NFKBIE is a predictive factor of survival and is correlated with immune infiltration and antigen processing and presentation in hepatocellular carcinoma.

The important role of the nuclear factor κB (NFκB) pathway in tumour development has long been recognized; however, the role of the NFκB inhibitor family in liver cancer has not been elucidated. Hepatocellular carcinoma (HCC) is a serious public health burden with a high incidence, poor prognosis, and early detection, especially in Asia, where hepatitis is prevalent. In the present study, the mRNA expression level of the NFκB inhibitor family was assessed in HCC and normal tissues using the Metabolic Gene Rapid Visualizer, University of Alabama at Birmingham Cancer Data Analysis Portal, and the Tumor Immune Estimation Resource database (TIMER). Survival curves of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor (NFKBI)E were obtained using the Kaplan-Meier method. Genes co-expressed with NFKBIE in HCC samples were studied using data from the LinkedOmics and the Hepatocellular Carcinoma Databases. Protein-protein interaction networks, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment pathway analyses were used to assess the NFKBIE mechanism in HCC. Using the TIMER database, the association between immune infiltration and NFKBIE was determined. RNA-sequencing (RNA-seq) was used to evaluate the function of NFKBIE in HCC and its impact on proliferation and migration. Western blotting was used to confirm the expression of NFKBIE in HCC cell lines. In addition, NFKBIE overexpression in HCC was demonstrated using tissue microarrays encompassing 80 pairs of HCC and normal liver tissues. NFKBIE was the only NFκB inhibitor with high expression and an improved prognosis in HCC compared with other NFκB inhibitors. NFKBIE was correlated with clinical characteristics, such as tumour grade, tumour protein P53 mutation status and tumour stage. Data obtained from Gene Set Cancer Analysis suggested that NFKBIE may inhibit the PI3K/AKT, RAS/MAPK, RTK and TSC/mTOR pathways. In addition, NFKBIE was significantly associated with B-cell immune infiltration and the RNA-seq data demonstrated that knockdown of NFKBIE significantly affected 'Antigen processing and presentation' and 'hepatocellular carcinoma' pathways. Immunohistochemistry of microarrays of tissue samples revealed that NFKBIE was overexpressed in several stages of HCC. Finally, inhibition of NFKBIE decreased the proliferation and migration of HCC cells. In conclusion, due to its prognostic value and overexpression in HCC, NFKBIE distinguished itself from other NFκB inhibitors. As such, it may provide a novel prognostic indicator and immunotherapeutic target for HCC.

Disrupting methyl-CpG-binding protein 2 expression induces the transformation of induced pluripotent stem cell cardiomyocytes into pacemaker-like cells by insulin gene enhancer binding protein 1.

BACKGROUND: The experiment will explore whether interfering with the expression of methyl-CpG-binding protein 2 (MecP2) can enhance the ability of insulin gene enhancer binding protein 1 (ISL1) to induce iPSC-CMs to differentiate into pacemaker-like cells. METHODS: Differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) can be induced via the regulation of the Wnt signaling pathway. Real-time quantitative PCR (qPCR), western blotting, immunofluorescence staining, and patch-clamp technique were used to analyze the ability of ISL1 to induce the transformation of iPSC-CMs into pacemaker-like cells. Calcium spark, patch-clamp technique, and real-time qPCR were used to verify whether disrupting the expression of MeCP2 enhanced this ability of ISL1 to induce the differentiation of iPSC-CMs into pacemaker cells. Transplant pacemaker-like cardiomyocytes into the myocardium of mice to observe whether the pacemaker cells can survive in the tissue for a long time. RESULTS: RT-qPCR and patch-clamp analyses showed that overexpression of ISL1 induced the successful differentiation of iPSC-CMs into pacemaker cells. ISL1-overexpressing pacemaker-like cells possessed typical characteristics of pacemaker morphology, including action potential and If inward current. Chromatin immunoprecipitation results showed that MeCP2 bound to the promoter region of HCN4. Following disruption of MeCP2 expression, the gene expression of sinoatrial node-specific transcription factors, If inward current, and cardiac rhythm changes in iPSC-CMs resembled those of sinoatrial node pacemaker cells. Therefore, ISL1 induced the differentiation of iPSC-CMs into pacemaker-like cells, and knockdown of MeCP2 increased this effect. Frozen section results showed that surviving pacemaker-like cells could still be observed in myocardial tissue after 45 days. CONCLUSIONS: Experiments have found that interfering with the expression of MeCP2 can increase the ability of ISL1 to induce iPSC-CM cells to differentiate into pacemaker-like cells. And the pacemaker-like cells obtained in this experiment can survive in myocardial tissue for a long time.

Effect of histone methylase EZH2 on proliferation and apoptosis of hypertrophic myocardial cells of AC16 / 国际生物医学工程杂志

Objective:To explore the molecular mechanism of the effect of the histone methylase zeste gene enhancer homolog 2 (EZH2) on the proliferation and apoptosis of human hypertrophic cardiomyocytes AC16.Methods:The AC16 hypertrophic cardiomyocyte model was constructed by adding angiotensin Ⅱ to the AC16 cell culture medium. The cells were divided into four groups, including the blank control group, the angiotensin Ⅱ group, the empty vector + angiotensin Ⅱ group, and the EZH2 overexpression + angiotensin Ⅱ group. The expression levels of EZH2 and brain natriuretic peptide ( BNP) genes were measured using fluorescent quantitative PCR. The EZH2, trimethylation of lysine at position 27 of histone H3 (H3K27me3), and BNP proteins expression were detected by Western Blot. The MTS method was used to detect the proliferation of AC16 cell. The Annexin V-FITC/PI double staining method was used to detect the apoptosis of AC16 cell. Results:Compared with the blank control group, the expression levels of EZH2 and H3K27me3 in the angiotensin Ⅱ group were decreased, the expression level of BNP was increased, cell proliferation was decreased, and apoptosis was increased (all P < 0.001). Compared with the empty vector + angiotensin Ⅱ group, the expression levels of EZH2 and H3K27me3 in the EZH2 overexpression + angiotensin Ⅱ group were increased, the expression level of BNP was decreased, the cell proliferation level was increased, and the apoptosis level was decreased (all P < 0.001). There was no significant difference between the angiotensin Ⅱ group and the empty vector + angiotensin Ⅱ group (all P > 0.05). Conclusions:Histone methylase EZH2 has an effect on the proliferation and apoptosis of AC16 cell, providing a reference for the treatment of myocardial hypertrophy and revealing the exact pathogenesis of myocardial hypertrophy.

Interplay between exosomes and autophagy machinery in pain management: State of the art.

Despite recent progress regarding inexpensive medical approaches, many individuals suffer from moderate to severe pain globally. The discovery and advent of exosomes, as biological nano-sized vesicles, has revolutionized current knowledge about underlying mechanisms associated with several pathological conditions. Indeed, these particles are touted as biological bio-shuttles with the potential to carry specific signaling biomolecules to cells in proximity and remote sites, maintaining cell-to-cell communication in a paracrine manner. A piece of evidence points to an intricate relationship between exosome biogenesis and autophagy signaling pathways at different molecular levels. A close collaboration of autophagic response with exosome release can affect the body's hemostasis and physiology of different cell types. This review is a preliminary attempt to highlight the possible interface of autophagy flux and exosome biogenesis on pain management with a special focus on neuropathic pain. It is thought that this review article will help us to understand the interplay of autophagic response and exosome biogenesis in the management of pain under pathological conditions. The application of therapies targeting autophagy pathway and exosome abscission can be an alternative strategy in the regulation of pain.

Neuroprotective effects of Korean White ginseng and Red ginseng in an ischemic stroke mouse model.

Background: Stroke is a neurological disorder characterized by brain tissue damage following a decrease in oxygen supply to brain due to blocked blood vessels. Reportedly, 80% of all stroke cases are classified as cerebral infarction, and the incidence rate of this condition increases with age. Herein, we compared the efficacies of Korean White ginseng (WG) and Korean Red Ginseng (RG) extracts (WGex and RGex, respectively) in an ischemic stroke mouse model and confirmed the underlying mechanisms of action. Methods: Mice were orally administered WGex or RGex 1 h before middle cerebral artery occlusion (MCAO), for 2 h; the size of the infarct area was measured 24 h after MCAO induction. Then, the neurological deficit score was evaluated and the efficacies of the two extracts were compared. Finally, their mechanisms of action were confirmed with tissue staining and protein quantification. Results: In the MCAO-induced ischemic stroke mouse model, WGex and RGex showed neuroprotective effects in the cortical region, with RGex demonstrating superior efficacy than WGex. Ginsenoside Rg1, a representative indicator substance, was not involved in mediating the effects of WGex and RGex. Conclusion: WGex and RGex could alleviate the brain injury caused by ischemia/reperfusion, with RGex showing a more potent effect. At 1,000 mg/kg body weight, only RGex reduced cerebral infarction and edema, and both anti-inflammatory and anti-apoptotic pathways were involved in mediating these effects.

The expression and potential mechanism of EGFR and EZH2 in breast cancer.

BACKGROUND: The purpose of our research was to investigate the expression of epidermal growth factor receptor (EGFR) and zeste gene enhancer homolog 2 (EZH2) in breast cancer, and to explore their potential common pathways. METHODS: Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the protein and corresponding mRNA expression of EGFR and EZH2 in breast cancer tissues and benign tissues. Then, the relationship between EGFR and EZH2 along with the corresponding clinicopathological parameters were also analyzed. Bioinformatics tools were applied to explore the possible common pathways. RESULTS: The results showed that both EGFR and EZH2 protein and mRNA were highly expressed in breast cancer tissues, and there was a positive correlation between EGFR and EZH2. Moreover, we found that increased mRNA expression was correlated with lymph node metastasis and clinical stage (P<0.05). Furthermore, the enrichment results of co-expressed genes indicated that EGFR and EZH2 may work together in the FOXO signaling pathway, affecting the growth and metastasis of breast cancer cells. CONCLUSIONS: The high expression of both EGFR and EZH2 mRNA in breast cancer was related to lymph node metastasis and clinical staging. The FOXO signaling pathway may be their common signaling pathway that affects tumor cell invasion and metastasis.

Stress-induced transcriptional memory accelerates promoter-proximal pause release and decelerates termination over mitotic divisions.

Heat shock instantly reprograms transcription. Whether gene and enhancer transcription fully recover from stress and whether stress establishes a memory by provoking transcription regulation that persists through mitosis remained unknown. Here, we measured nascent transcription and chromatin accessibility in unconditioned cells and in the daughters of stress-exposed cells. Tracking transcription genome-wide at nucleotide-resolution revealed that cells precisely restored RNA polymerase II (Pol II) distribution at gene bodies and enhancers upon recovery from stress. However, a single heat exposure in embryonic fibroblasts primed a faster gene induction in their daughter cells by increasing promoter-proximal Pol II pausing and by accelerating the pause release. In K562 erythroleukemia cells, repeated stress refined basal and heat-induced transcription over mitotic division and decelerated termination-coupled pre-mRNA processing. The slower termination retained transcripts on the chromatin and reduced recycling of Pol II. These results demonstrate that heat-induced transcriptional memory acts through promoter-proximal pause release and pre-mRNA processing at transcription termination.

Bone marrow-derived mesenchymal stem cells improve rat islet graft revascularization by upregulating ISL1.

Revascularization of the islet transplant is a crucial step that defines the success rate of patient recovery. Bone marrow-derived mesenchymal stem cells (BMSCs) have been reported to promote revascularization; however, the underlying cellular mechanism remains unclear. Moreover, our liquid chromatography-tandem mass spectrometry results showed that BMSCs could promote the expression of insulin gene enhancer binding protein-1 (ISL1) in islets. ISL1 is involved in islets proliferation and plays a potential regulatory role in the revascularization of islets. This study identifies the ISL1 protein as a potential modulator in BMSCs-mediated revascularization of islet grafts. We demonstrated that the survival rate and insulin secretion of islets were increased in the presence of BMSCs, indicating that BMSCs promote islet revascularization in a coculture system and rat diabetes model. Interestingly, we also observed that the presence of BMSCs led to an increase in ISL1 and vascular endothelial growth factor A (VEGFA) expression in both islets and the INS-1 rat insulinoma cell line. In silico protein structure modeling indicated that ISL1 is a transcription factor that has four binding sites with VEGFA mRNA. Further results showed that overexpression of ISL1 increased both the abundance of VEGFA transcripts and protein accumulation, while inhibition of ISL1 decreased the abundance of VEGFA. Using a ChIP-qPCR assay, we demonstrated that direct molecular interactions between ISL1 and VEGFA occur in INS-1 cells. Together, these findings reveal that BMSCs promote the expression of ISL1 in islets and lead to an increase in VEGFA in islet grafts. Hence, ISL1 is a potential target to induce early revascularization in islet transplantation.

Insulin gene enhancer protein 1 mediates glycolysis and tumorigenesis of gastric cancer through regulating glucose transporter 4.

BACKGROUND: Insulin gene enhancer protein 1, (ISL1), a LIM-homeodomain transcription factor, is involved in multiple tumors and is associated with insulin secretion and metabolic phenotypes. However, the role of ISL1 in stimulating glycolysis to promote tumorigenesis in gastric cancer (GC) is unclear. In this study, we aimed to characterize the expression pattern of ISL1 in GC patients and explore its molecular biological mechanism in glycolysis and tumorigenesis. METHODS: We analyzed the expression and clinical significance of ISL1 in GC using immunohistochemistry and real-time polymerase chain reaction (PCR). Flow cytometry and IncuCyte assays were used to measure cell proliferation after ISL1 knockdown. RNA-sequencing was performed to identify differentially expressed genes, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) to reveal key signaling pathways likely regulated by ISL1 in GC. Alteration of the glycolytic ability of GC cells with ISL1 knockdown was validated by measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) and by detecting glucose consumption and lactate production. The expression of glucose transporter 4 (GLUT4) and ISL1 was assessed by Western blotting, immunohistochemistry, and immunofluorescent microscopy. The luciferase reporter activity and chromatin immunoprecipitation assays were performed to determine the transcriptional regulation of ISL1 on GLUT4. RESULTS: High levels of ISL1 and GLUT4 expression was associated with short survival of GC patients. ISL1 knockdown inhibited cell proliferation both in vitro and in vivo. KEGG analysis and GSEA for RNA-sequencing data indicated impairment of the glycolysis pathway in GC cells with ISL1 knockdown, which was validated by reduced glucose uptake and lactate production, decreased ECAR, and increased OCR. Mechanistic investigation indicated that ISL1 transcriptionally regulated GLUT4 through binding to its promoter. CONCLUSION: ISL1 facilitates glycolysis and tumorigenesis in GC via the transcriptional regulation of GLUT4.

microRNA-9 and -29a regulate the progression of diabetic peripheral neuropathy via ISL1-mediated sonic hedgehog signaling pathway.

In this study, we tested the hypothesis that overexpression of miR-9 and miR-29a may contribute to DPN development and progression. We performed a meta-analysis of miR expression profile studies in human diabetes mellitus (DM) and the data suggested that miR-9 and miR-29a were highly expressed in patients with DM, which was further verified in serum samples collected from 30 patients diagnosed as DM. Besides, ISL1 was confirmed to be a target gene of miR-9 and miR-29a. Lentivirus-mediated forced expression of insulin gene enhancer binding protein-1 (ISL1) activated the sonic hedgehog (SHH) signaling pathway, increased motor nerve conduction velocity and threshold of nociception, and modulated expression of neurotrophic factors in sciatic nerves in rats with DM developed by intraperitoneal injection of 0.45% streptozotocin, suggesting that ISL1 could delay DM progression and promote neural regeneration and repair after sciatic nerve damage. However, lentivirus-mediated forced expression of miR-9 or miR-29a exacerbated DM and antagonized the beneficial effect of ISL1 on DPN. Collectively, this study revealed potential roles of miR-9 and miR-29a as contributors to DPN development through the SHH signaling pathway by binding to ISL1. Additionally, the results provided an experimental basis for the targeted intervention treatment of miR-9 and miR-29a.

Siglec1 enhances inflammation through miR-1260-dependent degradation of IκBα in COPD.

It has been documented that sialic acid-binding Ig-like lectin 1 (Siglec1) is a cell surface protein with a variety of functions in the immune system. In the present study, we evaluated whether Siglec1 plays a role in chronic obstructive pulmonary disease (COPD). Results show that the expression of Siglec1 was increased in the lung of COPD rats, and that Siglec1 overexpression greatly enhanced the expression of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and IL-6 in cigarette smoke extract (CSE)-treated NR8383 cells, a rat lung-derived macrophage cell line. Notably, the proinflammatory effect of Siglec1 was totally inhibited by overexpression of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α (IκBα). Importantly, Siglec1 overexpression increased miR-1260, which then degraded IκBα through its 3' untranslated region (3'UTR). Further study demonstrated that miR-1260 inhibitor attenuated inflammation in CSE-induced rat COPD lung and in CSE-treated NR8383 cells. Finally, the inhibitory effect of miR-1260 on inflammation was totally lost when IκBα was inhibited. In summary, the present study demonstrated that Siglec1 exerts its proinflammatory effects through increasing miR-1260, leading to decreased expression of IκBα.

Heterozygous Deletion of the SHOX Gene Enhancer in two Females With Clinical Heterogeneity Associating With Skewed XCI and Escaping XCI.

Skewed X-chromosome inactivation (XCI) plays an important role in the phenotypic heterogeneity of X-linked disorders. However, the role of skewed XCI in XCI-escaping gene SHOX regulation is unclear. Here, we focused on a heterozygous deletion of SHOX gene enhancer with clinical heterogeneity. Using SNP array, we detected that the female proband with Leri-Weill dyschondrosteosis (LWD) carried an 857 kb deletion on Xp22.3 (encompassing SHOX enhancer) and a 5,707 kb large-fragment deletion on Xq25q26. XCI analysis revealed that the X-chromosome with the Xq25q26 large-fragment deletion was completely inactivated, which forced the complete activation of the other X-chromosome carrying SHOX enhancer deletion. While the Xp22.3 deletion locates on the escaping XCI region, under the combined action of skewed XCI and escaping XCI, transcription of SHOX gene was mainly from the activated X-chromosome with SHOX enhancer defect, involving in the formation of LWD phenotype. Interestingly, this SHOX enhancer deletion was inherited from her healthy mother, who also demonstrated completely skewed XCI. However, the X-chromosome with SHOX enhancer deletion was inactivated, and the normal X-chromosome was activated. Combing with escaping XCI, her phenotype was almost normal. In summary, this study was a rare report of SHOX gene enhancer deletion in a family with clinical heterogeneity due to skewed inactivation of different X-chromosomes, which can help in the genetic counseling and prenatal diagnosis of disorders in females with SHOX defect.

Genome-Wide Comparative Analysis of HIF Binding Sites in Cyprinus Carpio for In Silico Identification of Functional Hypoxia Response Elements.

Cyprinus carpio is world's most widely distributed freshwater species highly used in aquaculture. It is a hypoxia-tolerant species as it lives in oxygen-deficient environment for a long period. The tolerance potential of an animal against hypoxia relates it to induced gene expression, where a hypoxia-inducible factor (HIF) binds to a transcriptionally active site, hypoxia response element (HRE), a 5-base short motif that lies within the promoter/enhancer region of a certain gene, for inducing gene expression and preventing/minimizing hypoxia effects. HRE is functionally active when it contains another motif, the hypoxia ancillary sequence (HAS), which is typically adjacent to downstream of HRE within 7- to 15-nt space. Here, an attempt was made for mining HRE and identifying functional HIF binding sites (HBS) in a genome-wide analysis of C. carpio. For this, gene information along with the 5,000-nt upstream (-4,900 to +100) sequences of 31,466 protein coding genes was downloaded from "Gene" and "RefSeq" databases. Analysis was performed after filtration of the impracticable genes. A total of 116,148 HRE consensus sequences were mined from 29,545 genes in different promoter regions. HRE with HAS consensus motifs were found in the promoter region of 9,589 genes. Further, the already reported genes for hypoxia response in humans and zebrafish were reanalyzed for detecting HRE sites in their promoters and used for comparative analysis with gene promoters of C. carpio for providing support to identify functional HBS in the gene promoter of C. carpio. An interactive user interface HREExplorer was developed for presenting the results on the World Wide Web and visualizing possible HBS in protein coding genes in C. carpio and displaying the comparative results along with the reported hypoxia-responsive genes of zebrafish and reported hypoxia-inducible genes in humans. In this study, a set of Perl program was written for the compilation and analysis of information that might be used for a similar study in other species. This novel work may provide a workbench for analyzing the promoter regions of hypoxia-responsive genes.

Mismatch repair-signature mutations activate gene enhancers across human colorectal cancer epigenomes.

Commonly-mutated genes have been found for many cancers, but less is known about mutations in cis-regulatory elements. We leverage gains in tumor-specific enhancer activity, coupled with allele-biased mutation detection from H3K27ac ChIP-seq data, to pinpoint potential enhancer-activating mutations in colorectal cancer (CRC). Analysis of a genetically-diverse cohort of CRC specimens revealed that microsatellite instable (MSI) samples have a high indel rate within active enhancers. Enhancers with indels show evidence of positive selection, increased target gene expression, and a subset is highly recurrent. The indels affect short homopolymer tracts of A/T and increase affinity for FOX transcription factors. We further demonstrate that signature mismatch-repair (MMR) mutations activate enhancers using a xenograft tumor metastasis model, where mutations are induced naturally via CRISPR/Cas9 inactivation of MLH1 prior to tumor cell injection. Our results suggest that MMR signature mutations activate enhancers in CRC tumor epigenomes to provide a selective advantage.

Discovery platform for inhibitors of IgH gene enhancer activity.

Immunoglobulin heavy chain (IgH) translocations are common and early oncogenic events in B cell and plasma cell malignancies including B cell non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). IgH translocations bring oncogenes into close proximity with potent enhancer elements within the IgH locus, leading to oncogene up-regulation. As IgH enhancer activity is tightly controlled by B cell lineage-specific signaling and transcriptional networks, we hypothesized that IgH enhancers are potentially druggable targets/elements. To test this, we developed a molecular imaging-based high-throughput screening platform for discovering inhibitors of IgH enhancer-driven transcriptional activity. As proof of concept, we identified a low micromolar potency molecule (compound 30666) that inhibited immunoglobulin production by MM cells and blocked expression of an array of IgH translocation-induced oncogenes (CCND1, FGFR3/MMSET, and MYC) in MM and NHL cell lines. Prolonged exposure to 30666 significantly reduced the viability of IgH translocation-positive NHL and MM cells, but was less effective against cells lacking IgH translocations. Compound 30666 exhibited suitable pharmacological properties, including metabolic stability in liver microsomes and oral bioavailability in mice, and demonstrated preclinical anti-MM activity in a plasmacytoma mouse model. Our work suggests that IgH enhancers are attractive and potentially druggable targets for IgH translocation driven malignancies.

Polyphenolic extract from extra virgin olive oil inhibits the inflammatory response in IL-1ß-activated synovial fibroblasts.

The polyphenolic extract (PE) from extra virgin olive oil (EVOO) has been shown to possess important anti-inflammatory and joint protective properties in murine models of rheumatoid arthritis (RA). This study was designed to evaluate the effects of PE on IL-1ß-activated human synovial fibroblasts SW982 cell line. PE from EVOO treatment inhibited IL-1ß-induced matrix metalloproteases (P<0·001), TNF-α and IL-6 production (P<0·001). Similarly, IL-1ß-induced cyclo-oxygenase-2 and microsomal PGE synthase-1 up-regulations were down-regulated by PE (P<0·001). Moreover, IL-1ß-induced mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation were ameliorated by PE (P<0·001). These results suggest that PE from EVOO reduces the production of proinflammatory mediators in human synovial fibroblasts; particularly, these protective effects could be related to the inhibition of MAPK and NF-κB signalling pathways. Taken together, PE from EVOO probably could provide an attractive complement in management of diseases associated with over-activation of synovial fibroblasts, such as RA.

Novel Clinical Criteria Allow Detection of Short Stature Homeobox-Containing Gene Haploinsufficiency Caused by Either Gene or Enhancer Region Defects.

INTRODUCTION: Short stature homeobox-containing gene (SHOX) haploinsufficiency is associated with short stature, Madelung deformity and mesomelia. Current clinical screening tools are based on patients with intragenic variants or deletions. However, recent discoveries showed that deletions of the enhancer elements are quite common. The majority of these patients show less body disproportion and respond better to recombinant human growth hormone treatment. We redefined clinical criteria for genetic analysis to facilitate detection of the full spectrum of SHOX haploinsufficiency. METHODS: We analyzed 51 children with SHOX variants or deletions and 25 children with a deletion in its enhancer region. Data were compared to 277 children referred for suspicion of growth failure without endocrine or genetic pathology. RESULTS: Only half of the patients with an enhancer region deletion fulfilled any of the current screening criteria. We propose new clinical criteria based on sitting height to height ratio >1 SDS or arm span ≥3 cm below height, with a sensitivity of 99%. When these criteria are combined with obligatory short stature, the sensitivity to detect SHOX haploinsufficiency is 68.1%, the specificity 80.6%, and the number needed to screen 21 patients. CONCLUSION: Novel clinical criteria for screening for SHOX haploinsufficiency allow the detection of patients within the full genetic spectrum, that is, intragenic variants and enhancer region deletions.

Palmitate-induced C/EBP homologous protein activation leads to NF-κB-mediated increase in BACE1 activity and amyloid beta genesis.

The etiology of Alzheimer's disease (AD) is egregiously comprehended, but epidemiological studies have posited that diets rich in the saturated fatty acid palmitic acid (palmitate) are a significant risk factor. The production and accumulation of amyloid beta peptide (Aß) is considered the core pathological molecular event in the pathogenesis of AD. The rate-limiting step in Aß genesis from amyloid-ß precursor protein (AßPP) is catalyzed by the enzyme ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), the expression and enzymatic activity of which is significantly up-regulated in the AD brain. In this study, we determined the molecular mechanisms that potentially underlie the palmitate-induced up-regulation in BACE1 expression and augmented Aß production. We demonstrate that a palmitate-enriched diet and exogenous palmitate treatment evoke an increase in BACE1 expression and activity leading to enhanced Aß genesis in the mouse brain and SH-SY5Y-APPSwe cells, respectively, through the activation of the transcription factor NF-κB. Chromatin immunoprecipitation (ChIP) assays and luciferase reporter assays revealed that palmitate enhances BACE1 expression by increasing the binding of NF-κB in the BACE1 promoter followed by an enhancement in the transactivation of the BACE1 promoter. Elucidation and delineation of upstream molecular events unveiled a critical role of the endoplasmic reticulum stress-associated transcription factor, C/EBP homologous protein (CHOP) in the palmitate-induced NF-κB activation, as CHOP knock-down cells and Chop-/- mice do not exhibit the same degree of NF-κB activation in response to the palmitate challenge. Our study delineates a novel CHOP-NF-κB signaling pathway that mediates palmitate-induced up-regulation of BACE1 expression and Aß genesis.

Incontinentia pigmenti in a male (XY) infant with long-term follow up over 8 years.

Incontinentia pigmenti (IP) is an X-linked genodermatosis affecting the skin and other sites, including the teeth, nails, hair, eyes and nervous system defects in female patients. Generally lethal in males, there are only a few known cases of males surviving this condition. Nuclear factor (NF)-κB essential modulator (NEMO), also known as inhibitor of kappa light polypeptide gene enhancer in B cells, kinase gamma (IKBKG), constitutes an essential activator of NF-κB. Over 80% of female patients with IP carry a common deletion mutation involving exons 4-10 of the IKBKG/NEMO gene. We present the case of a male infant (XY) with IP with no concomitant complications. Polymerase chain reaction (PCR) assay showed that the exon 4-10 deletion band was significantly stronger in the skin sample than in blood. Subsequently, long-range PCR was performed periodically to confirm the spontaneous regression of mutant cells from his blood. Over a period of 6 years, the 2.6-kb mutant band gradually became weaker, but we did not confirm complete regression. Our patient was a healthy, 8-year-old male child with no complications despite the presence of a 2.6-kb mutant band in his blood. Further follow up is necessary to assess for complications that may develop later.

Acquired and Innate Immunity Impairment and Severe Disseminated Mycobacterium genavense Infection in a Patient With a NF-κB1 Deficiency.

Background: NF-κB1 is a master regulator of both acquired and innate responses. NFKB1 loss-of-function mutations elicit a wide clinical phenotype with asymptomatic individuals at one end of the spectrum and patients with common variable immunodeficiency, combined immunodeficiency or autoinflammation at the other. Impairment of acquired and innate immunity and disseminated Mycobacterium genavense infection expands the clinical and immunological phenotype of NF-κB1 deficiency. Objective: Functional and molecular characterization of a patient with a novel phenotype of NF-κB1 deficiency. Methods: Circulating T, B, dendritic cell subsets and innate or unconventional T-cells were quantified. The cytokine production in stimulated whole blood samples was assessed and molecular characterization by next generation sequencing and gene expression assays were also performed. Results: We report a patient presenting with features of combined immunodeficiency (CID) and disseminated Mycobacterium genavense infection. Sequencing of genomic DNA identified a novel synonymous mutation (c.705G > A) in NFKB1 gene which resulted in exon 8 skipping and haploinsufficiency of the NF-κB1 subunit p50. The susceptibility to atypical mycobacterial infection has not been previously reported and may be the result of a dendritic cell deficiency. A selective deficiency of circulating follicular helper T (cTFH) cells responsible for mediating the differentiation of naive B cells into memory and plasma cells was also present in the patient. It could affect the maturation of innate or unconventional T cells where NF-κB1 could also be involved. Conclusion: These findings showed that the role of NF-κB1 in humans could be critical for the development of acquired and innate immunity and further highlights the role of human T cells in anti-mycobacterial immunity.