Maximum Fold Change Research Articles

Selection and validation of reference genes for RT-qPCR analysis in Desmodium styracifolium Merr.

The online version contains supplementary material available at 10.1007/s13205-021-02954-x.

Increased plasma levels of damage-associated molecular patterns during systemic anticancer therapy in patients with advanced lung cancer.

BackgroundImmunogenic cell death (ICD) characterized by the release of damage-associated molecular patterns (DAMPs) from dying cancer cells may contribute to the synergistic antitumor effect of cytotoxic chemotherapy combined with an immune checkpoint inhibitor. The kinetics of circulating DAMP levels in cancer patients have remained largely uncharacterized, however.MethodsWe evaluated the possible effects of various systemic anticancer therapy modalities on the kinetics of plasma DAMP concentrations in a prospective observational study of patients with advanced lung cancer. The plasma concentrations of high-mobility group box 1 (HMGB1), calreticulin (CRT), heat shock protein 70 (HSP70), annexin A1, and histone H3 were thus determined in 121 such patients at four time points during the first cycle of treatment.ResultsThe mean of the maximum fold change in HMGB1, HSP70, or annexin A1 concentration observed during treatment was significantly greater than the corresponding baseline value (P<0.005). The maximum fold changes in HMGB1 and CRT concentrations tended to be associated with clinical response as evaluated by RECIST criteria, although the changes in the levels of these two DAMPs were not correlated, suggestive of differential induction mechanisms. Among the various treatment modalities administered, platinum-based combination or single-agent chemotherapy tended to elicit robust increases in the concentrations of HMGB1 and CRT.ConclusionsSerial monitoring of plasma revealed that systemic anticancer therapy increased the circulating levels of HMGB1 and CRT and that these changes tended to be associated with clinical response, suggesting that agents capable of releasing these DAMPs into plasma might induce ICD in advanced lung cancer patients.

Novel bHLH and WD40 transcription factors from turmeric (Curcuma longa L.) as putative regulators of curcumin biosynthesis

Turmeric, the golden spice belonging to the family Zingiberaceae, is enriched with biologically active curcuminoids composed of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids are phenylpropanoid derivatives, and the biosynthetic pathway is controlled by several transcription factors (TFs). bHLH, WD40 and MYB TFs are the most important TFs regulating phenylpropanoid biosynthesis in plants. Through comparative transcriptome analysis of high and low curcumin germplasm accessions, 20 TFs belonging to the classes bHLH, WD 40, NAC, WRKY and bZIP, which showed differential expression with respect to curcumin, were identified. Among these, two bHLH and one WD40 TFs showed maximum comparative fold change and negative correlation vis-a-vis curcumin content in quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The results of comparative transcriptome and qRT-PCR analyses were in congruence, indicating their putative role as negative regulators.

Allele-defined genome reveals biallelic differentiation during cassava evolution

Allele-defined genome reveals biallelic differentiation during cassava evolution

Analysis of differences between unifloral honeys from different botanical origins based on non-targeted metabolomics by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry

不同的蜜源植物具有结构多样的次生代谢产物。该研究以8种不同蜜源单花蜜(洋槐蜜、枣花蜜、荆条蜜、椴树蜜、荞麦蜜、麦卢卡蜜、枸杞蜜、益母草蜜)为研究对象,建立了基于超高效液相色谱-四极杆飞行时间质谱技术(UPLC-Q-TOF-MSE)的非靶向代谢组学方法,考察了不同蜜源中次生代谢产物的差异。该研究采用固相萃取前处理方法和UPLC-Q-TOF-MSE方法,获得不同蜜源单花蜜的植物代谢组信息,并构建了多变量统计分析模型,对不同来源的单花蜜进行模式识别和差异分析,发现洋槐蜜、枣花蜜、荆条蜜、椴树蜜、荞麦蜜、麦卢卡蜜相互间存在不同程度的显著差异。结合模型的变量重要性投影、方差分析与最大差异倍数值,根据精确前体离子和碎片离子质量信息检索Chemspider、HMDB数据库,该研究筛选并鉴定出32个代谢差异化合物,其中黄酮类化合物18个、酚酸类化合物7个、苯苷与萜苷类化合物6个、甾体类化合物1个;研究发现麦卢卡蜜和荞麦蜜以黄酮类化合物为主要差异代谢物,荆条蜜中酚酸类化合物为特征性表达,苯苷与萜苷类化合物主要为椴树蜜的特征代谢物。该研究从植物代谢组学角度初步揭示了不同单花蜜的代谢产物差异性以及特征化合物,为基于化学分析技术的蜂蜜溯源识别与质量评价提供了有效的研究策略。

Quantitative analysis of proteome dynamics in a mouse model of asthma

Asthma is an inflammatory disease of the respiratory system, and a major factor of increasing health care costs worldwide. The molecular actors leading to the development of chronic asthma are not fully understood and require further investigation. The aim of this study was to monitor the proteome dynamics during asthma development from early inflammatory to late fibrotic stages. A mouse asthma model was used to analyse the lung proteome at four time points during asthma development (0weeks=control, 5, 8 and 12weeks of treatment, n=6 each). The model was analysed using lung function tests, immune cell counting and histology. Furthermore, a multi-fraction mass spectrometry-based proteome analysis was performed to achieve a comprehensive coverage and quantification of the lung proteome. At early stages, the mice showed predominant eosinophilic inflammation of the airways, which disappeared at later stages and was replaced by marked airway hyper-reactivity and fibrosis of the airways. 3325 proteins were quantified with 435 proteins found to be significantly differentially abundant between the experimental groups (ANOVA p-value ≤.05, maximum fold change ≥1.5). We applied hierarchical clustering to identify common protein abundance profiles along the asthma development and analysed these clusters using gene ontology annotation and enrichment analysis. We demonstrate the correlation of protein clusters with the course of asthma development, that is eosinophilic inflammation and fibrotic remodelling of the airways. Proteome analysis revealed proteins that were previously described to be important during asthma chronification. Moreover, we identified additional proteins previously not described in the context of asthma. We provide a comprehensive data set of a long-term mouse model of asthma that may contribute to a better understanding and allow new insights into the progression and development of chronic asthma. Data are available via ProteomeXchange with identifier PXD011159.

Biostimulation of Rhodovulum sp., for enhanced degradation of di-n-butyl phthalate under optimum conditions

Di-butyl phthalate (DBP) is an extensively applied synthetic plasticizer, toxic organic compound with elevated concentrations in aquatic and terrestrial ecosystem that cause serious risk to the human health. A marine bacterium Rhodovulum sp. DBP07 isolated from sea water with proficient of efficiently degrading DBP. The maximum DBP degradation (70.2%) and the cell growth (1.3 OD600nm) were observed at 600 mg/L. The DBP degradation characteristics of the isolate Rhodovulum sp. DBP07 with diverse preliminary concentrations of DBP was found to be 200 ˃ 400 ˃ 600 ˂ 800 ˂ 1000 mg/L DBP. Glucose was identified as most favorable nutrient factor for the enhanced growth and showed 79.8 and 77.4% of degradation rate at 5.0 and 2.0 g/L respectively. The influence of the carbon sources on DBP degradation was found to be Glucose ˃ fructose ˃ sucrose ˃ maltose ˃ lactose ˃ citric acid ˃ starch. Box-Behnken (BBD) statistical optimization results showed enhanced DBP biodegradation rate (91.1%) at pH 7.0, 3% of NaCl concentration with 3 days of incubation. Two intermediate compounds were observed in the retention times of 10.8 and 12.2 which are identified as diethyl phthalate (DEP) and mono-nbutyl phthalate (MBP) using Gas chromatography mass spectroscopy (GC-MS). Furthermore, the phthalate (pht) gene expression pattern under DBP stress was analyzed using RT-qPCR and the maximum fold change (5.7 fold) was observed at 3 day of incubation. Overall, the observed results indicate the possibility of utilizing Rhodovulum sp. for remediation of DBP contaminated environment.

The pseudogene problem and RT-qPCR data normalization; SYMPK: a suitable reference gene for papillary thyroid carcinoma

In RT-qPCR, accuracy requires multiple levels of standardization, but results could be obfuscated by human errors and technical limitations. Data normalization against suitable reference genes is critical, yet their observed expression can be confounded by pseudogenes. Eight reference genes were selected based on literature review and analysis of papillary thyroid carcinoma (PTC) microarray data. RNA extraction and cDNA synthesis were followed by RT-qPCR amplification in triplicate with exon-junction or intron-spanning primers. Several statistical analyses were applied using Microsoft Excel, NormFinder, and BestKeeper. In normal tissues, the least correlation of variation (CqCV%) and the lowest maximum fold change (MFC) were respectively recorded for PYCR1 and SYMPK. In PTC tissues, SYMPK had the lowest CqCV% (5.16%) and MFC (1.17). According to NormFinder, the best reference combination was SYMPK and ACTB (stability value = 0.209). BestKeeper suggested SYMPK as the best reference in both normal (r = 0.969) and PTC tissues (r = 0.958). SYMPK is suggested as the best reference gene for overcoming the pseudogene problem in RT-qPCR data normalization, with a stability value of 0.319.

Expression of transforming growth factor beta in oral submucous fibrosis

BackgroundOral submucous fibrosis (OSMF) is a premalignant condition mainly caused by areca nut chewing and is characterized by progressive fibrosis of submucosal tissues and epithelial atrophy. Activation of transforming growth factor beta (TGF-β) signaling is considered main causative event for increased collagen production and fibrosis. In this study, molecular pathogenesis of OSMF was investigated based on the expression of the TGF-β genes in OSMF tissues compared to normal controls. MethodsA total of 33 OSMF and 10 normal tissues were collected from patients and their clinic-epidemiological data was recorded. The expression of TGF-β isoform genes- TGF β1, TGF β2, TGF β3 and its receptor TGF βR1, TGF βR2 was studied by real time polymerase chain reaction (PCR). Comparison of the expression of these genes among normal controls and OSMF patients was done. The PCR results were confirmed by histopathological and immunohistochemical staining. ResultsThe histological changes included atrophic epithelium, loss of rete ridges, presence of inflammatory cells and dense collagen bundles in connective tissue. PCR showed statistically significant upregulation of TGF-β isoforms in OSMF as compared to normal tissues. Of the three isoforms, maximum fold change was observed in TGF-β1. Similarly, both TGF-βR1 and TGF-βR2 were found to be elevated in OSMF tissues compared to normal. The semi-quantitative analysis by immunohistochemical staining revealed statistically significant difference between normal and OSMF tissues. ConclusionTGF-β signaling plays a major role in the molecular pathogenesis of OSMF as shown by increased mRNA expression of all the three TGF-β isotypes and their receptors.

Biological variability of cell-free DNA in healthy females at rest within a short time course.

Alterations in cell-free DNA concentration (cfDNA) over time have been studied in diseased or injured patients or analyzed in athletes during exhaustive exercise. However, no fluctuations have been examined over a short time course in healthy humans at rest so far, wherefore the aim of this study was to examine individual variations at different time points within 75min. Serial blood drawing was performed in 14 healthy female volunteers at rest within 75min. Plasma DNA was quantified by real-time qPCR, and absolute levels were analyzed together with relative variations. cfDNA alterations were moreover analyzed in consideration of potential volunteer-related impact factors (e.g., pulse) and were compared to alterations of plasma CK and AST. Absolute cfDNA concentration ranged from 0.6 to 3.4ng/ml. Regarding alterations over time, positive and negative variations were identified, whereby the interdecile range of fold changes was from 0.5 to 1.4. The maximum fold change was determined at 10min. No relations were found between cfDNA levels and the analyzed individual factors. We evidenced the variability of cfDNA in healthy humans at rest within a short time course. The determined variations should serve in future studies to distinguish small cfDNA increases after minor trauma from natural fluctuations. Without such reference of intra-individual variation at rest, it would not be feasible to distinguish an injury from a fluctuation with certainty. Thus, a basis was established for the application of cfDNA as biomarker for the detection of mild injuries in forensic biomechanics.

Identification of gene biomarkers to predict responses to neoadjuvant chemoradiotherapy in patients with rectal cancer and pathways enrichment analysis

Objective: To screen out the potential gene biomarkers to predict responses to neoadjuvant chemoradiotherapy (CRT) in patients with rectal cancer and to explore the main downstream pathways of resistance. Methods: The gene expression profiles (GSE35452) of locally advanced rectal cancer undergoing neoadjuvant chemoradiotherapy from 46 specimens (24 responders, TRG 0/1, and 22 non-responders, TRG 2/3) were downloaded from the GEO database. The differentially expressed genes were identified to screen out the potential biomarkers by use of the GCBI platform. GO and KEGG pathways enrichment analysis were performed to integrate enrichment results of differentially expressed genes. Signal-signal interaction network was constructed and analyzed to screen out potential main downstream pathways. Results: A total of 1079 differentially expressed genes were screened, including 657 up-regulated and 422 down-regulated ones. Among these genes, REG4 had the maximum fold change value of -6.029 491. In GO term, these differentially expressed genes were mainly enriched in molecule metabolic process, cell cycle, DNA-dependent transcription, signal transduction and apoptotic process. The KEGG pathways enrichment analysis showed that the differentially expressed genes were enriched in 65 KEGG pathways, including metabolic pathways, cell cycle and metabolism pathways. Signal-signal interaction network analysis showed that MAPK signaling pathway and cell cycle pathway might play a determinant role in the development of neoadjuvant chemoradiotherapy resistance. Further analysis showed that CDKN1B, CDKN2A, RBL1, TFDP1, CCND2, CCNE2, CDC6 and CDK6 in cell cycle might induce chemoradiotherapy resistance by blocking G1/S phase cell cycle arrest, decreasing the apoptosis of tumor cells and increasing S phase ratio of chemoradiotherapy resistance. Conclusion: G1/S phase cell cycle arrest blocking plays an important role in the development of chemoradiotherapy resistance in patients with rectal cancer. Moreover, the key genes, such as REG4, may be useful in predicting responses to neoadjuvant chemoradiotherapy.

Weighted Gene Coexpression Network Analysis Identifies Cysteine-Rich Intestinal Protein 1 (CRIP1) as a Prognostic Gene Associated with Relapse in Patients with Acute Myeloid Leukemia.

BackgroundAcute myeloid leukemia (AML) is associated with a high relapse rate and poor prognosis. This study aimed to use weighted gene coexpression network analysis (WGCNA) of gene coexpression networks to identify candidate prognostic biomarker genes in patients with AML and to investigate the expression of these genes in the human U937 cell line in vitro.Material/MethodsRNA-seq data were retrieved from the Cancer Genome Atlas (TCGA) and included bone marrow samples and survival data of patients with AML (N=151), patients who did not relapse after treatment (N=119), and patients with relapse (N=40). Differentially expressed genes were identified, WGCNA was used to detect functional modules, and survival analysis was performed. The Cell Counting Kit-8 (CCK-8) assay investigated the proliferation of U937 cells transfected with short hairpin RNAs (shRNAs), shCRIP1, shHIST1H1C, and shHIST1H1E. RNA-seq analysis identified gene expression following CRIP1 knockdown.ResultsEighty-two genes were associated with both relapse and prognosis in patients with AML. There were two prognosis-related gene modules in the coexpression network. In the coexpression network, the histone cluster 1 H1 family member gene, HIST1H1C had the maximum relapse fold change, HIST1H1E had the lowest survival p-value, and the cysteine-rich intestinal protein 1 (CRIP1) gene had the most edge numbers and was significantly associated with poor prognosis (P=0.0165786). RNA-seq data showed that there was a significant difference in gene expression after CRIP1 knockdown in U937 cells.ConclusionsWGCNA of gene coexpression networks identified CRIP1 as a potential prognostic biomarker gene in patients with AML.

Transcriptome analysis of the effect of GA3 in sugarcane culm.

Our earlier studies have indicated that GA3, being a growth hormone, increases internodal length, in turn increasing sink strength and improving sucrose accumulation in sugarcane. In this study, transcriptomic level analysis was carried out on internodal samples of a high sugar accumulating variety (CoLk 94184) of sugarcane, to determine the effect of exogenous application of GA3 vis a vis functional analysis of differentially expressing transcripts. Overall, a total of 201,184 transcripts were identified, with median contig length of 450bp and N50 length of 1029bp. Analyzing the data from control and GA3-treated canes, at 0.01 significance, a total of 1516 differentially expressing transcripts were identified in bottom internodes and 1589 in top internodes. A KEGG (enrichment) analysis grouped the transcripts into 153 plant-related functional categories. From among these, the transcripts which were functionally relevant to sugar metabolism and photosynthesis were sieved out. Starch and sucrose metabolizing genes showed maximum fold change of 5.0 and 3.0 among top and bottom internodal samples. A homology match using Blastx analysis tool yielded 65 transcripts/differentially expressed genes (DEGs) which were found to share homology with C4 plants like Saccharum, Sorghum and Zea mays. Differentially expressing transcripts from both top and bottom internodes were validated by qRT-PCR, indicating their importance in such study. Results also enriched sugarcane transcriptome resources useful for omics study in genus Saccharum and family Poaceae.

Insights into the heavy metal-induced immunotoxic and genotoxic alterations as health indicators of Clarias gariepinus inhabiting a rivulet

There is a dire need to assess the quality of fishes transported for human consumption as lately, their health is challenged because of anthropogenic activities. Heavy metals with a long environmental persistence are toxic to fishes and the humans. The study was conducted to evaluate the impact of heavy metals on Clarias gariepinus inhabiting popular Ganges rivulet at Narora (28.18° E, 78.39° N). The limnological values deviated from the international Standards (USEPA, WHO). Higher total dissolved solids (859 mg/L), total suspended solids (406 mg/L), low dissolved oxygen (5.60 mg/L), and pH (5.21) indicated the presence of contaminants. Heavy metals estimated followed the order Cd > Ni > Cu > Cr. Serum enzymes (hepatic and renal markers) viz., aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were higher than the normal, whereas, creatine kinase (CK) was considerably low in both male and female fish. Stress induced was marked by elevation in cortisol and glucose. This had its impact on hematological parameters as well, as a decline in Total leucocyte count (TLC) & mean corpuscular volume (MCV) and increase in Mean cell hemoglobin (MCH) was observed. Erythrocytes also showed altered morphology. Marked histopathological alterations were observed in all immune organs (head-kidney, liver, spleen, thymus). Oxidative stress induced by heavy metals leads to the production of metal scavenging protein metallothionein (MT) and glutathione peroxidase (GPX). Maximum fold change in metallothionein (MT) gene expression was observed in the liver, followed by spleen, thymus, blood, and head-kidney. Glutathione peroxidase (GPX) gene expression was highest in the liver, followed by thymus, spleen, blood, and head-kidney. The gene expression studies further validated the increased level of heavy metals as potent contaminants of water and the non-condusive abiotic factors.

Comparative study of biochemical, histological and molecular biomarkers of heavy metal contamination in Cyprinus carpio collected from warm-monomictic lake and government culture pond.

The study investigated the metallothionein (MT) and glutathione peroxidase (GPX) genes expression in freshwater fish Cyprinus carpio dwelling in Warm-monomictic Lake (Dal) and Government culture pond. Oxidative stress induced by heavy metals in the fish body manipulates stress genes expression resulting in the production of scavenger protein (for free metal ions) metallothionein. It interacts with Cu, Cr, Ni and Cd via metal-thiolate bond relieving the metal load from fish body. Maximum fold change was observed in liver, muscle and midgut tissue. Similar rise seen in GPX indicates defence against lipid peroxidation. MT and GPX genes data were compared with beta-actin gene used as an internal control. Limnological studies of both the sites (temperature, dissolved oxygen, pH, total dissolved solids, conductivity), were essential to ensure the quality of water in which the dominant species (C. carpio) was thriving, as these fishes are transported for human consumption. Heavy metal concentration (water, tissues, gut content), bioindices, biochemical parameters and histological alterations were studied to observe the impact of elements selected.

PS1027 ASSESSMENT OF MOLECULAR MRD KINETICS BY ERROR‐CORRECTED NEXT‐GENERATION SEQUENCING PROVIDES INDEPENDENT PROGNOSTIC INFORMATION IN ADULT AML PATIENTS

Background:Identification of AML patients at high risk of relapse after achieving a complete remission (CR) with induction chemotherapy enables the use of personalized post‐remission treatment strategies to prevent relapse. Detection of molecular measurable residual disease (mMRD) by conventional next‐generation sequencing (NGS) at a single time point post‐induction has previously been associated with a higher incidence of relapse and inferior overall survival (OS).Aims:In this study, we used error‐corrected NGS (EC‐NGS) to evaluate whether changes in mMRD levels between two time points during remission provide additional prognostic information over single time point assessments.Methods:88 AML patients who received standard induction chemotherapy and achieved a CR were evaluated. Targeted NGS of 54 genes associated with myeloid malignancies was performed at diagnosis. We collected peripheral blood (PB) samples upon count recovery following induction chemotherapy and each cycle of consolidation chemotherapy. To detect mMRD, we used a custom 37‐gene hybrid‐capture panel and EC‐NGS based on the Duplex Sequencing approach. PB samples collected at two different time points during remission were analyzed for each patient. The Cox proportional hazards model was used to relate predictor variables to time to relapse and OS. P‐values &lt;0.05 were considered significant.Results:Sequencing analysis of diagnostic samples identified at least one putative oncogenic mutation in 82 of the 88 patients (93%). EC‐NGS of samples collected at the first remission time point (T1) identified at least one persistent mutation in 63 of the 82 patients (77%); 40% of the persistent mutations were in DNMT3A, TET2, and ASXL1 (DTA). The persistence of DTA or non‐DTA mutations, when considered separately, did not correlate with risk of relapse or OS. The co‐persistence of at least one DTA and one non‐DTA mutation was associated with a higher risk of relapse (HR: 2.61; 95% CI, 1.16 to 5.83; P = 0.02) but not OS (P = 0.56). To evaluate whether changes in mMRD level correlate with clinical outcomes, we analyzed the subset of mutations that were detected at diagnosis and persisted in both T1 and T2. We calculated the fold change (FC) in variant allele frequency (VAF) between T1 and T2 for each mutation and used the maximum FC among all mutations of each patient (maxFC) as a continuous variable for regression analysis. High maxFC values restricted to mutations found at diagnosis (maxFCD) were significantly correlated with a higher risk of relapse (P = 0.002) and inferior OS (P = 0.003). We extended the analysis to all non‐synonymous mutations that were detected in T1 and T2 including ones that were not identified at diagnosis. The mutations were predominantly in DNMT3A, TET2, ASXL1, and TP53. Higher maxFC values among mutations found in remission samples (maxFCR) was strongly associated with an increased risk of relapse (P &lt; 0.0001; Fig 1A) and inferior OS (P &lt; 0.0001; Fig 1B). In multivariable regression analysis, maxFCR remained an independent risk factor for relapse after adjustment for age, WBC, ELN 2017 risk group, maxFCD, MRD by flow cytometry, and persistence of mutations found at diagnosis (Table 1).Summary/Conclusion:Our analysis demonstrates that assessment of mMRD kinetics using EC‐NGS provides additional prognostic information over mMRD monitoring at a single time point. We developed an analytical framework to analyze mMRD results in remission and identified a novel parameter (maxFCR) that is independently associated with risk of relapse and OS.image

Curation and analysis of clinical pathology parameters and histopathologic findings from eTOXsys, a large database project (eTOX) for toxicologic studies

Large data sharing projects amongst the pharmaceutical industry have the potential to generate new insights using data on a scale that has not been previously available. A retrospective analysis of the preclinical toxicology data collected as part of the eTOX project was conducted with the aim to provide background rates and treatment-related value analysis on both clinical pathology and histopathology datasets. Incorporated into this analysis was an extensive data consolidation task to standardise all data. Reference intervals for common clinical pathology parameters in rat and dog were generated, alongside background histopathology incidence rates in the liver, heart and kidney. Systematically applied decision thresholds allowed consistent relabelling of data points considered anomalous, and maximum fold change estimates. Relabelling of anomalous data points was conducted for the histopathology data using a Bayesian model to identify dose-dependent increases in pathologies. The results of this study allow: newly generated data to be analysed using the same methodology, rates and distributions to be used when building predictive dose-response models, and the possibility to correlate clinical pathology findings with concurrent histopathology findings. In the first half of this paper we discuss data curation, in the second half we report on the analytical methods and results.

LC‑MS/MS proteomic analysis revealed novel associations of 37 proteins with T2DM and notable upregulation of immunoglobulins.

Type 2 diabetes mellitus (T2DM) is a disease associated with a number of metabolic disturbances, including protein metabolism. In the present study, blood samples were obtained from Bahraini subjects, including 6 patients with T2DM and 6 age- and sex-matched, non-diabetic, healthy controls. Depleted and non-depleted sera were prepared from the collected blood, and the global protein expression changes were evaluated by liquid chromatography tandem mass spectrometry. Only significantly and markedly differentially-expressed proteins (P<0.05, analysis of variance; maximum fold change ≥1.5) were considered as candidate proteins for informatics analysis. Accordingly, a total of 62 proteins were identified to be differentially expressed in T2DM, compared with control subjects, and they were grouped functionally into 16 classes of proteins. The largest class was that of the immune-associated proteins. Additionally, ~25 of these proteins (40%) had previously been associated with DM; however, the association of the other 37 proteins with T2DM was a novel observation. The majority of the identified proteins were upregulated in T2DM. The identified proteins could be involved in the pathogenesis of the disease or serve as disease biomarkers. Further validation of the identified proteins in a large study cohort is required, in order to fully access their potential clinical usefulness.

Genome-wide identification and expression analysis of new cytokinin metabolic genes in bread wheat (Triticum aestivum L.).

Cytokinins (CKs) are involved in determining the final grain yield in wheat. Multiple gene families are responsible for the controlled production of CKs in plants, including isopentenyl transferases for de novo synthesis, zeatin O-glucosyltransferases for reversible inactivation, β-glucosidases for reactivation, and CK oxidases/dehydrogenases for permanent degradation. Identifying and characterizing the genes of these families is an important step in furthering our understanding of CK metabolism. Using bioinformatics tools, we identified four new TaIPT, four new TaZOG, and 25 new TaGLU genes in common wheat. All of the genes harbored the characteristic conserved domains of their respective gene families. We renamed TaCKX genes on the basis of their true orthologs in rice and maize to remove inconsistencies in the nomenclature. Phylogenetic analysis revealed the early divergence of monocots from dicots, and the gene duplication event after speciation was obvious. Abscisic acid-, auxin-, salicylic acid-, sulfur-, drought- and light-responsive cis-regulatory elements were common to most of the genes under investigation. Expression profiling of CK metabolic gene families was carried out at the seedlings stage in AA genome donor of common wheat. Exogenous application of phytohormones (6-benzylaminopurine, salicylic acid, indole-3-acetic acid, gibberellic acid, and abscisic acid) for 3 h significantly upregulated the transcript levels of all four gene families, suggesting that plants tend to maintain CK stability. A 6-benzylaminopurine-specific maximum fold-change was observed for TuCKX1 and TuCKX3 in root and shoot tissues, respectively; however, the highest expression level was observed in the TuGLU gene family, indicating that the reactivation of the dormant CK isoform is the quickest way to counter external stress. The identification of new CK metabolic genes provides the foundation for their in-depth functional characterization and for elucidating their association with grain yield.

Differential expression of exosomal miRNAs in osteoblasts in osteoarthritis

To analyze the differentially expressed exosomal miRNAs in subchondral osteoblasts in patients with osteoarthritis (OA) and to investigate the key miRNAs potentially involved in the occurrence and progression of OA. Methods: Subchondral bones were harvested from 6 patients with OA. All subjects were divided into two groups which was based on the severity of joint wear: An OA group, severely worn side of subchondral bone, and a control group, less worn side of subchondral bone. The exosomes were extracted from osteoblast cells and their characteristics were identified. Then exosomal miRNAs were extracted and sequencing analysis was conducted to compare the expression in the two groups. The most differentially expressed ones (log2Ratio≥2) were subject to miRNA target prediction and quantitative reverse transcription PCR (RT-qPCR) to further quantify the difference. Results: Osteoblast extractions were confirmed to be exosomes, which were small double-membranous vesicles with 30-200 nm in diameter and 50-150 nm in peak value of particle size under the scanning microscope. High-throughput sequencing revealed 124 miRNAs whose expression significantly increased in the OA group. The most differentially expressed one with maximum fold change was hsa-miR-4717-5p and its target gene was RGS2. RT-qPCR demonstrated hsa-miR-4717-5p expression in the OA group was relatively higher than that in the control group (2.243 vs 0.480, P<0.01). Conclusion: There is distinct difference in expression profiles of exosomal miRNAs in subchondral osteoblasts between patients with OA and normal subjects. Up-regulated expression of miRANs might participate in OA occurrance and progression.