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Hedgehog (HH) signaling is a major driver of tissue patterning during embryonic development through the regulation of a multitude of cell behaviors including cell fate specification, proliferation, migration, and survival. HH ligands signal through the canonical receptor PTCH1 and three co-receptors, GAS1, CDON and BOC. While previous studies demonstrated an overlapping and collective requirement for these co-receptors in early HH-dependent processes, the early embryonic lethality of Gas1;Cdon;Boc mutants precluded an assessment of their collective contribution to later HH-dependent signaling events. Specifically, a collective role for these co-receptors during limb development has yet to be explored. Here, we investigate the combined contribution of these co-receptors to digit specification, limb patterning and long bone growth through limb-specific conditional deletion of Cdon in a Gas1;Boc null background. Combined deletion of Gas1, Cdon and Boc in the limb results in digit loss as well as defects in limb outgrowth and long bone patterning. Taken together, these data demonstrate that GAS1, CDON and BOC are collectively required for HH-dependent patterning and growth of the developing limb.

Lymph node (LN) metastasis affects both the management and prognosis of head and neck squamous cell carcinoma (HNSCC). Here, we explored the relationship between lymphatic metastasis and CEA family member 5 (CEACAM5), including its possible regulatory role in HNSCC. The levels of CEACAM5 in tissues from patients with HNSCC, with and without LN metastases, were assessed by transcriptome sequencing. The associations between CEACAM5 and the N stage of LN metastasis in HNSCC were predicted through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and a pan-cancer analysis of CEACAM5 expression in 33 common human tumors was conducted. CEACAM5 levels were analyzed in tumor and normal tissue specimens from HNSCC patients and the correlation between CEACAM5 levels and prognosis was evaluated. The influence of CEACAM5 on cell proliferation, invasion, migration, and apoptosis was investigated in HNSCC cell lines, as were the downstream regulatory mechanisms. A mouse model of LN metastasis was constructed. CEACAM5 levels were significantly higher in HNSCC tissue without LN metastasis than in that with LN metastasis. Similar findings were obtained for the clinical specimens. CEACAM5 levels were associated with better clinical prognosis. CEACAM5 was found to inhibit the proliferation and migration and promote the apoptosis of HNSCC cells. A mouse xenograft model showed that CEACAM5 inhibited LN metastasis. In conclusions, CEACAM5 inhibited epithelial–mesenchymal transition (EMT) in HNSCC by reducing murine double minute 2 (MDM2) expression and thereby suppressing LN metastasis. CEACAM5 has potential as both a prognostic marker and a therapeutic target in HNSCC.

The human cytomegalovirus (HCMV) UL24 and UL43 are tegument proteins that have recently been shown to interact with each other in a yeast two-hybrid system. By their overexpression in MRC5 cells, we demonstrate that these viral proteins interact with several important host proteins, especially Dicer and trans-activation response RNA binding protein. As these hots proteins are involved in regulating the production of cellular micro-RNAs, the cytomegalovirus (CMV) proteins could interfere with their actions to favor viral replication directly or through an immune escape mechanism. Double knockout of UL24 and UL43 does not show a remarkable effect on CMV entry or replication, but it significantly downregulates the expression of CMV-encoded miR-UL59, which is thought to regulate the expression of a downstream target UL16 binding protein 1 (ULBP1). Interestingly, the double knockout increases the expression of the ULBP1 recognized by the NKG2D activating receptor of natural killer cells. This study investigates the potential role of several proteins encoded by HCMV in regulating the host cellular environment to favor escape from immunity, and it also provides some basis for the future development of RNA-targeted small molecules to control HCMV infection.

Pattern recognition receptors (PRRs) sense ligands in pattern-triggered immunity (PTI). Plant PRRs include numerous receptor-like proteins (RLPs), but many RLPs remain functionally uncharacterized. Here, we examine an Arabidopsis thaliana RLP, RLP53, which positively regulates immune signaling. Our forward genetic screen for suppressors of enhanced disease resistance1 (edr1) identified a point mutation in RLP53 that fully suppresses disease resistance and mildew-induced cell death in edr1 mutants. The rlp53 mutants showed enhanced susceptibility to virulent pathogens, including fungi, oomycetes, and bacteria, indicating that RLP53 is important for plant immunity. The ectodomain of RLP53 contains leucine-rich repeat (LRR) motifs. RLP53 constitutively associates with the LRR receptor-like kinase SUPPRESSOR OF BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE (BAK1)-INTERACTING RECEPTOR KINASE1 (SOBIR1) and interacts with the co-receptor BAK1 in a pathogen-induced manner. The double mutation sobir1-12 bak1-5 suppresses edr1-mediated disease resistance, suggesting that EDR1 negatively regulates PTI modulated by the RLP53-SOBIR1-BAK1 complex. Moreover, the glycosylphosphatidylinositol (GPI)-anchored protein LORELEI-LIKE GPI-ANCHORED PROTEIN1 (LLG1) interacts with RLP53 and mediates RLP53 accumulation in the plasma membrane. We thus uncovered the role of a novel RLP and its associated immune complex in plant defense responses and revealed a potential new mechanism underlying regulation of RLP immune function by a GPI-anchored protein.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Highly penetrant copy number variants (CNVs) and genes related to the etiology of TOF likely exist with differences among populations. We aimed to identify CNV contributions to sporadic TOF cases in Han Chinese. Genomic DNA was extracted from peripheral blood in 605 subjects (303 sporadic TOF and 302 unaffected Han Chinese [Control] from cardiac centers in China) and analyzed by genome-wide association study (GWAS). The GWAS results were compared with existing Database of Genetic Variants. These CNVs were further validated by qPCR. Bioinformatics analyses were performed with protein-protein interaction (PPI) network and KEGG pathway enrichment. Across all chromosomes 119 novel "TOF-specific CNVs" were identified with prevalence of CNVs of 21.5% in chromosomes 1-20 and 37.0% including Chr21/22. In chromosomes 1-20, CNVs on 11q25 (encompasses genes ACAD8, B3GAT1, GLB1L2, GLB1L3, IGSF9B, JAM3, LOC100128239, LOC283177, MIR4697, MIR4697HG, NCAPD3, OPCML, SPATA19, THYN1, and VPS26B) and 14q32.33 (encompasses genes THYN1, OPCML, and NCAPD3) encompass genes most likely to be associated with TOF. Specific CNVs found on the chromosome 21 (6.3%) and 22(11.9%) were also identified in details. PPI network analysis identified the genes covering the specific CNVs related to TOF and the signaling pathways. This study for first time identified novel TOF-specific CNVs in the Han Chinese with higher frequency than in Caucasians and with 11q25 and 14q32.33 not reported in TOF of Caucasians. These novel CNVs identify new candidate genes for TOF and provide new insights into genetic basis of TOF.

The cumulative cancerous rate of colitis-associated cancer (CAC) has increased exponentially in patients with ulcerative colitis (UC). We have investigated the factors involved in the carcinogenic processes of CAC among UC patients.A total of 42 UC patients who underwent surgical treatments between January 2001 and December 2010 at Kurume University Hospital (Fukuoka, Japan) were enrolled. We conducted this study using 3 cases of CAC out of 42 UC cases and 1 case of colorectal cancer. cDNA microarray analyses were performed using normal, inflamed, and cancerous tissues from surgical CAC specimens and protein expression was confirmed by immunohistochemical analyses.cDNA microarray revealed 32 genes that were dominantly expressed in tumorous regions of CAC. Gene ontology analysis revealed that these genes were involved in inflammatory responses and cytokine-cytokine receptor interactions. Chitinase 3-like1 (CHI3L1), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), and Claudin-2 (CLND2) were selected from CAC-related genes as candidate molecules. Immunostaining revealed strong expression of each protein in cancerous regions.In this study, we identified CAC-related genes and found that CHI3L1, CEACAM6, and CLND2 were expressed in patient samples. All the above genes were associated with adherent invasive Escherichia coli (AIEC), which suggested that these molecules are likely involved in AIEC infection. Further analyses would be required to reveal unknown mechanisms of CAC-related genes in the tumor microenvironment.

Lung cancer is the leading cause of global cancer-related mortality. Although immune checkpoint therapy has achieved remarkable results in lung cancer, EGFR-mutant or ALK-positive non-smallcell lung cancer patients show limited benefit. Besides the low tumor mutational burden, PD-L1 expression and CD8+ tumor-infiltrating T cells, upregulation of CD73/adenosine pathway also contributes to the immune-inert microenvironment of EGFR-mutant NSCLC. However, the detailed mechanism underlying the regulation of CD73 is unclear.TCGA data was used to analyze the CD73 expression in cancer patients. Western blotting, qPCR, and ChIP-PCR were performed in multiple NSCLC cancer cell lines and patient derived organoids were used to explore the regulation of CD73 expression using western blotting.CD73 expression was highly expressed in multiple cancer types. Pharmacological or genetic inhibition of EGFR, MEK, KRAS, or ALK dramatically reduced the CD73 mRNA and protein expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. C-Jun overexpression-induced CD73 mRNA and protein expression. ChIP assay showed that c-Jun bind to CD73 genomic regions.Higher CD73 expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. Mechanistically, CD73 is regulated by ERK-Jun pathway, wherein c-Jun regulates CD73 expression via binding to CD73 genomic regions.

The vanins are ectoenzymes with pantetheinase activity and are involved in recycling pantothenic acid (vitamin B5) from pantetheine. Elevated levels of vanin have been linked with the development and severity of several diseases, including steatosis, diabetes, skin diseases, cancer, inflammatory diseases etc. Therefore, vanins have previously been used as a potential drug target to combat related diseases. In this study, we used a molecular docking and molecular dynamic simulation-based approach to screen dual inhibitors of hVnn1, and hVnn2 from a library of 120 chemical candidates. Molecular docking of drug candidates with hVnn1, and hVnn2 using GOLD and MOE revealed that the chemical compound "methotrexate (CID: 126941)" has the highest binding affinity against both the target enzymes which was further validated through molecular dynamic simulation. Toxicity profiling of drug candidates evaluated using Lipinski's rule of five and Molsoft tool, and AdmetSar 2.0 confirms the drug suitability of methotrexate, therefore, suggesting its use as a potential therapeutic agent to inhibit the activity of vainin enzyme in related disease conditions.

One of the main causes of low back pain (LBP) and degenerative musculoskeletal disorders is intervertebral disc degeneration (IVDD). Inflammation-associated senescence of Human nucleus pulposus cells (HNPCs) plays an essential function in the disease progression of IVDD. Omentin-1 is an adipokine that has been recently reported to have anti-inflammatory potential. In our research, IL-1β was used to simulate the inflammatory environment in the IVDD. We investigated in vitro the effects of Omentin-1 on HNPCs, including the components of senescence, cell cycle and extracellular matrix (ECM) synthesis. The results showed that the addition of Omentin-1 improved IL-1β-induced senescence in HNPCs. G1 phase cell cycle arrest and reduced ECM synthesis in HNPCs. Furthermore, we demonstrated that the effect of Omentin-1 in reducing senescence of HNPCs is dependent on SIRT1. These findings suggest that Omentin-1 plays an important function in protecting HNPCs against senescence and has the potential for IVDD gene target therapy.

Preeclampsia is a grievous pregnancy-related complication with an incidence of approximately 5∼7% in pregnant women. Placental abnormalities and decreased placental perfusion associated with impaired trophoblast invasion are early pathological findings of preeclampsia. BST2 is a multifunctional transmembrane protein that plays critical roles in physiological and pathological processes, but its impacts and mechanisms of action in preeclampsia are inadequately understood. The aim of this manuscript was to investigate the functional impacts of BST2 and MMP2 on the biological behavior of trophoblast cells in preeclampsia. The expression of these proteins and their genes was analyzed by qRT-PCR, western blotting and immunohistochemistry. The results showed that the expression of BST2 and MMP2 was significantly downregulated in preeclampsia. The migration and invasion capacities of HTR-8/SVneo and JAR cells with overexpression or knockdown of BST2 were detected by wound healing assay and Transwell assays. It was found that BST2 overexpression could up-regulate

Melanoma is responsible for 90% of skin cancer-related deaths. Major therapeutic advances have led to a considerable improvement in the prognosis of patients, with the development of targeted therapies (BRAF or MEK inhibitors) and immunotherapy (anti-CTLA-4 or -PD-1 antibodies). However, the tumor constitutes an immunosuppressive microenvironment that prevents the therapeutic efficacy and/or promotes the development of secondary resistances. CD160 is an activating NK-cell receptor initially described as delineating the NK and CD8+T-cell cytotoxic populations. Three forms of CD160 have been described: (1) the GPI isoform, constitutively expressed and involved in the initiation of NK-cells' cytotoxic activity, (2) the transmembrane isoform, neo-synthesized upon cell activation, allowing the amplification of NK cells' cytotoxic functions and (3) the soluble form, generated after cleavage of the GPI isoform, which presents an immuno-suppressive activity. By performing immunohistochemistry analyses, we observed a strong expression of CD160 at the primary cutaneous tumor site of melanoma patients. We further demonstrated that melanoma cells express CD160-GPI isoform and constitutively release the soluble form (sCD160) into the tumor environment. sCD160 was shown to inhibit the cytotoxic activity of NK-cells towards their target cells. In addition, it was found in the serum of melanoma patients and associated with increased tumor dissemination. Altogether these results support a role for sCD160 in the mechanisms leading to the inhibition of anti-tumor response and immune surveillance in melanoma.

Mesothelin (MSLN) is a cell surface protein associated with tumor invasion and metastasis. This study aims to explore the biological function of MSLN in gastric cancer and to evaluate the association of MSLN polymorphism (rs3764247, rs3764246, rs12597489, rs1057147, rs3765319) with the risk and prognosis of gastric cancer. Small interfering RNA (siRNA) transfection and MSLN overexpression were performed in human gastric cancer cell lines, respectively. The proliferation of tumor cells was evaluated by Cell counting kit 8(CCK-8) and colony formation assay. Wound healing assay and transwell assay were used to elucidate gastric cancer cell migration and invasion rates. We conducted a case-control study involving 860 patients with gastric cancer and 870 controls. All mutation sites were genotyped by PCR-LDR sequencing. First, our study revealed the cancer-promoting role of MSLN in gastric cancer. Second, we also demonstrated that rs3764247 and rs3764246 were associated with a reduced risk of gastric cancer (OR = 0.83, p = 0.010; OR = 0.84, p = 0.011; respectively). The clinicopathological analysis further showed that rs3764247 was closely related to T stage, vascular infiltration, and HER2 expression. In addition, in the survival analysis of 392 patients with gastric cancer, patients with rs3764247 CC genotype had poorer survival than patients with AA + AC genotype after adjusting for age, sex, TNM stage, and Lauren classification (HR = 2.07, p = 0.029). Our findings indicated that MSLN could be an oncogene whose polymorphisms were closely related to the risk and prognosis of gastric cancer.

Background. Lymphocyte antigen 6 complex, locus E (LY6E) is abnormally expressed in several cancers and is associated with poor outcomes. However, the biological role of LY6E in colorectal cancer (CRC) remains largely unknown. Hence, we aimed to evaluate the expression levels, prognostic value, biological functions, and immune effects of LY6E via pan-cancer and CRC analyses using multiple databases. Methods. We analyzed the expression pattern of LY6E in various cancers. The prognostic value of LY6E expression was identified using the Kaplan–Meier analysis and the Cox regression models. We used gene set enrichment analysis (GSEA) to identify the potential functions of LY6E. Correlations between the LY6E expression and various factors, including LY6E methylation level, copy number variation (CNV), microsatellite instability (MSI), and immune checkpoint genes, were also analyzed. The levels of LY6E expression and immune infiltration were analyzed using CIBERSORT. We constructed a regulatory network that was in compliance with the competing endogenous RNA (ceRNA) hypothesis. A ceRNA expression-based nomogram was established. Real-time PCR (qRT-PCR) was applied to validate the expression of LY6E-related ceRNA in CRC cell lines. Results. LY6E is overexpressed in several tumor types, including CRC, and patients with high expression levels of LY6E have a poor prognosis. The Kaplan–Meier analysis and Cox regression analysis showed that LY6E could be considered a favorable prognostic factor in TCGA and GEO cohort. The results of GSEA showed that high LY6E expression levels were associated with immune-related pathways, such as those involved in antigen processing and presentation and the intestinal immune network for IgA production. Six methylation sites of LY6E that were associated with prognostic survival were screened. Moreover, the high levels of LY6E expression were correlated with copy number gain, microsatellite instability high, and immunotherapy response. The results of CIBERSORT analysis demonstrated that the LY6E expression levels were correlated with the infiltration of multiple immune cells, especially T cells. Then, we constructed a ceRNA network (LINC00963/miR-92a-3p/LY6E) and validated it using qRT-PCR. A predictive ceRNA-based nomogram was established and validated. Conclusion. The oncogenic LY6E may serve as a promising marker for the diagnosis and treatment of CRC.

The risk stratification of B-acute lymphoblastic leukemia (B-ALL) is based on clinical and biological factors. However, B-ALL has significant biological and clinical heterogeneity and 50% of B-ALL patients do not have defined prognostic markers. In this sense, the identification of new prognostic biomarkers is necessary. Considering different cohorts of childhood B-ALL patients, gene (DPP4/CD38/ENTPD1/NT5E) and protein (CD38/CD39/CD73) expressions of ectonucleotidases were analyzed in silico and ex vivo and the association with prognosis was established. In univariate analyses, expression of NT5E was significantly associated with worse progression-free survival (PFS) in bone marrow (BM) samples. In multivariate analyses, Kaplan–Meier analysis, and log-rank test, higher NT5E expression predicted unfavorable PFS in BM samples. Considering minimal residual disease (MRD), higher levels of cellularity were associated with the high NT5E expression at day 8 of induction therapy. In addition, we observed that white blood cells (WBC) of childhood B-ALL patients had more CD38 compared to the same cell population of healthy donors (HD). In fact, MRD > 0.1% patients had higher CD38 protein expression on WBC in comparison to HD. Noteworthy, we observed higher CD38 expression on WBC than blasts in MRD > 0.1% patients. We suggest that NT5E gene and CD38 protein expression, of the ectonucleotidases family, could provide interesting prognostic biomarkers for childhood B-ALL.

The prevalence of metabolic syndrome (MetS) is more common in patients with hypertension and is associated with an increased risk of target organ damage and/or cardiovascular disease (CVD). Omentin-1 is a beneficial adipokine considered to play a role in MetS and MetS-related states such as obesity, diabetes, and coronary artery disease. The aim of this study was to determine the relationship between circulating omentin-1 levels and MetS uncomplicated by diabetes or CVD (nascent MetS) in patients with hypertension. In this study, 110 patients (54 men, 49%; average age: 49.72±11.32 years) treated for hypertension but without overt diabetes and/or CVD were enrolled. 66 patients were stratified into MetS (+) (group 1) and 44 patients into MetS (−) (group 2) according to the American Heart Association/National Heart, Lung, and Blood Institute criteria. The triglyceride glucose (TyG) index was used to assess insulin resistance. Circulating omentin-1 levels in venous blood samples were measured by an ELISA kit. Circulating omentin-1 levels in patients with MetS were significantly lower than in patients without MetS (46.35 ng/mL (42.70–57.70 ng/mL) vs 130.95 ng/mL (62.83–236.48 ng/mL), p

BACKGROUND Coronary artery disease (CAD) is the primary cause of death worldwide. It is mainly caused by atherosclerosis that initiates from a genetic-environmental interaction. Studies highlighted the association of numerous gene polymorphisms with CAD. Omentin-1 is secreted from visceral adipose tissues, intestine, and others; it has anti-inflammatory and insulin sensitivity improving roles. AIM To explore the association of the omentin-1 gene polymorphisms (rs2274907 and rs2274908) with serum lipid concentrations and CAD in a sample of the Iraqi population. METHODS A case-control study was followed, in which CAD patients were analyzed versus a group of healthy persons. Serum lipid concentrations were measured by enzymatic methods. Genotyping of the omentin-1 gene for rs2274907 SNP was achieved by ARMS-PCR, while for rs2274908 SNP by allele-specific PCR (AS-PCR) techniques. RESULTS Atherogenic serum lipid concentrations increased significantly in CAD patients relative to the control group. Genotyping of the omentin-1 gene for rs2274907 SNP revealed a significant (OR = 4.11, P = 0.035) elevation of the AT genotype carriers in CAD versus the control groups. The genotype analysis of the rs2274908 SNP failed to exhibit a significant variation. The two analyzed SNPs were indicated to be in linkage disequilibrium (r = 0.772, P

Study of human monocytic Myeloid-Derived Suppressor cells Mo-MDSC (CD14(+) HLA-DR(neg/low)) has been hampered by the lack of positive cell-surface markers. In order to identify positive markers for Mo-MDSC, we performed microarray analysis comparing Mo-MDSC cells from healthy subjects versus CD14(+) HLA-DR(high) monocytes. We have identified the surface ectoenzyme Vanin-2(VNN2) protein as a novel biomarker highly-enriched in healthy subjects Mo-MDSC. Indeed, healthy subjects Mo-MDSC cells expressed 68% VNN2, whereas only 9% VNN2 expression was observed on CD14(+) HLA-DR(high) cells (n=4 p

Background Immune checkpoint inhibitors prolong the survival of non-small cell lung cancer (NSCLC) patients. Although it has been acknowledged that there is some correlation between the efficacy of anti-programmed cell death-1 (PD-1) antibody therapy and immunohistochemical analysis, this technique is not yet considered foolproof for predicting a favorable outcome of PD-1 antibody therapy. We aimed to predict the efficacy of nivolumab based on a comprehensive analysis of RNA expression at the gene level in advanced NSCLC. Methods This was a retrospective study on patients with NSCLC who were administered nivolumab at the Kansai Medical University Hospital. To identify genes associated with response to anti-PD-1 antibodies, we grouped patients into responders (complete and partial response) and non-responders (stable and progressive disease) to nivolumab therapy. Significant genes were then identified for these groups using Welch’s t-test. Results Among 42 analyzed cases (20 adenocarcinomas and 22 squamous cell carcinomas), enhanced expression of MAGE-A4, BBC3, and OTOA genes was observed in responders with adenocarcinoma, and enhanced expression of DAB2, HLA-DPB,1 and CDH2 genes was observed in responders with squamous cell carcinoma. Conclusions This study predicted the efficacy of nivolumab based on a comprehensive analysis of mRNA expression at the gene level in advanced NSCLC. We also revealed different gene expression patterns as predictors of the effectiveness of anti PD-1 antibody therapy in adenocarcinoma and squamous cell carcinoma.

RECK encodes a membrane-anchored protease-regulator which is often downregulated in a wide variety of cancers, and reduced RECK expression often correlates with poorer prognoses. In mouse models, forced expression of RECK in tumor xenografts results in suppression of tumor angiogenesis, invasion, and metastasis. RECK mutations, however, are rare in cancer genomes, suggesting that agents that re-activate dormant RECK may be of clinical value. We found a potent RECK-inducer, DSK638, that inhibits spontaneous lung metastasis in our mouse xenograft model. Induction of RECK expression involves SP1 sites in its promoter and may be mediated by KLF2. DSK638 also upregulates MXI1, an endogenous MYC-antagonist, and inhibition of metastasis by DSK638 is dependent on both RECK and MXI1. This study demonstrates the utility of our approach (using a simple reporter assay followed by multiple phenotypic assays) and DSK638 itself (as a reference compound) in finding potential metastasis-suppressing drugs.

Body mass index (BMI) is a complex disease risk factor known to be influenced by genes acting via both metabolic pathways and appetite regulation. In this study, we aimed to gain insight into the phenotypic consequences of BMI-associated genetic variants, which may be mediated by their expression in different tissues. First, we harnessed meta-analyzed gene expression datasets derived from subcutaneous adipose (n = 1257) and brain (n = 1194) tissue to identify 86 and 140 loci, respectively, which provided evidence of genetic colocalization with BMI. These two sets of tissue-partitioned loci had differential effects with respect to waist-to-hip ratio, suggesting that the way they influence fat distribution might vary despite their having very similar average magnitudes of effect on BMI itself (adipose = 0.0148 and brain = 0.0149 standard deviation change in BMI per effect allele). For instance, BMI-associated variants colocalized with TBX15 expression in adipose tissue (posterior probability [PPA] = 0.97), but not when we used TBX15 expression data derived from brain tissue (PPA = 0.04) This gene putatively influences BMI via its role in skeletal development. Conversely, there were loci where BMI-associated variants provided evidence of colocalization with gene expression in brain tissue (e.g., NEGR1, PPA = 0.93), but not when we used data derived from adipose tissue, suggesting that these genes might be more likely to influence BMI via energy balance. Leveraging these tissue-partitioned variant sets through a multivariable Mendelian randomization framework provided strong evidence that the brain-tissue-derived variants are predominantly responsible for driving the genetically predicted effects of BMI on cardiovascular-disease endpoints (e.g., coronary artery disease: odds ratio = 1.05, 95% confidence interval = 1.04–1.07, p = 4.67 × 10(−14)). In contrast, our analyses suggested that the adipose tissue variants might predominantly be responsible for the underlying relationship between BMI and measures of cardiac function, such as left ventricular stroke volume (beta = 0.21, 95% confidence interval = 0.09–0.32, p = 6.43 × 10(−4)).

Oxidative stress is becoming increasingly implicated in the development of a variety of neurological disorders. However, the underlying mechanism remains elusive. In the present study, we investigated the function and related signal pathway which Cpg15, a neuronal-specific expressed neurotrophic factor, plays in the oxidative stress of neurons using a H

Classic T cell subsets are defined by a small set of cell surface markers, while single-cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq clustered populations (scCPops) and cell surface marker–defined classic T cell subsets remains unclear. In this article, we integrated six bead-enriched T cell subsets with 62,235 single-cell transcriptomes from human PBMCs and clustered them into nine scCPops. Bead-enriched CD4+/CD45RA+/CD25− naive T and CD8+/CD45RA+ naive T cells were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops, including mucosal-associated invariant T cells and NKT cells. The multiple T cell subsets forming one scCPop exhibit similar expression patterns, but not vice versa, indicating scCPop is a more homogeneous cell population with similar cell states. Interestingly, we discovered and named IFN signaling–associated gene (ISAG) high T (ISAGhi T) cells, a T cell subpopulation that highly expressed ISAGs. We further enriched ISAGhi T cells from human PBMCs by FACS of BST2 for scRNA-seq analyses. The ISAGhi T cell cluster disappeared on t-distributed stochastic neighbor embedding plot after removing ISAGs, whereas the ISAGhi T cell cluster showed up by analysis of ISAGs alone, indicating ISAGs are the major contributor of the ISAGhi T cell cluster. BST2+ and BST2− T cells showing different efficiencies of T cell activation indicate that a high level of ISAGs may contribute to quick immune responses.

Intelectin-1 is an anti-inflammatory adipokine encoded by the Intelectin 1 (ITLN1) gene. Genetic variations in the ITLN1 gene affect the risk of coronary artery disease (CAD) and related CAD risk factors. In this study, we aimed to investigate whether the ITLN1 gene Val109Asp polymorphism has an effect on the severity of CAD and serum lipid levels in both men and women.A total of 493 subjects who underwent coronary angiography (43.5% women, mean age 63.1±9.5 years) were grouped as individuals with critical CAD (≥70% stenosis, n=202), non-critical CAD (31%-69% stenosis, n=90), and non-CAD (control group) (1%-30% stenosis, n=201). Genotyping was performed using LightSNiP assay in Real-Time PCR.The frequency of the Val allele was significantly different among all the patients with critical CAD (n=41) and non-CAD control (n=51) groups in women (p=0.033) but not in men (n=77 and n=38). Women with the Val allele had a 1.69-fold increased risk for critical CAD (p=0.033). In addition, the presence of Val allele was associated with higher coronary stenosis after adjustment for several confounders only in women with critical CAD (p=0.025). Furthermore, carriers of the Val allele exhibited an increased low-density lipoprotein cholesterol (LDL-C) in men with critical CAD than in those with non-CAD (p0.05).These results suggest that the Val allele of the ITLN1 Val109Asp polymorphism is associated with critical CAD and high LDL-C levels in our study population. Further studies are required to elucidate the effect of Val109Asp polymorphism on CAD pathogenesis.

The α5-nicotinic acetylcholine receptor (α5-nAChR) is closely associated with nicotine-related lung cancer, offering a novel perspective for investigating the molecular pathogenesis of this disease. However, the mechanism by which α5-nAChR functions in lung carcinogenesis remains to be elucidated. Lymphocyte antigen 6 (Ly6) proteins, like snake three-finger alpha toxins such as α-bungarotoxin, can modulate nAChR signaling. Ly6E, a member of the Ly6 family, is a biomarker of poor prognosis in smoking-induced lung carcinogenesis and is involved in the regulation of TGF-β1/Smad signaling. Here, we explored the underlying mechanisms linking α5-nAChR and Ly6E in non-small cell lung cancer (NSCLC). The expression of α5-nAChR was correlated with Ly6 expression, smoking status and lower survival in NSCLC tissues. In vitro, α5-nAChR mediated Ly6E, the phosphorylation of the TGF-β1 downstream molecule Smad3 (pSmad3, a key mediator of TGF-β1 signaling), the epithelial-mesenchymal transition (EMT) markers Zeb1, N-cadherin and vimentin expression in NSCLC cells. The downregulation of Ly6E reduced α5-nAChR, pSmad3, Zeb1, N-cadherin and vimentin expression. Functionally, silencing both α5-nAChR and Ly6E significantly inhibited cell migration compared to silencing α5-nAChR or Ly6E alone. Furthermore, the functional effects of α5-nAchR and Ly6E were confirmed in chicken embryo chorioallantoic membrane (CAM) and mouse xenograft models. Therefore, our findings uncover a new interaction between α5-nAChR and Ly6E that inhibits cancer cell migration by modulating the TGF-β1/Smad signaling pathway in NSCLC, which may serve as a novel target for therapeutic intervention.

Introduction: Asthma is a common chronic respiratory disease. This study aimed to explore the expression level of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in induced sputum supernatant, induced sputum cells, and serum of asthma patients. Methods: The protein levels of CEACAM5 in induced sputum supernatant and serum were detected by enzyme-linked immunosorbent assay. The expression of CEACAM5 in induced sputum cells was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We analyzed the correlations between CEACAM5 expression and the clinical characteristics (FeNO and IgE) of asthma. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of CEACAM5 in asthma. The expression level of CEACAM5 in 16HBE and BEAS-2B cells was detected by qRT-PCR. Results: The expression of CEACAM5 in induced sputum supernatant, induced sputum cells, and serum of asthma patients was significantly upregulated. Asthma patients with high CEACAM5 expression in induced sputum supernatant had higher levels of FeNO, IgE, and IL-13. The expression levels of CEACAM5 in induced sputum supernatant and induced sputum cells were positively correlated with FeNO and IgE. The ROC curve showed that CEACAM5 had a good diagnostic value in asthma. CEACAM5 expression was upregulated in BEAS-2B and 16HBE cells after IL-4 or IL-13 stimulation for 48 h. Conclusion: The expression levels of CEACAM5 in induced sputum supernatant, induced sputum cells, and serum of asthma patients were significantly increased. CEACAM5 may be involved in eosinophilic inflammation of asthma and may be used as a diagnostic biomarker and therapeutic target of asthma.

Neuro-endocrine prostate cancer (NEPC) accounts for about 20% of lethal metastatic castration-resistant prostate cancer (CRPC). NEPC has the most aggressive biologic behavior of all prostate cancers and is associated with poor patient outcome. Effective treatment for NEPC is not available because NEPC exhibit distinct cell-surface expression profiles compared to other types of prostate cancer. Recently, the carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) (known as CEA or CD66e) was suggested to be a specific surface protein marker for NEPC. Therefore, we identified a new, fully-human anti-CEACAM5 monoclonal antibody, 1G9, which bound to the most proximal membrane domains, A3 and B3, of CEACAM5 with high affinity and specificity. It shows no off-target binding to other CEACAM family members, membrane distal domains of CEACAM5, or 5800 human membrane proteins. IgG1 1G9 exhibited CEACAM5-specific ADCC activity toward CEACAM5-positive prostate cancer cells in vitro and in vivo. Chimeric antigen receptor T cells (CAR-T) based on scFv 1G9 induced specific and strong antitumor activity in a mouse model of prostate cancer. Our results suggest that IgG1 and CAR-T cells based on 1G9 are promising candidate therapeutics for CEACAM5-positive NEPC and other cancers.

Background Ovarian cancer is a common gynecological malignant disease in women. Our work aimed to study the specific functions of ZNF252P antisense RNA 1 (ZNF252P-AS1) in ovarian cancer. Methods ZNF252P-AS1, miR-324-3p, and lymphocyte antigen 6 family member K (LY6K) expression were analyzed by bioinformatics tools in ovarian cancer tissues and was quantified by qRT-PCR in ovarian cancer cells. The effect of ZNF252P-AS1 knockdown, miR-324-3p suppression, and LY6K over-expression on apoptosis, cell viability, invasion, migration, and epithelial to mesenchymal transition (EMT) was determined in vitro by using colony formation and EdU assays, flow cytometry, transwell assay, and Western blot. The interactions between ZNF252P-AS1 and miR-324-3p and between miR-324-3p and LY6K were validated by luciferase assays. The effects of restraining ZNF252P-AS1 in vivo were studied using BALB/c male nude mice. Results ZNF252P-AS1 and LY6K levels were up-regulated, while miR-324-3p was declined in ovarian cancer tissues and cells. ZNF252P-AS1 knockdown reduced ovarian cancer cell proliferation, invasion, migration, and EMT, whereas promoted its apoptosis. Besides, ZNF252P-AS1 interacted with miR-324-3p and reversely regulated its level, and miR-324-3p was directly bound to LY6K and negatively regulated its expression. Moreover, ZNF252P-AS1 knockdown reversed the effect of miR-324-3p on cancer cell apoptosis, growth, migration, invasion, and EMT. Similar results were discovered in the rescue experiments between miR-324-3p and LY6K. Additionally, mouse models in vivo experiments further validated that ZNF252P-AS1 knockdown distinctly inhibited tumor growth. Conclusion ZNF252P-AS1 mediated miR-324-3p/LY6K signaling to facilitate progression of ovarian cancer.

We describe a novel system for examining the mechanisms of AAV traversal of the BBB using a multi-chamber microplate wherein astrocytes and HBMECs are co-cultured, and subjected to shear fluid force similar to that under physiological conditions.

The microRNA (miRNA) miR-576-5p was reported to facilitate tumor progression, but its underlying regulatory impacts on colon adenocarcinoma remain unknown. This work therefore attempted to examine the biological effects of miR-576-5p in colon adenocarcinoma. The Cancer Genome Atlas (TCGA) database was introduced to analyze miR-576-5p and NEGR1 messenger RNA (mRNA) expression levels between normal and cancer tissues. miR-576-5p and NEGR1 expression levels in colon adenocarcinoma cells and colon cells were evaluated with quantitative reverse transcription polymerase chain reaction (qRT-PCR). NEGR1 protein expression was examined by Western blot. Furthermore, colon adenocarcinoma cell behaviors were evaluated via CCK-8, wound-healing, Transwell, and hanging drop experiments. The interaction between miR-576-5p and NEGRI was verified by dual-luciferase assay. miR-576-5p was upregulated in colon adenocarcinoma, and miR-576-5p overexpression notably facilitated the proliferative, migratory, invasive abilities of colon adenocarcinoma cells. NEGR1 was newly identified as one target of miR-576-5p, and, miR-576-5p/NEGR1 axis was subsequently verified to modulate cell proliferative, migratory, invasive, and aggregate abilities. miR-576-5p facilitated aggressive progression of colon adenocarcinoma cells by targeting NEGR1, which could be an underlying therapeutic target for colon adenocarcinoma.

The identification of biomarkers allowing diagnostics, prognostics and patient classification is still a challenge in oncological research for patient management. Improvements in patient survival achieved with immunotherapies substantiate that biomarker studies rely not only on cellular pathways contributing to the pathology, but also on the immune competence of the patient. If these immune molecules can be studied in a non-invasive manner, the benefit for patients and clinicians is obvious. The immune receptor Natural Killer Group 2 Member D (NKG2D) represents one of the main systems involved in direct recognition of tumor cells by effector lymphocytes (T and Natural Killer cells), and in immune evasion. The biology of NKG2D and its ligands comprises a complex network of cellular pathways leading to the expression of these tumor-associated ligands on the cell surface or to their release either as soluble proteins, or in extracellular vesicles that potently inhibit NKG2D-mediated responses. Increased levels of NKG2D-ligands in patient serum correlate with tumor progression and poor prognosis; however, most studies did not test the biochemical form of these molecules. Here we review the biology of the NKG2D receptor and ligands, their role in cancer and in patient response to immunotherapies, as well as the changes provoked in this system by non-immune cancer therapies. Further, we discuss the use of NKG2D-L in liquid biopsy, including methods to analyse vesicle-associated proteins. We propose that the evaluation in cancer patients of the whole NKG2D system can provide crucial information about patient immune competence and risk of tumor progression.

Gastric cancer (GC) is one of the most common malignant tumors. Circular RNA (circRNA) has been shown to be involved in the progression of GC. However, the function of circ_0008035 in GC has not been studied. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_0008035, microRNA-1256 (miR-1256) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2’-deoxyuridine (EdU) assay, flow cytometry, and transwell assay were used to detect cell function. Western blot examined the protein levels of Ki67, Bax, MMP-2, and CEACAM6. The relationship between miR-1256 and circ_0008035 or CEACAM6 was verified by dual-luciferase reporter assays and RNA pull down. The xenotransplantation model was established in BALB/c nude mice to study the role of circ_0008035 in vivo. Circ_0008035 and CEACAM6 were significantly high-expressed in GC tissues and cells. Silencing of circ_0008035 reduced GC cell proliferation, migration, and invasion while enhancing apoptosis. MiR-1256 was a target of circ_0008035. The inhibition effect of circ_0008035 knockdown on the malignant behavior of GC cells could be reversed by miR-1256 inhibitor. In addition, CEACAM6 was a target of miR-1256. Overexpression of CEACAM6 partially restored the inhibitory effect of miR-1256 on cell progression. Animal experiments confirmed the anti-tumor effect of circ_0008035 knockdown in vivo. Collectively, circ_0008035 regulated the expression of CEACAM6 by sponging miR-1256, thereby promoting the development of GC. Our data provided a novel targeted therapy for GC.

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

The competing endogenous RNA (ceRNA) activity of circular RNAs (circRNAs) has been implicated in the development of gastric cancer. Here, we sought to explore the ceRNA function of circRNA Jupiter microtubule associated homolog 1 (circ_HN1) in gastric tumorigenesis. Circ_HN1, microRNA (miR)-628-5p, and NT5E expression levels were quantified by qRT-PCR and western blot. Dual-luciferase reporter assays were used to assess the direct relationship between miR-628-5p and circ_HN1 or NT5E. Our data showed that circ_HN1 expression was enhanced in human gastric cancer. Depletion of circ_HN1 impeded cell proliferation, spheroid formation, invasion, and migration and promoted apoptosis in vitro, as well as diminished tumor growth in vivo. NT5E was a downstream effector of circ_HN1 function. NT5E was targeted and inhibited by miR-628-5p through the perfect complementary site in NT5E 3ʹUTR, and circ_HN1 affected NT5E expression through miR-628-5p competition. Moreover, depletion of miR-628-5p reversed the effects of circ_HN1 silencing on regulating cell functional behaviors. Our findings identify a novel ceRNA network, the circ_HN1/miR-628-5p/NT5E axis, for the oncogenic activity of circ_HN1 in gastric cancer, highlighting circ_HN1 inhibition as a promising targeted treatment against gastric cancer.

Ruofei Chen,1,&ast; Yaqin Zhang,1,&ast; Honggang Xu,2 Huaqing Hu,3 Mingwei Chen,4 Zongwen Shuai1 1Department of Rheumatology and Immunology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, Anhui Province, People’s Republic of China; 2Department of Sports Injury and Arthroscopic Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, Anhui Province, People’s Republic of China; 3Health Management Center, The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, Anhui Province, People’s Republic of China; 4Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, Anhui Province, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Mingwei Chen; Zongwen Shuai Email chmw1@163.com; amushuaizw@163.comObjective: To investigate the correlation of the Val109Asp polymorphism of the omentin-1 gene with the risk and severity of knee osteoarthritis (KOA) in a Chinese Han population.Methods: This study enrolled 383 patients with primary KOA and 460 healthy controls. The genotypes were determined by the detection of single nucleotide polymorphism. To explore the interaction between omentin-1 gene polymorphism and obesity and age, the body mass index (BMI) of 25 kg/m2 and the age of 55 years old were preset as the cut-off value of stratified analysis. Furthermore, enzyme-linked immunosorbent assay was used to determine the levels of omentin-1, interleukin (IL)-1β, IL-6 in peripheral blood and synovial fluid and the contents of IL-1β, IL-6, metalloproteinase (MMP)-13 and collagen (COL)-II in the supernatant of knee joint cartilage tissue.Results: The Val109Asp polymorphism of the omentin-1 gene showed no obvious correlation with KOA. Compared with Asp/Asp genotype carriers with BMI < 25 kg/m2 and age < 55 years old, Val109 allele carriers with BMI≥ 25 kg/m2 and age ≥ 55 years old had obviously increased risk of KOA (adjusted OR = 1.416, p = 0.042; adjusted OR = 1.735, p = 0.038, respectively). In the KOA group, only the omentin-1 levels were significantly lower in the plasma and synovial fluid of Ala/Ala genotype carriers than in those of Asp/Asp genotype carriers. Meanwhile, the proportion of patients with moderate–severe K-L Classification, the levels of IL-1β, IL-6 in synovial fluid and the expression levels of IL-1β, IL-6 and MMP-13 in cartilage tissue significantly increased (p < 0.05). By contrast, the expression level of COL-II in cartilage tissue significantly decreased (p < 0.05).Conclusions: The Val109Asp polymorphism of the omentin-1 gene may not be the primary pathogenic factor of KOA in Chinese. The Val/Val genotype can be regarded as a potential biomarker for the risk of KOA progression.Keywords: omentin-1, single nucleotide polymorphism, knee osteoarthritis, biomarker, Chinese, obesity

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) infection induces elevated levels of inflammatory cytokines, which are mainly produced by the innate response to the virus. The role of NK cells, which are potent producers of IFN-γ and cytotoxicity, has not been sufficiently studied in the setting of SARS-CoV-2 infection. We confirmed a different distribution of NK cell subsets in hospitalized COVID-19 patients despite their NK cell deficiency. The impairment of this innate defense is mainly focused on the cytotoxic capacity of the CD56dim NK cells. On the one hand, we found an expansion of the CD56dimCD16neg NK subset, lower cytotoxic capacities, and high frequencies of inhibitory 2DL1 and 2DL1/S1 KIR receptors in COVID-19 patients. On the other hand, the depletion of CD56dimCD16dim/bright NK cell subsets, high cytotoxic capacities, and high frequencies of inhibitory 2DL1 KIR receptors were found in COVID-19 patients. In contrast, no differences in the distribution of CD56bright NK cell subsets were found in this study. These alterations in the distribution and phenotype of NK cells might enhance the impairment of this crucial innate line of defense during COVID-19 infection.

Background: By the end of 2019, the sudden outbreak of the novel coronavirus disease (COVID-19) has become a global threat. It is called COVID-19 because it was caused by the novel coronavirus (SARS-COV-2) in 2019. A total of 1.9 M deaths and 87.9 M cases have been reported all over the world, where 49M cases have recovered so far. Scientists are working hard to find chemotherapeutics and vaccines for COVID-19. Mutations in SARS-CoV-2 have been observed in a combination of several hazardous stresses, making them more resistant and beneficial. So to break down the viral system, the disease targets are examined. Objective: In today's review, a comprehensive study of spike protein explains the main purpose of the novel coronavirus and how to prevent the spread of the disease virus cross-transmission from infected to a healthy person. Methods: Covid-19 has already been declared a pandemic by the World Health Organization (WHO) due to its result in causing death and severe illness globally. SARS-CoV-2 is highly contagious; however, the intermediate host of the novel coronavirus is not clear. To explore the mechanisms of disease, one of the viral targets, such as the spike protein that binds to human cells and causes the disease by altering its genetic structure which is considered along with potential inhibitors. Results: It has been shown that the interaction of receptor-binding domain (RBD) protein of SARS- CoV-2 spike and the angiotensin-converting enzyme 2 (ACE2) host receptor and further replication of coronavirus spike protein causes its invasion in the host cell. The human Lymphocyte antigen 6 complex, Locus E (LY6E), inhibits the entry of CoV into host cells by interfering with the human gene, inducing spike protein-mediated membrane fusion. Some natural formulations have also been shown to prevent spike protein from binding to the host cell. Conclusion: With the development of the LY6E gene activator that can inhibit spike protein- ACE2-mediated membrane fusion, new opportunities for SARS-CoV-2 treatment may emerge. Existing antiviral fusion inhibitors and natural compounds targeting spike resistance can serve as a template for further SARS-CoV-2 drug formulation.

Circular RNAs (circRNAs) have been reported to play vital regulatory roles in human diseases. However, the functions of circRNAs in ischemic stroke (IS) are limited. In this study, we aimed to explore the functions and mechanisms of circRNA DLG associated protein 4 (circDLGAP4) in IS development. Oxygen-glucose deprivation (OGD)-treated HCN-2 cells were used to mimic IS environment in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect the levels of circDLGAP4, microRNA-503-3p (miR-503-3p) and neuronal growth regulator 1 (NEGR1) mRNA. RNase R assay was conducted to analyze the stability of circDLGAP4. Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis were adopted for cell viability and death, respectively. Western blot assay was performed for protein levels. Enzyme-linked immunosorbent assay (ELISA) kits were used to examine the concentrations of inflammatory cytokines. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were employed to analyze the relationships among circDLGAP4, miR-503-3p and NEGR1. CircDLGAP4 level was declined in HCN-2 cells after OGD treatment. CircDLGAP4 overexpression promoted cell viability and suppressed cell death and inflammatory cytokine concentrations in OGD-treated HCN-2 cells. CircDLGAP4 acted as the sponge for miR-503-3p and the impacts of circDLGAP4 overexpression on cell viability, death and inflammation in OGD-treated HCN-2 cells were reversed by miR-503-3p elevation. Furthermore, NEGR1 was the target gene of miR-503-3p. MiR-503-3p inhibition ameliorated OGD-induced HCN-2 cell impairments, but NEGR1 knockdown abolished the effects. CircDLGAP4 alleviated OGD-induced HCN-2 cell damage by regulating miR-503-3p/NEGR1 axis.

Data about the influence of short-term lifestyle intervention in children with obesity on long-term follow-up body weight, adipokines and cardiometabolic risk parameters is scarce.In a subgroup of the LOGIC-trial (Long-term Effects of Lifestyle Intervention in Obesity and Genetic Influence in Children), we assessed anthropometry (BMI, BMI-SDS (Standard Deviation Score), adipokines (omentin-1, chemerin, leptin, adiponectin) and cardiometabolic risk parameters, (e.g. hsCRP) in children with overweight/obesity after 4 weeks of lifestyle intervention (n = 156, 14.0 ± 1.8 yrs) and after one year follow-up (n = 50). Data were compared to normal weight children (JuvenTUM school cohort; n = 152, 13.3 ± 0.7 yrs).Short-term lifestyle intervention was associated with a significant reduction in BMI and BMI-SDS (p 0.001), with significant reductions in hsCRP, leptin, and chemerin levels, and an increase in adiponectin and omentin-1 levels (p 0.001 for all). After one year follow-up a significant reduction in BMI and BMI-SDS was observed in children from the LOGIC-trial (p 0.001). Improvements in adiponectin (p = 0.025) and chemerin levels (p = 0.027) were seen in children with clear weight loss success (BMI-SDS reduction ≥ 0.2), whereas children with no or only mild weight loss success showed an increase in leptin levels (p 0.001). An increase in omentin-1 levels was observed after 1 year independent of weight change (p 0.001).Effects of short-term weight reduction on mean BMI and BMI-SDS persist over one year. Improvements in omentin-1 levels were independent of short-term or long-term weight loss.ClinicalTrials.gov: LOGIC-trial: NCT01067157, JuvenTUM-trial: NCT00988754.

Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss–Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation., Nicotinamide riboside (NR) is a NAD + precursor exhibiting beneficial effects against aging. Here the authors demonstrate that orally administered NR increases NAD + levels in a diphasic manner and that bone marrow stromal cell antigen 1 plays a crucial role for NAD + synthesis from NR.

Parkinson’s disease (PD) is a disease that involves brain damage and is associated with neuroinflammation, mitochondrial damage, and cell aging. However, the pathogenic mechanism of PD is still unknown. Sequencing data and proteomic data can describe the fluctuation of molecular abundance in diseases at the mRNA level and protein level, respectively. In order to explore new targets in the pathogenesis of PD, the study analyzed molecular changes from the database by combining transcriptomic and proteomic analysis. Differentially expressed genes and differentially abundant proteins were summarized and analyzed. Enrichment and cluster analysis emphasized the importance of neurotransmitter release, mitochondrial damage, and vesicle transport. The molecular network revealed a subnetwork of 9 molecules related to SCNA and TH and revealed hub gene with differential expression at both mRNA and protein levels. It found that ACHE and CADPS could be used as new targets in PD, emphasizing that impaired nerve signal transmission and vesicle transport affect the pathogenesis of PD. Our research emphasized that the joint analysis and verification of transcriptomics and proteomics were devoted to understanding the comprehensive views and mechanism of pathogenesis in PD.

Introduction:\ud Malignant pleural mesothelioma (MPM) is difficult to diagnose. An accurate blood biomarker could prompt specialist referral or be deployed in future screening. In earlier retrospective studies, SOMAscan proteomics (Somalogic, Boulder, CO) and fibulin-3 seemed highly accurate, but SOMAscan has not been validated prospectively and subsequent fibulin-3 data have been contradictory.\ud \ud Methods:\ud A multicenter prospective observational study was performed in 22 centers, generating a large intention-to-diagnose cohort. Blood sampling, processing, and diagnostic assessment were standardized, including a 1-year follow-up. Plasma fibulin-3 was measured using two enzyme-linked immunosorbent assays (CloudClone [used in previous studies] and BosterBio, Pleasanton, CA). Serum proteomics was measured using the SOMAscan assay. Diagnostic performance (sensitivity at 95% specificity, area under the curve [AUC]) was benchmarked against serum mesothelin (Mesomark, Fujirebio Diagnostics, Malvern, PA). Biomarkers were correlated against primary tumor volume, inflammatory markers, and asbestos exposure.\ud \ud Results:\ud A total of 638 patients with suspected pleural malignancy (SPM) and 110 asbestos-exposed controls (AECs) were recruited. SOMAscan reliably differentiated MPM from AECs (75% sensitivity, 88.2% specificity, validation cohort AUC 0.855) but was not useful in patients with differentiating non-MPM SPM. Fibulin-3 (by BosterBio after failed CloudClone validation) revealed 7.4% and 11.9% sensitivity at 95% specificity in MPM versus non-MPM SPM and AECs, respectively (associated AUCs 0.611 [0.557–0.664], p = 0.0015) and 0.516 [0.443–0.589], p = 0.671), both inferior to mesothelin. SOMAscan proteins correlated with inflammatory markers but not with asbestos exposure. Neither biomarker correlated with tumor volume.\ud \ud Conclusions:\ud SOMAscan may prove useful as a future screening test for MPM in asbestos-exposed persons. Neither fibulin-3 nor SOMAscan should be used for diagnosis or pathway stratification.

The early identification of bacteremia is critical for ensuring appropriate treatment of nosocomial infections in intensive care unit (ICU) patients. The aim of this study was to use flow cytometric data of myeloid cells as a biomarker of bloodstream infection (BSI). An eight-color antibody panel was used to identify seven monocyte and two dendritic cell subsets. In the learning cohort, immunophenotyping was applied to (1) control subjects, (2) postoperative heart surgery patients, as a model of noninfectious inflammatory responses, and (3) blood culture-positive patients. Of the complex changes in the myeloid cell phenotype, a decrease in myeloid and plasmacytoid dendritic cell numbers, increase in CD14+CD16+ inflammatory monocyte numbers, and upregulation of neutrophils CD64 and CD123 expression were prominent in BSI patients. An extreme gradient boosting (XGBoost) algorithm called the “infection detection and ranging score” (iDAR), ranging from 0 to 100, was developed to identify infection-specific changes in 101 phenotypic variables related to neutrophils, monocytes and dendritic cells. The tenfold cross-validation achieved an area under the receiver operating characteristic (AUROC) of 0.988 (95% CI 0.985–1) for the detection of bacteremic patients. In an out-of-sample, in-house validation, iDAR achieved an AUROC of 0.85 (95% CI 0.71–0.98) in differentiating localized from bloodstream infection and 0.95 (95% CI 0.89–1) in discriminating infected from noninfected ICU patients. In conclusion, a machine learning approach was used to translate the changes in myeloid cell phenotype in response to infection into a score that could identify bacteremia with high specificity in ICU patients.

Summary Introduction Water jet-assisted liposuction has gained popularity due to favourable fat grafting outcomes. In this study, we compared stem cells obtained from fat isolated with manual or the water jet-assisted procedure. Methods Liposuction of abdominal fat was performed using the two methods on each donor (n = 10). Aspirate samples were collagenase digested and the isolated cells seeded in vitro prior to proliferation, adipogenic differentiation and angiogenic activity analyses. Results Cells from either procedure proliferated at similar rates and exhibited a similar colony-forming ability. The cells expressed stem cell markers CD73, CD90 and CD105. In the water jet cell preparations, there were higher numbers of cells expressing CD146. Robust adipogenic differentiation was observed in cultures expanded from both manual and water jet lipoaspirates. Gene analysis showed higher expression of the adipocyte markers aP2 and GLUT4 in the adipocyte-differentiated water jet cell preparations, and ELISA indicated increased secretion of adiponectin from these cells. Both cell groups expressed vasculogenic factors and the water jet cells promoted the highest levels of in vitro angiogenesis. Given these positive results, we further characterised the water jet cells when prepared using an automated closed cell processing unit, the Sepax-2 system (Cytiva). The growth and stem cell properties of the Sepax-processed cells were similar to the standard centrifugation protocol, but there was evidence for greater adipogenic differentiation in the Sepax-processed cells. Conclusions Water jet lipoaspirates yield cells with high adipogenic potential and angiogenic activity, which may be beneficial for use in cell-assisted lipotransfers.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy., Regulatory T (Treg) cells are important modulators of the tumor microenvironment. Here the authors perform transcriptome profiling of immune cells from patients with renal clear cell carcinoma to find a Treg signature that correlates with poorer prognosis, with CD177 being implicated as the main mediator for related alterations in Treg activity and tumor outcome.

BACKGROUND AND OBJECTIVES Isoantibodies to human neutrophil antigen 2 (CD177) have been associated with several clinical conditions but to date the molecular basis for altered or non-expression has not been determined. Reliance on phenotyping and crossmatch to investigate these neutropenic clinical cases are inconvenient for the patients and demanding of resources within the laboratory. Therefore, a molecular approach has been introduced to address both issues. MATERIALS AND METHODS A DNA panel of 100 randomly selected blood donors were collected and supplemented with 18 DNA samples from blood donors previously shown to be CD177 null. All DNA samples were sequence-based typed for all exons and observed polymorphisms recorded. The DNA from two families previously investigated for neonatal alloimmune neutropenia due to CD177 isoantibodies were also analysed. RESULTS The incidence of CD177 null could be associated with a known exon 7 single-nucleotide polymorphism in 16/21 known CD177 null samples, which is consistent with previously published findings. Two additional mutations that may lead to null expression were also identified, of which one may be novel. In both family investigations, this same mutation could also be observed in the maternal DNA sample. CONCLUSION Based on these observations, introduction of CD177 genotyping into routine use would identify null expression in over 75% (16/21) of associated cases. In turn, this could significantly reduce the need for supplementary testing and associated inconvenience to patients while permitting increased efficiency of laboratory testing. An added benefit would potentially elucidate other clinically relevant mutations and associated antigenic targets.

Background. Cytokeratin 19 fragment 21-1 (CYFRA21-1) and carcinoembryonic antigen (CEA) are effective prognostic biomarkers for lung cancer. This study investigated the predictive effects of change rates of CYFRA21-1 and CEA before and after the first cycles of chemotherapy on advanced IIIb/IIIc or IV stage non-small-cell lung cancer (NSCLC) patients. Methods. Data of 103 NSCLC patients who received chemotherapy in Zhejiang Provincial People’s Hospital from February 2018 to November 2020 were retrospectively analyzed. All patients received platinum doublet chemotherapy for at least 2 cycles. CYFRA21-1 and CEA levels of patients were detected before and after the first chemotherapy cycle, respectively. After the second cycle, the efficacy was evaluated, and patients were divided into the disease control (DC) and progressive disease (PD) groups. The generalized linear model (GLM) and linear trend test assessed the relationship between change rates of CYFRA21-1 and CEA levels and chemotherapeutic efficacy before and after chemotherapy. Moreover, the receiver operating characteristic (ROC) curve determined the predictive value of change rates of CYFRA21-1 and CEA on chemotherapeutic efficacy. Results. After the second chemotherapeutic cycle, there were 92 patients in the DC group and 11 in the PD group. GLM and linear trend test both indicated that change rates of CYFRA21-1 and CEA were inversely correlated with chemotherapeutic efficacy for NSCLC. Change rates of CYFRA21-1 and CEA were used to predict area under the ROC curve of chemotherapeutic efficacy (0.87, 0.71-1.00), which is better than single index prediction of CYFRA21-1 (0.71, 0.49-0.94) or CEA change rate (0.85, 0.69-1.00) ( p < 0.001 ). Conclusion. Before and after chemotherapy of the first cycle for advanced NSCLC patients, combining serum CYFRA21-1 and CEA levels could increase sensitivity and specificity to predict the chemotherapeutic efficacy and guide the following therapy of advanced NSCLC patients.

Background The FcγRs genotypes have been reported to play a key role in the defence against malaria parasites through both cellular and humoral immunity. This study aimed to investigate the possible correlation between FcγR (IIa, IIIa, and IIIb) genes polymorphism and the clinical outcome for anti‐malarial antibody response of Plasmodium falciparum infection among Saudi children. Methods A total of 600 volunteers were enrolled in this study, including 200 malaria-free control (MFC) subjects, 218 patients with uncomplicated malaria (UM) and 182 patients with severe malaria (SM). The FcγR genotypes were analysed using PCR amplification methods, and measurements of immunoglobulin were determined using enzyme-linked immunosorbent assay (ELISA) technique. Results The data revealed that the FcγRIIa-R/R131 showed a statistically significant association with SM patients when compared to UM patients. Furthermore, higher levels of IgG1, IgG2, and IgG4 were associated with the FcγRIIa-H/H131 genotype among UM patients. Although the FcγRIIa-F/V176 genotype was not associated with UM, it showed a significant association with severe malaria. Interestingly, the FcγRIIIa-V/V176 genotype offered protection against SM. Moreover, SM patients carrying the FcγRIIIa-F/F genotype showed higher levels of AMA-1-specific IgG2 and IgG4 antibodies. The FcγRIIIb-NA1/NA1 and FcγRIIIb-NA2/NA2 genotypes did not show significant differences between the UM and the MFC groups. However, the genotype FcγRIIIb-NA2/NA2 was statistically significantly associated with SM patients. Conclusions The data presented in this study suggest that the influence of the FcγRIIa-R/R131, FcγRIIIa-F/F176 and FcγRIIIb-NA2/NA2 genotypes are statistically significantly associated with SM patients. However, the FcγRIIa-H/H13 and FcγRIIIa-V/V176 genotypes have demonstrated a protective effect against SM when compared to UM patients. The impact of the FcyR (IIa, IIIa and IIIb) gene variants and anti-malaria IgG subclasses play an important role in susceptibility to malaria infection and disease outcome in Saudi children.

The rBC2LCN lectin, known as a stem cell marker probe that binds to an H type 3 fucosylated trisaccharide motif, was recently revealed to also bind to pancreatic ductal adenocarcinoma (PDAC) cells. A lectin‐drug conjugate was generated by fusing rBC2LCN with a cytocidal toxin, and it showed a strong anticancer effect in in vitro and in vivo PDAC models. However, it is unclear which molecules are carrier proteins of rBC2LCN on PDAC cells. In this study, we identified a rBC2LCN‐positive glycoprotein expressed in PDAC. Tumor lysates of PDAC patient‐derived xenografts (PDXs) were coprecipitated with rBC2LCN lectin and analyzed by liquid chromatography–mass spectrometry. A total of 343 proteins were initially identified. We used a web‐based database to select five glycoproteins and independently evaluated their expression in PDAC by immunohistochemistry (IHC). Among them, we focused on carcinoembryonic antigen 5 (CEA) as the most cancer‐specific carrier protein in PDAC, as it showed the most prominent difference in expression rate between PDAC cells (74%) and normal pancreatic duct cells (0%, P > .0001). rBC2LCN lectin and CEA colocalization in PDAC samples was confirmed by double‐staining analysis. Furthermore, rBC2LCN‐precipitated fractions were blotted with an anti‐CEA polyclonal antibody (pAb), and CEA pAb–precipitated fractions were blotted with rBC2LCN lectin. The results demonstrate that CEA is in fact a ligand of rBC2LCN lectin., This study reports that carcinoembryonic antigen 5 (CEA) is a cancer‐specific rBC2LCN‐positive glycoprotein expressed in pancreatic ductal adenocarcinoma (PDAC). The rBC2LCN lectin, reported as a stem cell marker probe, has been detected in some cancers, and a glycan epitope of rBC2LCN, H type 3 fucosylated motif (Fucα1‐2Galβ1‐3GalNAc), could be a therapeutic target for cancers. This study showed for the first time that CEA is modified with α1‐2 fucose.

Background Accumulating evidence indicates that circRNAs may serve as essential regulators in the progression of several human cancers, but the function and mechanism of circRNAs in intrahepatic cholangiocarcinoma (ICC) are largely unknown. Methods RNA-seq was used to assess differentially expressed circRNAs between 4 ICC and peritumor tissues. Quantitative RT-PCR and in situ hybridization were used to determine the circHMGCS1–016 expression in ICC tissues. The function and mechanism of circHMGCS1–016 were further identified via in vivo experiments. The clinical characteristics and prognostic significance of circHMGCS1–016 were analyzed by a retrospective study. The functions of circHMGCS1–016 were assessed via modifying circRNA expression in ICC cells. Moreover, the molecular mechanisms of circHMGCS1–016 in ICC cells were explored by circRNA precipitation, miRNA immunoprecipitation, SILAC and luciferase reporter assays. Results We identified that compared with peritumor tissues, ICC tissues expressed hsa_circ_0008621 (circHMGCS1–016) high by RNA-seq, which was further identified by qRT-PCR and in situ hybridization. Moreover, the expression of circHMGCS1–016 was revealed to be associated with survival and recurrence of ICC patients. By regulating circHMGCS1–016 expression, we found that elevated circHMGCS1–016 promoted ICC development both in vitro and in vivo. By SILAC and circRNA-pull down, we demonstrated that circHMGCS1–016 induced ICC cell invasion and reshaped the tumor immune microenvironment via the miR-1236-3p/CD73 and GAL-8 axis. In ICC tissues, we uncovered that a high level of circHMGCS1–016 was positively associated with CD73 and GAL-8 expression and negatively related to the CD8+ T cells infiltration, which was further validated by establishing a humanized mouse tumor model. Importantly, we displayed that ICC patients with high levels of circHMGCS1–016 in tumor tissues benefited less from anti-PD1 treatment compared to those with low levels of circHMGCS1–016. Conclusions CircHMGCS1–016 is a forceful contributor in ICC development and immune tolerance via miR-1236-3p/CD73 and GAL-8 axis. CircHMGCS1–016 can be explored as a new potential biomarker and therapeutic target for PD1-resistant ICC.

The main neurotransmitters in the brain—dopamine, γ-aminobutyric acid (GABA), glutamate, and opioids—are recognized to be the most important for the regulation of aggression and addiction. The aim of this work was to study differentially expressed genes (DEGs) in the main reward-related brain regions, including the ventral tegmental area (VTA), dorsal striatum (STR), ventral striatum (nucleus accumbens, NAcc), prefrontal cortex (PFC), and midbrain raphe nuclei (MRNs), in male mice with 20-day positive fighting experience in daily agonistic interactions. Expression of opioidergic, catecholaminergic, glutamatergic, and GABAergic genes was analyzed to confirm or refute the influence of repeated positive fighting experience on the development of “addiction-like” signs shown in our previous studies. High-throughput RNA sequencing was performed to identify differentially expressed genes in the brain regions of chronically aggressive mice. In the aggressive mice, upregulation of opioidergic genes was shown (Oprk1 in VTA, Pdyn in NAcc, Penk in PFC, and Oprd1 in MRNs and PFC), as was downregulation of genes Opcml and Oprk1 in STR and Pomc in VTA and NAcc. Upregulation of catecholaminergic genes in VTA (Ddc and Slc6a2) and in NAcc (Th and Drd2) and downregulation of some differentially expressed genes in MRNs (Th, Ddc, Dbh, Drd2, Slc18a2, and Sncg) and in VTA (Adra2c, Sncg, and Sncb) were also documented. The expression of GABAergic and glutamatergic genes that participate in drug addiction changed in all brain regions. According to literature data, the proteins encoded by genes Drd2, Oprk1, Oprd1, Pdyn, Penk, and Pomc are directly involved in drug addiction in humans. Thus, our results confirm our earlier claim about the formation of addiction-like signs following repeated positive fighting experience in mice, as shown previously in our biobehavioral studies.