FNDC3B 3'-UTR shortening escapes from microRNA-mediated gene repression and promotes nasopharyngeal carcinoma progression.

Alternative polyadenylation (APA), which induces shortening of the 3'-UTR, is emerging as an important feature in cancer development and progression. Nevertheless, the effects and mechanisms of APA-induced 3'-UTR shortening in nasopharyngeal carcinoma (NPC) remain largely unclear. Fibronectin type III domain containing 3B (FNDC3B) tended to use proximal polyadenylation site and produce shorter 3'-UTR according to our previous sequencing study. Herein, we found that FNDC3B with shorter 3'-UTR could escape from miRNA-mediated gene repression, and caused its increased expression in NPC. Knocking down of FNDC3B inhibited NPC cell proliferation, migration, invasion, and metastasis in vitro and in vivo. Overexpression of FNDC3B, especially those with shorter 3'-UTR, promoted NPC progression. Furthermore, the mechanism study revealed that FNDC3B could bind to and stabilize myosin heavy chain 9 (MYH9) to activate the Wnt/ß-catenin signaling pathway. In addition, MYH9 could reverse the inhibitory effects of FNDC3B knockdown in NPC. Altogether, our results suggested that the 3'-UTR shortening of FNDC3B mRNA mediated its overexpression in NPC and promoted NPC progression by targeting MYH9. This newly identified FNDC3B-MYH9-Wnt/ß-catenin axis could represent potential targets for individualized treatment in NPC.

Association Between Environmental Factors and Oral Epstein-Barr Virus DNA Loads: A Multicenter Cross-sectional Study in China.

Background: Oral Epstein-Barr virus (EBV) status reflects host EBV activity and potentially links to EBV-associated diseases, however, factors influencing oral EBV loads or reactivation, such as environmental exposures or host factors, are not fully understood. Methods: A 2-stage, multicenter, cross-sectional study of 6558 subjects from 21 administrative cities of southern China and 3 populations from representative geographical areas in China (referred to as the south, north, and northeastern populations) was performed. The relationships between demographical factors and environmental exposures to EBV loads were analyzed by logistic regression models. Results: Current smoking, with a dose-response effect, was found to be strongly associated with higher oral EBV loads in the pooled data, with an odds ratio of 1.58 (95% confidence interval, 1.39-1.79), as well as in each of the separate populations. The odds ratio increased to 3.06 when current smokers in southern China were compared to never smokers in northern China. Additionally, higher oral EBV loads tended to be detected in older participants, male participants, and participants in southern China. Conclusions: This study provided evidence linking the effect of host-environmental factors, particularly smoking, to oral EBV activity. It could strengthen our understanding of the possible causal roles of EBV-related diseases, which may help to prevent or mitigate EBV-associated diseases.

Differential genome-wide profiling of alternative polyadenylation sites in nasopharyngeal carcinoma by high-throughput sequencing.

BACKGROUND: Alternative polyadenylation (APA) is a widespread phenomenon in the posttranscriptional regulation of gene expression that generates mRNAs with alternative 3'-untranslated regions (3'UTRs). APA contributes to the pathogenesis of various diseases, including cancer. However, the potential role of APA in the development of nasopharyngeal carcinoma (NPC) remains largely unknown. METHODS: A strategy of sequencing APA sites (SAPAS) based on second-generation sequencing technology was carried out to explore the global patterns of APA sites and identify genes with tandem 3'UTRs in samples from 6 NPC and 6 normal nasopharyngeal epithelial tissue (NNET). Sequencing results were then validated using quantitative RT-PCR in a larger cohort of 16 NPC and 16 NNET samples. RESULTS: The sequencing data showed that the use of tandem APA sites was prevalent in NPC, and numerous genes with APA-switching events were discovered. In total, we identified 195 genes with significant differences in the tandem 3'UTR length between NPC and NNET; including 119 genes switching to distal poly (A) sites and 76 genes switching to proximal poly (A) sites. Several gene ontology (GO) terms were enriched in the list of genes with switched APA sites, including regulation of cell migration, macromolecule catabolic process, protein catabolic process, proteolysis, small conjugating protein ligase activity, and ubiquitin-protein ligase activity. CONCLUSIONS: APA site-switching events are prevalent in NPC. APA-mediated regulation of gene expression may play an important role in the development of NPC, and more detailed studies targeting genes with APA-switching events may contribute to the development of novel future therapeutic strategies for NPC.

ALS-linked mutations enlarge TDP-43-enriched neuronal RNA granules in the dendritic arbor.

Trans-activating response region (TAR) DNA-binding protein of 43 kDa (TDP-43) is an RNA-binding protein that is mutated in familial amyotrophic lateral sclerosis (ALS). Disease-linked mutations in TDP-43 increase the tendency of TDP-43 to aggregate, leading to a corresponding increase in formation of stress granules, cytoplasmic protein/RNA complexes that form in response to stress. Although the field has focused on stress granules, TDP-43 also forms other types of RNA granules. For example, TDP-43 is associated with RNA granules that are prevalent throughout the dendritic arbor in neurons. Because aggregation of TDP-43 is also important for the formation of these neuronal RNA granules, we hypothesized that disease-linked mutations might alter granule formation even in the absence of stress. We now report that ALS-linked mutations in TDP-43 (A315T and Q343R) increase the size of neuronal TDP-43 granules in the dendritic arbor of rat hippocampal neurons. The mutations correspondingly reduce the granule density, movement, and mobility of TDP-43 granules. Depolarization of rat hippocampal neurons with KCl stimulates TDP-43 granule migration into dendrites, but A315T and Q343R TDP-43 granules migrate shorter distances and into fewer dendrites than wild-type TDP-43. These findings highlight novel elements of TDP-43 biology that are affected by disease-linked mutations and suggest a neuronally selective mechanism through which TDP-43 mutations might elicit neuronal dysfunction.

The dual functions of the extreme N-terminus of TDP-43 in regulating its biological activity and inclusion formation.

TAR DNA-binding protein-43 (TDP-43) is the principal component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS) and the most common pathological subtype of frontotemporal dementia-frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). To date, the C-terminus of TDP-43, which is aggregation-prone and contains almost all ALS-associated mutations, has garnered much attention while the functions of the N-terminus of TDP-43 remain largely unknown. To bridge this gap in our knowledge, we utilized novel cell culture and computer-assisted models to evaluate which region(s) of TDP-43 regulate its folding, self-interaction, biological activity and aggregation. We determined that the extreme N-terminus of TDP-43, specifically the first 10 residues, regulates folding of TDP-43 monomers necessary for proper homodimerization and TDP-43-regulated splicing. Despite such beneficial functions, we discovered an interesting dichotomy: full-length TDP-43 aggregation, which is believed to be a pathogenic process, also requires the extreme N-terminus of TDP-43. As such, we provide new insight into the structural basis for TDP-43 function and aggregation, and we suggest that stabilization of TDP-43 homodimers, the physiologically active form of TDP-43, may be a promising therapeutic strategy for ALS and FTLD-TDP.

High expression of Talin-1 is associated with poor prognosis in patients with nasopharyngeal carcinoma.

BACKGROUND: Talin-1 is a cytoskeletal protein that plays an important role in tumourgenesis, migration and metastasis in several malignant tumors. The aim of this study was to evaluate the expression and prognostic value of Talin-1 in nasopharyngeal carcinoma (NPC). METHODS: Talin-1 mRNA and protein expression were examined in NPC cell lines and clinical nasopharyngeal tissues by quantitative RT-PCR, agarose gel electrophoresis and western blotting. The expression of Talin-1 was analyzed by immunohistochemical staining in 233 paraffin-embedded NPC specimens with clinical follow-up data and cox regression analysis was used to identify independent prognostic factors. The functional role of Talin-1 in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by the wound healing and transwell invasion assays. RESULTS: The expression of Talin-1 was significantly upregulated in most NPC cell lines and clinical tissues at both the mRNA and protein levels. High expression of Talin-1 was significantly associated with distant metastasis (P = 0.001) and patient death (P = 0.001). In addition, high expression of Talin-1 was associated with significantly poorer overall survival (OS: HR, 2.15; 95% CI, 1.28-3.63; P = 0.003) and poorer distant metastasis-free survival (DMFS: HR, 2.39; 95% CI, 1.38-4.15; P = 0.001). Cox regression analysis indicated that high expression of Talin-1 and TNM stage were independent prognostic indicators (both P < 0.05). Stratified analysis demonstrated that high expression of Talin-1 was associated with significantly poorer survival in patients with advanced stage disease (stage III-IV, HR, 1.91; 95% CI, 1.09-3.35; P = 0.02 for OS and HR, 2.22; 95% CI, 1.24-3.99; P = 0.006 for DMFS). Furthermore, the depletion of Talin-1 suppressed the migratory and invasive ability of NPC cells in vitro. CONCLUSIONS: Our data demonstrate that high expression of Talin-1 is associated with significantly poorer OS and poorer DMFS in NPC and depletion of Talin-1 expression inhibited NPC cell migration and invasion. Talin-1 may serve as novel prognostic biomarker in NPC.

Misregulation of human sortilin splicing leads to the generation of a nonfunctional progranulin receptor.

Sortilin 1 regulates the levels of brain progranulin (PGRN), a neurotrophic growth factor that, when deficient, is linked to cases of frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43)-positive inclusions (FTLD-TDP). We identified a specific splicing enhancer element that regulates the inclusion of a sortilin exon cassette (termed Ex17b) not normally present in the mature sortilin mRNA. This enhancer element is consistently present in sortilin RNA of mice and other species but absent in primates, which carry a premature stop codon within the Ex17b sequence. In the absence of TDP-43, which acts as a regulatory inhibitor, Ex17b is included in the sortilin mRNA. In humans, in contrast to mice, the inclusion of Ex17b in sortilin mRNA generates a truncated, nonfunctional, extracellularly released protein that binds to but does not internalize PGRN, essentially acting as a decoy receptor. Based on these results, we propose a potential mechanism linking misregulation of sortilin splicing with altered PGRN metabolism.

Overexpression of CIP2A is an independent prognostic indicator in nasopharyngeal carcinoma and its depletion suppresses cell proliferation and tumor growth.

BACKGROUND: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that acts as a prognostic marker for several human malignancies. In this study, we investigated the clinical significance of CIP2A and its function in nasopharyngeal carcinoma (NPC). METHODS: Quantitative RT-PCR, western blot, and immunohistochemistry analyses were used to quantify CIP2A expression in NPC cell lines and clinical samples. Kaplan-Meier curves were used to estimate the association between CIP2A expression and patient survival. The functional role of CIP2A in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation and xenograft growth. RESULTS: CIP2A levels were upregulated in NPC cell lines and clinical samples at both the mRNA and protein levels (P < 0.01). Patients with high CIP2A expression had poorer overall survival (HR, 1.98; 95% CI, 1.16-3.34; P = 0.01) and poorer disease-free survival (HR, 1.68; 95% CI, 1.07-2.62; P = 0.02) rates than patients with low CIP2A expression. In addition, CIP2A expression status was an independent prognostic indicator for NPC patients. The depletion of CIP2A expression inhibited c-Myc protein expression in NPC cell lines, suppressed cell viability, colony formation, and anchorage-independent growth in vitro, and inhibited xenograft tumor growth in vivo. CONCLUSIONS: Our data demonstrate that high CIP2A expression in patients was associated with poor survival in NPC, and depletion of CIP2A expression inhibited NPC cell proliferation and tumor growth. Thus, these results warrant further investigation of CIP2A as a novel therapeutic target for the treatment of NPC.

Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress.

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the "c9RAN proteins" thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.

MiR-451 inhibits cell growth and invasion by targeting MIF and is associated with survival in nasopharyngeal carcinoma.

BACKGROUND: MiRNAs play important roles in diverse biological processes including tumorigenesis. However, little is known about the function and mechanism of miR-451 in nasopharyngeal carcinoma (NPC). METHODS: Quantitative RT-PCR was used to quantify miR-451 expression in NPC cell lines and clinical tissues. Kaplan-Meier curves were used to estimate the association between miR-451 expression and survival. The MTT, colony formation, Transwell migration and invasion assays, and a xenograft model were performed. A miR-451 target was confirmed using luciferase reporter assays, quantitative RT-PCR, and Western blotting. RESULTS: MiR-451 was significantly downregulated in NPC cell lines and clinical tissues (P < 0.01). Patients with low expression of miR-451 had poorer overall survival (HR, 1.98; 95% CI, 1.16-3.34; P = 0.01) and disease-free survival (HR, 1.68; 95% CI, 1.07-2.62; P = 0.02) than patients with high expression. MiR-451 was an independent prognostic factor in NPC in multivariate Cox regression analysis. Ectopic expression of miR-451 suppressed cell viability, colony formation, and cell migration and invasion in vitro, and inhibited xenograft tumor growth in vivo. MIF was verified as a direct target of miR-451, and MIF regulated NPC cell growth and invasion. CONCLUSIONS: The newly identified miR-451/MIF pathway provides insight into NPC initiation and progression, and may represent a novel therapeutic target.

Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity.

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a approximately 25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitin- and phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies.

The Proinflammatory Cytokines IL-18, IL-21, and IFN-γ Differentially Regulate Liver Inflammation and Anti-Mitochondrial Antibody Level in a Murine Model of Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a cholestatic liver disease primarily featured by autoimmune-mediated damage of intrahepatic small- and medium-sized bile ducts. Elevated serum proinflammatory cytokines, serum anti-mitochondrial antibodies (AMAs), liver inflammation, and fibrosis are also hallmarks of PBC disease. However, whether the elevated proinflammatory cytokines play a role in autoimmune cholangitis remains unknown. Herein, we utilized the p40-/-IL-2Rα -/- PBC mouse model to investigate the roles of proinflammatory cytokines IL-18, IL-21, and IFN-γ in the onset and progression of PBC. IL-18-/-, IFN-γ -/-, and IL-21-/- mice were crossed with p40-/-IL-2Ra+/- mice, respectively, to produce corresponding cytokine-deficient PBC models. Autoantibody level, liver inflammation, and bile duct injury were analyzed. We found that livers from p40-/-IL-2Rα -/- mice exhibit similar transcriptomic characters of PBC patients. In p40-/-IL-2Rα -/- mice, deletion of IL-18 has no remarkable effect on disease progression, while deletion of IL-21 indicates that it is necessary for AMA production but independent of liver inflammation and cholangitis. IFN-γ is responsible for both AMA production and liver inflammation in our model. Our results demonstrate that different proinflammatory cytokines can regulate different effector functions in PBC pathogenesis and need to be considered in PBC treatment.

Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice.

Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose-dependent downregulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, and intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities, and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the CNS and for studying TDP-43-associated neurotoxicity.

Human genetic variants of homologous recombination repair genes first found to be associated with Epstein-Barr virus antibody titers in healthy Cantonese.

Epstein-Barr virus (EBV) infection is a major risk factor for nasopharyngeal carcinoma (NPC). Despite high prevalence of infection among the general population worldwide, only a small proportion of infected individuals presents with seropositivity for EBV-specific IgA antibodies. This seropositive subgroup of EBV carriers has an elevated cumulative risk for NPC during their lifetime. Previous studies reported that the host homologous recombination repair (HRR) system participates in EBV lytic replication, suggesting a potential mechanism to influence EBV reactivation status and thus seropositivity. To investigate whether genetic variants of HRR genes are associated with the serostatus in a healthy population, we investigated the association between seropositivity for anti-VCA-IgA and 156 tagging SNPs in 35 genes connected with HRR in an observational study among 755 healthy Cantonese speakers in southern China. Six variant alleles of MDC1, RAD54L, TP53BP1, RPA1, LIG3 and RFC1 exhibited associations with seropositivity (p(trend) from 0.0085 to 0.00027). Our study provides evidence that genetic variation within the HRR might affect an individual's propensity for EBV seropositive status of anti-VCA IgA antibody.

Accelerated lipofuscinosis and ubiquitination in granulin knockout mice suggest a role for progranulin in successful aging.

Progranulin (PGRN) is involved in wound repair, inflammation, and tumor formation, but its function in the central nervous system is unknown. Roles in development, sexual differentiation, and long-term neuronal survival have been suggested. Mutations in the GRN gene resulting in partial loss of the encoded PGRN protein cause frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions. We sought to understand the neuropathological consequences of loss of PGRN function throughout the lifespan of GRN-deficient ((-/+) and (-/-)) mice. An aged series of GRN-deficient and wild-type mice were compared by histology, immunohistochemistry, and electron microscopy. Although GRN-deficient mice were viable, GRN(-/-) mice were produced at lower than predicted frequency. Neuropathologically, GRN(-/+) were indistinguishable from controls; however, GRN(-/-) mice developed age-associated, abnormal intraneuronal ubiquitin-positive autofluorescent lipofuscin. Lipofuscin was noted in aged GRN(+/+) mice at levels comparable with those of young GRN(-/-) mice. GRN(-/-) mice developed microgliosis, astrogliosis, and tissue vacuolation, with focal neuronal loss and severe gliosis apparent in the oldest GRN(-/-) mice. Although no overt frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions type- or TAR DNA binding protein-43-positive lesions were observed, robust lipofuscinosis and ubiquitination in GRN(-/-) mice is strikingly similar to changes associated with aging and cellular decline in humans and animal models. Our data suggests that PGRN plays a key role in maintaining neuronal function during aging and supports the notion that PGRN is a trophic factor essential for long-term neuronal survival.

Akt and CHIP coregulate tau degradation through coordinated interactions.

A hallmark of the pathology of Alzheimer's disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PAR1/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimer's disease.

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis.

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

[Regulation of RNA Binding Protein Mbnl1 on Development of Mouse Embryonic Hematopoietic Stem Cells].

OBJECTIVE: To analyze the dynamic molecular expression characteristics of single cell RNA binding proteins (RBPs) in the development of mouse embryonic hematopoitic stem cells (HSCs), and obtain the functional research target RNA splicing factor--Mbnl1, to clarify the function of Mbnl1 involved in regulating mouse embryonic HSC development. METHODS: Bioinformatics was used to analyze the single-cell transcriptome data of mouse embryos during HSC development, and the single-cell RBP dynamic molecular expression maps in HSC development was obtained. Mbnl1 was obtained by combining differential analysis and literature research screening. The Mbnl1-knockout mouse model was constructed by the CRISPER/Cas9 technology. Aorta-gonad-mesonephros (AGM) and yolk sac (YS) tissue in two genotype embryos of Mbnl1+/+ and Mbnl1-/- at E11.5 were digested into single cells, and then a methylcellulose semi-solid culture system was used to perform an in vitro CFU-C of hematopoietic cells. The number and type of hematopoietic cell colonies in the two hematopoietic tissues in Mbnl1-knockout mice after 7 days were calculated. Furthermore, the whole AGM tissue cells of E11.5 Mbnl1+/+ and Mbnl1-/- donor mice were transplanted via tail vein injection respectively, and the function of HSC in AGM region of mice after Mbnl1 knockout was evaluated through the chimerism rate of peripheral blood in recipient mice at 4w and 8w and the kinetic experiment of hematopoietic system reconstruction. RESULTS: The in vitro CFU-C experiment of hematopoietic cells preliminarily indicated that there was no significant difference in the number of cell colonies in AGM region and YS transformed by the two genotypes of Mbnl1+/+ and Mbnl1-/- at E11.5, which suggested that HPC function in AGM and YS tissues had no abnormality after Mbnl1 knockout. Single cell transplantation in AGM region vin tail vein showed no significant difference in the hematopoietic reconstruction chimeric rate between Mbnl1+/+ and Mbnl1-/- mice, suggesting that there was no abnormality in HSC function in AGM tissues after Mbnl1 knockout. CONCLUSION: Through functional experiments in vivo and in vitro, it has been confirmed that knockout of the RNA splicing factor--Mbnl1 does not affect the development of HSPC in AGM region of mouse embryo.

Characterization of Organ-Specific Regulatory B Cells Using Single-Cell RNA Sequencing.

Regulatory B cells (Breg) are considered as immunosuppressive cells. Different subsets of Breg cells have been identified both in human beings and in mice. However, there is a lack of unique markers to identify Breg cells, and the heterogeneity of Breg cells in different organs needs to be further illuminated. In this study, we performed high-throughput single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) of B cells from the murine spleen, liver, mesenteric lymph nodes, bone marrow, and peritoneal cavity to better define the phenotype of these cells. Breg cells were identified based on the expression of immunosuppressive genes and IL-10-producing B (B10) cell-related genes, to define B10 and non-B10 subsets in Breg cells based on the score of the B10 gene signatures. Moreover, we characterized 19 common genes significantly expressed in Breg cells, including Fcrl5, Zbtb20, Ccdc28b, Cd9, and Ptpn22, and further analyzed the transcription factor activity in defined Breg cells. Last, a BCR analysis was used to determine the clonally expanded clusters and the relationship of Breg cells across different organs. We demonstrated that Atf3 may potentially modulate the function of Breg cells as a transcription factor and that seven organ-specific subsets of Breg cells are found. Depending on gene expression and functional modules, non-B10 Breg cells exhibited activated the TGF-ß pathway, thus suggesting that non-B10 Breg cells have specific immunosuppressive properties different from conventional B10 cells. In conclusion, our work provides new insights into Breg cells and illustrates their transcriptional profiles and BCR repertoire in different organs under physiological conditions.

Sequencing of DC-SIGN promoter indicates an association between promoter variation and risk of nasopharyngeal carcinoma in cantonese.

BACKGROUND: The dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) is an important pathogen recognition receptor of the innate immune system. DC-SIGN promoter variants play important role in the susceptibility to various infectious diseases. Nasopharyngeal carcinoma (NPC) is a malignancy that is common in southern China and whether DC-SIGN promoter variants have effects on susceptibility to NPC is still unknown. The aim of this study is to ascertain the potential involvement of DC-SIGN promoter single nucleotide polymorphisms (SNPs) in NPC susceptibility. METHODS: We conducted a case control study based on Cantonese population including 444 NPC patients and 464 controls matched on age and sex. The 1041 bp of DC-SIGN promoter region was directly sequenced for all samples. Sequence alignment and SNP search were inspected using DNAStar analysis programs and haplotype frequencies were estimated in Haploview V 4.0. The associations between the SNPs and the risk of NPC were analyzed using chi-square test and non-conditional logistic regression analysis with SPSS 13.0 software. RESULTS: A total of six variants were observed in the DC-SIGN promoter region and DC-SIGN -139 GG and -939 AA were significantly associated with NPC risk with adjusted Odds Ratios (ORs) of 2.10 (95% confidence interval [CI] = 1.23-3.59; P = 0.006) and 2.52 (1.29-4.93; P = 0.007) respectively and subjects carrying the risk allele DC-SIGN -871 G had 1.47-fold (95% CI = 1.14-1.90) increased risks of developing NPC (P = 0.003). Haplotype analysis revealed that h1 'AAAG' was significantly associated with protection against NPC (OR = 0.69; P = 0.0002) and the association was still significant when using 1000 permutation test runs (P = 0.001). CONCLUSIONS: Our study indicated that DC-SIGN promoter variants appear to be involved in the susceptibility to NPC and the detailed mechanism of this effect need further studies.