Mucosal Profiling of Pediatric-Onset Colitis and IBD Reveals Common Pathogenics and Therapeutic Pathways.

Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.

Time-Resolved Small RNA Sequencing Unravels the Molecular Principles of MicroRNA Homeostasis.

Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 105 copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.

Genetic mechanisms for impaired synaptic plasticity in schizophrenia revealed by computational modeling.

Schizophrenia phenotypes are suggestive of impaired cortical plasticity in the disease, but the mechanisms of these deficits are unknown. Genomic association studies have implicated a large number of genes that regulate neuromodulation and plasticity, indicating that the plasticity deficits have a genetic origin. Here, we used biochemically detailed computational modeling of postsynaptic plasticity to investigate how schizophrenia-associated genes regulate long-term potentiation (LTP) and depression (LTD). We combined our model with data from postmortem RNA expression studies (CommonMind gene-expression datasets) to assess the consequences of altered expression of plasticity-regulating genes for the amplitude of LTP and LTD. Our results show that the expression alterations observed post mortem, especially those in the anterior cingulate cortex, lead to impaired protein kinase A (PKA)-pathway-mediated LTP in synapses containing GluR1 receptors. We validated these findings using a genotyped electroencephalogram (EEG) dataset where polygenic risk scores for synaptic and ion channel-encoding genes as well as modulation of visual evoked potentials were determined for 286 healthy controls. Our results provide a possible genetic mechanism for plasticity impairments in schizophrenia, which can lead to improved understanding and, ultimately, treatment of the disorder.

Quantification of circulating HBV RNA expressed from intrahepatic cccDNA in untreated and NUC treated patients with chronic hepatitis B.

OBJECTIVE: A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg). DESIGN: Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR. RESULTS: cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(-) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(-) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(-) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA. CONCLUSION: Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients. TRIAL REGISTRATION NUMBER: NCT02602847.

In situ HER2 RNA expression as a predictor of pathologic complete response of HER2-positive breast cancer patients receiving neoadjuvant chemotherapy and anti-HER2 targeted treatment.

BACKGROUND: Immunohistochemistry (IHC) and in situ hybridization (ISH) remain standard biomarkers for therapeutic decisions in human epidermal growth factor 2 (HER2)-positive breast cancers (BCs); however, they are insufficient to explain the heterogeneous anti-HER2 response. METHODS: We aimed to investigate the correlation of in situ HER2 RNA expression (isHRE), using RNAscope, with HER2 biomarkers and the impact of isHRE on the pathological complete response (pCR) rates of 278 patients with HER2 IHC/fluorescence ISH (FISH)-positive BC receiving neoadjuvant chemotherapy and anti-HER2 targeted treatment (NCTT). RESULTS: We validated HER2 RNAscope scoring as a semiquantitative method to determine isHRE and showed a positive correlation between RNAscope scores and pCR rates, with particularly different rates between patients with a score of 5 versus 1-4 BCs (66.7% vs. 15.9%, p < 0.0001). There were higher RNAscope scores and pCR rates in patients with HER2 IHC 3 + versus IHC 2+/FISH + BCs and HER2 RNAscope scores and pCR rates showed similar non-linear positive correlations with HER2 copy numbers and HER2/centromere 17 ratios. Moreover, in each HER2-positive IHC/FISH category, higher pCR rates were observed in patients with RNAscope scores of 5 versus 1-4 BC. Patients achieving pCR had BCs with notably higher HER2 RNAscope scores. Multivariate analysis identified HER2 RNAscope 5 as a strong pCR predictor [odds ratio = 10.865, p < 0.001]. The combined impact of multivariate analysis-defined pCR predictors demonstrated that a higher pCR rate was observed in patients with a score of 5 versus a score of 1-4 BCs regardless of the status of hormone receptor and mono-or dual anti-HER2 blockade. CONCUSIONS: Our results demonstrated that high isHRE (RNAscope score 5) is a strong pCR predictor in patients with HER2-positive BCs receiving NCTT, highlighting the complementary role of isHRE in stratifying HER2 status in tissue. Such stratification is relevant to anti-HER2 therapeutic efficacy, particularly using the cutoff of score 1-4 versus 5.

Integrated molecular characterization of esophageal basaloid squamous cell carcinoma: a subtype with distinct RNA expression pattern and immune characteristics, but no specific genetic mutations.

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.

The ABC's of Alzheimer risk gene ABCA7.

Alzheimer's disease (AD) is a growing problem worldwide. Since ABCA7's identification as a risk gene, it has been extensively researched for its role in the disease. We review its recently characterized structure and what the mechanistic insights teach us about its function. We furthermore provide an overview of identified ABCA7 mutations, their presence in different ancestries and protein domains and how they might cause AD. For ABCA7 PTC variants and a VNTR expansion, haploinsufficiency is proposed as the most likely mode-of-action, although splice events could further influence disease risk. Overall, the need to better understand expression of canonical ABCA7 and its isoforms in disease is indicated. Finally, ABCA7's potential functions in lipid metabolism, phagocytosis, amyloid deposition, and the interplay between these three, is described. To conclude, in this review, we provide a comprehensive overview and discussion about the current knowledge on ABCA7 in AD, and what research questions remain. HIGHLIGHTS: Alzheimer's risk-increasing variants in ABCA7 can be found in up to 7% of AD patients. We review the recently characterized protein structure of ABCA7. We present latest insights in genetics, expression patterns, and functions of ABCA7.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

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.

Host RNA Expression Signatures in Young Infants with Urinary Tract Infection: A Prospective Study.

Early diagnosis of infections in young infants remains a clinical challenge. Young infants are particularly vulnerable to infection, and it is often difficult to clinically distinguish between bacterial and viral infections. Urinary tract infection (UTI) is the most common bacterial infection in young infants, and the incidence of associated bacteremia has decreased in the recent decades. Host RNA expression signatures have shown great promise for distinguishing bacterial from viral infections in young infants. This prospective study included 121 young infants admitted to four pediatric emergency care departments in the capital region of Denmark due to symptoms of infection. We collected whole blood samples and performed differential gene expression analysis. Further, we tested the classification performance of a two-gene host RNA expression signature approaching clinical implementation. Several genes were differentially expressed between young infants with UTI without bacteremia and viral infection. However, limited immunological response was detected in UTI without bacteremia compared to a more pronounced response in viral infection. The performance of the two-gene signature was limited, especially in cases of UTI without bloodstream involvement. Our results indicate a need for further investigation and consideration of UTI in young infants before implementing host RNA expression signatures in clinical practice.

Relationships between neurotransmitter receptor densities and expression levels of their corresponding genes in the human hippocampus.

Neurotransmitter receptors are key molecules in signal transmission, their alterations are associated with brain dysfunction. Relationships between receptors and their corresponding genes are poorly understood, especially in humans. We combined in vitro receptor autoradiography and RNA sequencing to quantify, in the same tissue samples (7 subjects), the densities of 14 receptors and expression levels of their corresponding 43 genes in the Cornu Ammonis (CA) and dentate gyrus (DG) of human hippocampus. Significant differences in receptor densities between both structures were found only for metabotropic receptors, whereas significant differences in RNA expression levels mostly pertained ionotropic receptors. Receptor fingerprints of CA and DG differ in shapes but have similar sizes; the opposite holds true for their "RNA fingerprints", which represent the expression levels of multiple genes in a single area. In addition, the correlation coefficients between receptor densities and corresponding gene expression levels vary widely and the mean correlation strength was weak-to-moderate. Our results suggest that receptor densities are not only controlled by corresponding RNA expression levels, but also by multiple regionally specific post-translational factors.

Identification of Potential Therapeutic Targets for Plasmablastic Lymphoma Through Gene Expression Analysis: Insights into RAS and Wnt Signaling Pathways.

Plasmablastic lymphoma (PBL) is a rare and aggressive B-cell lymphoma with overlapping characteristics with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. Hyperactive Wnt signaling derails homeostasis and promotes oncogenesis and chemoresistance in DLBCL and multiple myeloma. Evidence suggests active cross-talk between the Wnt and RAS pathways impacting metastasis in solid cancers in which combined targeted therapies show effective results. Recent genomic studies in PBL demonstrated a high frequency of mutations linked with the RAS signaling pathway. However, the role of RAS and Wnt signaling pathway molecule expression in PBL remained unknown. We examined the expression of Wnt and RAS pathway-related genes in a well-curated cohort of PBL. Because activated B cells are considered immediate precursors of plasmablasts in B cell development, we compared this data with activated B-cell type DLBCL (ABC-DLBCL) patients, employing NanoString transcriptome analysis (770 genes). Hierarchical clustering revealed distinctive differential gene expression between PBL and ABC-DLBCL. Gene set enrichment analysis labeled the RAS signaling pathway as the most enriched (37 genes) in PBL, including upregulating critical genes, such as NRAS, RAF1, SHC1, and SOS1. Wnt pathway genes were also enriched (n = 22) by gene set enrichment analysis. Molecules linked with Wnt signaling activation, such as ligands or targets (FZD3, FZD7, c-MYC, WNT5A, WNT5B, and WNT10B), were elevated in PBL. Our data also showed that, unlike ABC-DLBCL, the deranged Wnt signaling activity in PBL was not linked with hyperactive nuclear factor κB and B-cell receptor signaling. In divergence, Wnt signaling inhibitors (CXXC4, SFRP2, and DKK1) also showed overexpression in PBL. The high expression of RAS signaling molecules reported may indicate linkage with gain-in-function RAS mutations. In addition, high expression of Wnt and RAS signaling molecules may pave pathways to explore benefiting from combined targeted therapies, as reported in solid cancer, to improve prognosis in PBL patients.

Ex-vivo RNA expression analysis of vaccine candidate genes in COPD sputum samples.

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 ).

Molecular characterization of onychomatricoma: Spatial profiling reveals the role of onychofibroblasts in its pathogenesis.

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 ß-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.

Integrated DNA Methylation/RNA Profiling in Middle Temporal Gyrus of Alzheimer's Disease.

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.

Genetic and phenotypic spectrum in the NONO-associated syndromic disorder.

The non-POU domain-containing octamer-binding (NONO) protein is involved in multiple steps of gene regulation such as RNA metabolism and DNA repair. Hemizygous pathogenic variants in the NONO gene were confirmed to cause a rare X-linked syndromic disorder. Through our in-house diagnostics and subsequent matchmaking, we identified six unrelated male individuals with pathogenic or likely pathogenic NONO variants. For a detailed comparison, we reviewed all published characterizations of the NONO-associated disorder. The combined cohort consists of 16 live-born males showing developmental delay, corpus callosum anomalies, non-compaction cardiomyopathy and relative macrocephaly as leading symptoms. Seven prenatal literature cases were characterized by cardiac malformations. In this study, we extend the phenotypic spectrum through two more cases with epilepsy as well as two more cases with hematologic anomalies. By RNA expression analysis and structural modeling of a new in-frame splice deletion, we reinforce loss-of-function as the pathomechanism for the NONO-associated syndromic disorder.

Triticum aestivum: antioxidant gene profiling and morpho-physiological studies under salt stress.

BACKGROUND: Soil salinity drastically reduced wheat growth and production in Pakistan. It is a need of an hour to identify the best suitable salt tolerance or resistant wheat varieties which shows good growth under salinity affected areas. In presented study, two wheat varieties Johar (salt tolerant) and Sarsabaz (salt sensitive) were examined under NaCl stress conditions. METHODS: Antioxidant enzyme activities were investigated in 10-days old wheat seedlings under 200 mM NaCl stress in hydroponic conditions. To investigate the various growth parameters, antioxidant enzyme activities such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6) and ascorbate peroxidase (APX: EC 1.11.1.11) were monitored and studied. Besides this various growth parameters such as length of the roots, shoots, as well as Physiological parameters likes lipid peroxidation by malondialdehyde (MDA), hydrogen peroxide (H2O2), and proline contents and antioxidant enzyme activities were estimated. The effect of salinity was also observed on gene transcription level and eventually expression level. RESULTS: Shoot and root length were decreased in Sarsabaz variety while it showed opposite trend in johar at 200 mM salt concentration. The concentration of proline showed a noticeable rise in salt dependency. Higher concentrations of Proline in Johar were observed as compared to Sarsabaz. SOD showed the increase in activity for antioxidant enzymes. Significant increase of SOD levels were observed in shoot tissues as compared to root tissues. The results indicated that the shoots were more susceptible to salt stress. Activity of APX showed similar affects in both varieties. The production of CAT enzyme in the shoot and root tissues of both varieties showed substantial growth under increased salt stress. Furthermore, NaCl stress has increased the expression of certain genes coding for antioxidant enzymes such as catalase, superoxide dismutase, and peroxidase. Maximum expression of all the antioxidant enzyme coding genes were observed in Johar (tolerant) at 48 h exposure to salt. In contrast the expression of the all mentioned genes in Sarsabaz variety were found maximum at early hours (24 h) and gradually decreased at 48 h. CONCLUSION: The study showed that the selected salt tolerant wheat variety Johar is significantly resistant to 200 mM NaCl salt level as compared to Sarsabaz.

Do cord blood CD34 cells stay the same or change after expansion: A comprehensive analysis of gene expression results before and after seven days of cell culture.

INTRODUCTION: Hematopoietic stem cell transplantation is used to treat hematopoietic malignancies and bone marrow failure syndromes. Due to the difficulties of using these cells isolated from the bone marrow, an additional source for receiving essential hematopoietic cells is umbilical cord blood. But the main problem with using the umbilical cord is its insufficient blood volume. The ex-vivo reproductive system of hematopoietic stem cells can overcome this problem that has the ability for Transplantation and hematopoiesis in the long term. This study aimed to evaluate the expression profile of CD133 + umbilical cord blood microRNAs in different stages of hematopoietic stem cells before and after ex-vivo proliferation. MATERIALS AND METHODS: The expression profile of 1034 types of microRNA of CD34 + hematopoietic stem cells of the umbilical cord was analyzed before and after ex vivo proliferation. After isolating CD34 + cells from the umbilical cord blood of normal-born babies using MACS (Magnetic-activated cell sorting) column, these cells were cultured in a Stem span culture medium containing SCF, FLT3, and TPO cytokines in 24-well plates. The expression profile of microRNAs was investigated on days 0 and 7 days after cultivation by the Real-Time PCR method. RESULTS: The results showed that the production of two-thirds of the micro-RNAs was reduced during the proliferation process. The micro-RNA expression of hematopoietic stem cell proliferation was also lower. At the same time, micro-RNA expression related to differentiation was higher. CONCLUSION: The observed reduction in miRNA expression may be attributed to enhanced differentiation through proliferation. Therefore, miRNAs appear to be a viable option for regulating the proliferation processes of Hematopoietic stem cell transplantation.

ICOS gene polymorphisms in systemic lupus erythematosus: A case-control study.

The inducible T-cell costimulator (ICOS) may play an important role in adaptive immunity by regulating the interaction between T cells and antigen-presenting cells. Disruption of this molecule can lead to autoimmune diseases, in particular systemic lupus erythematosus (SLE). In this study, we aimed to explore the possible association between ICOS gene polymorphisms and SLE as well as their influence on disease susceptibility and clinical outcomes. A further objective was to assess the potential impact of these polymorphisms on RNA expression. A case-control study, including 151 patients with SLE, and 291 unrelated healthy controls (HC) matched in gender, and geographical origin, was performed to genotype two polymorphisms located in the ICOS gene: rs11889031 (-693 G/A) and rs10932029 (IVS1 + 173 T/C); using the polymerase chain reaction (PCR)-restriction fragment length polymorphism method. The different genotypes were validated by direct sequencing. The expression level of ICOS mRNA was assessed by quantitative PCR in peripheral blood mononuclear cells of SLE patients and HC. The results were analysed using Shesis and spss.20. Our results revealed a significant association between ICOS gene rs11889031 > CC genotype and SLE disease (codominant genetic model 1, (C/C vs. C/T), p = .001, odds ratio [OR] = 2.18 IC [1.36-3.49]); codominant genetic model 2, (C/C vs. T/T) p = .007, OR = 15.29 IC [1.97-118.5]); dominant genetic model, (C/C vs. C/T + T/T) p = .0001, OR = 2.44 IC [1.53-3.9]). Besides, there was a marginal association between rs11889031 > TT genotype and T allele with a protective role from SLE (recessive genetic model, p = .016, OR = 0.08 IC [0.01-0.63] and p = 7.6904E - 05, OR = 0.43 IC = [0.28-0.66], respectively). Moreover, statistical analysis indicated that the rs11889031 > CC genotype was linked with clinical and serological manifestations of SLE, including blood pressure, and anti-SSA antibodies production in SLE patients. However, the ICOS gene rs10932029 polymorphism was not associated with susceptibility to SLE. On the other side, we did not note any effect of the two selected polymorphisms on the level of ICOS mRNA gene expression. The study showed a significant predisposing association of the ICOS rs11889031 > CC genotype with SLE, in contrast to a protective effect of rs11889031 > TT genotype in Tunisian patients. Our results suggest that ICOS rs11889031 may act as a risk factor for SLE and could be used as a genetic susceptibility biomarker.

Circular RNA hsa_circ_0098181 inhibits metastasis in hepatocellular carcinoma by activating the Hippo signaling pathway via interaction with eEF2.

INTRODUCTION AND OBJECTIVES: The development of hepatocellular carcinoma (HCC) is a multi-step process that accumulates genetic and epigenetic alterations, including changes in circular RNA (circRNA). This study aimed to understand the alterations in circRNA expression in HCC development and metastasis and to explore the biological functions of circRNA. MATERIALS AND METHODS: Ten pairs of adjacent chronic hepatitis tissues and HCC tissues from patients without venous metastases, and ten HCC tissues from patients with venous metastases were analyzed using human circRNA microarrays. Differentially expressed circRNAs were then validated by quantitative real-time PCR. In vitro and in vivo assays were performed to assess the roles of the circRNA in HCC progression. RNA pull-down assay, mass spectrometry analysis, and RNA-binding protein immunoprecipitation were conducted to explore the protein partners of the circRNA. RESULTS: CircRNA microarrays revealed that the expression patterns of circRNAs across the three groups were significantly different. Among these, hsa_circ_0098181 was validated to be lowly expressed and associated with poor prognosis in HCC patients. Ectopic expression of hsa_circ_0098181 delayed HCC metastasis in vitro and in vivo. Mechanistically, hsa_circ_0098181 sequestered eukaryotic translation elongation factor 2 (eEF2) and dissociated eEF2 from filamentous actin (F-actin) to prevent F-actin formation, which blocked activation of the Hippo signaling pathway. In addition, the RNA binding protein Quaking-5 bound directly to hsa_circ_0098181 and induced its biogenesis. CONCLUSIONS: Our study reveals changes in circRNA expression from chronic hepatitis, primary HCC, to metastatic HCC. Further, the QKI5-hsa_circ_0098181-eEF2-Hippo signaling pathway exerts a regulatory role in HCC.

New Insights via RNA Profiling of Formalin-Fixed Paraffin-Embedded Lung Tissue of Pulmonary Fibrosis Patients.

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 (