Search

Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.

As an immune-regulator, Astragalus polysaccharides (APS) could effectively modulate the activity of toll-like receptor 4 (TLR4) signaling pathway, and induce anti-inflammatory response in intestinal. Our research before indicated that toll/interleukin 1 receptor-domain-containing adapter-inducing interferon-b (TRIF) might be a critical regulator for APS. So, in this experiment, we analyzed the effects of APS on lipopolysaccharide (LPS)-infected Caco2 cells in the circumstances of TRIF knockout. By using qRT-PCR and flow cytometry method, we analyzed the genes expression at transcriptional and translational level, respectively. The results of genes expression at both transcription and translation level showed that LPS could activate the myeloid differentiation factor 88 (MyD88)-TNF receptor associated factor (TRAF) pathway downstream from TLR4, and induce the high expression of pro-inflammatory cytokines. However, APS could effectively suppress the LPS induced inflammatory response. While, in the context of TRIF knockout, APS couldn't effectively attenuate the LPS activated MyD88-TRAF6 pathway, as well as the expression of pro-inflammatory cytokines. Above all, we concluded that APS could antagonize the LPS induced inflammatory response by a TRIF-dependent manner.

The emergence of a coordinated network of cognitive and speech genes in the human lineage performing overlapping functions is a great evolutionary puzzle. Prior studies on the speech gene FOXP2 are inconclusive on the nature of selection operating on this gene in the human lineage. Here, I show that the evolution of FOXP2 is accelerated in the human lineage due to relaxation of purifying selection (relaxed selection). Five potential genes associated with human-specific intelligence and speech genes have evolved under the impact of positive selection and three genes including FOXP2 have undergone relaxation of purifying selection in the human lineage. Overall, three evolutionary processes namely positive selection, relaxation of purifying selection and neutral evolution have contributed for the genomic evolution of extraordinary cognitive ability and speech in the hominin lineage. The cognitive and speech genes subjected to natural selection in the human lineage have demonstrated a coevolutionary trend.

Objective Adenoid cystic carcinoma (AdCC) and mucoepidermoid carcinoma (MEC) are the most frequent malignancies in the salivary glands. The purpose of this study was to explore the roles of sineoculis homeobox homolog 1 (SIX1), malic enzyme 2 (ME2) and AP-2 complex subunit mu (AP2M1) in AdCC and MEC, as well as the potential relationship between SIX1, ME2, AP2M1 and proliferation marker cyclin D1. Material and methods Immunohistochemistry was performed on human salivary gland tissue microarrays that contained 76 normal salivary glands (NSGs), 14 pleomorphic adenomas (PMAs), 81 AdCCs and 52 MECs. Results We observed that the expression levels of SIX1 and ME2 were significantly elevated in AdCC and MEC when compared with NSG and PMA. In addition, we detected that AP2M1 was overexpressed in AdCC and MEC when compared with NSG. We also found that SIX1 and AP2M1 were positively associated with cyclin D1. Conclusions These results may prove that SIX1 and AP2M1 are involved in the proliferation of AdCC and MEC to cause tumor growth.

The antiepileptic and mood stabilizer agent valproic acid (VPA) has been shown to exert anti-tumour effects and to cause neuronal damage in the developing brain through mechanisms not completely understood. In the present study we show that prolonged exposure of SH-SY5Y and LAN-1 human neuroblastoma cells to clinically relevant concentrations of VPA caused a marked induction of the protein and transcript levels of the common neurotrophin receptor p75NTR and its co-receptor sortilin, two promoters of apoptotic cell death in response to proneurotrophins. VPA induction of p75NTR and sortilin was associated with an increase in plasma membrane expression of the receptor proteins and was mimicked by cell treatment with several histone deacetylase (HDAC) inhibitors. VPA and HDAC1 knockdown decreased the level of EZH2, a core component of the polycomb repressive complex 2, and upregulated the transcription factor CASZ1, a positive regulator of p75NTR. CASZ1 knockdown attenuated VPA-induced p75NTR overexpression. Cell treatment with VPA favoured proNGF-induced p75NTR/sortilin interaction and the exposure to proNGF enhanced JNK activation and apoptotic cell death elicited by VPA. Depletion of p75NTR or addition of the sortilin agonist neurotensin to block proNGF/sortilin interaction reduced the apoptotic response to VPA and proNGF. Exposure of mouse cerebellar granule cells to VPA upregulated p75NTR and sortilin and induced apoptosis which was enhanced by proNGF. These results indicate that VPA upregulates p75NTR apoptotic cell signalling through an epigenetic mechanism involving HDAC inhibition and suggest that this effect may contribute to the anti-neuroblastoma and neurotoxic effects of VPA. Electronic supplementary material The online version of this article (10.1007/s10495-020-01626-0) contains supplementary material, which is available to authorized users.

Keratoconus is a common ocular disorder that causes progressive corneal thinning and is the leading indication for corneal transplantation. Central corneal thickness (CCT) is a highly heritable characteristic that is associated with keratoconus. In this two-stage genome-wide association study (GWAS) of CCT, we identified a locus for CCT, namely STON2 rs2371597 (P = 2.32 × 10−13), and confirmed a significant association between STON2 rs2371597 and keratoconus development (P = 0.041). Additionally, strong STON2 expression was observed in mouse corneal epithelial basal cells. We also identified SMAD3 rs12913547 as a susceptibility locus for keratoconus development using predictive analysis with IBM’s Watson question answering computer system (P = 0.001). Further GWAS analyses combined with Watson could effectively reveal detailed pathways underlying keratoconus development., Yoshikatsu Hosoda et al. study the genetic basis for central corneal thickness (CCT) that is associated with keratoconus. They identify two susceptibility loci, STON2 rs2371597 and SMAD3 rs12913547, using two-step genome-wide association study (GWAS) and predictive analysis with IBM’s Watson question answering computer system, respectively.

Ancestral sequence reconstruction has had recent success in decoding the origins and the determinants of complex protein functions. However, phylo­genetic analyses of remote homologues must handle extreme amino-acid se­quence diversity resulting from extended periods of evolutionary change. We exploited the wealth of protein structures to develop an evolutionary model based on protein secondary structure. The approach follows the differences between discrete secondary structure states observed in modern proteins and those hypothesised in their immediate ancestors. We implemented maximum likelihood-based phylogenetic inference to reconstruct ancestral secondary structure. The predictive accuracy from the use of the evolutionary model surpasses that of comparative modelling and sequence-based prediction; the reconstruction extracts information not available from modern structures or the ancestral sequences alone. Based on a phylogenetic analysis of multiple protein families, we showed that the model can highlight relationships that are evolutionarily rooted in structure and not evident in amino acid-based analysis.

Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1–3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4–6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event. Hsu et al. find that the protein kinase Akt acts as a co-adaptor in the clathrin coat complex and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling.

Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the main causes of death and nervous system damage in neonates. The aim of the present study was to investigate the effect of the Toll‑like receptor 4 (TLR4) antagonist TAK‑242 on HIE. The Rice‑Vannucci method was used for ligation of the left common carotid artery, followed by hypoxic treatment for 2.5 h to establish a neonatal HIE rat model. Rats were intraperitoneally injected with 7.5 ml/kg TAK‑242 after hypoxia‑ischemia. It was demonstrated that TAK‑242 significantly reduced the infarct volume and cerebral edema content of neonatal rats after HIE, alleviating neuronal damage and neurobehavioral function deficits. Furthermore, TAK‑242 decreased the protein expression levels of TLR4, MyD88, TIR‑domain‑containing adapter‑inducing interferon‑β (TRIF), NF‑κB, tumor necrosis factor α (TNF‑α) and interleukin‑1β in the hippocampus. The present results suggested that TAK‑242 may exert a neuroprotective effect after HIE by inhibiting the TLR4/MyD88/TRIF/NF‑κB signaling pathway, and reducing the release of downstream inflammatory cytokines.

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.

Purpose: To describe the presence of neurotrophic growth factors and histopathologic characteristics of internal limiting membrane (ILM) specimens obtained from large idiopathic full-thickness macu...

Metastatic cancer cells invade surrounding tissues by forming dynamic actin-based invadopodia, which degrade the surrounding extracellular matrix and allow cancer cell invasion. Regulatory RNAs, including circular RNA, have been implicated in this process. By microarray, we found that the circular RNA circSKA3 was highly expressed in breast cancer cells and human breast cancer tissues. We further found that the invasive capacity of breast cancer cells was positively correlated with circSKA3 expression, through the formation of invadopodia. Mechanistically, we identified Tks5 and integrin β1 as circSKA3 binding partners in these tumor-derived invadopodia. Ectopic circSKA3 expression conferred increased tumor invasiveness in vitro and in vivo. We further identified the RNA-protein binding sites between circSKA3, Tks5 and integrin β1. In tumor formation assays, we found that circSKA3 expression promoted tumor progression and invadopodium formation. Mutation of the circSKA3 binding sites or transfection with blocking oligos abrogated the observed effects. Thus, we provide evidence that the circular RNA circSKA3 promotes tumor progression by complexing with Tks5 and integrin β1, inducing invadopodium formation.

Activated epidermal growth factor receptors (EGFRs) are crucial for inducing metastasis in cancer cells by promoting matrix metalloproteinase (MMP) expression. The present study was designed to investigate the effects of 1‑palmitoyl‑2‑linoleoyl‑3‑acetyl‑rac‑glycerol (PLAG) on MMP expression in epidermal growth factor (EGF)‑stimulated breast cancer cells in vitro. EGF stimulation induced internalization of its cognate receptor, EGFR, for stimulus‑desensitization. These internalized receptors, complexed with the ubiquitin ligase c‑Cbl and EGFR pathway substrate 15 (EPS15) (for degradation), were evaluated by confocal microscopy at 5‑90 min time intervals. During intracellular trafficking of EGFRs, EGF‑induced signaling cascades were analyzed by examining EGFR and SHC phosphorylation. Modulation of MMP expression was assessed by evaluating the activity of transcription factor AP‑1 using a luciferase assay. PLAG accelerated the assembly of EGFRs with c‑Cbl and EPS15 and promoted receptor degradation. This faster intracellular EGFR degradation reduced AP‑1‑mediated MMP expression. PLAG stimulation upregulated thioredoxin‑interacting protein (TXNIP) expression, and this mediated the accelerated receptor internalization. This PLAG‑induced increase in EGFR trafficking was blocked in TXNIP‑silenced cells. By downregulating MMP expression, PLAG effectively attenuated EGF‑induced mobility and invasiveness in these cancer cells. These data suggest that PLAG may be a potential therapeutic agent for blocking metastasis.

Background: Brain-Derived Neurotrophic Factor (BDNF) plays critical roles during development of the central and peripheral nervous systems, as well as in neuronal survival after injury. Although proBDNF induces neuronal apoptosis after injury in vivo, whether it can also act as a death factor in vitro and in vivo under physiological conditions and after nerve injury, as well as its mechanism of inducing apoptosis, is still unclear. Objective: In this study, we investigated the mechanisms by which proBDNF causes apoptosis in sensory neurons and Satellite Glial Cells (SGCs) in Dorsal Root Ganglia (DRG) After Sciatic Nerve Transection (SNT). Methods: SGCs cultures were prepared and a scratch model was established to analyze the role of proBDNF in sensory neurons and SGCs in DRG following SNT. Following treatment with proBDNF antiserum, TUNEL and immunohistochemistry staining were used to detect the expression of Glial Fibrillary Acidic Protein (GFAP) and Calcitonin Gene-Related Peptide (CGRP) in DRG tissue; immunocytochemistry and Cell Counting Kit-8 (CCK8) assay were used to detect GFAP expression and cell viability of SGCs, respectively. RT-qPCR, western blot, and ELISA were used to measure mRNA and protein levels, respectively, of key factors in BDNF-TrkB, proBDNF-p75NTR/sortilin, and apoptosis signaling pathways. Results: proBDNF induced mitochondrial apoptosis of SGCs and neurons by modulating BDNF-TrkB and proBDNF-p75NTR/sortilin signaling pathways. In addition, neuroprotection was achieved by inhibiting the biological activity of endogenous proBDNF protein by injection of anti-proBDNF serum. Furthermore, the anti-proBDNF serum inhibited the activation of SGCs and promoted their proliferation. Conclusion: proBDNF induced apoptosis in SGCs and sensory neurons in DRG following SNT. The proBDNF signaling pathway is a potential novel therapeutic target for reducing sensory neuron and SGCs loss following peripheral nerve injury.

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.

Autophagy, a catabolic recycling process, has been implicated as a critical pathway in cancer. Its role in maintaining cellular homeostasis helps to nourish hypoxic, nutrient-starved tumors and protects them from chemotherapy-induced death. Recent efforts to target autophagy in cancer have focused on kinase inhibition, which has led to molecules that lack specificity due to the multiple roles of key kinases in this pathway. For example, the lipid kinase VPS34 is present in two multiprotein complexes responsible for the generation of phosphatidylinositol-3-phosphate. Complex I generates the autophagosome, and Complex II is crucial for endosomal trafficking. Molecules targeting VPS34 inhibit both complexes, which inhibits autophagy but causes undesirable defects in vesicle trafficking. The lack of specific autophagy modulators has limited the utility of autophagy inhibition as a therapeutic strategy. We hypothesize that disruption of the Beclin 1-ATG14L protein-protein interaction, which is required for the formation, proper localization, and function of VPS34 Complex I but not Complex II, will disrupt Complex I formation and selectively inhibit autophagy. To this end, a high-throughput, cellular NanoBRET assay was developed targeting this interaction. An initial screen of 2560 molecules yielded 19 hits that effectively disrupted the interaction, and it was confirmed that one hit disrupted VPS34 Complex I formation and inhibited autophagy. In addition, the molecule did not disrupt the Beclin 1-UVRAG interaction, critical for VPS34 Complex II, and thus had little impact on vesicle trafficking. This molecule is a promising new tool that is critical for understanding how modulation of the Beclin 1-ATG14L interaction affects autophagy. More broadly, its discovery demonstrates that targeting protein-protein interactions found within the autophagy pathway is a viable strategy for the discovery of autophagy-specific probes and therapeutics.

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection., Nature Communications, 11 (1), ISSN:2041-1723

Endothelial cell sprouting is a critical event in tumor-induced angiogenesis. In melanoma and lung cancer murine models, targeting RhoJ prevents endothelial sprouting, tumor growth and metastasis and enhances the effects of conventional anti-neoplastic therapy. Aiming to understand how RhoJ is activated, we used a gain of function approach to identify constitutively active Rho guanine nucleotide exchange factors (RhoGEFs) able to promote RhoJ-dependent actin-driven membrane protrusions. We demonstrate that a membrane-anchored Intersectin 1 (ITSN1) DH-PH construct promotes endothelial cell sprouting via RhoJ. Mechanistically, this is controlled by direct interaction between the catalytic ITSN1 DH-PH module and RhoJ, it is sensitive to phosphorylation by focal adhesion kinase (FAK) and to endosomal trapping of the ITSN1 construct by dominant negative RhoJ. This ITSN1/RhoJ signaling axis is independent of Cdc42, a previously characterized ITSN1 target and a RhoJ close homologue. In conclusion, our results elucidate an ITSN1/RhoJ molecular link able to promote endothelial cell sprouting and set the basis to explore this signaling pathway in the context of tumor-induced angiogenesis.

Integrin receptors orchestrate cell adhesion and cytoskeletal reorganization. The endocytic mechanism of integrin-β3 receptor at the podosome remains unclear. Using viscous RGD-membrane as the model system, here we show that the formation of podosome-like adhesion promotes Dab2/clathrin-mediated endocytosis of integrin-β3. Integrin-β3 and RGD ligand are endocytosed from the podosome and sorted into the endosomal compartment. Inhibitions of podosome formation and knockdowns of Dab2 and clathrin reduce RGD endocytosis. F-actin assembly at the podosome core exhibits protrusive contact towards the substrate and results in plasma membrane invaginations at the podosome ring. BIN1 specifically associates with the region of invaginated membrane and recruits DNM2. During the podosome formation, BIN1 and DNM2 synchronously enrich at the podosome ring and trigger clathrin dissociation and RGD endocytosis. Knockdowns of BIN1 and DNM2 suppress RGD endocytosis. Thus, plasma membrane invagination caused by F-actin polymerization promotes BIN1-dependent DNM2 recruitment and facilitate integrin-β3 endocytosis at the podosome., Cao et al. investigate the mechanism of integrin-β3 endocytosis on podosomes from cells on viscous RGD- membranes. By using live imaging, the authors monitor actin and membrane dynamics during podosome formation and show that integrin-β3/RGD endocytosis is DAB2/clathrin mediated and dynamin-2 and BIN1 dependent.

Ricin toxin (RT) is a natural plant-derived protein toxin from the seed of castor beans that belongs to a family of type II ribosome-inactivating proteins (RIPs). In addition to its main toxic mechanism of inhibiting the synthesis of cellular proteins, RT can induce the production of inflammatory cytokines and cause inflammatory injury. Macrophages play a crucial role in innate immunity and the adaptive immune response as the first line of host defense against bacterial infections and various types of invading pathogens. Upon activation, macrophages release types of cytokines to remove pathogens. However, the effect of RT on the immune response and its mechanism are not well characterized. In the current study, we investigated the activation of the TLR4-mediated signaling pathway by low-dose RT treatment and its interaction with signaling molecules in the transduction pathway. We found that low-dose RT can activate MyD88- and TRIF-dependent signaling pathways, revealing a possible mechanism by which low-dose RT-activates TLR4-mediated signaling pathways. We also confirmed that the TLR4-induced activation of the inflammatory signaling pathways was produced via its binding to RT. This study may help to identify the most important target molecules and clarify the mechanism of inflammatory injury of ricin.

Toll-like receptor 3 (TLR3)-mediated signaling is important for host defense against RNA virus. Upon viral RNA stimulation, toll and interleukin-1 receptor domain-containing adaptor inducing IFN-β (TRIF) is recruited to TLR3 and then undergoes oligomerization, which is required for the recruitment of downstream molecules to transmit signals. Here, we identified zinc finger CCHC-type containing 3 (ZCCHC3) as a positive regulator of TLR3-mediated signaling. Overexpression of ZCCHC3 promoted transcription of downstream antiviral genes stimulated by the synthetic TLR3 ligand poly(I:C). ZCCHC3-deficiency markedly inhibited TLR3- but not TLR4-mediated induction of type I interferons (IFNs) and proinflammatory cytokines. Zcchc3−/− mice were more resistant to poly(I:C)- but not lipopolysaccharide-induced inflammatory death. Mechanistically, ZCCHC3 promoted recruitment of TRIF to TLR3 after poly(I:C) stimulation. Our findings reveal that ZCCHC3 plays an important role in TLR3-mediated innate immune response by promoting the recruitment of TRIF to TLR3 after ligand stimulation., Graphical Abstract Graphical Abstract

We propose a novel variant set test for rare-variant association studies, which leverages multiple single-nucleotide variant (SNV) annotations. Our approach optimizes a convex combination of different sequence kernel association test (SKAT) statistics, where each statistic is constructed from a different annotation and combination weights are optimized through a multiple kernel learning algorithm. The combination test statistic is evaluated empirically through data splitting. In simulations, we find our method preserves type I error at α = 2.5 × 1 0 - 6 and has greater power than SKAT(-O) when SNV weights are not misspecified and sample sizes are large ( N ≥ 5 , 000 ). We utilize our method in the Framingham Heart Study (FHS) to identify SNV sets associated with fasting glucose. While we are unable to detect any genome-wide significant associations between fasting glucose and 4-kb windows of rare variants ( p 1 0 - 7 ) in 6,419 FHS participants, our method identifies suggestive associations between fasting glucose and rare variants near ROCK2 ( p = 2.1 × 1 0 - 5 ) and within CPLX1 ( p = 5.3 × 1 0 - 5 ). These two genes were previously reported to be involved in obesity-mediated insulin resistance and glucose-induced insulin secretion by pancreatic beta-cells, respectively. These findings will need to be replicated in other cohorts and validated by functional genomic studies.

Post‐transcriptional regulation of cytokine production is crucial to ensure appropriate immune responses. We previously demonstrated that poly‐rC‐binding protein‐1 (PCBP1) can act as a trans‐acting factor to stabilize transcripts encoding sortilin, which mediates cytokine trafficking. Here, we report that PCBP2, which strongly resembles PCBP1, can stabilize sortilin transcripts in macrophages using the same mechanism employed by PCBP1. PCBP2 recognized the C‐rich element in the 3′ UTR of sortilin mRNA, and PCBP2 knockdown decreased sortilin transcripts, indicating that PCBP2 stabilizes sortilin mRNA by binding to its 3′ UTR. Zn2+ reversibly inhibited the nucleotide binding ability of PCBP2 in vitro. These findings suggest that both PCBP2 and PCBP1 may control the stability of sortilin transcripts by sensing intracellular Zn2+ levels in immune cells., We analyzed the function of poly‐rC‐binding protein‐2 (PCBP2), a paralog of PCBP1, in stabilizing the mRNA of sortilin, a cytokine trafficking mediator. PCBP2 binds to C‐rich elements in the 3′ UTR of sortilin mRNA; Zn2+ interferes with this interaction, suggesting that both PCBP1 and PCBP2 may regulate the stability of sortilin mRNA by sensing the intracellular Zn2+ levels.

Objective The development of blood-based biomarkers for early diagnosis and treatment of Alzheimer’s disease (AD) is desirable. In AD model mouse brain and neuronal cells, Abelson helper integration site-1 (AHI1) protein is reduced. AHI1 facilitates intracellular amyloid precursor protein (APP) translocation to inhibit amyloidogenic pathology of AD , and thus may be an AD biomarker . Methods This study was conducted among 32 AD patients and 54 healthy control (HC) subjects. AHI1-related protein levels from initially collected serum samples in each group were screened using Western blotting . The protein concentrations of AHI1 and amyloid-β (Aβ), peptide(s) derived from APP, from all serum samples were analyzed using ELISA. Results In AD serum, AHI1 and a large truncated C-terminal APP fragment were significantly reduced. The average concentrations of serum AHI1 and Aβ in AD were significantly lower than those in HC. Notably, AHI1 concentration in HC serum was decreased in an age-dependent manner, while it was consistently low in AD serum and had no correlation with Aβ or mini-mental state examination score. The receiver operating characteristic analysis on all subjects demonstrated an area under curve (AUC) value of 0.7 for AHI1 on AD diagnosis, while the AUC increased to 0.82 on the subjects younger than 77 years old, suggesting a good diagnostic performance of serum AHI1 for AD especially at relatively young age. Conclusion An early event of AHI1 reduction in the body of AD patients was observed. Serum AHI1 may be valuable for early diagnosis of AD .

Experimental and clinical studies aimed at investigating the mechanism(s) underlying vascular complications of diabetes indicate that a great number of molecules are involved in the pathogenesis of these complications. Most of these molecules are inflammatory mediators or markers generated by immune or adipose tissue. Some of them, i.e. resistin and sortilin, have been shown to be involved in the cross talk between adipocytes and inflammatory cells. This interaction is an attractive area of research, particularly in type 2 diabetes and obesity. Other proteins, such as adiponectin and visfatin, appear to be more promising as possible vascular markers. In addition, some molecules involved in calcium/phosphorus metabolism, such as klotho and FGF23, have an involvement in the pathogenesis of diabetic vasculopathy, which appears to be dependent on the degree of vascular impairment. Inflammatory markers are a promising tool for treatment decisions while measuring plasma levels of adipokines, sortilin, Klotho and FGF23 in adequately sized longitudinal studies is expected to allow a more precise characterization of diabetic vascular disease and the optimal use of personalized treatment strategies.

The activation of C-C chemokine receptor type 1 (CCR1) has been shown to be pro-inflammatory in several animal models of neurological diseases. The objective of this study was to investigate the activation of CCR1 on neuroinflammation in a mouse model of intracerebral hemorrhage (ICH) and the mechanism of CCR1/tetratricopeptide repeat 1 (TPR1)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in CCR1-mediated neuroinflammation. Adult male CD1 mice (n = 210) were used in the study. The selective CCR1 antagonist Met-RANTES was administered intranasally at 1 h after autologous blood injection. To elucidate potential mechanism, a specific ERK1/2 activator (ceramide C6) was administered prior to Met-RANTES treatment; CCR1 activator (recombinant CCL5, rCCL5) and TPR1 CRISPR were administered in naïve mouse. Neurobehavioral assessments, brain water content, immunofluorescence staining, and western blot were performed. The endogenous expressions of CCR1, CCL5, TPR1, and p-ERK1/2 were increased in the brain after ICH. CCR1 were expressed on microglia, neurons, and astrocytes. The inhibition of CCR1 with Met-RANTES improved neurologic function, decreased brain edema, and suppressed microglia/macrophage activations and neutrophil infiltration after ICH. Met-RANTES treatment decreased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1β, which was reversed by ceramide C6. The brain CCR1 activation by rCCL5 injection in naïve mouse resulted in neurological deficits and increased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1β. These detrimental effects of rCCL5 were reversed by TPR1 knockdown using TPR1 CRISPR. Our study demonstrated that CCR1 activation promoted neuroinflammation through CCR1/TPR1/ERK1/2 signaling pathway after ICH in mice. CCR1 inhibition with Met-RANTES attenuated neuroinflammation, thereby reducing brain edema and improving neurobehavioral functions. Targeting CCR1 activation may provide a promising therapeutic approach in the management of ICH patients. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-019-00821-5) contains supplementary material, which is available to authorized users.

Vps35 (vacuolar protein sorting 35) is a key component of retromer that regulates transmembrane protein trafficking. Dysfunctional Vps35 is a risk factor for neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases. Vps35 is highly expressed in developing pyramidal neurons, and its physiological role in developing neurons remains to be explored. Here, we provide evidence that Vps35 in embryonic neurons is necessary for axonal and dendritic terminal differentiation. Loss of Vps35 in embryonic neurons results in not only terminal differentiation deficits, but also neurodegenerative pathology, such as cortical brain atrophy and reactive glial responses. The atrophy of neocortex appears to be in association with increases in neuronal death, autophagosome proteins (LC3-II and P62), and neurodegeneration associated proteins (TDP43 and ubiquitin-conjugated proteins). Further studies reveal an increase of retromer cargo protein, sortilin1 (Sort1), in lysosomes of Vps35-KO neurons, and lysosomal dysfunction. Suppression of Sort1 diminishes Vps35-KO-induced dendritic defects. Expression of lysosomal Sort1 recapitulates Vps35-KO-induced phenotypes. Together, these results demonstrate embryonic neuronal Vps35’s function in terminal axonal and dendritic differentiation, reveal an association of terminal differentiation deficit with neurodegenerative pathology, and uncover an important lysosomal contribution to both events.

Background: Sortilin, a pluripotent peptide hormone, plays a role in glucose and lipid metabolism. A link between sortilin and insulin sensitivity has been implicated. However, the clinical implications of this link remain elusive. Our aims were to investigate whether sortilin levels were altered in subjects with newly diagnosed type 2 diabetes mellitus (nT2DM) compared with subjects with normal glucose tolerance (NGT) and to determine whether a link exist between sortilin levels and metabolic parameters. Materials and Methods: A total of 150 subjects including 75 nT2DM patients and 75 subjects with NGT who were matched in age, body mass index, and sex were enrolled into this case-control study. the circulating levels of sortilin were measured using enzyme-linked immunosorbent assay. A 2-hour 75-g oral glucose tolerance test was used for diagnosis of T2DM. Metabolic parameters of enrolled subjects were also determined. Results: the circulating levels of sortilin were found to be significantly lower in subjects with nT2DM than in controls (138.44 +/- 38.39 vs. 184.93 +/- 49.67 pg/mL, P < 0.001). Sortilin levels showed a negative correlation with insulin resistance and unfavorable lipid profiles, while they were positively correlated with high-density lipoprotein cholesterol in subjects with nT2DM. Linear regression analysis showed an independent and inverse link between sortilin and insulin resistance and unfavorable lipid profiles. Moreover, logistic regression analysis revealed that the subjects with the lowest sortilin levels had an increased risk of nT2DM compared with those subjects with the highest sortilin levels. Conclusions: Decreased circulating levels of sortilin were associated with unfavorable metabolic profiles in subjects with nT2DM.

DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.

Intersectin 1 (ITSN1) contains two isoforms: ITSN1-S and ITSN1-L, which are highly regulated by alternative splicing. Our previous results showed that the two isoforms of ITSN1 displayed opposite functions: ITSN1-S promoted glioma development, while ITSN1-L exerted an inhibitory role in glioma progression. In this study, our transcriptome analysis using a large glioma cohort indicated that the ratio of ITSN1-S/ITSN1-L was positively correlated with glioma grading and poor prognosis. We identified the RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1) as an ITSN1 pre-mRNA interaction protein through RNA pull-down assay and RNA immunoprecipitation assay. Knockdown of PTBP1 decreased the ratio of ITSN1-S/ITSN1-L. Minigene reporter assay and mutation analyses further confirmed PTBP1 targeted polypyrimidine sequences on ITSN1 exon 30 (TTGCACTTCAGTATTTT) and promoted the inclusion of ITSN1 exon 30. Subsequently, silencing PTBP1 inhibited glioma cell proliferation, migration, and invasion by down-regulating the ratio of ITSN1-S/ITSN1-L. Taken together, our study provides a novel mechanism that PTBP1 modulates the alternative splicing of ITSN1 and promotes glioma proliferation and motility by up-regulating the ratio of ITSN1-S/ITSN1-L, thereby highlighting that PTBP1 may be an attractive therapeutic target for gliomas.

International audience; Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

TICAM-1/TRIF, a TLR3 adaptor molecule, associates with Act1 to inhibit the interaction between IL-17RA and Act1, resulting in attenuated IL-17-mediated inflammatory responses., TICAM-1 (also called TRIF) is the sole adaptor of TLR3 that recognizes double-stranded RNA. Here, we report that TICAM-1 is involved not only in TLR3 signaling but also in the cytokine receptor IL-17RA signaling. We found that TICAM-1 bound to IL-17R adaptor Act1 to inhibit the interaction between IL-17RA and Act1. Interestingly, TICAM-1 knockout promoted IL-17RA/Act1 interaction and increased IL-17A–mediated activation of NF-κB and MAP kinases, leading to enhanced expression of inflammatory cytokines and chemokines upon IL-17A stimulation. Moreover, Ticam-1 knockout augmented IL-17A–mediated CXCL1 and CXCL2 expression in vivo, resulting in accumulation of myeloid cells. Furthermore, Ticam-1 knockout enhanced delayed type hypersensitivity and exacerbated experimental autoimmune encephalomyelitis. Ticam-1 knockout promoted accumulation of myeloid and lymphoid cells in the spinal cord of EAE-induced mice. Collectively, these data indicate that TICAM-1 inhibits the interaction between IL-17RA and Act1 and functions as a negative regulator in IL-17A–mediated inflammatory responses.

Beauvericin (BEA), a mycotoxin of the enniatin family produced by various toxigenic fungi, has been attributed multiple biological activities such as anti-cancer, anti-inflammatory, and anti-microbial functions. However, effects of BEA on dendritic cells remain unknown so far. Here, we identified effects of BEA on murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow derived dendritic cells (BMDCs) and the underlying molecular mechanisms. BEA potently activates BMDCs as signified by elevated IL-12 and CD86 expression. Multiplex immunoassays performed on myeloid differentiation primary response 88 (MyD88) and toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon beta (TRIF) single or double deficient BMDCs indicate that BEA induces inflammatory cytokine and chemokine production in a MyD88/TRIF dependent manner. Furthermore, we found that BEA was not able to induce IL-12 or IFNβ production in Toll-like receptor 4 (

Aim The present study aimed to determine the folic acid supplement (FAS) effects on serum homocysteine and sortilin levels, glycemic indices, and lipid profile in type II diabetic patients. Method A double-blind randomized controlled clinical trial have been performed on 100 patients with T2DM randomly divided into two groups that received either placebo or folic acid 5 mg/d for 12 weeks. Results FAS caused a significant decrease in homocysteine and sortilin serum levels (28.2% and 33.7%, P P = 0.0005), 8.2% in HbA1c (P = 0.0002), 13.7% in serum insulin (P P r = 0.237, P = 0.018), homocysteine (r = 0.308, P = 0.002) and fasting blood glucose (r = 0.342, P = 0.000). There were no significant changes in lipid profile in both groups after 12 weeks. Conclusion FAS might be beneficial for reducing homocysteine and sortilin levels, enhancing glycemic control, and improved insulin resistance in patients with T2DM.

Physical activity relates to reduced dementia risk, but the cellular and molecular mechanisms are unknown. We translated animal andin vitrostudies demonstrating a causal link between physical activity and microglial homeostasis into humans. Decedents from Rush Memory and Aging Project completed actigraphy monitoring (average daily activity) and cognitive evaluation in life, and neuropathological examination at autopsy. Brain tissue was analyzed for microglial activation via immunohistochemistry (anti-human HLA-DP-DQ-DR) and morphology (% Stage I, II, or III), and synaptic protein levels (SNAP-25, synaptophysin, complexin-I, VAMP, syntaxin, synaptotagmin-1). Proportion of morphologically activated microglia (PAM) was estimated in ventromedial caudate, posterior putamen, inferior temporal (IT), and middle frontal gyrus. The 167 decedents averaged 90 years at death, two-thirds were nondemented, and 60% evidenced pathologic Alzheimer's disease (AD). Adjusting for age, sex, education, and motor performances, greater physical activity associated with lower PAM in the ventromedial caudate and IT. Relationships between physical activity and PAM in the ventromedial caudate or IT were particularly prominent in adults evidencing microinfarcts or AD pathology, respectively. Mediational analyses indicated that PAM IT mediated ∼30% of the relationships between (1) physical activity and synaptic protein in IT, and (2) physical activity and global cognition, in separate models. However, the size of the mediation depended on AD pathology ranging from >40% in adults with high AD burden, but SIGNIFICANCE STATEMENTPhysical activity relates to better cognitive aging and reduced risk of neurodegenerative disease, yet the cellular and molecular pathways linking behavior-to-brain in humans are unknown. Animal studies indicate that increasing physical activity leads to decreased microglial activation and corresponding increases in synaptogenesis and neurogenesis. We objectively monitored physical activity (accelerometer-based actigraphy) and cognitive performances in life, and quantified microglial activation and synaptic markers in brain tissue at death in older adults. These are the first data supporting microglial activation as a physiological pathway by which physical activity relates to brain heath in humans. Although more interventional work is needed, we suggest that physical activity may be a modifiable behavior leveraged to reduce pro-inflammatory microglial states in humans.

TLR3 plays a crucial role in innate immunity. In the present study, OnTLR3 was identified in the Nile tilapia Oreochromis niloticus, with a conserved LRR domain and a C-terminal TIR domain. OnTLR3 was broadly expressed in all tissues tested, with the highest expression levels in the blood and the lowest in the kidney. TLR3 mRNA could be detected from pharyngula (2.5 dpf) to late larva (8.5 dpf) during embryonic and larval development. Moreover, the expression level of OnTLR3 was clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, poly(I:C), S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. When OnTLR3 was overexpressed in 293 T cells, it was distributed in the cytoplasm and could significantly increase NF-κB activation. The pulldown assays showed that OnTLR3 interacted with both OnMyD88 and OnTRIF. The binding assays revealed the specificity of OnTLR3 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C), LPS and PGN. Taken together, these findings suggest that OnTLR3, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.

Increased neutrophil recruitment represents a hallmark event in myocardial ischemia/reperfusion (I/R) injury due to the ensuing inflammatory response. Circular RNAs (circRNAs) are important regulatory molecules involved in cell physiology and pathology. Herein, we analyzed the role of a novel circRNA circ_SMG6 in the regulation of neutrophil recruitment following I/R injury, which may associate with the miR-138-5p/EGR1/TLR4/TRIF axis. Myocardial I/R injury was modeled in vivo by ligation of the left anterior descending (LAD) artery followed by reperfusion in mice and in vitro by exposing a cardiomyocyte cell line (HL-1) to hypoxia/reoxygenation (H/R). Gain- and loss-of-function experiments were performed to evaluate the effect of the circ_SMG6/miR-138-5p/EGR1/TLR4/TRIF axis on cardiac functions, myocardial infarction, myocardial enzyme levels, cardiomyocyte activities, and neutrophil recruitment. We found that the EGR1 expression was increased in myocardial tissues of I/R mice. Knockdown of EGR1 was found to attenuate I/R-induced cardiac dysfunction and infarction area, pathological damage, and cardiomyocyte apoptosis. Mechanistic investigations showed that circ_SMG6 competitively bound to miR-138-5p and consequently led to upregulation of EGR1, thus facilitating myocardial I/R injury in mice and H/R-induced cell injury. Additionally, ectopic EGR1 expression augmented neutrophil recruitment and exacerbated the ensuing I/R injury, which was related to the activated TLR4/TRIF signaling pathway. Overall, our findings suggest that circ_SMG6 may deteriorate myocardial I/R injury by promoting neutrophil recruitment via the miR-138-5p/EGR1/TLR4/TRIF signaling. This pathway may represent a potential therapeutic target in the management of myocardial I/R injury.

Sortilin has been positively correlated with vascular disorders in humans. No study has yet evaluated the possible direct effect of sortilin on vascular function. We used pharmacological and genetic approaches coupled with study of murine and human samples to unravel the mechanisms recruited by sortilin in the vascular system. Sortilin induced endothelial dysfunction of mesenteric arteries through NADPH oxidase 2 (NOX2) isoform activation, dysfunction that was prevented by knockdown of acid sphingomyelinase (ASMase) or sphingosine kinase 1. In vivo, recombinant sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of sphingosine-1-phosphate receptor 3 (S1P3) and gp91phox/NOX2 resulted in preservation of endothelial function and blood pressure homeostasis after 14 days of systemic sortilin administration. Translating these research findings into the clinical setting, we detected elevated sortilin levels in hypertensive patients with endothelial dysfunction. Furthermore, in a population-based cohort of 270 subjects, we showed increased plasma ASMase activity and increased plasma levels of sortilin, S1P, and soluble NOX2-derived peptide (sNOX2-dp) in hypertensive subjects, and the increase was more pronounced in hypertensive subjects with uncontrolled blood pressure. Our studies reveal what we believe is a previously unrecognized role of sortilin in the impairment of vascular function and in blood pressure homeostasis and suggest the potential of sortilin and its mediators as biomarkers for the prediction of vascular dysfunction and high blood pressure.

Background. MicroRNA- (miR-) 657 has been shown to regulate immunological and inflammatory activity, and it has also been defined to be dysregulated in both non-small-cell lung cancer (NSCLC) and hepatocellular carcinoma. The mechanistic role whereby miR-657 influences NSCLC progression, however, has yet to be clarified. Methods. miR-657 and SRCIN1 expression levels were assessed via qPCR in the cell lines and tissues of NSCLC. Besides, correlations between the levels of miR-657 and NSCLC patient pathological characteristics were examined, and the Kaplan-Meier approach was employed for the evaluation of the prognostic utility of miR-657 in these patients. Moreover, the Pearson correlation analyses and dual-luciferase reporter assessments were used for detecting interactive relationships between miR-657 and SRCIN1. In addition, CCK-8, EdU, and Transwell assessments were employed for the appraisal of the ability of miR-657/SRCIN1 to regulate NSCLC cell proliferation and invasion. Western blotting was employed for the assessment of the levels of NSCLC cell proteins associated with the epithelial-mesenchymal transition (EMT) that were influenced by miR-657. The nude mice xenograft tumor model is established to observe the effect of miR-657 on NSCLC growth in vivo. Results. NSCLC patient tissues and cell lines exhibited upregulated miR-657 expression that was closely related to tumor differentiation, lymphoid metastasis, and TNM stage. High levels of miR-657 were predictive of a poorer NSCLC patient prognosis, and overexpressing miR-657 resulted in the more rapid growth of NCI-H1650 and A549 cells, with a concomitant increase in their invasion. In addition, miR-657 overexpression raised the levels of Slug, N-cadherin, and Vimentin in these two cell lines while promoting E-cadherin downregulation. Dual-luciferase reporter assays confirmed that miR-657 was capable of binding to the SRCIN1 gene, and SRCIN1 expression levels were negatively associated with those of miR-657, indicating that it acts as a negative regulator of this gene. Knocking down SRCIN1 was capable to reverse the influences of miR-657 inhibitor treatment on NSCLC cell behavior. Finally, in vivo studies showed that miR-657 promoted NSCLC cell growth. Conclusion. The obtained findings illuminate that miR-657 can promote the growth of tumors and the induction of the EMT in NSCLC cells by targeting SRCIN1 expression and modulating Slug pathway activation, highlighting this pathway as a promising therapeutic target in cases suffering from NSCLC.

Sortilin has been positively correlated with vascular disorders in humans. No study has yet evaluated the possible direct effect of sortilin on vascular function. We used pharmacological and genetic approaches coupled with study of murine and human samples to unravel the mechanisms recruited by sortilin in the vascular system. Sortilin induced endothelial dysfunction of mesenteric arteries through NADPH oxidase 2 (NOX2) isoform activation, dysfunction that was prevented by knockdown of acid sphingomyelinase (ASMase) or sphingosine kinase 1. In vivo, recombinant sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of sphingosine-1-phosphate receptor 3 (S1P3) and gp91phox/NOX2 resulted in preservation of endothelial function and blood pressure homeostasis after 14 days of systemic sortilin administration. Translating these research findings into the clinical setting, we detected elevated sortilin levels in hypertensive patients with endothelial dysfunction. Furthermore, in a population-based cohort of 270 subjects, we showed increased plasma ASMase activity and increased plasma levels of sortilin, S1P, and soluble NOX2-derived peptide (sNOX2-dp) in hypertensive subjects, and the increase was more pronounced in hypertensive subjects with uncontrolled blood pressure. Our studies reveal what we believe is a previously unrecognized role of sortilin in the impairment of vascular function and in blood pressure homeostasis and suggest the potential of sortilin and its mediators as biomarkers for the prediction of vascular dysfunction and high blood pressure.

The role of sortilin and omentin-1 in the pathogenesis of atherosclerosis and vascular disease is an emerging topic in recent years. These molecules can be found circulating in the blood. Recent studies have shown how these biomarkers appear to correlate with the severity of PAD. The levels of these molecules appear to be inversely proportional to each other. Their relationship may provide further insight into the management of the very old diabetic patients with PAD. This study aimed to assess the possible role of sortilin/omentin-1 ratio as easy-to-measure marker in peripheral artery disease (PAD) in type-2 diabetic patients.This study analyzed the association between sortilin and omentin-1 serum levels and the presence of clinically significant lower limb PAD in diabetic individuals. We enrolled 295 diabetic patients, including 179 with PAD. Serum levels were collected and correlated with clinical characteristics of the patients.Sortilin concentration was significantly higher in the latter group compared to the former and there was a trend toward increased sortilin levels as disease severity increased. Omentin-1 serum levels were significantly lower in diabetic patients with PAD than in diabetic controls and the levels gradually decreased in proportion to disease severity. The ratio of sortilin to omentin-1 was significantly higher in patients with PAD compared to the other group.The sortilin to omentin-1 ratio appears to be a predictive factor for PAD in patients with type-2 diabetes and it may be a promising marker for clinically significant atherosclerosis of the lower limbs. Further studies are needed to confirm this finding and to evaluate its clinical usefulness.

Background. Neuroinflammation is related with the inflammatory stress of brain tissue induced by the activation of microglial in the central nervous system (CNS), which is still an intractable disease for modern clinical system. Chlorogenic acid has multiple biological activities such as antivirus and anti-inflammation, while few researches have revealed its therapeutic functions in neuroinflammation. Methods. BV2 cells were treated with lipopolysaccharide (LPS) to establish neuroinflammation cell models, and the effects and mechanism of chlorogenic acid in improving the inflammatory progression were investigated. In brief, the toxicity of chlorogenic acid on BV2 cells was detected with MTT assay. The levels of the inflammatory factors including TNF-α, IL-6, IL-1β, and IFN-α were measured with ELISA, and the abundances of TLR4, MyD88, TRIF, and NF-κB were observed by qRT-PCR and western blot. Results. Chlorogenic acid did not exhibit obvious toxic and side effects on BV2 cells. The levels of TNF-α, IL-6, IL-1β, and IFN-α were observably upregulated in BV2 cells after treating with LPS. Chlorogenic acid significantly reduced the levels of TNF-α, IL-6, IL-1β, and IFN-α. Moreover, the abundances of TLR4, MyD88, TRIF, and NF-κB were increased in LPS-induced BV2 cells, while chlorogenic acid could obviously reduce their expressions. Conclusion. This study suggests that chlorogenic acid can improve the inflammatory stress of LPS-induced BV2 cell via interacting with the TLR4-mediated downstream pathway, which is a potential drug for neuroinflammation treatment.

BACKGROUND: Deficiency of Conserved Oligomeric Golgi (COG) subunits (COG1-8) is characterized by both N- and O-protein glycosylation defects associated with destabilization and mislocalization of Golgi glycosylation machinery components (COG-CDG). Patients with COG defects present with neurological and multisystem involvement and possible malformation occurrence. Eighteen patients with COG6-CDG (COG6 mutations) were reported to date. We describe a patient with COG6-CDG with novel variants and a novel clinical feature namely a congenital recto-vaginal fistula. METHODS: In-depth serum N- and O-glycosylation structural analyses were conducted by MALDI-TOF mass spectrometry. COG6 variants were identified by a gene panel and confirmed by Sanger sequencing. RESULTS: This female newborn presented with facial dysmorphism, distal arthrogryposis and recurrent stool discharges per vaginam. A double-contrast barium-enema X-ray study revealed a dehiscence (approximately 5 mm) at the anterior wall of the rectal ampoule communicating with the vagina consistent with a recto-vaginal fistula. She had developmental delay, corpus callosum dysgenesis, liver and gastrointestinal involvement, hyperthermia episodes and early demise. Serum N- and O-glycosylation analyses pointed to a profound Golgi disarrangement. We identified two novel variants in COG6: a deletion of 1 bp mutation c.823delA creating a shift in the reading frame and a premature stop codon and a 3 bp deletion (c.1141_1143delCTC) producing an in-frame deletion of 1 amino acid. CONCLUSION: The congenital recto-vaginal fistula is a rare type of anorectal malformation that, to our knowledge, has not been reported in patients with a COG6 defect nor in patients with other COG defects. This study broadens COG6-CDG genetic landscape and spectrum of malformations. ispartof: BIRTH DEFECTS RESEARCH vol:114 issue:5-6 pages:165-174 ispartof: location:United States status: published

ITSN1 plays an important role in brain development. Recent studies in large cohorts of subjects with neurodevelopmental disorders have identified de novo variants in ITSN1 gene thereby suggesting that this gene is involved in the development of such disorders. The aim of this study is to provide further proof of such a link. We performed trio exome sequencing in a patient presenting autism, intellectual disability, and severe behavioral difficulties. Additional affected patients with a neurodevelopmental disorder harboring a heterozygous variant in ITSN1 (NM_003024.2) were collected through a worldwide collaboration. All patients underwent detailed phenotypic and genetic assessment and data was collected and shared by healthcare givers. We identified ten novel patients from eight families with heterozygous truncating or missense variants in ITSN1 gene. In addition, four previously published patients from large meta-analysis studies were included. In total, 7/14 patients presented a de novo variant in ITSN1. All patients showed neurodevelopmental disorders from autism spectrum disorders (90%), intellectual disability (86%), and epilepsy (30%). We demonstrated that truncating variants are in the first half of ITSN1 whereas missense variants are clustered in C-terminal region. We suggest ITSN1 gene is involved in development of an autism spectrum disorder with variable additional neurodevelopmental deficiency, thus confirming the hypothesis that ITSN1 is important for brain development.

Complexin I (CPLX1), a presynaptic small molecule protein, forms SNARE complex in the central nervous system involved in the anchoring, pre-excitation, and fusion of axonal end vesicles. Abnormal expression of CPLX1 occurs in several neurodegenerative and psychiatric disorders that exhibit disrupted neurobehaviors. CPLX1 gene knockout induces severe ataxia and social behavioral deficits in mice, which has been poorly demonstrated. Here, to address the limitations of single-species models and to provide translational insights relevant to human diseases, we used CPLX1 knockout rats to further explore the function of the CPLX1 gene. The CRISPR/Cas9 gene editing system was adopted to generate CPLX1 knockout rats (CPLX1-/-). Then, we characterized the survival rate and behavioral phenotype of CPLX1-/- rats using behavioral analysis. To further explain this phenomenon, we performed blood glucose testing, Nissl staining, hematoxylin-eosin staining, and Golgi staining. We found that CPLX1-/- rats showed profound ataxia, dystonia, movement and exploratory deficits, and increased anxiety and sensory deficits but had normal cognitive function. Nevertheless, CPLX1-/- rats could swim without training. The abnormal histomorphology of the stomach and intestine were related to decreased weight and early death in these rats. Decreased dendritic branching was also found in spinal motor neurons in CPLX1-/- rats. In conclusion, CPLX1 gene knockout induced the abnormal histomorphology of the stomach and intestine and decreased dendritic branching in spinal motor neurons, causing different phenotypes between CPLX1-/- rats and mice, even though both of these phenotypes showed profound ataxia. These findings provide a new perspective for understanding the role of CPLX1.

Bone differentiation defects have been recently tied to Wnt signaling alterations occurring in vitro and in vivo Gaucher disease (GD) models. In this work, we provide evidence that the Wnt signaling multi-domain intracellular transducers Dishevelled 1 and 2 (DVL1 and DVL2) may be potential upstream targets of impaired beta glucosidase (GBA1) activity by showing their misexpression in different type 1 GD in vitro models. We also show that in Gba mutant fish a miR-221 upregulation is associated with reduced dvl2 expression levels and that in type I Gaucher patients single-nucleotide variants in the DVL2 3′ untranslated region are related to variable canonical Wnt pathway activity. Thus, we strengthen the recently outlined relation between bone differentiation defects and Wnt/β-catenin dysregulation in type I GD and further propose novel mechanistic insights of the Wnt pathway impairment caused by glucocerebrosidase loss of function.

ObjectivesPatients with lupus nephritis (LN) are in urgent need for early diagnosis and therapeutic interventions targeting aberrant molecular pathways enriched in affected kidneys.MethodsWe used mRNA-sequencing in effector (spleen) and target (kidneys, brain) tissues from lupus and control mice at sequential time points, and in the blood from 367 individuals (261 systemic lupus erythematosus (SLE) patients and 106 healthy individuals). Comparative cross-tissue and cross-species analyses were performed. The human dataset was split into training and validation sets and machine learning was applied to build LN predictive models.ResultsIn murine SLE, we defined a kidney-specific molecular signature, as well as a molecular signature that underlies transition from preclinical to overt disease and encompasses pathways linked to metabolism, innate immune system and neutrophil degranulation. The murine kidney transcriptome partially mirrors the blood transcriptome of patients with LN with 11 key transcription factors regulating the cross-species active LN molecular signature. Integrated protein-to-protein interaction and drug prediction analyses identified the kinases TRRAP, AKT2, CDK16 and SCYL1 as putative targets of these factors and capable of reversing the LN signature. Using murine kidney-specific genes as disease predictors and machine-learning training of the human RNA-sequencing dataset, we developed and validated a peripheral blood-based algorithm that discriminates LN patients from normal individuals (based on 18 genes) and non-LN SLE patients (based on 20 genes) with excellent sensitivity and specificity (area under the curve range from 0.80 to 0.99).ConclusionsMachine-learning analysis of a large whole blood RNA-sequencing dataset of SLE patients using human orthologs of mouse kidney-specific genes can be used for early, non-invasive diagnosis and therapeutic targeting of LN. The kidney-specific gene predictors may facilitate prevention and early intervention trials.

Herein, we report a lethal case of the ultra-rare COG6-congenital disorder of glycosylation (CDG) presenting with skin manifestations (scaling and erosions) and joint contractures in a neonate of Albanian origin. The patient was homozygous for a COG6 pathogenic variant, previously reported in another three individuals of Greek, Bulgarian and Turkish descent. The presence of a founder mutation in the geographical area is possible. The index case emphasizes the need to consider CDGs in neonatal patients with skin manifestations and joint contractures, particularly patients of Southeastern European or West Asian origin.

The hexameric HOPS (homotypic fusion and protein sorting) complex is a conserved tethering complex at the lysosome-like vacuole, where it mediates tethering and promotes all fusion events involving this organelle. The Vps39 subunit of this complex also engages in a membrane contact site between the vacuole and the mitochondria, called vCLAMP. Additionally, four subunits of HOPS are also part of the endosomal CORVET tethering complex. Here, we analyzed the partition of HOPS and CORVET subunits between the different complexes by tracing their localization and function. We find that Vps39 has a specific role in vCLAMP formation beyond tethering, and that vCLAMPs and HOPS compete for the same pool of Vps39. In agreement, we find that the CORVET subunit Vps3 can take the position of Vps39 in HOPS. This endogenous pool of a Vps3-hybrid complex is affected by Vps3 or Vps39 levels, suggesting that HOPS and CORVET assembly is dynamic. Our data shed light on how individual subunits of tethering complexes such as Vps39 can participate in other functions, while maintaining the remaining subcomplex available for its function in tethering and fusion.

BackgroundMultiple myeloma (MM) is one of the most frequently diagnosed hematological malignancy. Dysregulation of circular RNAs (circRNAs) has important impacts on MM process. Herein, this work aimed to investigate the role and mechanism of circ_0058058 in MM progression.MethodsLevels of genes and proteins were detected by real-time reverse transcription PCR (RT-qPCR) and Western blot. CCK-8 assay, colony formation assay, EdU assay, flow cytometry, tube formation assay, transwell assay and Western blot were utilized to detect the proliferation, apoptosis, angiogenesis and metastasis of MM cells. The target relationship between miR-338-3p and circ_0058058 or ATG14 (autophagy related 14) was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. In vivo experiments were performed using Xenograft assay.ResultsCirc_0058058 was up-regulated in MM bone marrow aspirates and cells, knockdown of circ_0058058 reduced MM cell proliferation, angiogenesis and metastasis, but induced apoptosis in vitro. In a MM xenograft mouse model, circ_0058058 silencing reduced MM tumor growth and cell proliferation. Mechanistically, circ_0058058 acted as a sponge for miR-338-3p to up-regulate ATG14 expression, which was validated to be a target of miR-338-3p. Rescue assay showed that miR-338-3p inhibition reversed the antitumor effects of circ_0058058 knockdown on MM cell. Moreover, forced expression of miR-338-3p suppressed MM cell malignant phenotype, which was abolished by ATG14 up-regulation.ConclusionCirc_0058058 functions as a sponge for miR-338-3p to elevate ATG14 expression to promote MM cell proliferation, metastasis and angiogenesis, affording a potential therapeutic target for MM prevention.