Major Markers Research Articles

Human Sarcopenic Myoblasts Can Be Rescued by Pharmacological Reactivation of HIF-1α.

Sarcopenia, an age-related decline in muscle mass and strength, is associated with metabolic disease and increased risk of cardiovascular morbidity and mortality. It is associated with decreased tissue vascularization and muscle atrophy. In this work, we investigated the role of the hypoxia inducible factor HIF-1α in sarcopenia. To this end, we obtained skeletal muscle biopsies from elderly sarcopenic patients and compared them with those from young individuals. We found a decrease in the expression of HIF-1α and its target genes in sarcopenia, as well as of PAX7, the major stem cell marker of satellite cells, whereas the atrophy marker MURF1 was increased. We also isolated satellite cells from muscle biopsies and cultured them in vitro. We found that a pharmacological activation of HIF-1α and its target genes caused a reduction in skeletal muscle atrophy and activation of PAX7 gene expression. In conclusion, in this work we found that HIF-1α plays a role in sarcopenia and is involved in satellite cell homeostasis. These results support further studies to test whether pharmacological reactivation of HIF-1α could prevent and counteract sarcopenia.

Seasonal and Spatial Variations of PM10 and PM2.5 Oxidative Potential in Five Urban and Rural Sites across Lombardia Region, Italy.

Oxidative potential (OP) of particulate matter (PM) is gaining strong interest as a promising health exposure metric. This study investigated OP of a large set of PM10 and PM2.5 samples collected at five urban and background sites near Milan (Italy), one of the largest and most polluted urban areas in Europe, afflicted with high particle levels. OP responses from two acellular assays, based on ascorbic acid (AA) and dithiothreitol (DTT), were combined with atmospheric detailed composition to examine any possible feature in OP with PM size fraction, spatial and seasonal variations. A general association of volume-normalized OP with PM mass was found; this association may be related to the clear seasonality observed, whereby there was higher OP activity in wintertime at all investigated sites. Univariate correlations were used to link OP with the concentrations of the major chemical markers of vehicular and biomass burning emissions. Of the two assays, AA was particularly sensitive towards transition metals in coarse particles released from vehicular traffic. The results obtained confirm that the responses from the two assays and their relationship with atmospheric pollutants are assay- and location-dependent, and that their combination is therefore helpful to singling out the PM redox-active compounds driving its oxidative properties.

Soluble PD-L1 in blood correlates positively with neutrophil and negatively with lymphocyte mRNA markers and implies adverse sepsis outcome

Sepsis causes a myriad of immunological reactions that result in life-threatening alterations in the human body. Immunosuppression in sepsis is partly attributed to the programmed death receptor (PD-1) and its associated ligand (PD-L1) via the regulation of lymphocytes and neutrophils. Although the soluble forms of these proteins (i.e., sPD-1 and sPD-L1, respectively) are recognized as possible sepsis biomarkers, their functional implications are yet to be elucidated. Our research assessed the correlation between sPD-1 and sPD-L1 and blood mRNA markers and sepsis outcome. Blood samples of septic patients of urogenital origin versus control patients (both groups: n = 18) were analyzed. Blood serum sPD-1 and sPD-L1 levels were determined using the enzyme-linked immunosorbent assay (ELISA). The whole blood mRNA concentrations of PD-1, PD-L1, neutrophil markers (CEACAM8 and MPO), and T-lymphocyte markers (TCRβ, CD4 and CD8) were determined via reverse transcriptase quantitative PCR (RT-qPCR). sPD-L1 levels were significantly increased in septic patients when compared to the controls, whereas sPD-1 levels were unaltered. Patients with high sPD-L1 levels, as dichotomized to the median, had a significantly shorter survival rate than those with low sPD-L1 levels. The sensitivity/specificity characteristics of sPD-L1 proved significant for sepsis detection. Furthermore, sPD-L1 correlated with the mRNA concentrations of PD-L1, CEACAM, and MPO, as well as major inflammatory markers (C-reactive protein and procalcitonin). However, sPD-L1 negatively correlated with TCRβ, CD4, and CD8 mRNAs. sPD-L1 was found to be significantly increased in septic patients. Notably, sPD-L1 correlated with PD-L1 mRNA and neutrophil markers and was indicative of adverse outcomes.

The Senescence Markers p16INK4A, p14ARF/p19ARF, and p21 in Organ Development and Homeostasis.

It is widely accepted that senescent cells accumulate with aging. They are characterized by replicative arrest and the release of a myriad of factors commonly called the senescence-associated secretory phenotype. Despite the replicative cell cycle arrest, these cells are metabolically active and functional. The release of SASP factors is mostly thought to cause tissue dysfunction and to induce senescence in surrounding cells. As major markers for aging and senescence, p16INK4, p14ARF/p19ARF, and p21 are established. Importantly, senescence is also implicated in development, cancer, and tissue homeostasis. While many markers of senescence have been identified, none are able to unambiguously identify all senescent cells. However, increased levels of the cyclin-dependent kinase inhibitors p16INK4A and p21 are often used to identify cells with senescence-associated phenotypes. We review here the knowledge of senescence, p16INK4A, p14ARF/p19ARF, and p21 in embryonic and postnatal development and potential functions in pathophysiology and homeostasis. The establishment of senolytic therapies with the ultimate goal to improve healthy aging requires care and detailed knowledge about the involvement of senescence and senescence-associated proteins in developmental processes and homeostatic mechanism. The review contributes to these topics, summarizes open questions, and provides some directions for future research.

Identification of a distal enhancer that determines the expression pattern of acute phase marker C-reactive protein

C-reactive protein (CRP) is a major acute phase protein and inflammatory marker, the expression of which is largely liver specific and highly inducible. Enhancers are regulatory elements critical for the precise activation of gene expression, yet the contributions of enhancers to the expression pattern of CRP have not been well defined. Here, we identify a constitutively active enhancer (E1) located 37.7 kb upstream of the promoter of human CRP in hepatocytes. By using chromatin immunoprecipitation, luciferase reporter assay, in situ genetic manipulation, CRISPRi, and CRISPRa, we show that E1 is enriched in binding sites for transcription factors STAT3 and C/EBP-β and is essential for the full induction of human CRP during the acute phase. Moreover, we demonstrate that E1 orchestrates with the promoter of CRP to determine its varied expression across tissues and species through surveying activities of E1-promoter hybrids and the associated epigenetic modifications. These results thus suggest an intriguing mode of molecular evolution wherein expression-changing mutations in distal regulatory elements initiate subsequent functional selection involving coupling among distal/proximal regulatory mutations and activity-changing coding mutations.

Abstract 2035: Imaging mass cytometry identifies structural and cellular composition of the mouse tissue microenvironment

Abstract Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to simultaneously assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits to highlight cellular composition of human tissues. Here, we showcase the recently released Maxpar OnDemand Antibodies for IMC application on mouse tissue. We introduced 11 additional biomarkers to our existing mouse antibody catalog, providing the basis for the use of high-multiplex imaging in preclinical investigations. To demonstrate the IMC workflow on mouse tissue, we analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers combined with our IMC Cell Segmentation Kit*. The IMC Cell Segmentation Kit facilitates identification of cellular borders using plasma membrane markers that lead to improved nucleus and plasma membrane demarcation. We generated a detailed spatial map of the heterogeneous tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix.Overall, this work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Information gained from IMC studies will enable in-depth high-throughput phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus accelerate preclinical discoveries. *The IMC Cell Segmentation Kit is part of the Innovative Solutions menu of custom-made reagents and workflows developed and tested by Fluidigm scientists to give faster access to new cutting-edge solutions for high-multiplex single-cell analysis. Innovative Solutions are not part of the Maxpar catalog. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Michael Cohen, Smriti Kala, Liang Lim, Geneve Awong, Andrew Quong, Christina Loh. Imaging mass cytometry identifies structural and cellular composition of the mouse tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2035.

Abstract 1722: Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer (NSCLC)

Abstract The rapid clinical expansion of immune checkpoint blockers (ICB) has transformed the therapeutic arsenal for non-small cell lung cancer (NSCLC). However, only a relatively small fraction of patients benefits from these treatments and those who respond can develop acquired resistance. This underlines the need to identify factors or biomarkers associated with treatment sensitivity and resistance to guide clinical decisions. It has been proposed that elevated levels of tumor infiltrating lymphocytes (TIL) in the tumor microenvironment could be associated with favorable clinical outcomes after treatment with ICB. However, few studies have explored this relationship and they used semi-quantitative scoring methods and/or analyzed small tumor areas. Here, we used multiplexed quantitative immunofluorescence (QIF) panels to study major TIL subsets (DAPI, CK, CD4, CD8 and CD20) and T-cell exhaustion markers (DAPI, CD3, LAG-3, PD-1, TIM-3) in full-face baseline whole-tumor slides from a large multi-institutional patient cohort (n=179). The analysis of numerous fields of view per case allowed us to examine the spatial distribution of immune cells and spatial immune heterogeneity. Our results demonstrate that CD8+ effector T-cells are the largest TIL subpopulation and that they are preferentially located in the stromal compartment. We also show that tertiary lymphoid structures have limited association with survival and their detection depends on the size of the tissue area analyzed. Additionally, higher levels of baseline CD8+ T-cells in the stroma were significantly associated with better survival in PD-L1 positive patients but not in PD-L1 negative. To validate these results, we used whole-exome sequencing to analyze the TCR-burden in an independent cohort of ICI-treated NSCLC patients. Increased expression of the exhaustion markers LAG-3 and TIM-3 in CD3 positive T-cells was associated with reduced survival. Finally, we used a novel multiparametric heterogeneity metric termed Rao’s quadratic entropy that enabled us to measure the spatial immune heterogeneity and was associated with worse survival. In summary, we quantitatively measured the density, functional properties, and spatial distribution of major TIL subsets in a large cohort of NSCLC patients treated with ICB. Our results highlight the prominent role of TILs in NSCLC and their potential role as a biomarker. These results could be easily translated into the clinic and used to guide optimal treatment options. Citation Format: Miguel Lopez de Rodas Gregorio, Venkata Nagineni, Arvind Ravi, Ila J. Datar, Mari Mino-Kenudson, German Corredor, Cristian Barrera, Lindsey Behlman, David L. Rimm, Roy S. Herbst, Anant Madabhushi, Jonathan W. Riess, Vamsidhar Velcheti, Matthew Hellmann, Justin F. Gainor, Kurt A. Schalper. Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1722.

Serum Untargeted Metabolomics Reveal Potential Biomarkers of Progression of Diabetic Retinopathy in Asians.

Purpose: To reveal molecular mechanisms of diabetic retinopathy (DR) in Asians and facilitate the identification of new therapeutic targets through untargeted metabolomics. To determine the differences in serum metabolites and metabolic pathways between different stages of diabetic retinopathy in patients with type 2 diabetic mellitus (T2DM) and proliferative DR (PDR) and non-proliferative DR (NPDR) and identify differential metabolites between T2DM and DR (NPDR and PDR) patients. Methods: This prospective observational registration study described the differential metabolites between 45 T2DM patients and 15 control cases with no significant differences in clinical characteristics. Their biospecimens and clinical information were collected and recorded in their medical reports. DR phenotypes of the subjects were verified by retina specialists. Serum metabolites were analyzed using high-resolution mass spectrometry with liquid chromatography. Untargeted metabolomics was performed on serum samples from 15 T2DM patients, 15 non-proliferative diabetic retinopathy patients, 15 proliferative diabetic retinopathy patients, and 15 diabetic controls. Discriminatory metabolic features were identified through partial least squares discriminant analysis (PLS-DA), hierarchical clustering analysis (HCA), and generalized linear regression models. Result: Through untargeted metabolomics, 931 features (523 in positive and 408 in negative modes) with 102 common metabolites highly relevant to the presence of DR were detected. In the adjusted analysis, 67 metabolic features differed significantly between T2DM and NPDR patients. Pathway analysis revealed alterations in metabolisms of amino acids and fatty acids. Glutamate, phosphatidylcholine, and 13-hydroperoxyoctadeca-9,11-dienoic acid (13-PHODE) were key contributors to these pathway differences. A total of 171 features distinguished PDR patients from T2DM patients, and pathway analysis revealed alterations in amino acid metabolism, fatty acid metabolism, nitrogen metabolism, and tricarboxylic acid cycle. Aspartate, glutamate, glutamine, ornithine, N-acetyl-l-glutamate, N-acetyl-l-aspartate, citrate, succinate, N-(L-arginino)succinate, 2-oxoglutarate, 13-hydroperoxyoctadeca-9,11-dienoic acid, methionine, lysine, threonine, phenylalanine, N(pi)-methyl-l-histidine, phosphatidylcholine, and linoleate were major contributors to the pathway differences. Between NPDR patients and PDR patients, there were 79 significant differential metabolites. Enrichment pathway analysis showed changes in amino acid metabolism, fatty acid metabolism, pantothenate, and CoA biosynthesis. Aspartate, glutamine, N-acetyl-l-glutamate, N-acetyl-l-aspartate, pantothenate, dihomo-gamma-linolenate, docosahexaenoic acid, and icosapentaenoic acid were key factors for the differences of these pathways. Conclusion: This study demonstrated that the pathways of arginine biosynthesis metabolism, linoleic acid metabolism, alanine, aspartate, and glutamate metabolism, as well as d-glutamine and d-glutamate metabolism, were dysregulated in DR patients of the Asian population. Increased levels of glutamate, aspartate, glutamine, N-acetyl-l-glutamate, and N-acetyl-l-aspartate and decreased levels of dihomo-gamma-linolenate, docosahexaenoic, and icosapentaenoic were considered as the metabolic profile that could distinguish PDR from NPDR in Asians. Phosphatidylcholine and 13-PHODE were identified as two major novel metabolite markers in advanced stages of DR in our study.

Identification and Validation of Quantitative Trait Loci for Grain Size in Bread Wheat (Triticum aestivum L.)

Grain width (GW) and grain length (GL) are crucial components affecting grain weight. Dissection of their genetic control is essential for improving yield potential in wheat breeding. Yangmai 12 (YM12) and Yanzhan 1 (YZ1) are two elite cultivars released in the Middle and Lower Yangtze Valleys Wheat Zone (MLYVWZ) and the Yellow-Huai River Valleys Wheat Zone (YRVWZ), respectively. One biparental population derived from YM12/YZ1 cross was employed to perform QTL mapping based on the data from four environments over two years to detect quantitative trait loci (QTL) for GW and GL. A total of eight QTL were identified on chromosomes 1B, 2D, 3B, 4B, 5A, and 6B. Notably, QGW.yz.2D was co-located with QGL.yz.2D, and QGW.yz.4B was co-located with QGL.yz.4B, respectively. QGW.yz.2D and QGL.yz.2D, with the increasing GW/GL allele from YZ1, explained 12.36–18.27% and 13.69–26.53% of the phenotypic variations for GW and GL, respectively. QGW.yz.4B and QGL.yz.4B, with the increasing GW/GL allele from YM12, explained 10.34–11.95% and 10.35–16.04% of the phenotypic variation for GW and GL, respectively. QGL.yz.5A, with the increasing GL allele from YM12, explained 10.04–12.48% of the phenotypic variation for GL. Moreover, the positive alleles of these three QTL regions could significantly increase thousand-grain weight, and QGW.yz.4B/QGL.yz.4B and QGL.yz.5A did not show significant negative effects on grain number per spike. QGL.yz.2D, QGW.yz.4B/QGL.yz.4B, and QGL.yz.5A have not been reported. These three QTL regions were then further validated using Kompetitive Allele-Specific PCR (KASP) markers in 159 wheat cultivars/lines from MLYVWZ and YRVWZ. Combining the positive alleles of the major QTL significantly increased GW and GL. Eleven candidate genes associated with encoding ethylene-responsive transcription factor, oleosin, osmotin protein, and thaumatin protein were identified. Three major QTL and KASP markers reported here will be helpful in developing new wheat cultivars with high and stable yields.

An integrated strategy to reveal the potential anti-asthma mechanism of peimine by metabolite profiling, network pharmacology, and molecular docking.

Peimine, one of the major quality markers in Fritillaria Cirrhosae Bulbus, was expected to become a new anti-asthma drug. However, its metabolic profiles and anti-asthma mechanism have not been clarified previously. In this study, a method was developed for the detection of peimine metabolites in vitro by ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry. The potential anti-asthma mechanism was predicted by an integrated analysis of network pharmacology and molecular docking. A total of 19 metabolites were identified with the aid of software and molecular networking. The metabolic profiles of peimine elucidated that the metabolism was a multi-pathway process with characteristics of species difference. The network pharmacology results showed that peimine and its metabolites could regulate multiple asthma-related targets. The above targets were involved in various regulatory pathways linked to asthma. Moreover, the results of molecular docking showed that both peimine and its metabolites had a certain affinity with the β2 adrenergic receptor. The results provided not only important references to understand the metabolism and pharmacodynamic changes of peimine in vitro, but also supporting data for further pharmacological evaluation. It also provided a new perspective for clarifying the functional changes of traditional Chinese medicine in vitro.

P171 EVALUATION OF ARRHYTHMIC RISK IN THE PSYCHOPHARMACOLOGIC–TREATED PATIENT

Abstract Background Neuroleptics (NRL) and Tricyclic Antidepressants (TCA) could be related to the QT prolongation although the risk of ventricular arrhythmias and sudden death caused by these drugs was not clearly described in literature. Corrected QT (QTc), is the major marker of arrhythmic risk; Qt dispersion (QTd) is defined as the dispersion of the QT interval on 12–lead electrocardiogram (ECG) and is predictive of mortality if > 58; the heart rate variability (HRV) is indicative of the sympathetic/vagal tone and of the arrhythmic risk. Aim of the study. Evaluation of the main effects of psychotropic drugs in psychiatric patients on the following parameters: QT interval, QTc using 4 formulas (Bazett, Fridericia, Framingham e Hodges), Qtd and HRV depending on time and heart rate. Materials and Methods Data from 167 patients treated with typical and atypical NRL, TCA, and mood stabilizers have been collected. The observational study consisted in these phases: 1) chart review of 84 patients admitted in day hospital to detect sex, age, diagnosis, therapy and Qtc; 2) evaluation of the same parameters in 83 of them, to whom an ECG was performed at the discharge; 3) ECG Holter in a subgroup of 17 patients who have given consent to the exam, to highlith the incidence of arrhythmias and to investigate the HRV. Results No significant QTc alterations related to therapy were detected at the admission or at the discharge. Moreover, no statistically significant differences emerged among NRL classes nor an increased arrhythmic risk in polypharmacy. The use of different formulas has confirmed an overestimation of the QTc according to the Bazett formula. QTd was altered in 67 patients ( QTd ≥50 in 51 subjects; QTd>60 in 12 subjects)and the HRV was lower compared to the general population regardless of the therapies. Conclusions The psychopharmacological therapy in a “real world setting” didn’t show a signiticative influence on the evaluated parameters of arrhythmic risk. The arrhythmic risk is low in absence of other risk factors or comorbidities. QT correction using FRI or HOD, the QTd if QTc >450–470 ms and the HRV in patients with arrhythmic risk factors, should be considered as parameters to evaluate in the clinic routine of follow–up of psychopharmacologic–treated patient.

Significance of preoperative peripheral blood neutrophil-lymphocyte ratio in predicting postoperative survival in patients with multiple myeloma bone disease.

BACKGROUNDThe neutrophil-lymphocyte ratio (NLR) is often used to predict a poor prognosis in patients with tumors. This study investigated the preoperative peripheral blood NLR in predicting postoperative survival (POS) in patients with multiple myeloma bone disease (MMBD).AIMTo evaluate whether NLR can be used to predict the prognosis of MMBD patients after surgery.METHODSThe clinical data of 82 MMBD patients who underwent surgical treatments in Beijing Chao-yang Hospital were collected. The NLR was obtained from the absolute number of neutrophils and lymphocytes, calculated by the number of neutrophils and divided by the number of lymphocytes. The peripheral blood lymphocyte percentage was used as the major marker to analyze the change in characteristics of the immune statuses of multiple myeloma patients.RESULTSThe NLR cut-off values of NLR ≥ 3 patients and NLR ≥ 4 patients were significantly correlated with POS. The 3- and 5-year cumulative survival rates of the high NLR group (NLR ≥ 3 patients) were 19.1% and 0.0%, respectively, which were lower than those of the low NLR group (NLR < 3 patients) (67.2% and 48.3%) (P = 0.000). In the high NLR group, POS (14.86 ± 14.28) was significantly shorter than that in the low NLR group (32.68 ± 21.76). Univariate analysis showed that the lymphocyte percentage 1 wk after the operation (19.33 ± 9.08) was significantly lower than that before the operation (25.72 ± 11.02). Survival analysis showed that postoperative chemotherapy, preoperative performance status and preoperative peripheral blood NLR ≥ 3 were independent risk factors for POS.CONCLUSIONThe preoperative peripheral blood NLR can predict POS in MMBD patients. MMBD patients with a high preoperative NLR (NLR ≥ 3) showed poor prognosis.

Effects of Nigella sativa supplementation on blood concentration and mRNA expression of TNF-α, PPAR-γ and adiponectin, as major adipogenesis-related markers, in obese and overweight women: a crossover, randomised-controlled trial.

Adipocyte expansion through adipogenesis can offset the adverse metabolic effects of obesity. Nigella sativa (NS) (black seed) oil is shown to have therapeutic features in the management of obesity. NS oil might have beneficial changes in obese populations through mediating serum levels of adipogenesis-related parameters and relative transcriptional gene-diet interactions (nutrigenomics), though no previous studies assessed this mechanism in overweight/obese participants. This study assessed the effects of NS oil supplements on blood concentration and mRNA expression levels of TNF-α, PPAR-γ and serum adiponectin and expression of AdipoR1, as major adipogenesis and obesity-related parameters, in overweight/obese women using a cross-over design. Eligible women were randomised to receive either NS oil supplements (2000 mg/d) or placebo. Two periods of interventions (8 weeks in each) were cross-changed by a 4-week washout period. An individualised diet plan without calorie deficits was given to participants to match their energy/macronutrient intakes. The Pkcross procedure and intention-to-treat analysis were performed using Stata. Cohen's d(d) was estimated to measure the magnitude of the effects. Forty-six participants were included. NS oil capsules reduced transcription levels ((d = -2·31), P < 0·001) and blood concentrations of TNF-α ((d = -0·29), P < 0·001). AdipoR1 expression (d = 2·24, P < 0·001) and serum adiponectin (d = 0·88, P < 0·001) showed a significant augmentation with a medium-high effect size, as did gene expression (d = 0·69, P < 0·001) and serum levels of PPAR-γ (d = 0·97, P < 0·001). There was a moderate but significant decrease in body weight (d = 0·6, P < 0·001). The present beneficial findings would provide strong information for future nutrigenomics/clinical trial studies assessing the role of NS in the management of obesity and other comorbidities.

MO737: Vascular Calcification and the Gut Microbiome in Chronic Kidney Disease Patients on Peritoneal Dialysis

Abstract BACKGROUND AND AIMS Cardiovascular disease (CVD) is the leading cause of death among patients with chronic kidney disease (CKD). In addition to diabetes, hypertension, dyslipidaemia and inflammation, vascular calcification and its severity have long been recognized as a major factor in the development of CVD in patients with CKD. CKD favours gut dysbiosis, and this dysbiosis may contribute to the aggravation of CVD, inflammation, and vascular calcification in CKD. Therefore, our aim was to study the potential interconnection between vascular calcification, mortality risk, and the gut microbiome in CKD patients on peritoneal dialysis (PD). METHOD A group of 44 CKD patients on PD was evaluated concerning major inflammatory markers and key players of CKD-mineral bone disorder. Vascular calcification severity was assessed by the Adragao score, and all-cause mortality risk was assessed using the Charlson comorbidity index. Gut microbiome profile was obtained by sequencing the V3–V4 region of 16S rRNA. RESULTS The global mean of the Adragao score of our CKD-PD patients was 2.98 ± 2.74, including 26.1% with no vascular calcification (Adragao score = 0), 30.4% with moderate vascular calcification (Adragao score of 1 or 2), and 39.1% presenting severe vascular calcification (Adragao score higher than 2). When comparing CKD-PD patients with moderate or severe vascular calcification with CKD-PD patients with no vascular calcification, we found statistically significant differences regarding age (47.7 ± 11.5 versus 59.4 ± 8.8; P &amp;lt; 0.01), sex (% male 33.3% versus 78.1%; P = 0.011), total Kt/V (urea) (2.6 ± 0.6 versus 2.1 ± 0.4; P = 0.04) and history of diabetes mellitus (8.3% versus 48.8%; P = 0.035). No differences were found regarding the expression of inflammation markers between the two groups. When vascular calcification severity was correlated with all-cause mortality risk scored by the Charlson comorbidity index, we observed a positive significant correlation between these two factors (Spearman correlation, correlation coefficient = 0.538; P &amp;lt; 0.001). When comparing PD patients with and without vascular calcification, no significant differences were found regarding gut microbiome profile. Nonetheless, relative changes of specific taxa were observed, namely regarding Coprobacter, Coprococcus 3, Lactobacillus and Eubacterium eligens group in the gut microbiome. Among these taxonomic differences, patients with different Charlson comorbidity index values also demonstrated changes in the Eubacterium eligens group in the gut microbiome. The Adragao score and the Charlson index were positively correlated. CONCLUSION There is an association between vascular calcification and mortality risk in CKD patients on PD. We have found small differences in some specific taxa when comparing the gut microbiomes of CKD-PD patients with and without vascular calcification. Future studies exploring the role of these bacterial groups in the increased risk of vascular calcification, mortality and CVD in CKD-PD patients are of extreme relevance to ensure the health and well-being of these patients.

Imaging Mass Cytometry identifies structural and cellular composition of the mouse tissue microenvironment

Abstract Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits on human tissues. Here, we showcase Maxpar OnDemand Antibodies for IMC application including 11 new highly relevant markers to construe cellular and molecular composition of mouse tissues. We analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers. We generated a detailed spatial map of the diverse tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix. This work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Future studies utilizing IMC in combination with Maxpar OnDemand Antibodies will enable in-depth phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus provide the basis for the use of high-multiplex imaging in preclinical investigations. For Research Use Only. Not for use in diagnostic procedures.

Elements of Biblical Exegesis: A Basic Guide for Students and Ministers

Elements of Biblical Exegesis: A Basic Guide for Students and Ministers

Effective inhibition of adipogenesis-mediated inflammation by a macular carotenoid, lutein in vitro.

An absolute interlinks between inflammation and obesity with scarce investigations on the role of lutein in inflammation-induced obesity motivated us to explore the protective mechanism of lutein on adipogenesis-mediated inflammation in vitro by culturing RAW264.7 macrophages in adipocyte conditioned medium. The RAW264 macrophage cells were cultured with adipocyte-conditioned media, and the potency of lutein on the expression of adipocyte inflammation-associated protein markers (IL-1β, MCP-1, TNF-α, IL-6, NF-κB, and IKKα/β) were analyzed by western blotting. The data revealed that lutein effectively reduces the protein levels of major inflammatory markers such as NF-κB, IL-1β, MCP-1, and TNF-α in differentiated adipocytes. Interestingly, lutein hampered inflammation in the RAW264 cells that were cultured in adipocyte-conditioned media by lowering the protein expression of IL-1β, MCP-1, and TNF-α. The blockage of inflammation by lutein in both differentiated adipocytes, and adipogenesis-induced macrophages is associated with suppression of IKK α/β phosphorylation. These data suggest that lutein potentially alters adipocyte differentiation-mediated inflammation by regulating the NF-κB signaling pathway. Thus, lutein could be utilized as a potent nutraceutical agent in the management of obesity and associated inflammation. PRACTICAL APPLICATIONS: Lutein isolated from a dietary source exhibited an inhibitory effect in adipogenesis-induced inflammations. The findings of this study authenticate the diversified prospective of lutein in regulating obesity and other inflammation-related diseases. Thus, it is understood that continuous intake of lutein-rich food or dietary intervention of lutein may reduce the risk of developing obesity.

HiIDDD: a high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells

DNA damage is a prominent biomarker for numerous diseases, including cancer, as well as for the aging process. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, quantitative and single-cell assay that measures DNA damage by high-throughput imaging using the two major DNA damage markers 53BP1 and upgamma-H2AX. We demonstrate sensitive detection with low inter-assay variability of DNA damage in various types of freshly isolated and cryopreserved primary human immune cells, including CD4 + and CD8 + T cells, B cells and monocytes. As proof of principle, we demonstrate parallel batch processing of several immune cell types from multiple donors. We find common patterns of DNA damage in multiple immune cell types of donors of varying ages, suggesting that immune cell properties are specific to individuals. These results establish a novel high-throughput assay for the evaluation of DNA damage in large-scale studies.

Role of platelet count in diagnosis of oral potentially malignant disorders and oral squamous cell carcinoma

Introduction: Platelets play an important role in the metabolic functions of malignant tissues. Platelet count is the major marker for malignancy and their increase in number can depict stages, severity and metastasis of cancer. Increase in number of platelet count can be used in early diagnosis of cancer. However, use of some chemotherapeutic agents is associated with low platelet count. Therefore, the platelet to cancer cell ratio varies through the course of the disease and is highly affected by treatment. Aim: To assess correlation between platelet count and progression of malignancy. Materials and method: Platelet counts were retrieved from patients diagnosed oral potentially malignat disorders and oral squamous cell carcinoma based on clinical data. The collected data is analyzed to correlate the platelet count in oral potentially malignat disorders and oral squamous cell carcinoma compared to healthy control patients. Result: Nonsignificant differnce was noted in platelet count in oral potentially malignat disorders and oral squamous cell carcinoma compared to healthy control patients. Conclusion: Thrombocystosis is a poor predicator for malignancy, platelet count alone cannot determine the severity of malignancy, correlation with histopathologic data can better establish the grading of malignancy.

Design and development of integrated TIRF and common-path quantitative phase microscopic health care system with high stability

We demonstrate a compact microscopy system that enables simultaneous cell imaging in both digital holographic and fluorescence mode thus providing valuable multi-modal information for biosciences applications. The digital holographic imaging system uses a speckle free illumination by employing a rotating diffuser. Further a compact shear plate based interferometer arrangement is used to significantly reduce the system size and increase its robustness when compared with Michelson or Mach-Zehnder interferometer configurations. The overall reduction in form factor of the system is achieved since we employ the shear plate configuration additionally as a beam-splitter for the fluorescence imaging part of the system. We have fabricated a compact total internal reflection fluorescence (TIRF) unit which is combined with the common path sheared interferometer for this purpose. Our experiments with this compact system show excellent temporal phase stability. The working of the system is illustrated with imaging of polystyrene beads and MG63 osteosarcoma cell line sample. The fluorescence image of MG63 cell shows high expression of vinculin protein around the nuclei when compared with the expression in the cell's filopodia. These proteins are among the major molecular markers for cell adhesion and play a vital role in sensing the mechanical stimulus of the microenvironment. Further the quantitative phase information about the cell also provides its optical thickness information that is spatially aligned with the fluorescence signal. The proposed system can find applications in bioscience laboratories as well as in diagnostics.