a quintessential autophagic regulator. Class III PI3-Kinase also has a role in regulating almost all membrane transport in cells. Blocking it not only inhibits autophagy, but also hampers all the membrane trades, including endosomal transport. The pharmacological inhibitors used to block autophagy by blocking class III PI3-Kinase further compound these limitations with their off-target effects. Knowing the limitations involved in blocking a target or using an autophagy-blocking tool is a prerequisite for designing the experiments meant for analyzing autophagic functions. This review attempts to provide a detailed overview about the practical constraints involved in using autophagic inhibition as a strategy to understand autophagy.

Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells.

We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBL-induced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells.

Extracellular vesicles in glioblastoma: a challenge and an opportunity.

Glioblastoma is a highly heterogeneous tumor whose pathophysiological complexities dictate both the diagnosis of disease severity as well as response to therapy. Conventional diagnostic tools and standard treatment regimens have only managed to achieve limited success in the management of patients suspected of glioblastoma. Extracellular vesicles are an emerging liquid biopsy tool that has shown great promise in resolving the limitations presented by the heterogeneous nature of glioblastoma. Here we discuss the contrasting yet interdependent dual role of extracellular vesicles as communication agents that contribute to the progression of glioblastoma by creating a heterogeneous microenvironment and as a liquid biopsy tool providing an opportunity to accurately identify the disease severity and progression.

Identification of novel endogenous control miRNAs in heart failure for normalization of qPCR data.

MicroRNAs (miRNAs), a class of non-coding RNAs, are utilized as biomarkers for a wide range of disorders. Circulating miRNAs are proposed as potential markers in the clinical identification of heart failure (HF). However, identifying miRNA biomarkers in HF requires identification of robust endogenous control miRNAs for normalization in differential expression analysis. Hence, this study aimed to identify circulating miRNAs that can be utilized as endogenous controls in HF. We evaluated the expression of eight miRNAs, which were previously reported as endogenous controls in different pathological conditions. Total RNA, including miRNA, was extracted from the serum samples of 30 HF patients (15 HFrEF and 15 HFpEF) and their matched controls (n = 15). We used quantitative PCR to determine the miRNA expression. The stability of the selected endogenous miRNAs was assessed and compared using a standard set of criteria with the RefFinder software. Six of the eight miRNAs analyzed showed consistent expression among all sample groups. Stability analysis ranked hsa-let-7i-5p, hsa-miR-148b-3p, and hsa-miR-484 as the most stable miRNAs, indicating their potential as reliable endogenous controls.

Soluble ST2 Predicts Poor Functional Outcome in Acute Ischemic Stroke Patients.

INTRODUCTION: There are very limited data on the role of biomarkers correlating with the outcome in acute ischemic stroke (AIS). We evaluated the predictive values of the plasma concentrations of soluble serum stimulation-2 (sST2), matrix metalloproteinase-9 (MMP-9), and claudin-5 in AIS. METHODS: The biomarker levels in the plasma samples of consecutive AIS patients collected at baseline, 12 h, and 24 h from stroke onset were quantified using immunoassays. Stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS) and functional outcome at 90 days using the modified Rankin Scale (mRS), with scores above 3 defined as poor outcome. Receiver operating characteristic curve analysis and multiple logistic regression were performed for evaluating the discriminative power of each marker. RESULTS: We included 108 patients in the study (mean age 62.3 ± 11.7 years). Median NIHSS score was 12 (interquartile range 8-18). High baseline glucose levels, systolic blood pressure, baseline NIHSS, low Alberta Stroke Program Early CT Score, and hemorrhagic transformation were associated with poor outcomes. Elevated sST2 at 12 h (50.4 ± 51.0 ng/mL; p = 0.047) and 24 h (81.8 ± 101.3 ng/mL; p = 0.001) positively correlated with poor outcomes. MMP-9 (p = 0.086) and claudin-5 (p = 0.2) were not significantly associated with the outcome, although increased expressions of both markers were observed at 12 h. Multiple logistic regression showed that sST2 levels ≥71.8 ng/mL at 24 h, with a specificity of 96.9%, emerged as an independent predictor of poor functional outcome (OR: 6.44; 95% CI: 1.40-46.3; p = 0.029). CONCLUSION: Evaluation of sST2 may act as a reliable biomarker of functional outcome in AIS.

Nimbolide retards tumor cell migration, invasion, and angiogenesis by downregulating MMP-2/9 expression via inhibiting ERK1/2 and reducing DNA-binding activity of NF-κB in colon cancer cells.

Nimbolide, a plant-derived limonoid has been shown to exert its antiproliferative effects in various cell lines. We demonstrate that nimbolide effectively inhibited proliferation of WiDr colon cancer cells through inhibition of cyclin A leading to S phase arrest. It also caused activation of caspase-mediated apoptosis through the inhibition of ERK1/2 and activation of p38 and JNK1/2. Further nimbolide effectively retarded tumor cell migration and invasion through inhibition of metalloproteinase-2/9 (MMP-2/9) expression, both at the mRNA and protein level. It was also a strong inhibitor of VEGF expression, promoter activity, and in vitro angiogenesis. Finally, nimbolide suppressed the nuclear translocation of p65/p50 and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9 and VEGF gene expression.