Significant Differences In Protein Levels Research Articles

Knockdown of the ZcVgR Gene Alters the Expression of Genes Related to Reproduction and Lifespan in Zeugodacus cucurbitae (Coquillett) Under Extreme Heat Conditions.

Zeugodacus cucurbitae (Coquillett) is an important migratory vegetable pest. Previous research has demonstrated that short-term high temperatures induce differential expression of the vitellogenin receptor (ZcVgR) gene, reducing the number of eggs laid and the lifespan of female Z. cucurbitae. In this paper, we used Tandem Mass Tags (TMT) quantitative proteomics and Illumina high-throughput sequencing to determine the proteomic and transcriptomic information of female Z. cucurbitae after siRNA-mediated silencing of the target gene (ZcVgR) to gain a comprehensive understanding of the molecular mechanism of this gene in the regulation of reproduction and lifespan. The findings demonstrated that following the target gene's silencing, the ZcVgR gene's transcriptional expression was significantly downregulated, and there was no significant difference in protein level. The transcriptome and proteome had a low correlation; when the ZcVgR gene was silenced, vitellogenin-1 (ZcVg1), juvenile hormone epoxide hydrolase (JHEH), troponin C (TnC), heat shock protein 70 (HSP70), and other related genes were downregulated at the transcriptional level. By silencing the ZcVgR gene, transcriptionally level immune-related pathways were activated and energy metabolism-related pathways were inhibited; protein-level glycometabolism and phagosome pathways were activated, while phototransduction-fly and autophagy-animal pathways were inhibited. The findings of this study might offer a theoretical foundation for integrated management of Z. cucurbitae in the summertime.

Gene amplification of chromatin remodeling factor SMARCC2 and low protein expression of ACTL6A are unfavorable factors in ovarian high‑grade serous carcinoma.

Ovarian high-grade serous carcinoma (OHGSC) is the most common type of ovarian cancer worldwide. Genome sequencing has identified mutations in chromatin remodeling factors (CRFs) in gynecological cancer, such as clear cell carcinoma, endometrioid carcinoma and endometrial serous carcinoma. However, to the best of our knowledge, the association between CRFs and OHGSC remains unexplored. The present study aimed to investigate the clinicopathological and molecular characteristics of CRF dysfunction in OHGSC. CRF alterations were analyzed through numerous methods, including the analysis of public next-generation sequencing (NGS) data from 585 ovarian serous carcinoma cases from The Cancer Genome Atlas (TCGA), immunohistochemistry (IHC), and DNA copy number assays, which were performed on 203 surgically resected OHGSC samples. In the public NGS dataset, the most frequent genetic alteration was actin-like protein 6A (ACTL6A) amplification at 19.5%. Switch/sucrose non-fermentable related, matrix associated, actin dependent regulator of chromatin subfamily c member 2 (SMARCC2) amplification (3.1%) was associated with significantly decreased overall survival (OS). In addition, chromodomain-helicase-DNA-binding protein 4 (CHD4) amplification (5.7%) exhibited unfavorable outcome trends, although not statistically significant. IHC revealed the protein expression loss of ARID1A (2.5%), SMARCA2 (2.5%) and SMARCA4 (3.9%). The protein expression levels of ACTL6A, SMARCC2 and CHD4 were evaluated using H-score. Patients with low protein expression levels of ACTL6A showed a significantly decreased OS. Copy number gain or gene amplification was demonstrated in ACTL6A (66.2%) and SMARCC2 (33.5%), while shallow deletion or deep deletion was demonstrated in CHD4 (70.7%). However, there was no statistically significant difference in protein levels of these CRFs, between the different copy number alterations (CNAs). Overall, OHGSC exhibited CNAs and protein loss, indicating possible gene alterations in CRFs. Moreover, there was a significant association between the protein expression levels of ACTL6A and poor prognosis. Based on these findings, it is suggested that CRFs could serve as prognostic markers for OHGSC.

Dynamics of inflammation-associated plasma proteins following faecal microbiota transplantation in patients with psoriatic arthritis and healthy controls: exploratory findings from the FLORA trial

ObjectivesThe gut microbiota can mediate both pro and anti-inflammatory responses. In patients with psoriatic arthritis (PsA), we investigated the impact of faecal microbiota transplantation (FMT), relative to sham transplantation, on...

Examining sex differences in Alzheimer Disease neuropathology in Down Syndrome

AbstractBackgroundPeople with Down syndrome (DS) have a higher risk of developing Alzheimer disease (AD), due to an extra copy of the amyloid precursor protein (APP) gene on chromosome 21. Overexpression of APP is associated with increased production of amyloid beta (Aβ) in DS, and contributes to early onset of AD. Some studies suggest women with DS are at a higher risk for AD, while others find the opposite. Sex differences may be mediated by estrogen, which is critically involved in neuronal survival and modulation. We hypothesized that lower levels of ERα protein would correlate with increasing AD pathology in DS.MethodWe examined 35 brains (frontal and occipital cortices) of people with DS and DS with AD. Serial sections were immunostained for phosphorylated tau (pTau ‐AT8) and Aβ (6E10) along with ERα and quantified using QuPath. Spearman rank correlations using log transformed % load data were calculated, Kruskal‐Wallis was used to compare men and women.ResultNo significant differences in protein levels of ERα, Aβ, or tau were observed when comparing men and women with DS. However, we found higher levels of ERα protein were associated with higher levels of Aβ in the gray matter of the occipital cortex (R = 0.7078; p = 0.0005) in all cases. This positive correlation was driven primarily by women. Females with DS showed a significant positive association with Aβ and ERα (R = 0.93; p = 0.00012) whereas men showed a weaker trend (R = 0.62; p = 0.057). In contrast, no significant associations between ERα, Aβ or pTau were observed in the frontal cortex. PTau measures in the occipital cortex in women with DS were positively associated with ERα (R = 0.61; p = 0.055), whereas men with DS showed a slight negative, though not significant association (R = ‐0.29; p = 0.41). There was no such trend in the frontal cortex.ConclusionThese results suggest ERα, in contrast to our original hypothesis, may be increasing in response to AD pathology to preserve neuronal health in the occipital cortex. Lack of similar positive associations in the frontal cortex may be related to AD progression, with the occipital cortex being affected later in disease, giving us the opportunity to observe increased ERα.

Investigating the Role of Estrogen Receptor Alpha in Alzheimer’s Disease Pathogenesis in Down Syndrome

AbstractBackgroundPeople with Down syndrome (DS) have a higher risk of developing Alzheimer disease (AD), due to an extra copy of the amyloid precursor protein (APP) gene on chromosome 21. Overexpression of APP is associated with increased production of amyloid beta (Aβ) in DS, and contributes to early onset of AD. Some studies suggest women with DS are at a higher risk for AD, while others find the opposite. Sex differences may be mediated by estrogen, which is critically involved in neuronal survival and modulation. We hypothesized that lower levels of ERα protein would correlate with increasing AD pathology in DS.MethodWe examined 34 brains (frontal and occipital cortices) of people with DS and DS with AD. Serial sections were immunostained for phosphorylated tau (pTau ‐AT8) and Aβ (6E10) along with ERα and quantified using QuPath. Spearman rank correlations using log transformed % load data were calculated, Kruskal‐Wallis was used to compare men (n = 19) and women(n = 15).ResultNo significant differences in protein levels of ERα, Aβ, or tau were observed when comparing men and women with DS. However, we found higher levels of ERα protein were associated with higher levels of Aβ in the gray matter of the occipital cortex (r = 0.7078; p = 0.0005) in all cases. This positive correlation was driven primarily by women. Females with DS showed a significant positive association with Aβ and ERα (r = 0.93; p = 0.00012) whereas men showed a weaker trend (r = 0.62; p = 0.057). In contrast, no significant associations between ERα, Aβ or pTau were observed in the frontal cortex. PTau measures in the occipital cortex in women with DS were positively associated with ERα (r = 0.61; p = 0.055), whereas men with DS showed a slight negative, though not significant association (r = ‐0.29; p = 0.41). There was no such trend in the frontal cortex.ConclusionThese results suggest ERα, in contrast to our original hypothesis, may be increasing in response to AD pathology to preserve neuronal health in the occipital cortex. Lack of similar positive associations in the frontal cortex may be related to AD progression, with the occipital cortex being affected later in disease, giving us the opportunity to observe increased ERα.

Prognosis of spontaneous bacterial peritonitis in patients with hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fourth leading cause of death from cancer worldwide. Spontaneous bacterial peritonitis (SBP) is associated with poor prognosis. This study aimed to evaluate risk factors, differences in clinical characteristics and prognosis of SBP in patients with HCC in comparison with non-HCC patients. MethodsThis study was conducted on patients with cirrhosis who were admitted to hospital with SBP. The patients were divided into two groups: SBP group with HCC (n = 150) and SBP group without HCC (n = 250). ResultsMen and women accounted for 72% and 28% (n = 108 and 42, respectively) of the population in SBP group with HCC with mean age 55.8 ± 13.1 years. They accounted for 68.4% and 31.6% (n = 171 and 79, respectively) in the SBP group without HCC with mean age 56.8 ± 10.5 years. In-hospital mortality was 25.3% in the SBP group with HCC and 18.8 % in SBP group without HCC. Gastrointestinal bleeding was the most common cause of death in both groups. No significant difference was observed in patient outcomes between the two studied groups. The deceased patients had significantly higher levels of leukocytes and neutrophils in ascitic fluid as well as a higher frequency of positive culture results than in patients who survived (p < 0.001). However, there was no significant difference in protein level in ascitic fluid or causative organism between patients who survived and those who died (p = 0.63 and 0.19, respectively). ConclusionsPrognosis of SBP in patients with HCC seemed similar to that in patients without HCC.

Protein profiling in plasma for biomarkers of seizure

PurposeA biochemical way to measure seizures would greatly benefit epilepsy research and clinical follow-up. Short-term biomarkers like lactate exist, and interest in biomarkers representative of longer-term seizure burden is growing. In this exploratory study, we aimed to identify markers in blood plasma that differentiate persons with recent seizures from persons with epilepsy and long-standing seizure freedom. MethodsA proteomic analysis was performed on plasma samples of 120 persons with seizures using the Olink Neuro-exploratory panel. Participants were selected from a regional biobank study in Västra Götaland (Sweden) and categorized into two groups: recent seizure and seizure-free. The panel contained 92 proteins linked to neurological diseases and processes, and levels of these proteins were compared between the patient groups to identify potential markers of seizure activity. ResultsWe identified significant differences in protein levels between the recent seizure and seizure-free patient groups for Cadherin-15 [(CDH15; p = 0.008)], Latent transforming growth factor beta-binding protein 3 [(LTBP3; p = 0.002)], Phosphoethanolamine/phosphocholine phosphatase 1 [(PHOSPHO1; p = 0.011)], and Progestagen associated endometrial protein [(PAEP; p = 0.0005)]. ConclusionThe findings in this study present CDH15, LTBP3, PHOSPHO1 and PAEP as candidate markers of seizure activity. Further confirmatory studies are needed.

Potential Role of Seven Proteomics Tissue Biomarkers for Diagnosis and Prognosis of Prostate Cancer in Urine.

As the currently available tests for the clinical management of prostate cancer (PCa) are still far from providing precise diagnosis and risk stratification, the identification of new molecular marker(s) remains a pertinent clinical need. Candidate PCa biomarkers from the published proteomic comparative studies of prostate tissue (2002-2020) were collected and systematically evaluated. AZGP1, MDH2, FABP5, ENO1, GSTP1, GSTM2, and EZR were chosen for further evaluation in the urine of 85 PCa patients and controls using ELISA. Statistically significant differences in protein levels between PCa and BPH showed FABP5 (p = 0.019) and ENO1 (p = 0.015). A biomarker panel based on the combination of FABP5, ENO1, and PSA provided the highest accuracy (AUC = 0.795) for PCa detection. The combination of FABP5, EZR, AZGP1, and MDH2 showed AUC = 0.889 in PCa prognosis, with 85.29% of the samples correctly classified into low and high Gleason score (GS) groups. The addition of PSA to the panel slightly increased the AUC to 0.914. AZGP1, FABP5, and EZR showed significant correlation with GS, stage, and percentage of positive biopsy cores. Although validation using larger patient cohorts will be necessary to establish the credibility of the proposed biomarker panels in a clinical context, this study opens a way for the further testing of more high-quality proteomics biomarkers, which could ultimately add value to the clinical management of PCa.

P–178 Mural granulosa cells of the human follicles indirectly show death molecular signals not depending on different ovarian stimulation protocols

Abstract Study question Could the expression of the anti-apoptotic molecules AKT, p-Akt and ERK1/2 in Mural Granulosa Cells (MGC) be considered as marker of oocyte quality? Summary answer MGCs activate cell death pathways in analyzed follicles and it is not influenced by different stimulation protocols and it is not correlated to oocyte competence. What is known already It has been previously demonstrated that apoptosis rate of mural granulosa or cumulus cells (CC) were correlated with follicular oocyte number, age, embryo numbers in IVF/ICSI and also clinical pregnancy. Moreover, our previous data demonstrated that in selected patients, who received recombinant LH associated with recombinant FSH (rFSH), the DNA fragmentation in cumulus cells was significantly lower and pregnancy rate was higher, compared to patients treated with rFSH alone. However, to date little is known about the differences between MGC and CC regarding death/survival pathways and whether the two cell types respond in the same way. Study design, size, duration Molecular study on MGCs to investigate the role of the surviving/apoptotic molecules AKT, p-Akt and ERK1/2 and their relationship with the administration of exogenous r-LH combined with r-FSH administration in ovarian stimulation comparing with r-FSH alone. We analyzed also the oocyte competence, for each follicle, according to the embryo development during in vitro culture and the pregnancy outcome. We included fifty-three normo-responder women undergoing ICSI in two years. Participants/materials, setting, methods Patients were divided into two groups: 1) 34 women were stimulated with r-FSH and used as control group, 2) 19 women were stimulated with r-FSH combined with r-LH. Mural granulosa cells isolated singularly from 255 MII oocytes of the 53 patients were used for the study. The study was conducted in public university laboratory. MGCs obtained from each single follicle were suspended in medium, without serum. For immunoreaction anti-AKT, p-AKT, ERK1/2 antibodies were used. Main results and the role of chance Out of 255 MII oocytes collected, 197 were fertilized and the derived embryos had the following evolution: 117 transferred, 57 vitrified and 23 arrested during in vitro culture. 58 oocytes were not analyzed because of failed fertilization or because of their immature condition (GV or MI). In the MGCs isolated from the follicle of each oocyte generating an embryo, the expression AKT, pAKT and ERK1/2 was analyzed and associated with embryo quality and pregnancy outcomes. Immunoblot analysis on granulose cells showed no statistically significant differences in protein level in MGCs isolated from oocytes that have generated transferred embryos (blastocyst at day 5 or 6) comparing with embryos who arrested during in vitro culture. No differences were found also in MGCs collected from the follicles derived from r-FSH ovarian stimulation compared to r-FSH+r-LH. Moreover, no difference was highlighted even between protein level and pregnancy outcomes. The results seem to demonstrate that the MGCs primarily have an endocrine function and support the growth of the follicle, and finally follow a specific death pathway. This condition is not influenced by different ovarian stimulation protocol, in contrast with CC, and is not correlated to oocyte competence, embryo quality and clinical outcomes. Limitations, reasons for caution Only a limited number of patients have been observed. Wider implications of the findings: Our current and past results suggest that the evaluation of cumulus cells and mural granulosa cells in the same follicle show different expression of molecules involved in the apoptotic pathway and therefore they cannot be used, as molecular markers, in the same way, to assess the competence of oocytes. Trial registration number Not applicable

Reduction of Autophagosome Overload Attenuates Neuronal Cell Death After Traumatic Brain Injury

Previous studies have shown that alterations in autophagy-related proteins exist extensively after traumatic brain injury (TBI). However, whether autophagy is enhanced or suppressed by TBI remains controversial. In our study, a controlled cortical impact was used to establish a model of moderate TBI in rats. We found that a significant increase in protein levels of LC3-II and SQSTM1 in the injured cortex group. However, there were no significant differences in protein levels of VPS34, Beclin-1, and phosphor-ULK1, which are the promoters of autophagy. Lysosome dysfunction after TBI might lead to autophagosome accumulation. In addition, the highly specific autophagy inhibitor SAR405 administration reduced TBI-induced apoptosis-related protein cleaved caspase-3 and cleaved caspase-9 levels in the ipsilateral cortex, as well as brain edema and neurological defects accessed by mNSS. Furthermore, chloroquine treatment reversed the beneficial effects of SAR405 by increasing the accumulation of autophagosomes. Finally, our data showed that autophagy inhibition by VPS34 gene knockout method attenuated cell death after TBI. Our findings indicate that impaired autophagosome degradation is involved in the pathological reaction after TBI, and the inhibition of autophagy contributes to attenuate neuronal cell death and functional defects.

Matrix Metalloproteinase-2 Isoforms Differ within the Aortic Wall of Ascending Aortic Aneurysms Associated with Bicuspid Aortic Valve.

The pathogenesis of ascending thoracic aortic aneurysm (aTAA) is thought to differ between patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV), and one of the causes is different hemodynamics. Influenced by hemodynamics, the tissue levels of proteins associated with aTAA might differ between aTAAs with BAV and TAV and between different localities within the aortic wall. We therefore analyzed aTAA tissue levels of MMP-2 (matrix metalloproteinase-2) isoforms (Pro-MMP-2, active MMP-2, and total MMP-2) and tissue levels of MMP-14, TIMP-2 (tissue inhibitor of metalloproteinase-2), MMP-9, and TIMP-1 in 19 patients with BAV and 23 patients with TAV via gelatin zymography and enzyme-linked immunosorbent assay (ELISA), respectively. TAV and BAV groups' protein levels did not differ significantly. Whereas the TAV group exhibited no significant differences in protein levels between the aneurysm's anterior and posterior parts, the BAV group revealed significantly higher levels of Pro-MMP-2, total MMP-2, and TIMP-2 in the aneurysm's posterior parts (mean Pro-MMP-2 200.52 arbitrary units (AU) versus 161.12 AU, p=0.007; mean total MMP-2 235.22 AU versus 193.68 AU, p=0.002; mean TIMP-2 26.90 ng/ml versus 25.36 ng/ml, p=0.009), whereas the other proteins did not differ significantly within the aortic wall. Thus, MMPs are distributed more heterogeneously within the aortic wall of aTAAs associated with BAV than in those associated with TAV, which is a new aspect for understanding the underlying pathogenesis. This heterogeneous protein level distribution might be attributable to differences in the underlying pathogenesis, especially hemodynamics. This result is important for further studies as it will be essential to specify the location of samples to ensure data comparability regarding the main goals of understanding the pathogenesis of aTAA, optimizing treatments, and establishing a screening method for its potentially deadly complications.

Identification and validation of differential plasma proteins levels in epithelial ovarian cancer

Diagnosis of Ovarian cancer (OC) has been a challenge, the purpose, therefore is to identify plasma proteins differentially expressed in epithelial ovarian cancer patients. Human plasma samples from patients with OC (n = 138), benign tumors (n = 20) and controls (n = 238) were used. Tandem Mass Tag (TMT) based quantitative analysis by high resolution mass spectrometry, was followed by validation using Quantibody array and ELISA techniques. 507 plasma proteins showed differential protein levels in OC plasma samples. 21 proteins were validated using Quantibody array. Further, nine proteins (CA125, CFD, CST3, ICAM1, IGFBP2, IGFBP3, SPP1, TSP1 and VEGFA) which showed significant differences in protein levels in Quantibody array analysis were validated using ELISA. In ELISA, the levels of CA125, IGFBP2, ICAM1 and SPP1 were significantly increased and levels of Adipsin and TSP1 were decreased in tumors compared to controls and benign group. Epithelial ovarian cancer diagnosis model combining five markers (CA125, IGFBP2, SPP1, TSP1 and ADI) showed 90.24% sensitivity and 94.87% specificity. In conclusion a panel of 5 plasma proteins has been found to be useful in distinguishing plasma samples from epithelial ovarian cancers from patients with benign tumors and healthy normal subjects. This has the potential as a diagnostic assay for epithelial ovarian cancer. SignificanceThe significance of this case-control study is based on the large and well defined ovarian cancer patient population (epithelial ovarian cancers including serous and mucinous subtypes), age matched controls and benign ovarian tumors. This study incorporates a discovery phase involving quantitative proteomic analysis of immune-depleted plasma followed by two levels of validation studies involving a selected list of proteins using antibody arrays and ELISA. The validations were performed on an independent set of samples comprising of epithelial ovarian cancer subtypes, controls and benign tumors. The multiple marker combination comprising of Adipsin, CA125, IGFBP2, SPP1 and TSP1 identified in the study by ELISA could enable rapid translation to a larger screening study.

Altered levels of CSF proteins in patients with FTD, presymptomatic mutation carriers and non-carriers

BackgroundThe clinical presentations of frontotemporal dementia (FTD) are diverse and overlap with other neurological disorders. There are, as of today, no biomarkers in clinical practice for diagnosing the disorders. Here, we aimed to find protein markers in cerebrospinal fluid (CSF) from patients with FTD, presymptomatic mutation carriers and non-carriers.MethodsAntibody suspension bead arrays were used to analyse 328 proteins in CSF from patients with behavioural variant FTD (bvFTD, n = 16) and progressive primary aphasia (PPA, n = 13), as well as presymptomatic mutation carriers (PMC, n = 16) and non-carriers (NC, n = 8). A total of 492 antibodies were used to measure protein levels by direct labelling of the CSF samples. The findings were further examined in an independent cohort including 13 FTD patients, 79 patients with Alzheimer’s disease and 18 healthy controls.ResultsWe found significantly altered protein levels in CSF from FTD patients compared to unaffected individuals (PMC and NC) for 26 proteins. The analysis show patterns of separation between unaffected individuals and FTD patients, especially for those with a clinical diagnosis of bvFTD. The most statistically significant differences in protein levels were found for VGF, TN-R, NPTXR, TMEM132D, PDYN and NF-M. Patients with FTD were found to have higher levels of TN-R and NF-M, and lower levels of VGF, NPTXR, TMEM132D and PDYN, compared to unaffected individuals. The main findings were reproduced in the independent cohort.ConclusionIn this pilot study, we show a separation of FTD patients from unaffected individuals based on protein levels in CSF. Further investigation is required to explore the CSF profiles in larger cohorts, but the results presented here has the potential to enable future clinical utilization of these potential biomarkers within FTD.

Serotonin transporter protein in autopsied brain of chronic users of cocaine.

The long-held speculation that the brain serotonin system mediates some behavioral effects of the psychostimulant cocaine is supported in part by the high affinity of cocaine for the serotonin transporter (SERT) and by reports that the serotonin transporter (SERT), estimated by SERT binding, is increased in brain of human chronic cocaine users. Excessive SERT activity and consequent synaptic serotonin deficiency might cause a behavioral (e.g., mood) abnormality in chronic users of the drug. Previous studies focused on changes in SERT binding, which might not necessarily reflect changes in SERT protein. Therefore, we compared levels of SERT protein, using a quantitative Western blot procedure, in autopsied brain (striatum, cerebral cortices) of chronic human cocaine users (n = 9), who all tested positive for the drug/metabolite in brain, to those in control subjects (n = 15) and, as a separate drug of abuse group, in chronic heroin users (n = 11). We found no significant difference in protein levels of SERT or the serotonin synthesizing enzyme tryptophan hydroxylase-2 among the control and drug abuse groups. In the cocaine users, no significant correlations were observed between SERT and brain levels of cocaine plus metabolites, or with levels of serotonin or its metabolite 5-hydroxyindoleacetic acid. Our postmortem data suggest that a robust increase in striatal/cerebral cortical SERT protein is not a common characteristic of chronic, human cocaine users.

Targeted multiplex proteomics for molecular prescreening and biomarker discovery in metastatic colorectal cancer

Protein biomarkers are widely used in cancer diagnosis, prognosis, and prediction of treatment response. Here we introduce the use of targeted multiplex proteomics (TMP) as a tool to simultaneously measure a panel of 54 proteins involved in oncogenic, tumour suppression, drug metabolism and resistance, in patients with metastatic colorectal cancer (mCRC). TMP provided valuable diagnostic information by unmasking an occult neuroendocrine differentiation and identifying a misclassified case based on abnormal proteins phenotype. No significant differences in protein levels between unpaired primary and metastatic samples were observed. Four proteins were found differentially expressed in KRAS-mutant as compared to wild-type tumours (overexpressed in mutant: KRAS, EGFR; overexpressed in wild-type: TOPO1, TOP2A). Survival analyses revealed the association between mesothelin expression and poor overall survival, whereas lack of PTEN protein expression associated with lower progression-free survival with anti-EGFR-based therapy in the first-line setting for patients with RAS wild-type tumour. Finally, outlier analysis identified putative targetable proteins in 65% of patients lacking a targetable genomic alteration. Our data show that TMP constitutes a promising, novel molecular prescreening tool in mCRC to identify protein expression alterations that may impact on patient outcomes and more precisely guide patient eligibility to clinical trials with novel targeted experimental therapies.

IDENTIFICATION AND BIOLOGICAL CHARACTERIZATION OF EXTRACELLULAR VESICLE PROTEINS FOR PROPAGATION AND SPREAD OF TAU

Accumulation of hyper-phosphorylated tau aggregates are pathological hallmark of Alzheimer's disease (AD). Extracellular vesicles (EVs) derived from AD derived microglia contribute to tau propagation as previously shown in a mouse model of human disease. The data sets, derived from the entorhinal cortex to the dentate gyrus, support a role for EVs in tau-associated pathobiology. A quantitative proteomics profile was made from EVs isolated from cortical grey matter of AD (n=20) and control (CTRL) (n=18) brain tissues. Here, we have identified differentially expressed proteins between these brain tissue cohorts using predictive machine learning. Expressed EV proteins were validated by ELISA and by biological function then linked to AD progression. Differentially expressed EV proteins, recovered from AD and CTRL brain samples, were observed by quantitative proteomics employing machine learning. The candidate proteins were selected then validated from an independent cohort by a commercially available ELISA. We evaluated the effect of the identified proteins on microglial EV uptake. AD brain-derived EVs were labeled with PKH26 and preincubated with recombinant target proteins then tested with murine microglia for uptake measurements. Annexin V (ANXA5), neurosecretory protein VGF, and neuronal membrane glycoprotein M6-a (GPM6A) were differentially expressed in AD EVs. Each of the proteins were confirmed with 88% accuracy by machine learning analyses. There was significant difference in protein levels of ANXA5 (p = 4.72E-06, fold change = 2.10), VGF (p = 7.49E-07, fold change = 0.23), GPM6A (p = 5.00E-04, fold change = 1.56) between AD and CTRL brain-derived EVs. Increased protein level of ANXA5 in AD compared CTRL EVs (p = 4.80E-03) was confirmed by ELISA. Moreover, the ANXA5 and VGF recombinant proteins inhibited EV microglial uptake. ANXA5 and VGF distinguished AD from CTRL EVs. Preincubation of AD brain-derived EVs with ANXA5 or VGF inhibited EV microglial uptake. These data suggest an ANXA5 and VGF-dependent spread of pathogenic tau protein in the brain, and these molecules can be a potential therapeutic target for AD.

Effects of chlorogenic acid, epicatechin gallate, and quercetin on mucin expression and secretion in the Caco-2/HT29-MTX cell model.

Mucins are a family of large glycoproteins that represent the major structural components of the mucus and are encoded by 20 different mucin genes. Mucin expression can be modulated by different stimuli. In this study, we analyzed four mucins (MUC2, MUC3, MUC13, and MUC17) in coculture of Caco‐2/HT29‐MTX cells to demonstrate the variation in gene expression in the presence of antioxidant compounds like chlorogenic acid, epicatechin gallate, and quercetin (apple, tea, and coffee polyphenols, respectively). coculture of Caco‐2/HT29‐MTX cells was treated with polyphenols, and the expression of four mucins was determined by reverse‐transcriptase PCR. In addition, the secretion levels of MUC2 were established by enzyme‐linked immunoassay (ELISA) analysis. The results showed that each polyphenol compound induces different expression patterns of the mucin genes. Statistically significant up‐regulation of MUC17 was observed following incubation with epicatechin gallate and quercetin. ELISA results did not prove any significant differences in protein levels of MUC2 after treatment by the polyphenol compounds. The polyphenols considered in this study may influence mucin secretion and act on diverse salivary substrates to change the barrier properties of mucins for mucus secretion in different ways.

Altered Purinergic Receptor Sensitivity in Type 2 Diabetes-Associated Endothelial Dysfunction and Up₄A-Mediated Vascular Contraction.

Purinergic signaling may be altered in diabetes accounting for endothelial dysfunction. Uridine adenosine tetraphosphate (Up4A), a novel dinucleotide substance, regulates vascular function via both purinergic P1 and P2 receptors (PR). Up4A enhances vascular contraction in isolated arteries of diabetic rats likely through P2R. However, the precise involvement of PRs in endothelial dysfunction and the vasoconstrictor response to Up4A in diabetes has not been fully elucidated. We tested whether inhibition of PRs improved endothelial function and attenuated Up4A-mediated vascular contraction using both aortas and mesenteric arteries of type 2 diabetic (T2D) Goto Kakizaki (GK) rats vs. control Wistar (WT) rats. Endothelium-dependent (EDR) but not endothelium-independent relaxation was significantly impaired in both aortas and mesenteric arteries from GK vs. WT rats. Non-selective inhibition of P1R or P2R significantly improved EDR in aortas but not mesenteric arteries from GK rats. Inhibition of A1R, P2X7R, or P2Y6R significantly improved EDR in aortas. Vasoconstrictor response to Up4A was enhanced in aortas but not mesenteric arteries of GK vs. WT rats via involvement of A1R and P2X7R but not P2Y6R. Depletion of major endothelial component nitric oxide enhanced Up4A-induced aortic contraction to a similar extent between WT and GK rats. No significant differences in protein levels of A1R, P2X7R, and P2Y6R in aortas from GK and WT rats were observed. These data suggest that altered PR sensitivity accounts for endothelial dysfunction in aortas in diabetes. Modulating PRs may represent a potential therapy for improving endothelial function.

Ketogenic diet increases mitochondria volume in the liver and skeletal muscle without altering oxidative stress markers in rats

Ketogenic diets (KD) consist of high fat, moderate protein and low carbohydrates. Studies have suggested that KD may influence oxidative stress by affecting mitochondrial quantity and/or quality, and perhaps lengthen lifespan. Therefore, we determined the effects of KD on multi-organ mitochondria volume and oxidative stress markers in rats. Ten month-old male Fisher 344 rats (n = 8 per group) were provided with one of two isocaloric diets: standard chow (SC) or KD. Rats were euthanized if: a) vitality scores exceeded a score of 16, b) rapid weight loss, or c) veterinarian deemed euthanasia necessary. The median lifespan of rats was higher in KD (762 days) compared to SC (624 days). Citrate synthase activity (i.e. estimate of mitochondria volume) was higher in the liver (p = 0.034) and gastrocnemius (p = 0.041) of KD compared to SC. Liver superoxide dismutase 1 and catalase antioxidant protein levels were higher in KD, albeit not significant (p = 0.094 and p = 0.062, respectively). No significant differences in protein levels of other antioxidants or markers of lipid and protein oxidative damage were observed in either the gastrocnemius, liver, or brain. In summary, KD increased mitochondria volume in liver and gastrocnemius and median lifespan in rats. Additionally, our data show that the increase in mitochondrial volume occurred without changes in oxidative damage or antioxidant protein levels in the gastrocnemius, liver, or brain.

Correlation of 18F-FDG uptake and thyroid cancer stem cells.

2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography (18F-FDG PET) has the potential to detect various types of cancers, including thyroid cancer (TC), at a potentially curable stage. Increased uptake of 18F-FDG was observed in anaplastic and poorly differentiated thyroid cancer cells, and PET-positive tumors are more likely to be resistant to 131I treatment. As cancer stem cells (CSCs) possess a dedifferentiated phenotype and are resistant to many anticancer therapies, we hypothesized that the expression of CSC-related markers is correlated with the ability of tumor cells in TC to uptake FDG. The present study cohort included 12 patients with TC, who underwent 18F-FDG PET/CT imaging before surgery. Quantitative polymerase chain reaction (QPCR) and immunohistochemical (IHC) staining were performed to analyze the expression patterns of gene markers related to embryonic stem (ES) cells and CSCs in TC. The mRNA expression levels of CSC- (CD133 and CD44) and ES-related genes (Oct4 and Nanog) were higher in TC tissue than in normal thyroid tissue, whereas the mRNA expression levels of thyroid-specific genes (Tg, TSHR, and TTF1) were higher in normal thyroid tissue than in TC tissue. There was a positive and statistically significant correlation between FDG uptake (SUV<inf>max</inf>) of tumor and relative mRNA levels of CD133, CD44, Oct4, and Nanog. The IHC results demonstrated that CD133 and Nanog were expressed in TC tissue but not in normal thyroid tissue, however, CD44 expression was observed in both TC and normal thyroid tissue. Comparisons of the clinicopathological parameters between TC tissues with low and high SUV<inf>max</inf> demonstrated significant differences in protein level of CD133 but not in that of Nanog. The pre-therapeutic tumor SUV<inf>max</inf> obtained from 18F-FDG PET/CT may be a potential predictor for evaluating the proportion of CSC population in individual patients with TC.