Whole Blood Samples Of Patients Research Articles

Circulating neutrophil extracellular trap-forming neutrophils in rheumatoid arthritis exacerbation are majority dual endothelin-1/signal peptide receptor+ subtype.

Neutrophil extracellular traps (NETs) are associated with rheumatoid arthritis pathogenesis and severity. Since homeostatic NET-forming neutrophils [NET+Ns] have beneficial roles in defense against pathogens, their distinction from pro-injury [NET+N] subtypes is important, especially if they are to be therapeutically targeted. Having identified circulating, pro-injury DEspR+CD11b+[NET+Ns] in patients with neutrophilic secondary tissue injury, we determined whether DEspR+[NET+Ns] are present in rheumatoid arthritis (RA) flares. Whole blood samples of patients with RA flares on maintenance therapy (n = 6) were analyzed by flow cytometry (FCM) and immunofluorescence cytology followed by semi-automated quantitative confocal microscopy (qIFC). We assessed clinical parameters, levels of neutrophils and [NET+Ns], and plasma S100A8/A9. qIFC detected circulating DEspR+CD11b+neutrophils and [NET+Ns] in RA-flare patients but not healthy controls. DEspR+[NET+Ns] were positive for citrullinated histone H3 (citH3+), extruded DNA, decondensed but recognizable polymorphic nuclei, and [NET+N] doublet interactions in mostly non-ruptured NET-forming neutrophils. Circulating DNA+/DEspR+/CD11b+/citH3+microvesicles (netMVs) were observed. FCM detected increased %DEspR+CD11b+neutrophils and DEspR+ cell-cell doublets whose levels trended with DAS28 scores, as did plasma S100A8/A9 levels. This study identifies circulating DEspR+/CD11b+neutrophils and [NET+Ns] in RA-flare patients on maintenance therapy. Detection of circulating DEspR+citH3+[NET+Ns] and netMVs indicate a systemic neutrophilic source of citH3-antigen concordant with multi-joint RA pathogenesis. Increased S100A8/A9 alarmin levels are associated with cell injury and released upon NET-formation. As a ligand for TLR4, S100A8/A9 forms a positive feedback loop for TLR4-induced DEspR+neutrophils. These data identify DEspR+neutrophils and [NET+Ns] in RA pathogenesis as a potential biomarker and/or therapeutic target.

Identification of Autoantigen Markers for SARS CoV-2 Infection with Machine Learning-based Feature Selection: An Insight into COVID Symptoms

Background and Introduction:: Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection has been shown to trigger autoimmunity, and the phenomenon leads to several chronic human diseases such as Type-1 diabetes, Crohn’s disease, vasculitis, Guillian-Barrė syndrome, etc. The mechanism underlying SARS CoV-2-induced autoimmune response is unknown and is an active area of interest for the researchers. Objective:: The primary objective of this study is to identify the autoantigen markers for the classification of SARS CoV-2 (COVID-19 positive and negative samples) that trigger an immune response leading to autoimmunity using a machine learning approach that provides information to obtain a more accurate diagnosis for COVID-induced diseases. Materials and Methods:: Our study reports the transcriptomic profile of the COVID patient's whole blood samples collected from 0 to 35th day of acute infection as described in the GSE215865 dataset. The binary classification algorithm from the sci-kit learn python library, namely logistic regression and random forest with 10-fold cross-validation, was applied to the processed data, followed by a selection of the 20 best gene features with recursive feature elimination from a set of 10,719 gene features to obtain the classification accuracy of 87%. Results:: The fidgetin, microtubule severing factor (FIGN), SH3 and cysteine-rich domain (STAC), Cadherin-6 (CDH6), docking protein 6 (DOK6), nuclear RNA export factor 3 (NXF3) and maternally expressed 3 (MEG3) are the autoantigens markers identified for classification of COVID-positive and negative samples. Conclusion:: The identified autoantigen markers from transcriptomic datasets using machine learning techniques provide a deeper understanding of COVID-induced diseases and may play an important role as potential diagnostic and drug targets for COVID-19.

Impact of BK polyomavirus viremia on the outcomes of allogeneic hematopoietic stem cell transplantation.

Although it is known that BK polyomavirus (BKPyV) causes hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT), the clinical significance of BKPyV viremia has not been fully evaluated. We retrospectively analyzed the results of quantitative polymerase chain reaction (PCR) evaluations for detecting BKPyV in the whole blood samples of patients undergoing allogeneic HSCT during the period from January 2010 to June 2020 at a single institute, Tokyo Medical and Dental University. BKPyV was detected in the blood of 28 of the 107 evaluated patients, and the cumulative incidence of was 27.9% (95%CI: 20.2-37.9%). HC due to BKPyV developed in four of the 28 patients with BKPyV viremia (14.3%) and in two of the 79 patients without it (2.5%; P < 0.05). BKPyV viremia itself did not affect the patients' post-transplant estimated glomerular filtration rate (eGFR), but BKPyV viremia with a high viral load was significantly associated with decreased eGFR values (P < 0.05). BKPyV viremia was also associated with significantly lower progression-free survival at 3 years (35.1% [95%CI: 17.8-53.1%] vs. 60.4% [95%CI: 48.4-70.5], P < 0.05). Our findings demonstrated that BKPyV viremia was associated with onset of HC, an early decline of renal function, and poorer survival after allogeneic HSCT. Further studies are needed to test these results and elucidate the mechanisms of renal dysfunction associated with BKPyV viremia.

Machine Learning-assisted immunophenotyping of peripheral blood identifies innate immune cells as best predictor of response to induction chemo-immunotherapy in head and neck squamous cell carcinoma – knowledge obtained from the CheckRad-CD8 trial

PurposeIndividual prediction of treatment response is crucial for personalized treatment in multimodal approaches against head-and-neck squamous cell carcinoma (HNSCC). So far, no reliable predictive parameters for treatment schemes containing immunotherapy have been identified. This study aims to predict treatment response to induction chemo-immunotherapy based on the peripheral blood immune status in patients with locally advanced HNSCC. MethodsThe peripheral blood immune phenotype was assessed in whole blood samples in patients treated in the phase II CheckRad-CD8 trial as part of the pre-planned translational research program. Blood samples were analyzed by multicolor flow cytometry before (T1) and after (T2) induction chemo-immunotherapy with cisplatin/docetaxel/durvalumab/tremelimumab. Machine Learning techniques were used to predict pathological complete response (pCR) after induction therapy. ResultsThe tested classifier methods (LDA, SVM, LR, RF, DT, and XGBoost) allowed a distinct prediction of pCR. Highest accuracy was achieved with a low number of features represented as principal components. Immune parameters obtained from the absolute difference (lT2-T1l) allowed the best prediction of pCR. In general, less than 30 parameters and at most 10 principal components were needed for highly accurate predictions. Across several datasets, cells of the innate immune system such as polymorphonuclear cells, monocytes, and plasmacytoid dendritic cells are most prominent. ConclusionsOur analyses imply that alterations of the innate immune cell distribution in the peripheral blood following induction chemo-immuno-therapy is highly predictive for pCR in HNSCC.

The composition of the blood microbiota and its relationship to osteoporosis-related clinical parameters

Osteoporosis, a systemic bone disease, is characterized by decreased bone mass, deterioration of skeletal structure, and increased bone susceptibility. Age, environment, hormone levels, nutrition, and immunity are all factors that influence bone mass. Currently, intestinal flora has been recently recognized as a key regulator of bone mass. The blood's microbiome role in bone health and in the pathogenesis of osteoporosis remains unknown. In this study, the abundance of various blood's microbial taxa in osteoporosis patients were analyzed. We investigated the associations between prominent bacterial taxa and other clinical indicators (i.e. biochemical, blood cell counts and CT scan). DNA was extracted from the whole blood samples of patients with degenerative bone diseases with or without osteoporosis (i.e. n = 8; ST and n = 12, T group) and healthy controls (n = 4, N group). The 16S rRNA gene sequencing technique was utilized to characterize the blood microbiome taxaThe Shannon–Winner and dilution curves revealed that all the characterized species in the sample and the sequencing data were reliable. The number of bacterial taxa in blood and annotated operational taxonomic units were positively correlated with neutrophils. This support that bacteria exist within or adhere to the neutrophil's membrane. The abundance of Yersinia ruckeri, Rhodanobacter_uncultured bacterium, Enterobacter spp., and Raoultella spp increased in the ST group as compared with the N group. Hence, indicate their potential role in the onset and progression of osteoporosis. These findings provide new insights into the association between blood microbiota and bone health. This study could open a new horizon in exploring the clinical application of blood microbiome to improve bone health.

Rs3761548 (C/A) and rs5902434 (del/ATT) polymorphisms of Foxp3 gene in Iranian patients with migraine

BackgroundMigraine is a neurovascular disorder; several studies have demonstrated the immune system plays a key role in migraine pathogenesis. The aim of this study was to investigate the association between FOXP3 gene polymorphism and susceptibility to migraine.MethodsIn a case–control study, 55 whole blood samples of patients with migraine and 80 healthy samples were collected. After DNA extraction, genotyping of the rs5902434 (del/ATT) and rs3761548 (C/A) FOXP3 was performed using sequence-specific primers method (PCR-SSP).ResultsThe results of this study showed that there were statistically significant differences between patient and control group in genotype frequencies of rs3761548. In addition, the frequency of heterozygous genotype AC at rs3761548 in patients was found to be significantly higher than controls. We also found no significant differences between cases and controls were found in the allelic and genotype distribution of the rs5902434 (del/ATT) polymorphism. None of the rs5902434 (del/ATT) genotypes showed any significant association with the migraine.ConclusionsAccording to finding of our study, polymorphism rs3761548 in FOXP3 gene were associated with susceptibility to migraine. Further studies with larger sample sizes and different populations in other parts of the world are needed to investigate relationship between this polymorphism on migraine susceptibility.

Filter-assisted smartphone colorimetry / ICP-MS dual-mode biosensor of butyrylcholinesterase in clinical samples

Clinically, butyrylcholinesterase (BChE) is an indispensable biochemical marker for evaluating liver function and organophosphorus poisoning. In this work, we proposed a novel smartphone readable colorimetry and inductively coupled plasma mass spectrometer (ICP-MS) dual-mode analytical strategy for BChE detection in clinical patients based on the phenomenon that the filter membrane can selectively separate MnO2 nanosheets (MnO2 NSs) and Mn2+. In the presence of BChE and acetylthiocholine (ATCh), the enzymatic hydrolysate (thiocholine) can efficiently trigger the decomposition of MnO2 NSs to Mn2+. After selective separation of MnO2 NSs and Mn2+ via the filter, colorimetric detection was achieved with a smartphone by identifying the RGB value of the MnO2 NSs coated membrane, while the Mn2+ in the filtrate was detected by ICP-MS. Under optimal conditions, the limits of detection (LODs) were as low as 1.3 U/L and 0.001 U/L for the smartphone-based RGB analysis and ICP-MS assay, respectively. This method was applied to quantify BChE in 40 clinical whole blood samples of patients with clinical liver disease. The levels of BChE detected in clinical samples by this method were consistent with clinical Ellman’s colorimetric findings. Therefore, this method can provide more options for clinical cholinesterase detection and is expected to be developed into a kit.

Long non-coding RNA LINC01793 as a potential diagnostic biomarker of hepatitis B virus-related hepatocellular carcinoma

BackgroundThere is growing evidence that long non-coding RNAs (lncRNAs) play important roles in the progression of hepatocellular carcinoma (HCC) and may serve as diagnostic markers. This study investigates the diagnostic efficiency of the long intergenic non-protein-coding RNA 1793 (LINC01793) in hepatitis B virus (HBV)-related HCC. MethodsBioinformatics methods were used to screen the aberrantly expressed lncRNAs in HCC tissues based on The Cancer Genome Atlas (TCGA). Quantitative reverse transcription polymerase chain reaction was performed to assess the expression of the candidate lncRNAs in tissues, cells and whole blood samples of patients with HBV-related HCC, liver cirrhosis (LC), chronic hepatitis (CHB), and healthy controls. Then, the correlations between LINC01793 and clinical characteristics were analyzed. Finally, the diagnostic value of LINC01793 was explored based on the receiver operating characteristic curve. ResultsLINC01793 was remarkably upregulated in the HCC tissues and cells. It was highly expressed in the whole blood of the HBV-related HCC patients, unlike in that of the healthy controls and of the CHB and LC patients. Subsequent analysis revealed that high LINC01793 was related to the Barcelona Clinic Liver Cancer (P = 0.007), tumor invasion (P = 0.042), the number of tumors (P = 0.031) and serum level of alanine aminotransferase(p = 0.022). The areas under the curve of LINC01793, for distinguishing HCC from healthy controls, CHB and LC patients, were 0.824, 0.767 and 0.756, respectively. In addition, the combination of LINC01793 with alpha fetoprotein (AFP) had a stronger diagnostic value than LINC01793 or AFP alone in AFP-negative HCC patients. ConclusionHigh expression of LINC01793 is correlated with adverse clinical characteristics and can serve as a non-invasive biomarker of HCC.

The Novel lncRNA ENST00000530525 Affects ANO1, Contributing to Blood-Brain Barrier Injury in Cultured hCMEC/D3 Cells Under OGD/R Conditions.

Ischemic stroke (IS) is a major neurological disease with high fatality and residual disability burdens. Long noncoding RNAs (lncRNAs) have been found to play an important role in IS. However, the roles and significance of most lncRNAs in IS are still unknown. This study was performed to identify differentially expressed (DE) lncRNAs using a lncRNA microarray in whole blood samples of patients suffering from acute cerebral ischemia. Bioinformatics analyses, including GO, KEGG pathway enrichment analysis, and proximity to putative stroke risk location analysis were performed. The novel lncRNA, ENST00000530525, significantly decreased after IS. Furthermore, we evaluated lncRNA ENST00000530525 expression in cultured hCMEC/D3 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions using fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (RT–qPCR) analysis. To investigate the function of lncRNA ENST00000530525, its over-expression (OE) and negative control (NC) plasmids were transfected into hCMEC/D3 cells, and cell viability was detected by a cell counting kit-8 (CCK-8) assay after OGD/R. LncRNA ENST00000530525 and ANO1 expression were investigated using RT–qPCR and immunofluorescence. For blood–brain barrier (BBB) permeability, FITC-dextran transendothelial permeability assay and tight junction (TJ) protein immunofluorescence assays were performed. There were 3352 DE lncRNAs in the blood samples of acute IS patients. The validation results were consistent with the gene chip data. The GO and KEGG results showed that these lncRNAs were mainly related to oxygen and glucose metabolism, leukocyte transendothelial migration, mitophagy and cellular senescence. Among these, lncRNA ENST00000530525 was the most highly downregulated lncRNA and it was mapped within the IS-associated gene anoctamin-1 (ANO1). We further found that lncRNA ENST00000530525 was downregulated in hCMEC/D3 cells under 4 h OGD and 20 h reoxygenation (OGD4/R20) conditions. Upregulating lncRNA ENST00000530525 by plasmid transfection decreased cell viability while increasing ANO1 expression and it contributed to BBB injury in hCMEC/D3 cells after OGD4/R20. The lncRNA ENST00000530525 might play deleterious roles in post-stroke pathogenesis. These results show that some DE lncRNAs in humans participate through characteristic roles in post-stroke pathogenesis; thus, the roles and significance of some novel lncRNAs in IS warrant further study.

Detection of Treponema pallidum in whole blood samples of patients with syphilis by the polymerase chain reaction.

Syphilis is caused by the bacterium Treponema pallidum. The diagnosis is based on clinical data and serological analysis; however, the sensitivity and specificity of such tests may vary depending on the type of test and stage of the infection. In order to overcome this premise, this study utilized the polymerase chain reaction (PCR) for the detection of T. pallidum DNA in whole blood samples of patients with syphilis. The blood samples from patients with or without symptoms of syphilis, but with positive results in enzyme-linked immunosorbent assay (ELISA), were included in this study. A venereal disease research laboratory (VDRL) test was performed for all collected sera samples. For PCR, the T. pallidum DNA was extracted from the collected blood samples and a specific primer set was designed to amplify 131 nucleotides of polA (Tp0105). The specificity of the primers was evaluated with the DNA of 17 different pathogens. From a total of 314 blood samples reactive in ELISA, 58.2% (183/314) of the samples were reactive in the VDRL test. In the PCR, 54% (168/314) of the ELISA-reactive samples were positive. In both tests (VDRL and PCR) 104 samples were positive. Of 104 positive samples for both tests, 71 were at the latent stage. Based on these results, it can be concluded that PCR with the designed set of primers can be utilized as a diagnostic method for T. pallidum detection in blood samples of patients with syphilis, especially those with latent infection. In addition, it can be utilized as a supplement for serological methods to improve the diagnosis of syphilis.

Effects of hydroxychloroquine and its metabolites in patients with connective tissue diseases.

Hydroxychloroquine has attracted attention in the treatment of COVID-19. Many conflicting findings have been reported regarding the efficacy and safety of this drug, which has been used safely in the rheumatological diseases for years. However, these studies lacked measurement methods that allow accurate assessment of hydroxychloroquine and its metabolite levels. The aim of this study was to measure hydroxychloroquine and its metabolite levels in whole blood samples of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS) and scleroderma (Scl) by a robust, simple and accurate validated tandem mass spectrometric method, and to investigate the relationship between these levels with drug-related adverse effects and disease activity scores. The validated LC–MS/MS method was applied to measure blood hydroxychloroquine and its metabolite levels of patients with RA, SLE, SS, Scl. Various haematological and biochemical parameters were measured with Beckman-Coulter AU 5800 and Beckman Coulter LH 780 analyzers, respectively. QTc intervals were calculated with Bazett’s formula, and the patients were followed up by clinicians in terms of clinical findings and adverse effects. Hydroxychloroquine levels of patients were similar to previous studies. There was a negative correlation between disease activity scores and hydroxychloroquine levels, while the highest correlation was between QTc interval, creatinine and GFR levels with desethylchloroquine. Bidetylchloroquine had the highest correlation with RBC count and liver function tests. Our findings showed that hydroxychloroquine and its metabolite levels were associated with disease activity scores, renal, hepatic function, QTc prolongation, and hematological parameters.

Prospective Monitoring of Circulating Epithelial Tumor Cells (CETC) Reveals Changes in Gene Expression during Adjuvant Radiotherapy of Breast Cancer Patients

Circulating epithelial tumor cells (CETC) are considered to be responsible for the formation of metastases. Therefore, their importance as prognostic and/or predictive markers in breast cancer is being intensively investigated. Here, the reliability of single cell expression analyses in isolated and collected CETC from whole blood samples of patients with early-stage breast cancer before and after radiotherapy (RT) using the maintrac® method was investigated. Single-cell expression analyses were performed with qRT-PCR on a panel of selected genes: GAPDH, EpCAM, NANOG, Bcl-2, TLR 4, COX-2, PIK3CA, Her-2/neu, Vimentin, c-Met, Ki-67. In all patients, viable CETC were detected prior to and at the end of radiotherapy. In 7 of the 9 (77.8%) subjects examined, the CETC number at the end of the radiotherapy series was higher than before. The majority of genes analyzed showed increased expression after completion of radiotherapy compared to baseline. Procedures and methods used in this pilot study proved to be feasible. The method is suitable for further investigation of the underlying molecular biological mechanisms occurring in cells surviving radiotherapy and possibly the development of radiation resistance.

Extensive Changes in Transcription Dynamics Reflected on Alternative Splicing Events in Systemic Lupus Erythematosus Patients.

In addition to increasing the complexity of the transcriptional output, alternative RNA splicing can lead to the reduction of mRNA translation or the production of non-functional or malfunctional proteins, thus representing a vital component of the gene regulation process. Herein, we set out to detect and characterize alternative splicing events that occur in whole-blood samples of patients with Systemic Lupus Erythematosus (SLE) as compared to healthy counterparts. Through the implementation of a computational pipeline on published RNA-sequencing data, we identified extensive changes in the transcription dynamics affecting a large number of genes. We found a predominance of intron retention events, with the majority introducing premature stop codons, suggestive of gene repression, in both inactive and active SLE patient samples. Alternative splicing affected a distinct set of genes from the ones detected as differentially expressed in the same comparisons, while alternatively spliced genes tended to reside in genome areas associated with increased gene co-expression. Functional analysis of genes affected by alternative splicing pointed towards particular functions related to metabolism and histone acetylation as of potential interest. Together, our findings underline the importance of incorporating alternative splicing analyses in the context of molecular characterization of complex diseases such as SLE.

Correlational analysis of chemokine and inflammatory cytokine expression in the intervertebral disc and blood in patients with lumbar disc disease.

The involvement of intervertebral disc (IVD) tissues, whole blood (WB) cytokines, and chemokines in pain in patients with lumbar degenerative disc disease (LDD) is unknown. We investigated the relationships between inflammatory cytokines and chemokines in human IVD tissues and WB samples and their association with pain. Expression levels of chemokines and cytokine gene expression were measured in samples from 20 patients with LDD and compared between IVD tissues and WB samples. The associations between WB chemokine and cytokine gene expression levels and pain intensity (numeric rating scale) were also analyzed. The mRNA of C-C chemokine ligand 20 (CCL20), C-C chemokine receptor 6 (CCR6), interleukin-6 (IL-6), IL-1β, IL-17, and tumor necrosis factor-α (TNF-α) was expressed in degenerated IVD tissues. Pearson's product-moment correlation analysis produced positive correlations between CCR6 and IL-6 expression levels in IVD tissues (r = 0.845, p < 0.001) and WB samples (r = 0.963, p < 0.001). WB IL-6 and CCR6 mRNA expression levels correlated significantly with present pain, maximum pain, and average pain. By contrast, low back pain (LBP) did not correlate with serum chemokine/cytokine expression. This is the first study to report correlations between chemokine and inflammatory cytokine gene expression levels in IVD tissues and WB samples in patients with LDD in relation to pain intensity. WB CCR6 and IL-6 gene expression levels correlated significantly with present pain, maximum pain, and average pain, but not with LBP. These data provide a new understanding of the role of chemokines and inflammatory cytokines in patients with LDD and may lead to new treatment strategies for pain.

Homogeneous Visual and Fluorescence Detection of Circulating Tumor Cells in Clinical Samples via Selective Recognition Reaction and Enzyme-Free Amplification.

Here we report a simple all-nucleic-acid enzyme-free catalyzed hairpin assembly assisted amplification strategy with quantum dots (QDs) as the nanoscale signal reporter for homogeneous visual and fluorescent detection of A549 lung cancer cells from clinical blood samples. This work was based on the phenomenon that CdTe QDs can selectively recognize Ag+ and C-Ag+-C and by using mucin 1 as the circulating tumor cells (CTCs) marker and aptamer as the recognition probe. Under optimized conditions, the limits of detections as low as 0.15 fg/mL of mucin 1 and 3 cells/mL of A549 cells were achieved with fluorescence signals. A 1 fg/mL concentration of mucin 1 and 100 cells/mL of A549 can be distinguished by the naked eye. This method was used to quantitatively analyze CTCs in 51 clinical whole blood samples of patients with lung cancer. The levels of CTCs detected in clinical samples by this method were consistent with those obtained using the folate receptor-polymerase chain reaction clinical test kit and correlated with radiologic and pathological findings.

The ElonginB/C-Cullin5-SOCS-Box-Complex Is a Potential Biomarker for Growth Hormone Disorders

Insulin-like growth factor 1 (IGF-1) is the standard biochemical marker for the diagnosis and treatment control of acromegaly and growth hormone deficiency (GHD). However, its limitations necessitate the screening for new specific and sensitive biomarkers. The elonginB/C-cullin5-SOCS-box-complex (ECS-complex) (an intracellular five-protein complex) is stimulated by circulating growth hormone (GH) and regulates GH receptor levels through a negative feedback loop. It mediates the cells’ sensitivity for GH and therefore, represents a potent new biomarker for those diseases. In this study, individual ECS-complex proteins were measured in whole blood samples of patients with acromegaly (n = 32) or GHD (n = 12) via ELISA and compared to controls. Hierarchical clustering of the results revealed that by combining the three ECS-complex proteins suppressor of cytokine signaling 2 (SOCS2), cullin-5 and ring-box protein 2 (Rbx-2), 93% of patient samples could be separated from controls, despite many patients having a normal IGF-1 or not receiving medical treatment. SOCS2 showed the best individual diagnostic performance with an overall accuracy of 0.93, while the combination of the three proteins correctly identified all patients and controls. This resulted in perfect sensitivity and specificity for all patient groups, which demonstrates potential benefits of the ECS-complex proteins as clinical biomarkers for the diagnostics of GH-related diseases and substantiates their important role in GH metabolism.

Methionine synthase A2756G polymorphism associated with an increased risk of head and neck cancer

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; The 5-methyltetrahydrofolate-homocysteine methyltransferase gene (&lt;em&gt;MTR&lt;/em&gt;) encodes the methionine synthase enzyme (OMIM 156570). Methionine synthase synthesizes methionine by re-methylation of homocysteine. A single nucleotide variation &lt;em&gt;MTR&lt;/em&gt;-A2756G may affects the function of methionine synthase enzyme, which could lead to the development of head and neck squamous cell carcinoma (HNSCC).&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; In current study, 292 HNSCC patients and 324 normal individuals without any history of cancer (control) were enrolled. EDTA whole blood samples of patients and control individuals were collected, and DNA was extracted. All samples were genotyped for &lt;em&gt;MTR&lt;/em&gt;-A2756G polymorphism using polymerase chain reaction-restriction fragment length polymorphism. Frequency of polymorphism was compared between HNSCC patients and control individuals. The association of &lt;em&gt;MTR&lt;/em&gt;-A2756G polymorphism with risk factors was statistically analysed through multivariate analysis (multiple logistic regression) whereas univariate analysis (chi square) was performed for group comparisons. &lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; Univariate analysis revealed that the frequency of groups like age, smoking and &lt;em&gt;MTR&lt;/em&gt;-A2756G genotype was different in HNC patients and controls (p value &amp;lt;0.05). Multivariate analysis showed that smoking (adjusted OR, 3.7; 95% CI, 2.3-6.0), age groups 41-50 years (adjusted OR, 3.6; 95% CI, .9-6.7) and &amp;gt;60 years (adjusted OR, 3.5; 95% CI, 1.7-7.3), &lt;em&gt;MTR&lt;/em&gt;-A2756G genotype (adjusted OR, 2.1; 95% CI, 1.3-3.5) is associated with increased risk of HNSCC.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; Our data suggests that the &lt;em&gt;MTR&lt;/em&gt;-A2756G polymorphism is associated with the occurrence of HNSCC in Pakistani population while the individuals between 40 to 50 years of age and tobacco smokers are at a greater risk of developing HNSCC.&lt;/p&gt;

Assessment of recent alcohol consumption by detecting ethyl glucuronide and ethyl sulphate level among traffic accident patients

Objective: This study aims to explain whether EtG and EtS, which are two non-oxidative ethanol metabolites secreted by the liver and used as markers of alcohol intake related to an incident, were also present in whole blood samples of patients with traffic accident injuries.Methods: EtG and EtS concentrations in all of the whole blood samples were sent to the Forensic Medicine Department of Cukurova University for testing Blood Alcohol Concentrations (BAC) via validated liquid chromatography-tandem mass spectrometer method.Results: The scope of the study covers 200 patients that were admitted to medical services within 12 h following the incident. Of all patients 16.5% were found to be BAC positive, while 35.5 and 23.5 were positive for EtG and EtS, respectively. All samples with a positive BAC result were also positive for EtG and EtS.Conclusion: Detection of EtG and EtS, minor metabolites along with ethanol in the blood can serve as an objective tool to provide further analysis regarding alcohol-related traffic accidents at emergency services.

Enantioanalysis of glutamine-a key factor in establishing the metabolomics process in gastric cancer.

Gastric cancer is the second leading cause of death in the world. Early detection will facilitate early treatment and full recovery of the patients. Metabolomics facilitated the detection of few amino acids able to be used as biomarkers. It was always assumed that the L-enantiomer of the chiral amino acid is part of the process, forgetting the twisting of DNA molecules which may also produce the D-enantiomer of the amino acid. Therefore, an enantioanalysis of amino acids such as glutamine which are part of the gastric cancer metabolomics is absolutely necessary. Four stochastic sensors based on immobilization of protoporphyrin IX, β-cyclodextrin, and 2,2-diphenyl-1-picrylhydrazyl on graphene materials were proposed for the enantioanalysis of glutamine in whole blood samples of patients with gastric cancer. Different signatures were obtained for the enantiomers for each stochastic sensor, making possible the enantioanalysis of glutamine in large concentration ranges-from fmol/L to mmol/L; these ranges facilitating the enantioanalysis of glutamine in healthy patients as well as in patients were found in early and later stages of gastric cancer. Graphical abstract.

Genome-wide association study of genetic variations associated with treatment failure after intravesical bacillus Calmette-Guérin therapy for non-muscle invasive bladder cancer.

Bacillus Calmette-Guérin (BCG) instillation is a key therapy to manage non-muscle invasive bladder cancer (NMIBC). However, intravesical BCG therapy fails in approximately half of the patients, leading to recurrence and progression. We aimed to reveal the genetic variations associated with treatment failure after intravesical BCG therapy for NMIBC. This study included 91 Japanese patients treated with BCG instillation for NMIBC. Genomic DNA was obtained from patient whole-blood samples, and a genome-wide association study and genotyping for target regions were performed. The association between genetic variation and treatment failure was analyzed by genome-wide association in 44 patients as the discovery cohort. As a validation study, candidate single nucleotide polymorphisms (SNPs) were examined among 47 patients in another cohort. The genome-wide association study indicated 19 candidate SNPs (rs1607282, rs7825442, rs1319325, rs3738088, rs4250, rs11894207, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs73520681, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, and rs61094339) associated with BCG failure. Following the cumulative analysis of candidate SNPs, 2-gene (rs73520681 and rs61094339) and 4-gene (rs4250, rs11894207, rs73520681, and rs61094339) models successfully predicted treatment failure after intravesical BCG therapy. This study showed that several SNPs were possibly associated with outcome after intravesical BCG therapy in a Japanese population with NMIBC. The cumulative models of these SNPs may have value in clinical applications, although this should be confirmed in future studies.