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The development of the Automated Agricultural Cabinet (AAC) prototype was driven by the goal of improving the aromatic qualities of locally grown rice. This compact prototype, designed with attention, employed Arduino programming to investigate genes related to the production of aromatic compounds in rice seedlings under different light conditions, including red, blue and white light. To ensure the reliability of experiments, a controlled environment was maintained at 25 °C and 75 % humidity, and rigorous testing confirmed precise light intensity measurements within the AAC. Results indicated specific light values: 9.25 ± 0.602 for red light, 11.47 ± 0.055 for blue light and 0.91 ± 0.027 µmol·m-²·s-1 for white light. The study focused on up-regulated genes in 7-day-old 'Hom Bon' rice seedlings, revealing a significant increase in P5CS and 1Δ-pyrroline gene expression. These genes are crucial in the biosynthesis pathway of 2-acetyl-1-pyrroline (2-AP), with P5CS acting as a precursor and 1Δ-pyrroline as the final step. Interestingly, the study discovered that when 7-day-old 'Hom Bon' rice seedlings were exposed to both red and blue light, gene expression rose dramatically, contrasting when exposed to white light. This finding demonstrates that the wavelength of light exposure influences gene expression in the 2-acetyl-1-pyrroline (2-AP) biosynthetic pathway of 'Hom Bon' rice seedlings throughout the 7-day growth stage. This study demonstrates the feasibility of improving the smell of indigenous rice varieties by changing pre-planting light conditions, giving a promising avenue for increased commercial appeal. Furthermore, this project aims to improve the local community's financial well-being by supporting better farming methods. [ABSTRACT FROM AUTHOR]

Sjögren's syndrome is a multisystemic autoimmune disease that mainly affects the exocrine glands, causing dryness of the eyes and the mouth as the principal symptoms. Non-coding RNAs (ncRNAs), once regarded as genomic "junk", are now appreciated as important molecular regulators of gene expression, not least in Sjögren's syndrome and other autoimmune diseases. Here we review research into the causative roles of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) on immunological responses, inflammation, and salivary gland epithelial cell function in Sjögren's syndrome patients. These ncRNAs represent promising new therapeutic targets for treating the disease and possibly as biomarkers for early diagnosis. [ABSTRACT FROM AUTHOR]

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at −80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies. [ABSTRACT FROM AUTHOR]

Pemphigus foliaceus (PF) is an autoimmune skin blistering disease characterized by antidesmoglein-1 IgG production, with an endemic form (EPF) in Brazil. Genetic and epigenetic factors have been associated with EPF, but its etiology is still not fully understood. To evaluate the genetic association of histone (de)acetylation-related genes with EPF susceptibility, we evaluated 785 polymorphisms from 144 genes, for 227 EPF patients and 194 controls. Carriers of HDAC4_rs4852054*A were more susceptible (OR = 1.79, p = 0.0038), whereas those with GSE1_rs13339618*A (OR = 0.57, p = 0.0011) and homozygotes for PHF21A_rs4756055*A (OR = 0.39, p = 0.0006) were less susceptible to EPF. These variants were not associated with sporadic PF (SPF) in German samples of 75 SPF patients and 150 controls, possibly reflecting differences in SPF and EPF pathophysiology. We further evaluated the expression of histone (de)acetylation-related genes in CD4+ T lymphocytes, using RNAseq. In these cells, we found a higher expression of KAT2B, PHF20, and ZEB2 and lower expression of KAT14 and JAD1 in patients with active EPF without treatment compared to controls from endemic regions. The encoded proteins cause epigenetic modifications related to immune cell differentiation and cell death, possibly affecting the immune response in patients with PF. [ABSTRACT FROM AUTHOR]

In sporadic idiopathic pulmonary fibrosis (sIPF) and pulmonary fibrosis caused by a mutation in telomere (TRG-PF) or surfactant related genes (SRG-PF), there are a number of aberrant cellular processes known that can lead to fibrogenesis. We investigated whether RNA expression of genes involved in these processes differed between sIPF, TRG-PF, and SRG-PF and whether expression levels were associated with survival. RNA expression of 28 genes was measured in lung biopsies of 26 sIPF, 17 TRG-PF, and 6 SRG-PF patients. Significant differences in RNA expression of TGFBR2 (p = 0.02) and SFTPA2 (p = 0.02) were found between sIPF, TRG-PF, and SRG-PF. Patients with low (

Background: Chronic obstructive pulmonary disease (COPD) is a lung disease characterised by airflow-limiting inflammation and mucus production. Acute exacerbations are a major cause of COPD-related morbidity and mortality and are mostly associated with bacterial or viral infections. A vaccine targeting non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat), the main bacteria associated with exacerbations, was tested in a Phase 2 trial. We assessed "ex-vivo" expression of vaccine candidate and housekeeping genes pd, pe, pilA, gapA, ompP6 of NTHi, and uspA2, parE, polA of Mcat in sputum samples of COPD patients and determined whether expression of the vaccine candidate genes pd, pe, pilA (NTHi) and uspA2 (Mcat) differed between stable and exacerbation samples. Methods: A single-centre, prospective, observational cohort study was conducted where 123 COPD patients were seen on enrolment, followed monthly for 2 years, and reviewed after onset of acute exacerbations. We selected 69 patients with sputum samples positive for NTHi or Mcat by PCR during at least one stable and one exacerbation visit. mRNA was isolated from the sputum, and expression of NTHi and Mcat genes was analysed with RT-PCR. Statistical analyses compared mRNA concentrations between stable and exacerbation samples and in relationship to COPD severity and exacerbation frequency. Results: The vaccine candidate genes were variably expressed in sputum samples, suggesting they are expressed in the lung. Absolute and relative expression of all NTHi vaccine candidate genes and Mcat uspA2 were similar between exacerbation and stable samples. Expression of pd and pilA was slightly associated with the number of exacerbations in the year before enrolment, and uspA2 with the disease severity status at enrolment. Conclusions: The NTHi-Mcat vaccine candidate genes were expressed in sputum samples, and each gene had a specific level of expression. No statistically significant differences in gene expression were detectable between stable and exacerbation samples. However, the history of COPD exacerbations was slightly associated with the expression of pd, pilA and uspA2. Trial registration NCT01360398 (https://www.clinicaltrials.gov) [ABSTRACT FROM AUTHOR]

Alzheimer's disease is a neurodegenerative disorder clinically defined by gradual cognitive impairment and alteration in executive function. We conducted an epigenome-wide association study (EWAS) of a clinically and neuropathologically characterized cohort of 296 brains, including Alzheimer's disease (AD) and non-demented controls (ND), exploring the relationship with the RNA expression from matched donors. We detected 5246 CpGs and 832 regions differentially methylated, finding overlap with previous EWAS but also new associations. CpGs previously identified in ANK1, MYOC, and RHBDF2 were differentially methylated, and one of our top hits (GPR56) was not previously detected. ANK1 was differentially methylated at the region level, along with APOE and RHBDF2. Only a small number of genes showed a correlation between DNA methylation and RNA expression statistically significant. Multiblock partial least-squares discriminant analysis showed several CpG sites and RNAs discriminating AD and ND (AUC = 0.908) and strongly correlated with each other. Furthermore, the CpG site cg25038311 was negatively correlated with the expression of 22 genes. Finally, with the functional epigenetic module analysis, we identified a protein–protein network characterized by inverse RNA/DNA methylation correlation and enriched for "Regulation of insulin-like growth factor transport", with IGF1 as the hub gene. Our results confirm and extend the previous EWAS, providing new information about a brain region not previously explored in AD DNA methylation studies. The relationship between DNA methylation and gene expression is not significant for most of the genes in our sample, consistently with the complexities in the gene expression regulation. [ABSTRACT FROM AUTHOR]

Onychomatricoma (OM) is a rare nail unit tumour with a characteristic presentation of finger‐like projections arising from the nail matrix. Due to the lack of transcriptome information, the mechanisms underlying its development are largely unknown. To characterize molecular features involved in the disease pathogenesis, we used digital spatial profiling (DSP) in 2 cases of OM and normal control nail units. Based on the histological evaluation, we selectively profiled 69 regions of interest covering epithelial and stromal compartments of each tissue section. Dermoscopic and histopathologic findings were reviewed in 6 cases. Single‐cell RNA sequencing of nail units and DSP were combined to define cell type contributions of OM. We identified 173 genes upregulated in stromal compartments of OM compared to onychodermis, specialized nail mesenchyme. Gene ontology analysis of the upregulated genes suggested the role of Wnt pathway activation in OM pathogenesis. We also found PLA2G2A, a known modulator of Wnt signalling, is strongly and specifically expressed in the OM stroma. The potential role of Wnt pathway was further supported by strong nuclear localization of β‐catenin in OM. Compared to the nail matrix epithelium, only a few genes were increased in OM epithelium. Deconvolution of nail unit cell types showed that onychofibroblasts are the dominant cell type in OM stroma. Altogether, integrated spatial and single‐cell multi‐omics concluded that OM is a tumour that derives a significant proportion of its origin from onychofibroblasts and is associated with upregulation of Wnt signals, which play a key role in the disease pathogenesis. [ABSTRACT FROM AUTHOR]

Recently, in vitro gene preservation has gained ground thanks to its lower cost and higher stability compared to in vivo techniques. One of the methods that can preserve female-specific W chromosome-linked genes is primordial germ cell (PGC) freezing. PGCs can be isolated from Hamburger–Hamilton stage 14–16 embryos via blood sampling. In our experiment, we used two newly established Black Transylvanian naked neck chicken cell lines and four cell lines from our gene bank. We compared two different freezing media (FAM1 and FAM2) in this study. The cell number and viability of the PGCs were measured before freezing (BF) and after thawing on Day 0, Day 1, and Day 7 of cultivation. We analyzed the germ cell-specific chicken vasa homologue (CVH) expression profile in PGCs using RT-qPCR. We found that on Day 0, immediately after thawing, the cell number in cell lines frozen with the FAM2 medium was significantly higher than in the FAM1-treated ones. On Day 1 and Day 7, the cell number and viability were also higher in most cell lines frozen with FAM2, but the difference was insignificant. The freezing also affected the chicken vasa homologue gene expression in male lines treated with both freezing media. [ABSTRACT FROM AUTHOR]

Background: Sex chromosome aneuploidies (SCAs) give rise to a broad range of phenotypic traits and diseases. Previous studies based on peripheral blood samples have suggested the presence of ripple effects, caused by altered X chromosome number, affecting the methylome and transcriptome. Whether these alterations can be connected to disease-specific tissues, and thereby having clinical implication for the phenotype, remains to be elucidated. Methods: We performed a comprehensive analysis of X chromosome number on the transcriptome and methylome in blood, fat, and muscle tissue from individuals with 45,X, 46,XX, 46,XY, and 47,XXY. Results: X chromosome number affected the transcriptome and methylome globally across all chromosomes in a tissue-specific manner. Furthermore, 45,X and 47,XXY demonstrated a divergent pattern of gene expression and methylation, with overall gene downregulation and hypomethylation in 45,X and gene upregulation and hypermethylation in 47,XXY. In fat and muscle, a pronounced effect of sex was observed. We identified X chromosomal genes with an expression pattern different from what would be expected based on the number of X and Y chromosomes. Our data also indicate a regulatory function of Y chromosomal genes on X chromosomal genes. Fourteen X chromosomal genes were downregulated in 45,X and upregulated in 47,XXY, respectively, in all three tissues (AKAP17A, CD99, DHRSX, EIF2S3, GTPBP6, JPX, KDM6A, PP2R3B, PUDP, SLC25A6, TSIX, XIST, ZBED1, ZFX). These genes may be central in the epigenetic and genomic regulation of sex chromosome aneuploidies. Conclusion: We highlight a tissue-specific and complex effect of X chromosome number on the transcriptome and methylome, elucidating both shared and non-shared gene-regulatory mechanism between SCAs. [ABSTRACT FROM AUTHOR]

Simple Summary: A mesiodens is an extra tooth located in the midline of the upper jaw. To investigate the genetic cause of mesiodens, clinical and radiographic examination were performed on 23 family members of a two-generation Hmong family. Whole exome or Sanger sequencing were performed in 22 family members. We found an extremely rare mutation (c.1807G>A;p.Glu603Lys) in the PTPN23 gene in seven family members who had mesiodens. However, six family members who did not have mesiodens also carried the mutation. The mode of inheritance appears to be autosomal dominance with incomplete penetrance (53.84%). The finding of a PTPN23 mutation as a cause of mesiodens phenotype is supported by the findings of two additional rare PTPN23 mutations in two unrelated Thai patients with mesiodens. PTPN23 is a regulator of endosomal trafficking functioning to move activated membrane receptors, such as EGFR, from the endosomal sorting complex for multivesicular body biogenesis, lysosomal degradation and subsequent downregulation of receptor signaling. Our immunohistochemical study and RNAscope on developing mouse embryos showed broad expression of PTPN23 in oral tissues, while immunofluorescence showed that EGFR was specifically concentrated in the midline epithelium. Our study showed for the first time that genetic variants in PTPN23 caused reduced phosphatase activity, disrupted midline signaling, and subsequent mesiodens formation. A mesiodens is a supernumerary tooth located in the midline of the premaxilla. To investigate the genetic cause of mesiodens, clinical and radiographic examination were performed on 23 family members of a two-generation Hmong family. Whole exome sequencing (WES) or Sanger sequencing were performed in 22 family members and two unrelated Thai patients with mesiodens. WES in the Hmong family revealed a missense mutation (c.1807G>A;p.Glu603Lys) in PTPN23 in seven affected members and six unaffected members. The mode of inheritance was autosomal dominance with incomplete penetrance (53.84%). Two additional mutations in PTPN23, c.2248C>G;p.Pro750Ala and c.3298C>T;p.Arg1100Cys were identified in two unrelated patients with mesiodens. PTPN23 is a regulator of endosomal trafficking functioning to move activated membrane receptors, such as EGFR, from the endosomal sorting complex towards the ESCRT-III complex for multivesicular body biogenesis, lysosomal degradation, and subsequent downregulation of receptor signaling. Immunohistochemical study and RNAscope on developing mouse embryos showed broad expression of PTPN23 in oral tissues, while immunofluorescence showed that EGFR was specifically concentrated in the midline epithelium. Importantly, PTPN23 mutant protein was shown to have reduced phosphatase activity. In conclusion, mesiodens were associated with genetic variants in PTPN23, suggesting that mesiodens may form due to defects in endosomal trafficking, leading to disrupted midline signaling. [ABSTRACT FROM AUTHOR]

Esophageal basaloid squamous cell carcinoma (bSCC) is a subtype of squamous cell carcinoma (SCC) with a different behavior and poor prognosis. Exploring bSCC's molecular characteristics and treatment strategies are of great clinical significance. We performed multi‐omics analysis of paired bSCC and common SCC (cSCC) using whole exome sequencing and a NanoString nCounter gene expression panel. Immunohistochemistry was used for verification of candidate biomarkers. Different treatment response was analyzed on both patients receiving neoadjuvant treatment and late‐stage patients. The common genetically‐clonal origin of bSCC and cSCC was confirmed. No significant differences between their genetic alterations or mutation spectra were observed. Mutation signature 15 (associated with defective DNA damage repair) was less prominent, and tumor mutational burden (TMB) was lower in bSCC. bSCC with an RNA expression pattern resembling cSCC had a better survival than other bSCCs. Moreover, bSCC showed significant upregulation of expression of genes associated with angiogenesis response, basement membranes, and epithelial–mesenchymal transition, and downregulation of KRT14 (squamous differentiation) and CCL21 (associated with immune response). Immunohistochemistry for SFRP1 was shown to be highly sensitive and specific for bSCC diagnosis (p < 0.001). In addition, bSCC receiving neoadjuvant immuno‐chemotherapy had a worse pathological response than bSCC receiving neoadjuvant chemotherapy (but without statistical significance), even in bSCC positive for PD‐L1. Our results demonstrated the molecular characteristics of esophageal bSCC as a subtype with a distinct RNA expression pattern and immune characteristics, but no specific genetic mutations. We provided a useful biomarker, SFRP1, for diagnosis. After outcome analysis for six bSCCs with neoadjuvant immunotherapy treatment and four late‐stage bSCCs with immunotherapy, we found that immunotherapy may not be an effective treatment option for most bSCCs. This may also provide a clue for the same subtypes of lung and head and neck cancer. Our study highlighted the heterogeneity among bSCC patients, and might explain the conflicting results of bSCC outcomes in existing studies. © 2022 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing. [ABSTRACT FROM AUTHOR]

The immune response is critical for the elimination of pathogens. However, uncontrolled immune responses can lead to tissue damage and inflammatory diseases. The generation of an immune response requires the activation, differentiation and proliferation of various immune cells and the production of cytokines that are regulated directly or indirectly by multiple pathways, including posttranscriptional regulation by RNA‐binding proteins (RBPs). Heterogeneous ribonucleoprotein D (hnRNPD), an RBP that is also known as AU‐rich elements/poly(U)‐binding/degradation factor 1 (AUF1), can regulate the expression of various target RNAs, including mRNAs, miRNAs, lncRNAs, by binding to their Au‐rich elements (AREs) or U‐/GU‐rich sequences. The target RNAs of hnRNPD range from cytokines, inflammatory receptors and growth factors to G protein‐coupled receptors; thus, hnRNPD influences various physiological processes, including the immune response. In addition, hnRNPD is strongly expressed in multiple lymphocytic tissues, suggesting that it may play an important role in the immune system. In this review, we discuss the roles of hnRNPD in the immune system and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Background and Objective: Understanding the tumor microenvironment (TME) and immune cell infiltration (ICI) may help guide immunotherapy efforts for colon cancer (COAD). However, whether ARID1B is truly regulated by hypermethylation or linked to immune infiltration remains unknown. The current work focused on the ARID1B gene expression and methylation in COAD, as well as its relation with ICI. Methods and Results: Multiple tools based on TCGA were used to analyze the differences in the expression of the ARID1B gene, DNA methylation, and its association with various clinicopathological features, somatic mutations, copy number variation, and the prognosis of patients with COAD. According to the analysis results, patients with high mRNA, low methylation levels showed better overall survival than patients with low mRNA, high methylation levels. The correlation analysis of immune cell infiltration and immune checkpoint gene expression showed that the infiltration rates of the main ICI subtypes, cancerassociated fibroblast, and myeloid cells were significantly enriched and correlated with ARID1B in COAD. An association between ARID1B expression and immune infiltration in COAD was found by correlating ICI indicators with ARID1B expression in the immune cell composition of the COAD microenvironment. Notably, M2 chemokines were related to ARID1B expression, while M1 chemokines were not. Conclusion: This study provided evidence that ARID1B may have a role in the pathogenesis of COAD. The specific underlying mechanisms that could be responsible for ARID1B’s downregulation in COAD will need to be investigated in the future. [ABSTRACT FROM AUTHOR]

The culmination of conventional yield improving parameters has widened the margin between food demand and crop yield, leaving the potential yield productivity to be bridged by the manipulation of photosynthetic processes in plants. Efficient strategies to assess photosynthetic capacity in crops need to be developed to identify suitable targets that have the potential to improve photosynthetic efficiencies. Here, we assessed the photosynthetic capacity of the Japanese soybean mini core collection (GmJMC) using a newly developed high-throughput photosynthesis measurement system "MIC-100" to analyze physiological mechanisms and genetic architecture underpinning photosynthesis. K-means clustering of light-saturated photosynthesis (Asat) classified GmJMC accessions into four distinct clusters with Cluster2 comprised of highly photosynthesizing accessions. Genome-wide association analysis based on the variation of Asat revealed a significant association with a single nucleotide polymorphism (SNP) on chromosome 17. Among the candidate genes related to photosynthesis in the genomic region, variation in expression of a gene encoding G protein alpha subunit 1 (GPA1) showed a strong correlation (r = 0.72, p < 0.01) with that of Asat. Among GmJMC accessions, GmJMC47 was characterized by the highest Asat , stomatal conductance (gs), stomatal densit y (SDensity), electron transfer rate (ETR), and light use efficiency of photosystem II (Fv'/Fm′) and the lowest non-photochemical quenching [ NPQ (t)], indicating that GmJMC47 has greater CO2 supply and efficient light-harvesting systems. These results provide strong evidence that exploration of plant germplasm is a useful strategy to unlock the potential of resource use efficiencies for photosynthesis. [ABSTRACT FROM AUTHOR]

Purpose: Docosahexaenoyl difluorodeoxycytidine (DHA–dFdC) is an amide with potent, broad-spectrum antitumor activity. In the present study, DHA–dFdC's ability to induce immunogenic cell death (ICD) was tested using CT26 mouse colorectal cancer cells, an established cell line commonly used for identifying ICD inducers, as well as Panc-02 mouse pancreatic cancer cells. Methods: The three primary surrogate markers of ICD (i.e., calreticulin (CRT) surface translocation, ATP release, and high mobility group box 1 protein (HMGB1) release) were measured in vitro. To confirm DHA–dFdC's ability to induce ICD in vivo, the gold standard mouse vaccination studies were conducted using both CT26 and Panc-02 models. Additionally, the effect of DHA–dFdC on tumor response to anti-programmed cell death protein 1 monoclonal antibody (anti-PD-1 mAb) were tested in mice with pre-established Panc-02 tumors. RNA sequencing experiments were conducted on PANC-1 human pancreatic cancer cells treated with DHA–dFdC, dFdC, or vehicle control in vitro. Results: DHA–dFdC elicited CRT surface translocation and ATP and HMGB1 release in both cell lines. Immunization of mice with CT26 or Panc-02 cells pretreated with DHA–dFdC prevented or delayed the development of corresponding secondary live challenge tumor. DHA–dFdC enabled Panc-02 tumors to respond to anti-PD-1 mAb. RNA sequencing experiments revealed that DHA–dFdC and dFdC differentially impacted genes related to the KRAS, TP53, and inflammatory pathways, and DHA–dFdC enriched for the unfolded protein response (UPR) compared to control, providing insight into DHA–dFdC's potential mechanism of inducing ICD. Conclusion: DHA–dFdC is a bona fide ICD inducer and can render pancreatic tumors responsive to anti-PD-1 mAb therapy. [ABSTRACT FROM AUTHOR]

Objectives: Mutations in the STXBP1 gene have been associated with epileptic encephalopathy. Previous studies from in vitro neuroblastoma 2A cells showed that haploinsufficiency of STXBP1 is the mechanism for epileptic encephalopathy. In this ex vivo study, STXPB1 DNA mutations and RNA expression were assessed from two patients to help understand the impact of STXBP1 mutations on the disease etiology and mechanism. Methods: Microarray analysis and DNA sequencing were performed on two children with development delay, one with and one without infantile spasms. Different pathogenic mutations of STXBP1 were identified in the patients and RNA expression of STXPB1 was then performed by RT-Q-PCR on RNA extracted from blood samples of each patient. Results: Pathogenic deletion [of exons 13–20 and 3′ downstream of STXBP1 ] and nonsense mutation [c.1663G>T (p.Glu555X) in exon 18 of STXBP1 ] were detected from the two patients, respectively. RNA analysis showed that 1) the deletion mediated RNA decay, and that 2) no RNA decay was identified for the nonsense mutation at codon 555 which predicts a truncated STXBP1 protein. Significance: Our RNA expression analyses from the patient blood samples are the first ex vivo studies to support that both haploinsufficiency and truncation of STXBP1 protein (either dominant negative or haploinsufficiency) are causative mechanisms for epileptic encephalopathies, intellectual disability and developmental delay. The RNA assay also suggests that escape from nonsense-mediated RNA decay is possible when the nonsense mutation resides <50 nucleotides upstream of the last coding exon-exon junction even in the presence of additional non-coding exons that are 3′ downstream of the last coding exon. [ABSTRACT FROM AUTHOR]

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and treatment of advanced disease is challenging. Clinical trials with multi-tyrosine kinase inhibitors in the past have yielded disappointing results. Here, we investigated fibroblast growth factor (FGF) receptors and their pathways in adrenocortical tumors as potential treatment targets. We performed real-time RT-PCR of 93 FGF pathway related genes in a cohort of 39 fresh frozen benign and malignant adrenocortical, 9 non-adrenal tissues and 4 cell lines. The expression of FGF receptors was validated in 166 formalin-fixed paraffin embedded (FFPE) tissues using RNA in situ hybridization (RNAscope) and correlated with clinical data. In malignant compared to benign adrenal tumors, we found significant differences in the expression of 16/94 FGF receptor pathway related genes. Genes involved in tissue differentiation and metastatic spread through epithelial to mesechymal transition were most strongly altered. The therapeutically targetable FGF receptors 1 and 4 were upregulated 4.6- and 6-fold, respectively, in malignant compared to benign adrenocortical tumors, which was confirmed by RNAscope in FFPE samples. High expression of FGFR1 and 4 was significantly associated with worse patient prognosis in univariate analysis. After multivariate adjustment for the known prognostic factors Ki-67 and ENSAT tumor stage, FGFR1 remained significantly associated with recurrence-free survival (HR=6.10, 95%CI: 1.78 – 20.86, p=0.004) and FGFR4 with overall survival (HR=3.23, 95%CI: 1.52 – 6.88, p=0.002). Collectively, our study supports a role of FGF pathways in malignant adrenocortical tumors. Quantification of FGF receptors may enable a stratification of ACC for the use of FGFR inhibitors in future clinical trials. [ABSTRACT FROM AUTHOR]

Estrogen deficiency has been shown to negatively influence rotator cuff tendon healing. Therefore, the addition of an estrogen‐like‐compound (ELC) in a nonestrogen‐deficient animal may improve the quality of a rotator cuff repair. The purpose of this study was to evaluate the effects of an ELC, diethylstilbestrol (DES), on tendon healing in a murine rotator cuff repair model. Thirty‐three male wild‐type mice (C57BL/6NJ) were randomly divided into three study groups. Group 1—unoperated mice with normal rotator cuff tendons. Groups 2 and 3 consisted of surgically repaired rotator cuff tendons; Group 2 (repair‐only) was the standard repair group (no DES injected), whereas Group 3 (repair + DES) was the experimental repair group (injected with DES). Comparing the maximal thickness of calcified fibrocartilage to uncalcified fibrocartilage, the ratios for the control (intact tendon), repair‐only, and repair + DES groups were 2:1, 0.9:1, and 1.7:1. RNA expression data demonstrated upregulation of chondrogenic, angiogenic, and tendon modulation genes in the repair‐ only group compared to the control (intact tendon) group (p < 0.04 for all), and that addition of DES further increased the osteogenic, angiogenic, and tendon modulation gene expression compared to the repair‐only group (p < 0.02). Immunohistochemical analysis indicated that the addition of DES further increased osteogenic, angiogenic, and tendon maturation protein expression at the enthesis compared to standard repairs. [ABSTRACT FROM AUTHOR]

Aim: Splicing factor proline and glutamine rich (SFPQ) is an RNA–DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case–control cohorts. Methods: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA‐sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. Results: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ‐positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. Conclusions: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology. [ABSTRACT FROM AUTHOR]

Interfering RNA technology has been established as an effective strategy to protect plants against viral infection. Despite this success, interfering RNA (RNAi) has rarely been applied due to the regulatory barriers that confront genetically engineered plants and concerns over possible environmental and health risks posed by non-endogenous small RNAs. 'HoneySweet' was developed as a virus-resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by the plum pox virus. 'HoneySweet' has been approved for cultivation in the United States but not in countries where the plum pox virus is endemic. In this study, we evaluated the long-term efficacy of virus resistance in 'HoneySweet,' the nature and stability of its sRNA profile, and the potential health risks of consuming 'HoneySweet' plums. Graft-challenged 'HoneySweet' trees carrying large non-transgenic infected limbs remained virus-free after more than 10 years in the field, and the viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and, on average, were more than 10 times lower than those present in symptom-less fruits from virus-infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice, and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene-derived sRNAs to the mouse genome. Collectively, the data confirmed that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing. [ABSTRACT FROM AUTHOR]

Purpose: To characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of partial (PG) and complete globozoospermic (CG) men in order to identify the best reproductive treatment. Methods: We assessed spermatozoa from 14 consenting men ultrastructurally, as well as for histone content, sperm chromatin integrity, and sperm aneuploidy. Additional genomic, transcriptomic, and proteomic evaluations were carried out to further characterize the CG cohort. The presence of oocyte-activating sperm cytosolic factor (OASCF) was measured by a phospholipase C zeta (PLCζ) immunofluorescence assay. Couples were treated in subsequent cycles either by conventional ICSI or by ICSI with assisted gamete treatment (AGT) using calcium ionophore (Ionomycin, 19657, Sigma-Aldrich, Saint Louis, MO, USA). Results: Ultrastructural assessment confirmed complete acrosome deficiency in all spermatozoa from CG men. Histone content, sperm chromatin integrity, and sperm aneuploidy did not differ significantly between the PG (n = 4) and CG (n = 10) cohorts. PLCζ assessment indicated a positive presence of OASCF in 4 PG couples, who underwent subsequent ICSI cycles that yielded a 36.1% (43/119) fertilization with a 50% (2/4) clinical pregnancy and delivery rate. PLCζ assessment failed to detect OASCF for 8 CG patients who underwent 9 subsequent ICSI cycles with AGT, yielding a remarkable improvement of fertilization (39/97; 40.2%) (P = 0.00001). Embryo implantation (6/21; 28.6%) and clinical pregnancies (5/7; 71.4%) were also enhanced, resulting in 4 deliveries. Gene mutations (DPY19L2, SPATA16, PICK1) were identified in spermatozoa from CG patients. Additionally, CG patients unable to sustain a term pregnancy had gene mutations involved in zygote development (NLRP5) and postnatal development (BSX). CG patients who successfully sustained a pregnancy had a mutation (PIWIL1) related to sperm phenotype. PLCZ1 was both mutated and underexpressed in these CG patients, regardless of reproductive outcome. Conclusions: Sperm bioassays and genomic studies can be used to characterize this gamete's capacity to support embryonic development and to tailor treatments maximizing reproductive outcome. [ABSTRACT FROM AUTHOR]

The treatment and early outcome of patients with acute myocardial infarction (MI) have dramatically improved the past decades, but the incidence of left ventricular (LV) dysfunction post-MI remains high. Peripheral blood RNAs reflect pathophysiological changes during acute MI and the inflammatory process. Therefore, these RNAs are promising new markers to molecularly phenotype patients and improve the early identification of patients at risk of subsequent LV dysfunction. We here discuss the coding and long non-coding RNAs that can be measured in peripheral blood of patients with acute MI and list the advantages and limitations for implementation in clinical practice. Although some studies provide preliminary evidence of their diagnostic and prognostic potential, the use of these makers has not yet been implemented in clinical practice. The added value of RNAs to improve treatment and outcome remains to be determined in larger clinical studies. International consortia are now catalyzing renewed efforts to investigate novel RNAs that may improve post-MI outcome in a precision-medicine approach. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL; Leishmania donovani) cases produce interferon-γ and tumor necrosis factor in response to soluble leishmanial antigen (SLA) in whole-blood assays. Using transcriptional profiling, we demonstrate the impact of interleukin-10 (IL-10), a cytokine implicated in VL, on this response. SLA stimulation identified 28 differentially expressed genes (DEGs), 17/28 in a single network with TNF as hub. SLA plus anti-IL-10 produced 454 DEGs, 292 in a single network with TNF, IFNG, NFKBIA, IL6, and IL1B as hubs in concert with a remarkable chemokine/cytokine storm. Our data demonstrate the singular effect of IL-10 as a potent immune modulator in VL. [ABSTRACT FROM AUTHOR]

Background: DNA methylation is likely a key mechanism regulating changes in gene transcription in traits that show temporal fluctuations in response to environmental conditions. To understand the transcriptional role of DNA methylation we need simultaneous within-individual assessment of methylation changes and gene expression changes over time. Within-individual repeated sampling of tissues, which are essential for trait expression is, however, unfeasible (e.g. specific brain regions, liver and ovary for reproductive timing). Here, we explore to what extend between-individual changes in DNA methylation in a tissue accessible for repeated sampling (red blood cells (RBCs)) reflect such patterns in a tissue unavailable for repeated sampling (liver) and how these DNA methylation patterns are associated with gene expression in such inaccessible tissues (hypothalamus, ovary and liver). For this, 18 great tit (Parus major) females were sacrificed at three time points (n = 6 per time point) throughout the pre-laying and egg-laying period and their blood, hypothalamus, ovary and liver were sampled. Results: We simultaneously assessed DNA methylation changes (via reduced representation bisulfite sequencing) and changes in gene expression (via RNA-seq and qPCR) over time. In general, we found a positive correlation between changes in CpG site methylation in RBCs and liver across timepoints. For CpG sites in close proximity to the transcription start site, an increase in RBC methylation over time was associated with a decrease in the expression of the associated gene in the ovary. In contrast, no such association with gene expression was found for CpG site methylation within the gene body or the 10 kb up- and downstream regions adjacent to the gene body. Conclusion: Temporal changes in DNA methylation are largely tissue-general, indicating that changes in RBC methylation can reflect changes in DNA methylation in other, often less accessible, tissues such as the liver in our case. However, associations between temporal changes in DNA methylation with changes in gene expression are mostly tissue- and genomic location-dependent. The observation that temporal changes in DNA methylation within RBCs can relate to changes in gene expression in less accessible tissues is important for a better understanding of how environmental conditions shape traits that temporally change in expression in wild populations. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing. [ABSTRACT FROM AUTHOR]

Background: Vertebral fractures (VFs) are the most common clinical manifestation of osteoporosis associated with high morbimortality. A personal/familiar history of fractures increases the risk of fractures. The purpose of this study is to identify possible molecular markers associated with osteoporotic VFs in elderly women from community. Methods: Transcriptomic analysis using Affymetrix HTA2 microarray was performed using whole blood samples of 240 subjects from a population‐based survey (Sao Paulo Ageing & Health [SPAH] study). Only elderly women with osteoporosis diagnosis by densitometry were analyzed, and divided in two groups: VF: women with osteoporosis and VFs versus no vertebral fracture (NVF): women with osteoporosis and NVFs. They were matched for age, chronic disease, medication use, and bone mineral density (BMD). The logistic regression model adjusted for age was applied for transcriptome data analysis. SYBR green‐based quantitative polymerase chain reaction (qPCR) was used to validate the most significant expression changes obtained in the microarray experiment. Results: Microarray analysis identified 142 differentially expressed genes (DEGs, p <.01), 57 upregulated and 85 downregulated, compared VF versus NVF groups. The DEG with the greatest expression difference was the Gamma2‐Syntrophin (SNTG2) (β = 31.88, p =.005). Validation by qPCR confirmed increased expression in VF group of Syntrophin (SNTG2, fold change = 2.79, p =.009), TRAF3 Interacting Protein2 (TRAF3IP2, fold change = 2.79, p =.020), and Integrin Subunit Alpha 6 (ITGA6, fold change = 2.86, p =.038). Conclusion: Our data identified and validated the association of SNTG2 (608715), TRAF3IP2 (607043), and ITGA6 (147556) with osteoporotic VF in elderly women, independently of BMD. These results suggest that these transcripts have potential clinical significance and may help to explain the molecular mechanisms and biological functions of vertebral fracture. [ABSTRACT FROM AUTHOR]

Background/aim: Bone morphogenetic protein-15 (BMP-15) is one of the maturation indicators of the ovarian follicular pool. The aim of this study was to investigate the possible difference of follicular fluid (FF) BMP-15 RNA expression among low, normal, and high responder women attending controlled ovarian hyperstimulation-intracytoplasmic sperm injection (COH-ICSI) cycles. Materials and methods: This cross-sectional study was conducted with 75 FFs of COH-ICSI cycles performed at the IVF Unit of University Hospital. Twenty FF from low response (group 1), 27 FF from normal response (group 2), and 28 FF from high response (group 3) were recruited for the study between September 2014 and February 2015. Cycle parameters were collected from patient files. FF BMP-15 RNA expression was evaluated with real-time polymerase chain reaction analysis. Statistical analysis was done with SPSS 16.0 version (SPSS Chicago, IL, USA). Results: The mean age, infertility duration, and body mass index (BMI) of patients were 31.1 ± 4.4 years, 7.4 ± 4.5 years, and 25.6 ± 4.1 kg/m2, respectively. There was no significant difference among groups for age, infertility duration, and BMI. There was no significant difference among groups for fertilization rate, implantation rate, pregnancy rate, and live birth rate. Among the 3 groups, FF BMP-15 RNA overexpression and lower expression rates were not significantly different. In all groups, overexpression showed dominance. The pregnancy rate was 45% among women with lower expression and the pregnancy rate among women with overexpression was 26% (P = 0.02). BMP-15 overexpression showed impact on becoming pregnant (OR = 3.7, 95% CI = 1.245–11.299, P = 0.019). Conclusion: In this study, there was no significant difference in FF BMP-15 RNA expression levels among low, normal, and high responder women. However, overexpression of FF BMP-15 RNA showed a negative impact on pregnancy rates of women who attended COH-ICSI cycles. [ABSTRACT FROM AUTHOR]

Stretch and tachycardia are common triggers for cardiac remodelling in various conditions, but a comparative characterization of their role in the excitation‐transcription coupling (ETC) and early regulation of gene expression and structural changes is lacking. Here, we show that stretch and tachycardia directly induced hypertrophy of neonatal rat cardiac myocytes and also of non‐myocytes. Both triggers induced similar patterns of hypertrophy but had largely distinct gene expression profiles. ACTA1 served as good hypertrophy marker upon stretch, while RCAN1 was found increased in response to tachycardia in a rate‐dependent fashion. Mechanistically, several calcium‐handling proteins, including the sodium‐calcium exchanger (NCX), contributed to ETC. Phosphorylation of the calcium/calmodulin‐dependent protein kinase II (CaMKII) was elevated and occurred downstream of NCX activation upon tachycardia, but not stretch. Microarray profiling revealed that stretch and tachycardia regulated around 33% and 20% genes in a NCX‐dependent manner, respectively. In conclusion, our data show that hypertrophy induction by stretch and tachycardia is associated with different gene expression profiles with a significant contribution of the NCX. [ABSTRACT FROM AUTHOR]

Sleep regulation involves interleukin-1β (IL1) family members, TNF, and circadian clock genes. Previously, we characterized spontaneous sleep and sleep after 8 h of sleep deprivation (SD) ending at zeitgeber time (ZT)4 and ZT16 in wild-type (WT) and IL1 receptor accessory protein (AcP)- and brain-specific AcP (AcPb)-knockout (KO) mice. Here, we applied quantitative reverse transcriptase polymerase chain reaction and Spearman gene pair expression correlation methods to characterize IL1, IL1 receptor 1 (IL1R1), AcP, AcPb, Period 1 (Per1), Clock, adenosine deaminase (Ada), peptidoglycan recognition protein 1 (Pglyrp1), and TNF mRNA expressions under conditions with distinct sleep phenotypes. In WT mice, IL1, IL1R1, AcP, Ada, and Clock mRNAs were higher at ZT4 (mid-sleep period) than at ZT16. mRNA expressions differed substantially in AcP and AcPb KO mice at those times. After SD ending at ZT4, only WT mice had a non-rapid eye movement sleep (NREMS) rebound, and AcPb and IL1R1 mRNA increases were unique to WT mice. In AcPb KO mice, which have spontaneous high EEG slow wave power, AcP and Pglyrp1 mRNAs were elevated relative to WT mice at ZT4. At ZT4, the AcPb KO- WT Spearman correlation difference networks showed high positive correlations between IL1R1 and IL1, Per1, and Clock and high negative correlations between TNF and Pglyrp1 and Ada. At ZT16, the WT mice gene pair expression network was mostly negative, whereas in AcP KO mice, which have substantially more rapid eye movement sleep than WT mice, it was all positive. We conclude that gene pair expression correlations depend on the presence of AcP and AcPb. [ABSTRACT FROM AUTHOR]

The outcome in esophageal adenocarcinoma (EAC) is still poor with only 20% of patients in Western populations surviving for more than 5 years. Almost nothing is known about the precise composition of immune cells and their gene expression profiles in primary resected EACs and also nothing compared to neoadjuvant treated EACs. This study analyzes and compares immune profiles of primary resected and neoadjuvant treated esophageal adenocarcinoma and unravels possible targets for immunotherapy. We analyzed 47 EAC in total considering a set of 30 primary treatment-naive EACs and 17 neoadjuvant pretreated (12 × CROSS, 5 × FLOT) using the Nanostring's panel-based gene expression platform including 770 genes being important in malignant tumors and their immune micromileu. Most of the significantly altered genes are involved in the regulation of immune responses, T-and B cell functions as well as antigen processing. Chemokine-receptor axes like the CXCL9, -10,-11/CXCR3- are prominent in esophageal adenocarcinoma with a fold change of up to 9.5 promoting cancer cell proliferation and metastasis. ARG1, as a regulator of T-cell fate is sixfold down-regulated in untreated primary esophageal tumors. The influence of the currently used neoadjuvant treatment revealed a down-regulation of nearly all important checkpoint markers and inflammatory related genes in the local microenvironment. We found a higher expression of checkpoint markers like LAG3, TIM3, CTLA4 and CD276 in comparison to PD-L1/PD-1 supporting clinical trials analyzing the efficacy of a combination of different checkpoint inhibitors in EACs. We found an up-regulation of CD38 or LILRB1 as examples of additional immune escape mechanism. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is a complex disorder caused by a combination of genetic and non-genetic risk factors. In addition, an increasing evidence suggests that epigenetic mechanisms also accompany AD. Genetic and epigenetic factors are not independent, but multiple loci show genetic-epigenetic interactions, the so-called quantitative trait loci (QTLs). Recently, we identified the first QTL association with AD, namely Peptidase M20 Domain Containing 1 (PM20D1). We observed that PM20D1 DNA methylation, RNA expression, and genetic background are correlated and, in turn, associated with AD. We provided mechanistic insights for these correlations and had shown that by genetically increasing and decreasing PM20D1 levels, AD-related pathologies were decreased and accelerated, respectively. However, since the PM20D1 QTL region encompasses also other genes, namely Nuclear Casein Kinase and Cyclin Dependent Kinase Substrate 1 (NUCKS1); RAB7, member RAS oncogene family-like 1 (RAB7L1); and Solute Carrier Family 41 Member 1 (SLC41A1), we investigated whether these genes might also contribute to the described AD association. Results: Here, we report a comprehensive analysis of these QTL genes using a repertoire of in silico methods as well as in vivo and in vitro experimental approaches. First, we analyzed publicly available databases to pinpoint the major QTL correlations. Then, we validated these correlations using a well-characterized set of samples and locus-specific approaches—i.e., Sanger sequencing for the genotype, cloning/sequencing and pyrosequencing for the DNA methylation, and allele-specific and real-time PCR for the RNA expression. Finally, we defined the functional relevance of the observed alterations in the context of AD in vitro. Using this approach, we show that only PM20D1 DNA methylation and expression are significantly correlated with the AD-risk associated background. We find that the expression of SLC41A1 and PM20D1—but not NUCKS1 and RAB7L1—is increased in mouse models and human samples of AD, respectively. However, SLC41A1 and PM20D1 are differentially regulated by AD-related stressors, with only PM20D1 being upregulated by amyloid-β and reactive oxygen species, and with only PM20D1 being neuroprotective when overexpressed in cell and primary cultures. Conclusions: Our findings reinforce PM20D1 as the most likely gene responsible of the previously reported PM20D1 QTL association with AD. [ABSTRACT FROM AUTHOR]

Background: Treatment of urethral trauma is currently done after urethral stricture occurs. Stricture therapy after occurrence gives unsatisfactory success rates. Several genes, such as transforming growth factor beta 1 (TGF-β1), matrix metalloproteinase 1 (MMP-1), connective tissue growth factor (CTGF), and plasminogen activator inhibitor 1 (PAI-1), have a proven role in urethral stricture development. The purpose of this study was to assess the effect docetaxel and/or captopril on the RNA expression of those genes. Methods: The subjects of this research were 26 male New Zealand rabbits aged 230 ± 20 days weighing 4-5 kg that underwent urethral rupture by endoscopic resection under anesthetized conditions. Subjects were divided into five groups; control, stricture, captopril (captopril 0.05 mg/rabbit/day), docetaxel (docetaxel 0.1 mg/rabbit/day), and docetaxel-captopril (docetaxel 0.1 mg/rabbit/day and captopril 0.05 mg/rabbit/day). Each group consisted of 4-6 rabbits. Each rabbit received a water-soluble transurethral gel containing drug according to its group for 28 days. After the treatment period, rabbits were sacrificed with 200 mg Pentothal, and the corpus spongiosum was then prepared for real-time PCR examination. Results: TGF-β1 RNA expression in the stricture group was statistically different from that in the control, docetaxel and docetaxel-captopril groups (p = 0.016; p = 0.016; p = 0.004). The stricture group did not exhibit any statistical difference from the captopril group (p = 0.190). The control group did not show any statistically difference from the captopril, docetaxel, and docetaxel-captopril groups (p = 0.114; p = 0.190; p = 1.000). Docetaxel-captopril suppresses expression of TGF-β1 RNA most significantly. MMP-1 RNA expression showed no significant differences among groups (p = 0.827). The docetaxel group and stricture group pair was most significant (p = 0.247), compared with other pairs of stricture groups in MMP-1 RNA expression. CTGF RNA expression in the stricture group was statistically different from that of control, captopril, docetaxel, and docetaxel-captopril groups (p = 0.003; p = 0.019; p = 0.005; p = 0.005). The control group did not exhibit any statistically difference from the captopril, docetaxel, and docetaxel-captopril groups (p = 0.408; p = 0.709; p = 0.695). There was no statistical difference among treatment groups. Docetaxel and docetaxel-captopril groups suppress the most significant expression of CTGF RNA expression. PAI-1 RNA expression in the stricture group differed statistically significantly from the control and docetaxel groups (p = 0.044; p = 0.016). The stricture group did not show any statistically significant difference from the captopril and docetaxel-captopril groups (p = 0.763; p = 0.086). The control group did not exhibit any statistical difference with any of the treatment groups (p = 0.101; p = 0.637; p = 0.669). Conclusion: Docetaxel-captopril gel proved to be able to inhibit RNA expression of TGF-β1 and CTGF significantly. Captopril gel proved to be able to inhibit RNA expression of CTGF significantly. Docetaxel gel proved to be able to inhibit RNA expression of TGF-β1, CTGF, and PAI-1 significantly. There were no differences in MMP-1 expression among all study groups. Longer follow up after therapy discontinuation and greater sample size is needed to determine the therapeutic effect. [ABSTRACT FROM AUTHOR]

Converging evidence suggests a role of serotonin (5-hydroxytryptamine, 5-HT) and tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme of 5-HT synthesis in the brain, in modulating long-term, neurobiological effects of early-life adversity. Here, we aimed at further elucidating the molecular mechanisms underlying this interaction, and its consequences for socio-emotional behaviors, with a focus on anxiety and social interaction. In this study, adult, male Tph2 null mutant (Tph2 -/-) and heterozygous (Tph2 +/-) mice, and their wildtype littermates (Tph2 +/+) were exposed to neonatal, maternal separation (MS) and screened for behavioral changes, followed by genome-wide RNA expression and DNA methylation profiling. In Tph2 -/- mice, brain 5-HT deficiency profoundly affected socio-emotional behaviors, i.e., decreased avoidance of the aversive open arms in the elevated plus-maze (EPM) as well as decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Tph2 +/- mice showed an ambiguous profile with context-dependent, behavioral responses. In the EPM they showed similar avoidance of the open arm but decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Notably, MS effects on behavior were subtle and depended on the Tph2 genotype, in particular increasing the observed avoidance of EPM open arms in wildtype and Tph2 +/- mice when compared to their Tph2 -/- littermates. On the genomic level, the interaction of Tph2 genotype with MS differentially affected the expression of numerous genes, of which a subset showed an overlap with DNA methylation profiles at corresponding loci. Remarkably, changes in methylation nearby and expression of the gene encoding cholecystokinin, which were inversely correlated to each other, were associated with variations in anxiety-related phenotypes. In conclusion, next to various behavioral alterations, we identified gene expression and DNA methylation profiles to be associated with TPH2 inactivation and its interaction with MS, suggesting a gene-by-environment interaction-dependent, modulatory function of brain 5-HT availability. [ABSTRACT FROM AUTHOR]

Lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the two major subtypes of lung cancer. To explore mitochondrial respiratory gene expression profiles in LUSC and LUAD, RNA sequencing data from The Cancer Genome Atlas was used for comprehensive analyses to establish the molecular characteristics of LUSC and LUAD. To elucidate expression profiles, subtypes were defined using unsupervised clustering of mitochondrial gene expression data. Differences in nuclear gene expression levels, signaling pathways and tumor microenvironments between subtypes were investigated. The analysis revealed that mitochondrial respiratory genes were generally expressed at lower levels in tumor tissues compared with matched control tissues. The expression of mitochondrially encoded NADH dehydrogenase 5 or 6 was associated with tumor progression in LUAD and LUSC. Patients were clustered into three subgroups based on the expression profile of 13 mitochondrial protein‑encoding genes, and patients in Cluster 3 exhibited poor survival rates compared with patients from Cluster 1. Furthermore, this association was also observed in another independent data set. Further analyses of the expression of nuclear‑encoded genes in the three clusters revealed the enrichment of several cancer‑associated signaling pathways in Cluster 3, particularly the apoptotic signaling pathway, suggesting a potential association between the decreased expression of mitochondrial DNA genes and increased tumor aggressiveness. Furthermore, the analyses of immune cell compositions in the tumor microenvironment detected a significant increase in the proportion of CD4+ T cells and a decrease in the proportion of macrophages in LUAD compared with LUSC (P=0.0000104 and P=0.0000105, respectively). In conclusion, the present study revealed an association between the expression patterns of mitochondrial‑encoded genes and lung cancer, which may contribute to novel therapeutic strategies for patients with LUSC and LUAD. [ABSTRACT FROM AUTHOR]

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Investigating the expression of RNAs that differ by short or single nucleotide sequences at a single-cell level in tissue has been limited by the sensitivity and specificity of in situ hybridization (ISH) techniques. Detection of short isoform-specific sequences requires RNA isolation for PCR analysis—an approach that loses the regional and cell-type-specific distribution of isoforms. Having the capability to distinguish the differential expression of RNA variants in tissue is critical because alterations in mRNA splicing and editing, as well as coding single nucleotide polymorphisms, have been associated with numerous cancers, neurological and psychiatric disorders. Here we introduce a novel highly sensitive single-probe colorimetric/fluorescent ISH approach that targets short exon/exon RNA splice junctions using single-pair oligonucleotide probes (~ 50 bp). We use this approach to investigate, with single-cell resolution, the expression of four transcripts encoding the neuregulin (NRG) receptor ErbB4 that differ by alternative splicing of exons encoding two juxtamembrane (JMa/JMb) and two cytoplasmic (CYT-1/CYT-2) domains that alter receptor stability and signaling modes, respectively. By comparing ErbB4 hybridization on sections from wild-type and ErbB4 knockout mice (missing exon 2), we initially demonstrate that single-pair probes provide the sensitivity and specificity to visualize and quantify the differential expression of ErbB4 isoforms. Using cell-type-specific GFP reporter mice, we go on to demonstrate that expression of ErbB4 isoforms differs between neurons and oligodendrocytes, and that this differential expression of ErbB4 isoforms is evolutionarily conserved to humans. This single-pair probe ISH approach, known as BaseScope, could serve as an invaluable diagnostic tool to detect alternative spliced isoforms, and potentially single base polymorphisms, associated with disease. [ABSTRACT FROM AUTHOR]

Major depressive disorder (MDD) is a complex illness caused by both genetic and environmental factors. Antidepressant resistance also has a genetic component. To date, however, very few genes have been identified for major depression or antidepressant resistance. In this study, we investigated whether outbred heterogeneous stock (HS) rats would be a suitable model to uncover the genetics of depression and its connection to antidepressant resistance. The Wistar Kyoto (WKY) rat, one of the eight founders of the HS, is a recognized animal model of juvenile depression and is resistant to fluoxetine antidepressant treatment. We therefore hypothesized that adolescent HS rats would exhibit variation in both despair‐like behavior and response to fluoxetine treatment. We assessed heritability of despair‐like behavior and response to sub‐acute fluoxetine using a modified forced swim test (FST) in 4‐week‐old HS rats. We also tested whether blood transcript levels previously identified as depression biomarkers in adolescent human subjects are differentially expressed in HS rats with high vs. low FST immobility. We demonstrate heritability of despair‐like behavior in 4‐week‐old HS rats and show that many HS rats are resistant to fluoxetine treatment. In addition, blood transcript levels of Amfr, Cdr2 and Kiaa1539, genes previously identified in human adolescents with MDD, are differentially expressed between HS rats with high vs. low immobility. These data demonstrate that FST despair‐like behavior will be amenable to genetic fine‐mapping in adolescent HS rats. The overlap between human and HS blood biomarkers suggest that these studies may translate to depression in humans. [ABSTRACT FROM AUTHOR]

Background: Despite increased understanding of the pathophysiology of rotator cuff tears and the evolution of rotator cuff repair, healing failure remains a substantial problem. The critical roles played by biological factors have been emphasized, but little is known of the implications of gene expression profile differences at the time of repair. Purpose: To document the relationship between the perioperative gene expression of healed and unhealed rotator cuffs by RNA microarray analysis. Study Design: Case-control study; Level of evidence, 3. Methods: Superior (supraspinatus involvement) and posterosuperior (supraspinatus and infraspinatus involvement) tears were included in the study. Samples of rotator cuff tendons were prospectively collected during rotator cuff surgery. Three samples were harvested at the tendon ends of tears from the anterior, middle (apex), and posterior parts using an arthroscopic punch. Seven patients with an unhealed rotator cuff were matched one-to-one with patients with a healed rotator cuff by sex, age, tear size, and fatty degeneration of rotator cuff muscles. mRNA microarray analysis was used to identify genetic differences between healed and unhealed rotator cuff tendons. Gene ontology and gene association files were obtained from the Gene Ontology Consortium, and the Gene Ontology system in DAVID was used to identify enhanced biological processes. Results: Microarray analyses identified 262 genes that were differentially expressed by at least 1.5-fold between the healed and unhealed groups. Overall, in the healed group, 103 genes were significantly downregulated, and 159 were significantly upregulated. DAVID Functional Annotation Cluster analysis showed that in the healed group, the genes most upregulated were related to the G protein–coupled receptor protein signaling pathway and to the neurological system. On the other hand, the genes most downregulated were related to immune and inflammatory responses. BMP5 was the gene most upregulated in the healed group, and the majority of downregulated genes were involved in the immune/inflammatory response. Conclusion: The downregulation of inflammatory response genes and the upregulation of cell differentiation genes in torn rotator cuffs at the time of surgery are related to rotator cuff healing. These results provide useful baseline information for future biological studies on rotator cuff healing. [ABSTRACT FROM AUTHOR]

Acute ischemic stroke affects over 800,000 US adults annually, with hundreds of thousands more experiencing a transient ischemic attack. Emergent evaluation, prompt acute treatment, and identification of stroke or TIA (transient ischemic attack) etiology for specific secondary prevention are critical for decreasing further morbidity and mortality of cerebrovascular disease. The Biomarkers of Acute Stroke Etiology (BASE) study is a multicenter observational study to identify serum markers defining the etiology of acute ischemic stroke. Observational trial of patients presenting to the hospital within 24 h of stroke onset. Blood samples are collected at arrival, 24, and 48 h later, and RNA gene expression is utilized to identify stroke etiology marker candidates. The BASE study began January 2014. At the time of writing, there are 22 recruiting sites. Enrollment is ongoing, expected to hit 1000 patients by March 2017. The BASE study could potentially aid in focusing the initial diagnostic evaluation to determine stroke etiology, with more rapidly initiated targeted evaluations and secondary prevention strategies. Clinical Trial Registration NCT02014896 [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer (PC) is one of the deadliest cancers worldwide for which little clinical progress has been made in the last decades. Furthermore, increased trends of PC mortality rates have been reported in Westernised countries. PC is usually diagnosed in advanced stages, precluding patients of an effective treatment. Identifying high-risk populations and early detection markers is the first and crucial step to impact on these figures and change the PC horizon. Aims/Objectives: To discuss the published body of evidence on host and tumor genomics promising markers for primary and/or secondary personalised PC prevention, as well as the future perspectives in the field. Methods: A review of the literature was performed to identify germline and tumor DNA and RNA markers that showed potential usefulness in defining the high-risk population, diagnosing the disease early, and identifying new carcinogens associated with PC. Results: Only high-penetrance inherited mutations are used, at present, to define the high-risk PC population. Although there are some promising genomics markers to be used as early detection tests, none has been validated adequately to be integrated into the clinics routine. Conclusions: Despite of important efforts made in the recent time, little progress has been made to better characterise high-risk PC populations and to identify genomics-based markers for its early diagnosis. PC rates continue to rise, and this disease is becoming a real public health problem in the Westernised world. International and multidisciplinary strategies to identify new markers and properly validate the promising ones are urgently needed to implement cost-efficient primary and secondary prevention interventions in PC. [ABSTRACT FROM AUTHOR]

Drug-induced vascular injury (DIVI) in preclinical studies can delay, if not terminate, a drug development program. Clinical detection of DIVI can be very difficult as there are no definitive biomarkers known to reliably detect this disorder in all instances. The preclinical identification of DIVI requires detailed microscopic examination of a wide range of tissues although one of the most commonly affected areas in rats is the mesenteric vasculature. The reason for this predisposition of mesenteric arteries in rats as well as the exact mechanism and cell types involved in the initial development of these lesions have not been fully elucidated. We hypothesized that by using a mixed culture of cells from rat mesenteric tissue, we would be able to identify an RNA expression signature that could predict the in vivo development of DIVI. Five compounds designed to inhibit Phosphodiesterase 4 activity (PDE4i) were chosen as positive controls. PDE4i's are well known to induce DIVI in the mesenteric vasculature of rats and there is microscopic evidence that this is associated, at least in part, with a proinflammatory mechanism. We surveyed, by qRT-PCR, the expression of 96 genes known to be involved in inflammation and using a Random-Forest model, identified 12 genes predictive of in vivo DIVI outcomes in rats. Using these genes, we were able to cross-validate the ability of the Random-Forest modeling to predict the concentration at which PDE4i caused DIVI in vivo. [ABSTRACT FROM AUTHOR]

Psoralea corylifolia L. (Fructus Psoraleae) is widely used in Asia, but there are concerns about hepatotoxicity caused by constituents such as psoralens and bakukiol. Bakuchiol (BAK) has antiinflammatory, antipyretic, antibacterial antiviral, anticancer, and estrogenic activity but appears to be hepatotoxic in in vitro tests. This study investigated the hepatotoxicity in vivo in rats. Using intragastrically administered bakuchiol at doses of 52.5 and 262.5 mg/kg for 6 weeks. Bodyweight, relative liver weight, biochemical indicators, histopathology, mRNA expression of CYP7A1, HMG-CoA reductase, BSEP, PPARα, SREBP-2, and MRP3 were measured. Many abnormalities were observed in the bakuchiol-treated groups including suppression of weight gain and food intake, change of some parameters in serum biochemistry, and increased weight of liver. The mRNA expression of CYP7A1, HMG-CoA reductase, PPARα, and SREBP-2 decreased in bakuchiol-treated group, the expression of BSEP increased in bakuchiol-treated low dosage, and the expression of BSEP decreased in bakuchiol-treated high dosage. In conclusion, we provide evidence for the first time that bakuchiol can induce cholestatic hepatotoxicity, suggesting potential hepatotoxicity. The mechanism may be related to effects on liver lipid metabolism, but further investigation is necessary. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis ( VL) in Sudan caused by Leishmania donovani is fatal in susceptible individuals if untreated. Treatment with sodium stibogluconate ( SSG) leads to post-kala-azar dermal leishmaniasis ( PKDL) in 58% of patients. Here, Affymetrix microarrays were used to identify genes differentially expressed in lymph nodes (N=9 paired samples) pre- and post-treatment with SSG. Using the Bioconductor package limma, 438 genes from 28 869 post-quality-control probe sets were differentially expressed ( Pnominal≤.02) post- vs pretreatment. Canonical pathway analysis using Ingenuity Pathway Analysis™ identified 'role of nuclear factor of activated T-cell in regulation of immune response' ( Pnominal=1.35×10−5; PBH-adjusted=4.79×10−3), 'B-cell development' ( Pnominal=2.04×10−4; PBH-adjusted=.024), 'Fcγ receptor-mediated phagocytosis in macrophages and monocytes' ( Pnominal=2.04×10−4; PBH-adjusted=.024) and ' OX40 signalling' ( Pnominal=2.82×10−4; PBH-adjusted=.025) as pathways differentially regulated post- vs pretreatment. Major network hub genes included TP53, FN1, MYC, BCL2, JUN, SYK, RUNX2, MMP1 and ACTA2. Top endogenous upstream regulators included IL-7 ( P=2.28×10−6), TNF ( P=4.26×10−6), Amyloid Precursor Protein ( P=4.23×10−5) and SPI1/ PI.1 ( P=1.17×10−7). Top predicted chemical drug regulators included the flavonoid genistein ( P=4.56×10−7) and the quinoline alkaloid camptothecin ( P=5.14×10−5). These results contribute to our understanding of immunopathology associated with VL and response to SSG treatment. Further replication could identify novel therapeutic strategies that improve on SSG treatment and reduce the likelihood of progression to PKDL. [ABSTRACT FROM AUTHOR]

Objectives To determine the basic biochemical features of microparticles ( MP) in patients with venous malformation ( VM) of the head and neck. Methods Microparticles were isolated from peripheral venous blood of VM patients or healthy subjects and from lesional fluid of VM patients. Flow cytometry and real-time polymerase chain reaction were employed to determine the concentration, cellular origin, and RNA expression of obtained MP. A functional coagulation test was applied to measure the coagulant activity of MP. Results Circulating levels of total MP, platelet-derived MP, and endothelial MP were significantly elevated in VM patients and were consistently increased in VM patients with more extensive lesions. Lesional MP ( MP from lesional fluid of VM) in VM patients were more abundant than circulating MP from VM patients or healthy subjects. Moreover, MP from VM patients displayed markedly distinct mRNA and micro RNA expression compared with healthy subjects. Furthermore, MP from VM patients exhibited enhanced procoagulant activity, as evidenced by significantly shorter coagulation time. Conclusions This study demonstrates for the first time that patients with VM have an altered MP profile and MP may be associated with VM-associated thrombogenesis. Further studies are required to explore the precise pathophysiological roles of MP in VM. [ABSTRACT FROM AUTHOR]

Background: Alterations in gene expression are key events in disease etiology and risk. Poor reproducibility in detecting differentially expressed genes across studies suggests individual genes may not be sufficiently informative for complex diseases, such as myocardial infarction (MI). Rather, dysregulation of the 'molecular network' may be critical for pathogenic processes. Such a dynamic network can be built from pairwise non-linear interactions. Results: We investigate non-linear interactions represented in mRNA expression profiles that integrate genetic background and environmental factors. Using logistic regression, we test the association of individual GWAS-based candidate genes and non-linear interaction terms (between these mRNA expression levels) with MI. Based on microarray data in CATHGEN (CATHeterization in GENetics) and FHS (Framingham Heart Study), we find individual genes and pairs of mRNAs, encoded by 41 MI candidate genes, with significant interaction terms in the logistic regression model. Two pairs replicate between CATHGEN and FHS (CNNM2∣GUCY1A3 and CNNM2∣ZEB2). Analysis of RNAseq data from GTEx (Genotype-Tissue Expression) shows that 20 % of these disease-associated RNA pairs are co-expressed, further prioritizing significant interactions. Because edges in sparse co-expression networks formed solely by the 41 candidate genes are unlikely to represent direct physical interactions, we identify additional RNAs as links between network pairs of candidate genes. This approach reveals additional mRNAs and interaction terms significant in the context of MI, for example, the path CNNM2∣ACSL5∣SCARF1∣GUCY1A3, characterized by the common themes of magnesium and lipid processing. Conclusions: The results of this study support a role for non-linear interactions between genes in MI and provide a basis for further study of MI systems biology. mRNA expression profiles encoded by a limited number of candidate genes yield sparse networks of MI-relevant interactions that can be expanded to include additional candidates by co-expression analysis. The non-linear interactions observed here inform our understanding of the clinical relevance of gene-gene interactions in the pathophysiology of MI, while providing a new strategy in developing clinical biomarker panels. [ABSTRACT FROM AUTHOR]

Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. [ABSTRACT FROM AUTHOR]

We studied HERV expression in cell lines after hypoxia, mitogenic stimulation, and demethylation, to better understand if hypoxia may play a role in ERV activation also within the nervous system, as represented by neuroblastoma cell lines. The level of RNA of four human ERV groups ( HERVs) ( HERVE, I/T, H, and W), and three housekeeping genes, of different cell lines including A549, COS-1, Namalwa, RD-L and Vero-E6, as well as human neuroblastoma cel l lines SH- SY5Y, SK-N- DZ, and SK-N- AS were studied using reverse transcription and real-time quantitative PCR ( QPCR). During the course of recovery from hypoxia a pronounced and selective activation of RNA expression of HERVW-like sequences, but not of HERVE, I/T, H, and three housekeeping genes, was found in the neuroblastoma cell lines, most pronounced in SK-N- DZ. In the SK-N- DZ cell line, we also tested the expression of HERVs after chemical treatments. HERVW-like sequences were selectively upregulated by 5-azacytidine, a demethylating agent. Some HERVW loci seem especially responsive to hypoxia and demethylation. HERV expression in neuroblastoma cells is selectively and profoundly influenced by some physiological and chemical stimuli. [ABSTRACT FROM AUTHOR]