One-pot hydrothermal synthesis of silicon, nitrogen co-doped carbon dots for enhancing enzyme activity of acid phosphatase (ACP) to dopamine and for cell imaging.

Developing water-soluble nanomaterials with high photoluminescence emission and high yield for biological analysis and imaging is urgently needed. Herein, water-soluble blue emitting silicon and nitrogen co-doped carbon dots (abbreviated as Si-CDs) of a high photoluminescence quantum yield of 80 % were effectively prepared with high yield rate (59.1 %) via one-step hydrothermal treatment of N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) and trans-aconitic acid. Furthermore, the Si-CDs demonstrate environmental robustness, photo-stability and biocompatibility. Given the importance of the potentially abnormal levels of acid phosphatase (ACP) in cancer diagnosis, developing a reliable and sensitive ACP measurement method is of significance for clinical research. The Si-CDs unexpectedly promote the catalytic oxidation of ACP on dopamine (DA) to polydopamine under acidic conditions through the produced reactive oxygen species (ROS). Correspondingly, a fluorescence response strategy using Si-CDs as the dual functions of probes and promoting enzyme activity of ACP on catalyzing DA was constructed to sensitively determine ACP. The quantitative analysis of ACP displayed a linear range of 0.1-60 U/L with a detection limit of 0.056 U/L. The accurate detection of ACP was successfully achieved in human serum through recovery tests. As a satisfactory fluorescent probe, Si-CDs were successfully applied to fluorescent imaging of A549 cells in cytoplasmic with long-term and safe staining. The Si-CDs have the dual properties of outstanding fluorescent probes and auxiliary oxidase activity, indicating their great potential in multifunctional applications.

Xijiao Dihuang Decoction Protects Against Murine Sepsis-Induced Cardiac Inflammation and Apoptosis via Suppressing TLR4/NF-κB and Activating PI3K/AKT Pathway.

Background: Xijiao Dihuang decoction (XJDHT), a traditional Chinese medicine, is widely used to treat patients with sepsis. However, the mechanisms underlying the effects of XJDHT on cardiac dysfunction have yet to be fully elucidated. The present study evaluated the potential utility of XJDHT in protecting against sepsis-induced cardiac dysfunction and myocardial injury. Methods: The mice were randomly divided into 3 groups and administered Lipopolysaccharide (LPS,10 mg/kg) or equivalent saline solution (control) and treated with XJDHT (10 g/kg/day) or saline by gavage for 72 hours. XJDHT was dissolved in 0.9% sodium chloride and administered at 200 µL per mouse. Transthoracic echocardiography, RNA-seq, TUNEL assays and hematoxylin and eosin (H&E) staining of cardiac tissues were performed. Results: Treatment with XJDHT significantly enhanced myocardial function and attenuated pathological change, infiltration of inflammatory cells, levels of TNF-α, IL-1ß and expression of TLR4 and NF-κB in mice with sepsis. RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analyses identified 531 differentially expressed genes and multiple enriched signaling pathways including the PI3K/AKT pathway. Further, XJDHT attenuated cardiac apoptosis and decreased Bax protein expression while increasing protein levels of Bcl-2, PI3K, and p-AKT in cardiac tissues of mice with sepsis. Conclusion: In summary, XJDHT improves cardiac function in a murine model of sepsis by attenuating cardiac inflammation and apoptosis via suppressing the TLR4/NF-κB pathway and activating the PI3K/AKT pathway.

Two Novel Frameshift Mutations in the GLI3 Gene Underlie Non-Syndromic Polydactyly in Chinese Families.

Objective: Polydactyly is characterized by multiple distinct heterogeneous phenotypes, the etiologies of which involve several genes. This study aimed to explore the genetic defects and further clarify the molecular mechanism of polydactyly in several Chinese families. Methods: Three families with diverse phenotypes of non-syndromic polydactyly were analyzed: two were cases of familial disease, whereas one was sporadic. PCR and Sanger sequencing were used to screen for pathogenic mutations in two known disease-associated genes, GLI3 and HOXD13, while bioinformatic analyses predicted the pathogenicity of the identified variants. Reverse transcription PCR was used to analyze the splicing effect of an intronic variant. Results: Two novel heterozygous frameshift mutations (c.4478delG/p.S1493Tfs*18; c.846_c.847insC/p.R283Qfs*21) were identified in the GLI3 gene from two of the pedigrees. Both c.4478delG and c.846_c.847insC were later confirmed in affected and unaffected members and normal controls, to truncate and disrupt the integrity of the GLI3 protein, reduce its level of expression, and disrupt its biological function through nonsense-mediated mRNA decay (NMD). In addition, a deep intron mutation (c.125-47 C>A) was detected in the GLI3 gene from the sporadic case, however, both bioinformatics analysis (HSF, splice AI, and CBS) and RT-PCR indicated that the variant c.125-47 C>A had minimal if any impact on splicing of the GLI3 gene. Conclusion: Two newly identified heterozygous frameshift mutations in the GLI3 gene were detected in two families with non-syndromic polydactyly, further extending the mutational spectrum of the GLI3 gene in non-syndromic polydactyly. Moreover, our study further expanded the phenotypic spectrum of non-syndromic polydactyly.

A Novel Intronic Splicing Mutation in the EXT2 Gene of a Chinese Family with Multiple Osteochondroma.

Background: Multiple osteochondroma (MO), an autosomal dominant genetic disease, is caused by heterozygous mutations in the EXT1 and EXT2 genes. Approximately 80% of pathogenic mutations are nonsense/missense mutations, small indels, and splicing mutations. Splicing mutations, particularly at the 3' and 5' splice sites, disrupt normal mRNA processing and cause exon skipping or aberrant splicing, ultimately resulting in protein truncation and loss of function. Methods: Polymerase chain reaction (PCR) and Sanger sequencing were applied to detect subtle mutations in a Chinese family with MO, the pathogenicity of a splicing variant was predicted by bioinformatics and further verified using a minigene splicing assay. Results: A novel and heterozygous splicing mutation, c.626 + 2_626 + 5delTAGG, was identified in the EXT2 gene of the proband and the father by PCR and Sanger sequencing, whereas the unaffected mother and brother had wild-type alleles at the same site. Bioinformatics predicted that the 5' splicing site of exon 3 in the EXT2 gene was destroyed due to this mutation. A hybrid minigene splicing assay (HMSA) indicated that the mutation disturbed the normal splicing of the EXT2 gene mRNA and led to a deletion of 79 bp at the 5' end of exon 3, which resulted in aberrant splicing of exon 3 and introduced an earlier stop codon in the EXT2 gene. Conclusion: A novel splicing mutation was identified that produced the MO phenotype through aberrant splicing in a Chinese family. This observation, expands our knowledge of the spectrum of molecular pathogenic mechanisms leading to aberrant mRNA splicing.

[Analysis of genetic variants in a pedigree affected with hereditary multiple osteochondroma].

OBJECTIVE: To explore the genetic basis for a pedigree affected with hereditary multiple osteochondroma (HMO). METHODS: Peripheral blood samples were collected from the proband and members of his pedigree with informed consent. Following extraction of genomic DNA, all coding exons and flanking intronic sequences (-10 bp) of the EXT1 and EXT2 genes were subjected to targeted capture and next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing. RESULTS: A heterozygous nonsense variant (c.1911C>A) was found in exon 10 of the EXT1 gene in the proband and his affected father but not in a healthy sister and normal controls. The variant was classified as a pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (PVS1+PM2+PP1). Bioinformatic analysis predicted that the c.1911C>A variant may be disease-causing via nonsense-mediated mRNA decay and anomalous splicing. CONCLUSION: The c.1911C>A variant probably underlay the disease in this pedigree. Discovery of this variant enriched the variant spectrum of HMO.

A Nonsense Mutation in HOXD13 Gene from A Chinese Family with Non-Syndromic Synpolydactyly.

Synpolydactyly is a congenital limb malformation characterized by incomplete separation and duplication in fingers and/or toes, which is mainly caused by mutations in the homeobox D13 (HOXD13) gene. Here, a four-generation family with variant phenotypes of synpolydactyly was analyzed, in which the proband had bilateral preaxial synpolydactyly in toes with normal fingers, the father had clinodactyly in the fifth fingers, while the mother and grandma was normal. Trio whole-exome sequencing (trio-WES) is a high throughput sequencing targeting whole genome for detecting exonic variants from the proband and the parents in a family. Through trio-WES followed by Sanger sequencing and enzyme digestion, a heterozygous nonsense mutation (c.859 C>T/p.Gln287Ter) was newly identified in the homeodomain of the HOXD13 gene from the proband and the affected father, but not from the unaffected mother, the unaffected grandma, or the normal control. Mutation Taster, Human Splicing Finder and EX-SKIP predicted that the heterozygous mutation (c.859 C>T) would result in haploinsufficiency of HOXD13 protein through nonsense-mediated mRNA decay (NMD) and splicing abnormality, which might disrupt the integrity and reduce the expression level of the HOXD13 protein (loss-of-function). In short, a heterozygous nonsense mutation in the HOXD13 gene was newly identified in two patients with mild phenotypes of synpolydactyly, which extends the mutation spectrum in HOXD13 gene. Moreover, the findings we presented here deepen our understanding of the clinical consequences of non-syndromic synpolydactyly and may provide a new clue for further studies of the pathogenic mechanism of the mutation that causes aberrant splicing of HOXD13 gene.

A novel splice mutation induces exon skipping of the EXT1 gene in patients with hereditary multiple exostoses.

The molecular mechanism of hereditary multiple exostoses (HME) remains ambiguous and a limited number of studies have investigated the pathogenic mechanism of mutations in patients with HME. In the present study, a novel heterozygous splice mutation (c.1284+2del) in exostosin glycosyltransferase 1 (EXT1) gene was identified in a three­generation family with HME. Bioinformatics and TA clone­sequencing indicated that the splice site mutation would result in exon 4 skipping. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) revealed that the expression levels of wild­type EXT1/EXT2 mRNA in patients with HME were significantly decreased, compared with normal control participants (P<0.05). Abnormal EXT1 transcript lacking exon 4 (EXT1­DEL) and full­length EXT1 mRNA (EXT1­FL) were overexpressed in 293­T cells and Cos­7 cells using lentivirus infection. RT­qPCR demonstrated that the expression level of EXT1­DEL was significantly increased, compared with EXT1­FL (17.032 vs. 6.309, respectively; P<0.05). The protein encoded by EXT1­DEL was detected by western blot analysis, and the level was increased, compared with EXT1­FL protein expression. Immunofluorescence indicated that the protein encoded by EXT1­DEL was located in the cytoplasm of Cos­7 cells, which was consistent with the localization of the EXT1­FL protein. In conclusion, the present study identified a novel splice mutation that causes exon 4 skipping during mRNA splicing and causes reduced expression of EXT1/EXT2. The mutation in EXT1­DEL generated a unique peptide that is located in the cytoplasm in vitro, and it expands the mutation spectrum and provides molecular genetic evidence for a novel pathogenic mechanism of HME.

Targeted Next-Generation Sequencing Newly Identifies Mutations in Exostosin-1 and Exostosin-2 Genes of Patients with Multiple Osteochondromas.

Multiple osteochondromas (MO) is one of the most common benign bone tumors in humans with an autosomal dominant hereditary mode. MO is a genetic heterogeneity disease with variable number and size of osteochondromas, as well as changeable number and location of diseased bones. Mutations in Exostosin-1/Exostosin-2 (EXT1/EXT2) genes are the main molecular basis of MO. EXT1 and EXT2 genes encode exostosin 1 and exostosin 2, respectively, both of which are transmembrane glycosyltransferases that elongate the chains of heparin sulfate (HS) at HS proteoglycans (HSPGs). HSPGs are considered to be involved in regulating the proliferation and differentiation of chondrocytes. Owing to large size of EXT1/EXT2 genes and lack of mutation hotspots, molecular diagnosis of MO is challenging. Here, we applied targeted next-generation sequencing (t-NGS) in mutation screening of EXT1/EXT2 genes for 10 MO patients. The results were compared and validated with Sanger sequencing. Overall, nine mutations identified by t-NGS were confirmed with Sanger sequencing, excluding two variants of false positive, suggesting the reliability of mutation screening by t-NGS. The nine mutations identified by t-NGS include two missense mutations (EXT1: c.1088G>A and c.2120C>T), one splicing mutation (EXT2: c.744-1G>T), and six nonsense mutations (EXT1: c.351C>G, c.1121G>A, and c.1843_1846dup; EXT2: c.67C>T, c.561delG, and c.575T>A). In summary, our paper provides the primary data of the application of t-NGS in MO molecular diagnosis, including six newly identified mutations (EXT1: c.1843_1846dup, c.1088G>A, c.351C>G, and c.2120C>T and EXT2: c.744-1G>T and c.575T>A), which further enrich the mutation database of MO from the Chinese population.

[Analysis of a multiple osteochondroma case caused by novel splice mutation (c.1164+1G to A) of EXT1 gene].

OBJECTIVE: To detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism. METHODS: The coding regions and their flanking sequences of the EXT1/EXT2 genes were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified by excluding possible single nucleotide polymorphisms and bioinformatics analysis. Transcripts of the EXT1 gene in the proband were analyzed by TA clone-sequencing, with its abundance compared with that of healthy controls. RESULTS: DNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G to A) at the 5'splice sites of intron 3 of the EXT1 gene. The same mutation was not found in the healthy controls. Bioinformatics analysis indicated that the mutation is highly conserved and can lead to skipping of exon 3 or aberrant splicing. TA clone-sequencing indicated that the numbers of transcripts with skipping of exon 3 has significantly increased in the proband (< 0.05) compared with the controls. CONCLUSION: The c.1164+1G to A mutation has resulted in skipping of exon 3 in a proportion of EXT1 gene transcripts. As the result, the number of transcripts with tumor suppressing function is relatively reduced and has ultimately led to the tumors.

[The effect of ulinastatin on gene expression of brain tissue in septic rat].

OBJECTIVE: To detect the differences in gene expression of brain tissue in septic rats with ulinastatin (UTI) preconditioning with DNA microarray. METHODS: Forty-five male Wistar rats were equally divided into control group, sepsis group, and UTI group by means of random number table. In UTI group the rats were treated with intramuscular injection of UTI (100 kU/kg) 1 hour before cecal ligation and puncture (CLP). In sepsis group and control group intramuscular balanced solution (5 ml/kg) instead of UTI was given. Septic rat model was reproduced by CLP. The control group underwent a simulated operation without CLP. Gene expression profile was studied by using RatRef-12 rat gene expression profile microarray to detect the changes in gene expression pattern of rat brain tissue after CLP. Then related computer software was used to screen and analyze the relationship between the sepsis/UTI group and control group. Finally, the difference between the sepsis group and UTI group was analyzed. RESULTS: In 22 523 genes, 55 differential genes were found between sepsis group and control group, accounting for 0.244%. Among them 47 genes showed down-regulation, with 23 known functional genes; 8 genes showed up-regulation, with 6 known functional genes. Eighty-two differential genes were found between UTI group and control group, accounting for 0.364%. Among them 66 genes showed down-regulation, with 39 known functional genes; 16 genes showed up-regulation, with 8 known functional genes. When sepsis group and UTI group compared with control group, 19 genes showed the same degree of regulation,with 18 genes (Adora2a, Avp, Cart, Gng7, Myh7, Oxt, Pde1b, Pdyn, Prkcd, Prkch, Rgs9, Rxrg, Six3, Slc17a6, Slco1a5, Sostdc1, Tac1, Ttr) showed down-regulation and 1 gene (S100a8) showed up-regulation. CONCLUSION: UTI preconditioning can partly adjust abnormal expression of genes in the brain tissue of rat with sepsis in the presence of excessive inflammation and immune suppression. UTI has some degree of protective effect on brain at the genetic level. Meanwhile, there is certain self regulation in the body during sepsis.

[Effect of ulinastatin preconditioning on gene expression profile of heart tissue in a rat sepsis model].

OBJECTIVE: To observe the regulatory effect of ulinastatin (UTI) preconditioning on gene expression of heart tissue in septic rats by DNA microarrays. METHODS: Forty-five male Wistar rats were equally divided into control group, sepsis group and UTI group by means of random number table. Cecal ligation and puncture (CLP) was used to reproduce rat sepsis model. The control group only experienced a simulated operation without CLP. In UTI group the rats were treated with intramuscular injection of UTI 100 kU/kg 1 hour before CLP. In sepsis group and control group balanced electrolyte solution (5 ml/kg) was given. Gene expression spectrum was studied with RatRef-12 rat gene expression profile microarray to detect the changes in gene expression pattern of rat heart tissue after CLP. Genes with fluorescent signal of Cy3/Cy5 of ratio average (RA)>2.0 or RA<0.5 were identified as differential genes, and those highly correlated to sepsis and UTI groups were screened by means of related computer software to analyze their relationship. RESULTS: In 22 523 genes, 418 differential genes were found in sepsis group compared with control group, accounting for 1.856%, and among them 200 genes showed up-regulation, with 84 known functional genes, and 43 of which only showed up-regulation in sepsis group, but normal in UTI group. Two hundred and eighteen genes showed down-regulation, with 74 known functional genes, 37 of which only showed down-regulation in sepsis group, but normal in UTI group. Two hundred and two differential genes were found in UTI group compared with control group, accounting for 0.897%, and among them 111 genes showed up-regulation, with 57 known functional genes, and 17 of which only showed up-regulation in UTI group, but normal in sepsis group. Ninety-one genes showed down-regulation, with 48 known functional genes, 18 of which only showed down-regulation in UTI group, but normal in sepsis group. Compared with the control group, in both UTI group and sepsis group, 41 of known functional genes showed up-regulation, and 37 showed down-regulation. CONCLUSION: UTI preconditioning can ameliorate the damage to heart tissue in rat sepsis model, thus it has a protective effect on heart, and its mechanism may be attributable to regulatory effect of UTI on expression of stress reaction, cell signal transduction, energy metabolism, immune reaction and other related genes.