Methylome profiling identifies TCHH methylation in CfDNA as a noninvasive marker of liver metastasis in colorectal cancer.

Methylation of circulating free DNA (CfDNA) has emerged as an efficient marker of tumor screening and prognostics. However, no efficient methylation marker has been developed for monitoring liver metastasis (LM) in colorectal cancer (CRC). Utilizing methylome profiling and bisulfite sequencing polymerase chain reaction of paired primary and LM sites, significantly increased methylation of TCHH was identified in the process of LM in CRC in the present study. Methylight analysis of TCHH methylation in CfDNA displayed a promisingly discriminative power between CRC with and without LM. Besides, significant coefficient of TCHH methylation and LM tumor volume was also validated. Together, these results indicated the potential of TCHH methylation in CfDNA as a monitoring marker of LM in CRC.

Negative auto-regulation of sulfur dioxide generation in vascular endothelial cells: AAT1 S-sulfenylation.

Recently, endogenous sulfur dioxide (SO2) has been found to exert an important function in the cardiovascular system. However, the regulatory mechanism for SO2 generation has not been entirely clarified. Hence, we aimed to explore the possible auto-regulation of endogenous SO2 generation and its mechanisms in vascular endothelial cells. We showed that SO2 did not affect the protein expression of aspartate aminotransferase 1 (AAT1), a major SO2 synthesis enzyme, but significantly inhibited AAT activity in primary human umbilical vein endothelial cells (HUVECs) and porcine purified AAT1 protein. An AAT1 enzymatic kinetic study showed that SO2 reduced the Vmax (1.89 ± 0.10 vs 2.55 ± 0.12, µmol/mg/min, P < 0.05) and increased the Km (35.97 ± 9.54 vs 19.33 ± 1.76 µmol/L, P < 0.05) values. Furthermore, SO2 induced S-sulfenylation of AAT1 in primary HUVECs and purified AAT1 protein. LC-MS/MS analysis indicated that SO2 sulfenylated AAT1 at Cys192. Mechanistically, thiol reductant DTT treatment or C192S mutation prevented SO2-induced AAT1 sulfenylation and the subsequent inhibition of AAT activity in purified AAT1 protein and primary HUVECs. Our findings reveal, for the first time, a mechanism of auto-regulation of SO2 generation through sulfenylation of AAT1 at Cys192 to suppress AAT activity in vascular endothelial cells. These findings will greatly deepen the understanding of regulatory mechanisms in the cardiovascular homeostasis.

The novel long noncoding RNA CRART16 confers cetuximab resistance in colorectal cancer cells by enhancing ERBB3 expression via miR-371a-5p.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been shown to participate in multiple biological processes and confer drug resistance. However, it remains unclear whether lncRNAs are involved in conferring cetuximab resistance in colorectal cancer (CRC) cells. METHODS: Cell Counting Kit-8 (CCK-8) assays were performed to assess the sensitivity of CRC cell lines to cetuximab treatment. We incubated Caco-2 cells, which are partially responsive to cetuximab, with increasing concentrations of cetuximab for approximately 6 months to generate Caco-2 cetuximab-resistant (Caco-2 CR) cells. Microarray analysis comparing Caco-2 CR with Caco-2 cells was used to identify lncRNAs that were potentially related to cetuximab resistance. Caco-2 cells were stably transduced with cetuximab resistance-associated RNA transcript 16 (CRART16) or an empty vector using lentiviral infection; the cells were designated Caco-2-CRART16 and Caco-2-NC, respectively, and were analyzed with RNA sequencing (RNA-seq). Quantitative real-time PCR (qRT-PCR) was performed to investigate RNA expression. Flow cytometry and TUNEL assays were used to assess apoptosis levels induced by cetuximab. The cell cycle, stemness biomarkers and membrane proteins of CRC cells were assessed via flow cytometry. RNA fluorescence in situ hybridization (FISH) was used to examine CRART16 localization and expression. Bioinformatics analysis was performed to predict the potential mechanism of CRART16, which was further validated by a dual-luciferase reporter assay. Differences in measurement data were compared using Student's t test, one-way ANOVA followed by Dunnett's test and two-way ANOVA. RESULTS: The novel lncRNA CRART16 was upregulated in Caco-2 CR cells. CRART16 overexpression reversed the effects of cetuximab on cell viability and reduced cetuximab-induced apoptosis. Meanwhile, CRART16 overexpression led to increases in the proportion of CD44+/CD133+ cells. In addition, CRART16 acts as a competing endogenous RNA (ceRNA) for miR-371a-5p to regulate V-Erb-B2 Erythroblastic Leukemia Viral Oncogene Homolog 3 (ERBB3) expression. MiR-371a-5p mimics counteracted the cetuximab resistance induced by CRART16 overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that after CRART16 was overexpressed, the resulting differentially expressed mRNAs were mainly enriched in the MAPK signaling pathway. CONCLUSIONS: CRART16 overexpression may contribute to cetuximab resistance through the miR-371a-5p/ERBB3/MAPK pathway. Additionally, CRART16 contributes to the acquisition of stemness properties.

A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells.

Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

Endogenous hydrogen sulfide sulfhydrates IKKß at cysteine 179 to control pulmonary artery endothelial cell inflammation.

BACKGROUND: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. METHODS: Purified recombinant human inhibitor of κB kinase subunit ß (IKKß) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. RESULTS: We showed that hydrogen sulfide (H2S) inhibited IKKß activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKß activity directly via sulfhydrating IKKß at cysteinyl residue 179 (C179) in purified recombinant IKKß protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKß inactivation. Furthermore, to demonstrate the significance of IKKß sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKß. In purified IKKß protein, C179S mutation of IKKß abolished H2S-induced IKKß sulfhydration and the subsequent IKKß inactivation. In human PAECs, C179S mutation of IKKß blocked H2S-inhibited IKKß activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKß abolished the inhibitory effect of H2S on IKKß activation and pulmonary vascular inflammation and remodeling. CONCLUSION: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKß via sulfhydrating IKKß at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

Decrease of galectin-3 in keratinocytes: A potential diagnostic marker and a critical contributor to the pathogenesis of psoriasis.

Psoriasis-specific proteins dysregulated in keratinocytes and involved in the pathophysiological process of psoriasis remains elusive. We report here that epidermal galectin-3 expression is significantly downregulated in lesional skin, but not in non-lesional skin in psoriasis patients, nor in a group of diseases known as psoriasiform dermatitis clinically and histologically similar to psoriasis. The deficiency of epidermal galectin-3 is sufficient to promote development of psoriatic lesions, as evidenced by more severe skin inflammation in galectin-3 knockout (gal3-/-) mice, compared to wild-type mice, after imiquimod treatment, and in skin from gal3-/- mice grafted onto wildtype mice. The development of psoriatic-like lesions is attributable to 1) the spontaneously tuning up of psoriasis signatures in keratinocytes through JNK pathway; and 2) neutrophil accumulation caused by the enhanced leukocyte-recruiting capacity associated with overexpression of S100A7-9 and CXCL-1, 8 in keratinocytes with impaired galectin-3 expression. Psoriasis-like skin inflammation is significantly improved in gal-3-/- mice both by inhibition of neutrophils accumulation with a selective CXCR2 antagonist of SB225002, and by intracutaneous injection of recombinant galectin-3. Overall, these findings offer promising galectin-3-related diagnostic and therapeutic resolutions of psoriasis.

IP3R-Grp75-VDAC1-MCU calcium regulation axis antagonists protect podocytes from apoptosis and decrease proteinuria in an Adriamycin nephropathy rat model.

BACKGROUND: The mechanism of podocyte apoptosis is not fully understood. In addition, the role of the inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (Grp75)/voltage-dependent anion channel 1 (VDAC1)/mitochondrial calcium uniporter (MCU) calcium regulation axis, which is located at sites of endoplasmic reticulum (ER) mitochondria coupling, in the mechanism of podocyte apoptosis is unclear. This study aimed to understand the roles of this axis in podocyte apoptosis and explore potential targets for podocyte protection. METHODS: The expression of IP3R, Grp75, VDAC1, and MCU and mitochondrial Ca2+ were analyzed during Adriamycin- or angiotensin II-induced apoptosis in cultured mouse podocytes. The interaction between IP3R, Grp75, and VDAC1 was investigated using co-immunoprecipitation experiments. The effects of IP3R, Grp75, and MCU agonists and antagonists on mitochondrial Ca2+ and apoptosis were investigated in cultured podocytes. The podocyte-protective effects of an MCU inhibitor were further investigated in rats with Adriamycin-induced nephropathy. RESULTS: Increased expression of IP3R, Grp75, VDAC1 and MCU, enhanced interaction among the IP3R-Grp75-VDAC1 complex, mitochondrial Ca2+ overload, and increased active caspase-3 levels were confirmed during Adriamycin- or angiotensin II-induced mouse podocyte apoptosis. Agonists of this axis facilitated mitochondrial Ca2+ overload and podocyte apoptosis, whereas specific antagonists against IP3R, Grp75, or MCU prevented mitochondrial Ca2+ overload and podocyte apoptosis. A specific MCU inhibitor prevented Adriamycin-induced proteinuria and podocyte foot process effacement in rats. CONCLUSIONS: This study identified a novel pathway in which the IP3R-Grp75-VDAC1-MCU calcium regulation axis mediated podocyte apoptosis by facilitating mitochondrial Ca2+ overload. Antagonists that inhibit Ca2+ transfer from ER to mitochondria protected mouse podocytes from apoptosis. An MCU inhibitor protected podocytes and decreased proteinuria in rats with Adriamycin-induced nephropathy. Therefore, antagonists to this pathway have promise as novel podocyte-protective drugs.

Hydrogen sulfide suppresses oxidized low-density lipoprotein (ox-LDL)-stimulated monocyte chemoattractant protein 1 generation from macrophages via the nuclear factor κB (NF-κB) pathway.

This study was designed to examine the role of hydrogen sulfide (H2S) in the generation of oxidized low-density lipoprotein (ox-LDL)-stimulated monocyte chemoattractant protein 1 (MCP-1) from macrophages and possible mechanisms. THP-1 cells and RAW macrophages were pretreated with sodium hydrosulfide (NaHS) and hexyl acrylate and then treated with ox-LDL. The results showed that ox-LDL treatment down-regulated the H2S/cystathionine-ß-synthase pathway, with increased MCP-1 protein and mRNA expression in both THP-1 cells and RAW macrophages. Hexyl acrylate promoted ox-LDL-induced inflammation, whereas the H2S donor NaHS inhibited it. NaHS markedly suppressed NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter in ox-LDL-treated macrophages. Furthermore, NaHS decreased the ratio of free thiol groups in p65, whereas the thiol reductant DTT reversed the inhibiting effect of H2S on the p65 DNA binding activity. Most importantly, site-specific mutation of cysteine 38 to serine in p65 abolished the effect of H2S on the sulfhydration of NF-κB and ox-LDL-induced NF-κB activation. These results suggested that endogenous H2S inhibited ox-LDL-induced macrophage inflammation by suppressing NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter. The sulfhydration of free thiol group on cysteine 38 in p65 served as a molecular mechanism by which H2S inhibited NF-κB pathway activation in ox-LDL-induced macrophage inflammation.

Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.

Olmsted syndrome (OS) is a rare congenital disorder characterized by palmoplantar and periorificial keratoderma, alopecia in most cases, and severe itching. The genetic basis for OS remained unidentified. Using whole-exome sequencing of case-parents trios, we have identified a de novo missense mutation in TRPV3 that produces p.Gly573Ser in an individual with OS. Nucleotide sequencing of five additional affected individuals also revealed missense mutations in TRPV3 (which produced p.Gly573Ser in three cases and p.Gly573Cys and p.Trp692Gly in one case each). Encoding a transient receptor potential vanilloid-3 cation channel, TRPV3 is primarily expressed in the skin, hair follicles, brain, and spinal cord. In transfected HEK293 cells expressing TRPV3 mutants, much larger inward currents were recorded, probably because of the constitutive opening of the mutants. These gain-of-function mutations might lead to elevated apoptosis of keratinocytes and consequent skin hyperkeratosis in the affected individuals. Our findings suggest that TRPV3 plays essential roles in skin keratinization, hair growth, and possibly itching sensation in humans and selectively targeting TRPV3 could provide therapeutic potential for keratinization or itching-related skin disorders.

[Analysis of the relationship between mitochondrial DNA deletion and clinical complexity of mitochondrial disease].

OBJECTIVE: To analyze the relationship between proportion of mitochondrial DNA 4 977 bp deletion (ΔmtDNA(4977)) or copy number in blood and the clinical complexity to find the pathogenesis of mitochondrial disease. METHODS: A total of 160 patients with mitochondrial disease and 101 healthy controls of Peking University First Hospital from December 2003 to December 2013 were collected in this study. Their peripheral blood showed no hot-point mutation which detected by polymerase chain reaction-restriction fragment length polymorphism. All the patients were divided into younger group (age<10y) and elder group (10y≤age<20y). The incidence of ΔmtDNA(4977) was detected by real-time quantitative PCR. Internal gene was used to calculate the number of mitochondrial DNA in each cell. Statistical analysis were carried out by the independent t-test, one-way ANOVA and Spearman's bivariate correlation analysis. RESULTS: ΔmtDNA (4977) proportion in the younger group was (2.66 ± 0.63)% and in the elder group was (3.09 ± 0.74)%, both of them were higher than that of healthy control group with the same age (the younger group: t=8.57, P<0.01; the elder group: t=4.38, P<0.01); ΔmtDNA(4977) copy number per cell in the younger group was (2.79 ± 0.50) copy and in the elder group was (2.97 ± 0.48) copy, both of them were higher than that of healthy control group with the same age (the younger group: t=4.50, P<0.01; the elder group: t=-3.67, P<0.01). The ΔmtDNA (4977) proportion was positively correlated with the complexity of the mitochondrial disease(the younger group: r=0.519, P<0.01; the elder group: r=0.772, P<0.01). The ΔmtDNA (4977) copy number per cell was positively correlated with the complexity of the mitochondrial disease(the younger group: r=0.389, P<0.01; the elder group: r=0.607, P<0.05). However, the total mtDNA copy number per cell was negatively correlated with the complexity of the mitochondrial disease (the younger group: r=-0.260, P<0.01; the elder group: r=-0.430, P<0.05). CONCLUSIONS: The proportion or copy number of ΔmtDNA (4977) or total mtDNA copy number in blood are correlated with the complexity of mitochondrial diseases, especially the proportion of ΔmtDNA (4977).

Brg1-dependent epigenetic control of vascular smooth muscle cell proliferation by hydrogen sulfide.

Hydrogen sulfide (H2S) can modulate the proliferation of vascular smooth muscle cells. This study was designed to investigate the epigenetic control of vascular smooth muscle cell proliferation in response to H2S. Microarray analysis indicated that Brahma-related gene 1 (Brg1) and proliferation-related genes including proliferating cell nuclear antigen (Pcna), neurotrophin 3 (Ntf3) and platelet-derived growth factor subunit A (Pdgfα) were significantly downregulated by H2S in endothelin-1-stimulated proliferative vascular smooth muscle cells. Brg1 is the central catalytic subunit of the SWI/SNF apparatus (an ATP-dependent chromatin remodeling complex). Overexpression and knockdown of Brg1 confirmed that Brg1 was crucial for H2S-induced inhibition of vascular smooth muscle cell proliferation. A luciferase reporter assay, real-time PCR and Western blotting demonstrated that H2S inhibited Brg1 transcription and expression. A DNase I hypersensitivity assay revealed that H2S reversed endothelin-1-stimulated Pcna, Ntf3 and Pdgfα chromatin remodeling and vascular smooth muscle cell proliferation. A chromatin immunoprecipitation assay indicated that H2S inhibited the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoters. The results of this study indicate that H2S inhibits vascular smooth muscle cell proliferation via an epigenetic mechanism involving the inhibition of Brg1 transcription and expression, and by reducing the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoter regions.

SERPINB7 Deficiency Increases Legumain Activity and Impairs the Epidermal Barrier in Nagashima-type Palmoplantar Keratoderma.

Nagashima-type palmoplantar keratoderma (NPPK) is an autosomal recessive genodermatosis caused by loss-of-function variants in SERPINB7 and is the most prevalent form of inherited palmoplantar keratodermas among Asians. However, there is currently no effective therapy for NPPK because its pathogenesis remains unclear. In this study, Serpinb7-/- mice were generated and spontaneously developed a disrupted skin barrier, which was further exacerbated by acetone-ether-water treatment. The skin of these Serpinb7-/- mice showed weakened cytoskeletal proteins. Additionally, SERPINB7 deficiency consistently led to decreased epidermal differentiation in a three-dimensional human epidermal model. We also demonstrated that SERPINB7 was an inhibitory serpin that mainly inhibited the protease legumain. SERPINB7 bound directly with legumain and inhibited legumain activity both in vitro and in vivo. Furthermore, we found that SERPINB7 inhibited legumain in a 'protease-substrate' manner and identified the cleavage sites of SERPINB7 as Asn71 and Asn343. Overall, we found that SERPINB7 showed the nature of a cysteine protease inhibitor, and identified legumain as a key target protease of SERPINB7. Loss of SERPINB7 function led to overactivation of legumain, which might disrupt cytoskeletal proteins, contributing to the impaired skin barrier in NPPK. These findings may lead to the development of therapeutic strategies for NPPK.

Common genetic variants of the human uromodulin gene regulate transcription and predict plasma uric acid levels.

Uromodulin (UMOD) genetic variants cause familial juvenile hyperuricemic nephropathy, characterized by hyperuricemia with decreased renal excretion of UMOD and uric acid, suggesting a role for UMOD in the regulation of plasma uric acid. To determine this, we screened common variants across the UMOD locus in one community-based Chinese population of 1000 individuals and the other population from 642 American twins and siblings of European and Hispanic ancestry. Transcriptional activity of promoter variants was estimated in luciferase reporter plasmids transfected into HEK-293 cells and mIMCD3 cells. In the primary Chinese population, we found that carriers of the GCC haplotype had higher plasma uric acid, and three promoter variants were associated with plasma uric acid. UMOD promoter variants displayed reciprocal effects on urine uric acid excretion and plasma uric acid concentration, suggesting a primary effect on renal tubular handling of urate. These UMOD genetic marker-on-trait associations for uric acid were replicated in the independent American cohort. Site-directed mutagenesis at trait-associated UMOD promoter variants altered promoter activity in transfected luciferase reporter plasmids. Thus, UMOD promoter variants seem to initiate a cascade of transcriptional and biochemical changes influencing UMOD secretion, leading to altered plasma uric acid levels.

[Mutational analysis of the methyl-CpG-binding protein 2 (MECP2) gene in male autism patients].

OBJECTIVE: To investigate mutations in the methyl-CpG-binding protein 2 (MECP2) gene in male autism patients by PCR, denaturing high-performance liquid chromatography (DHPLC) and sequencing to explore the role of mutations in MECP2 in autism patients. METHODS: We recruited DNA samples from 44 male autism patients who matched the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DMS-IV) standards. DHPLC was used to screen the mutations in MECP2 gene, and DNA sequencing was performed for the samples with positive DHPLC results. The family members were further investigated in the patients with missense mutations in MECP2 gene. RESULTS: Four cases were found to have mutations in MECP2 gene, including missense mutations of c.590C>T(T197M)in one case and c.602C>T(A201V)in one case, and synonymous mutations of c.1053C>G in one case and c.897C>T in one case. In addition, we found C>T variation in intron 3 at the +74 bp before exon 4, a SNP (rs2071569) usually detected in Chinese population. In the case with c.602C>T(A201V)mutation, his mother and maternal grandfather had the same mutation. His mother had normal phenotype, but his maternal grandfather had depressive disease. CONCLUSION: Mutations in MECP2 are present in male autism patients with relatively higher prevalence, suggesting that these mutations may play roles in the pathogenesis of autism.

CBS-H2S axis preserves the intestinal barrier function by inhibiting COX-2 through sulfhydrating human antigen R in colitis.

INTRODUCTION: Lipopolysaccharide (LPS) causes lesions of the epithelial barrier, which allows translocation of pathogens from the intestinal lumen to the host's circulation. Hydrogen sulfide (H2S) regulates multiple physiological and pathological processes in colonic epithelial tissue, and CBS-H2S axis involved in multiple gastrointestinal disorder. However, the mechanism underlying the effect of the CBS-H2S axis on the intestinal and systemic inflammation in colitis remains to be illustrated. OBJECTIVES: To investigate the effect of CBS-H2S axis on the intestinal and systematic inflammation related injuries in LPS induced colitis and the underlying mechanisms. METHODS: Wild type and CBS-/+ mice were used to evaluate the effect of endogenous and exogenous H2S on LPS-induced colitis in vivo. Cytokine quantitative antibody array, western blot and real-time PCR were applied to detect the key cytokines in the mechanism of action. Biotin switch of S-sulfhydration, CRISPR/Cas9 mediated knockout, immunofluorescence and ActD chase assay were used in the in vitro experiment to further clarify the molecular mechanisms. RESULTS: H2S significantly alleviated the symptoms of LPS-induced colitis in vivo and attenuated the increase of COX-2 expression. The sulfhydrated HuR increased when CBS express normally or GYY4137 was administered. While after knocking kown CBS, the expression of COX-2 in mice colon increased significantly, and the sulfhydration level of HuR decreased. The results in vitro illustrated that HuR can increase the stability of COX-2 mRNA, and the decrease of COX-2 were due to increased sulfhydration of HuR rather than the reduction of total HuR levels. CONCLUSION: These results indicated that CBS-H2S axis played an important role in protecting intestinal barrier function in colitis. CBS-H2S axis increases the sulfhydration level of HuR, by which reduces the binding of HuR with COX-2 mRNA and inhibited the expression of COX-2.

The T788G mutation in the cyp51C gene confers voriconazole resistance in Aspergillus flavus causing aspergillosis.

With voriconazole (VRC) being approved as the first choice in treating invasive aspergillosis (IA) and its increasing use in treatment, a VRC-resistant strain of Aspergillus flavus, the second leading cause of IA after Aspergillus fumigatus, has emerged. The VRC-resistant strain of A. flavus was isolated for the first time from the surgical lung specimen of an IA patient with no response to VRC therapy. In order to ascertain the mechanism of VRC resistance, the azole target enzyme genes in this strain of A. flavus were cloned and sequenced, and 4 mutations generating amino acid residue substitutions were found in the cyp51C gene. To further determine the role of this mutated gene for VRC resistance in A. flavus, an Agrobacterium tumefaciens-mediated gene replacement approach was applied. Consequently, the mutated cyp51C gene from this A. flavus strain was proven to confer the VRC resistance. Finally, to discern the one out of the four mutations in the cyp51C gene that is responsible for contributing to VRC resistance, a site-directed gene mutagenesis procedure combined with a gene replacement method was performed. As a result, the T788G missense mutation in the cyp51C gene was identified as responsible for VRC resistance in A. flavus. These findings indicated that the detection of this mutation in A. flavus could serve as an indicator for physicians to avoid the use of VRC during IA treatment. Further comprehensive surveillance for antifungal susceptibility, as well as intensive study on the mechanism of azole resistance in A. flavus causing IA, would be required to fully understand this mechanism.

Family history of von Hippel-Lindau disease was uncommon in Chinese patients: suggesting the higher frequency of de novo mutations in VHL gene in these patients.

Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome caused by germline mutations in VHL tumor suppressor gene. It is characterized by hemangioblastoma in central nervous system and retina, renal cell carcinoma or cyst, pheochromocytoma, pancreatic cyst and tumor, endolymphatic-sac tumor, and papillary cystadenoma in epididymis and broad ligament. Here, we used PCR-direct sequencing and universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR) to detect VHL mutations in 16 patients clinically diagnosed with VHL disease. PCR-direct sequencing detected 12 germline mutations (75%, 12/16), in which a novel mutation of c.451A>T/p.Ile151Phe found in one proband had not been reported previously. UPQFM-PCR found two large deletions (12.5%, 2/16). The two remaining patients carried non-typical disease-causing mutations, including one silent mutation (c.481C>A/p.Arg161Arg) and one mutation in 3'-UTR (c.642+70C>A). Remarkably, 56.3% (9/16) probands did not have family history of VHL disease, suggesting the higher frequency of de novo mutations in Chinese patients. We also summarized Chinese VHL disease patients with VHL mutation findings published in the literature to provide information about the spectrum of VHL mutations in Chinese VHL disease patients.

Recombinant jurkat cells (HMGN2-T cells) secrete cytokines and inhibit the growth of tumor cells.

High Mobility Group Chromosomal Protein N2 (HMGN2) can recognize tumor cells and enhance the anti-tumor effect of immune cells. This study aimed to establish a lentiviral vector of recombinant HMGN2 gene, establish recombinant T cells (HMGN2-T cells), and observe their anti-tumor effects. Total RNA was isolated from peripheral blood mononuclear cells. HMGN2, cluster of differentiation (CD) 8 A, CD28, CD137, and CD3ζ genes were amplified and connected. Jurkat cells were transfected with the recombinant lentivirus vector. The viability, apoptosis, and cell cycle of HMGN2-T cells were detected using Cell Counting Kit-8 assay and flow cytometry. The co-culture was performed by adding HMGN2-T cells to tumor cells with different effect-to-target (E:T) ratios. The cytotoxic activity was measured by lactate dehydrogenase (LDH) releasing assay. The sequences of HMGN2, CD8A, CD28, CD137, and CD3ζ gene plasmids were confirmed using gene sequencing. After the lentiviral transfection for 72 h, green fluorescence cells (HMGN2-T cells) could be seen. Cell viability and apoptosis were increased in HMGN2-T cells. The cytokine levels of interleukin 2 (IL-2) and tumor necrosis factor α (TNF-α) increased in cell supernatants of HMGN2-T cells. The percentage of G0/G1 phase cells was lower, the rate of S phase cells was higher in HMGN2-T cells than control cells. The co-culture of HMGN2-T cells and tumor cells could promote the cytokines' release. The LDH level was increased with the elevation of E:T ratios. In conclusion, the HMGN2-T cells were well-established and have the effect of secreting cytokines and killing tumor cells.

Paraneoplastic Pemphigus Autoantibodies Against C-terminus of Desmoplakin Induced Acantholysis In Vitro and In Vivo.

Paraneoplastic pemphigus (PNP) is an autoimmune bullous disease associated with underlying neoplasms and characterized by antibodies against desmoglein 3 (Dsg 3) and plakins. Autoantibodies against desmoglein 3 in sera of patients with PNP have been proven to cause acantholysis in vivo in neonatal mice. As a member of the plakin family, autoantibodies against desmoplakin were detected frequently by immunoprecipitation in the sera of PNP. The recombinant C-terminus of desmoplakin was expressed and purified to adsorb the specific autoantibodies against the C-terminus of desmoplakin. In vitro dispase-dependent keratinocyte dissociation assay and in vivo IgG passive transfer into neonatal mice assay were performed, followed by the electronic microscopy examination and TUNEL assay. We found that anti-C terminus of desmoplakin autoantibodies caused blisters and acantholysis in mice skin at a dose-dependent manner. Moreover, dissociated fragments were observed after incubation with the purified IgG against desmoplakin, compared with normal human IgG (P-value =0.0207). The electronic microscopy examination showed the disconnection of keratin intermediate filaments from desmosomes. Lastly, apoptosis of keratinocytes in the TUNEL assay was all detected in the skins of neonatal mice after injection of the anti-C terminus of desmoplakin autoantibodies. Taken together, the study suggests that autoantibodies against the C-terminus of desmoplakin might be pathogenic in PNP.