Inhibition of endogenous hydrogen sulfide production suppresses the growth of nasopharyngeal carcinoma cells.

Hydrogen sulfide (H2 S) has been widely recognized as one of gasotransmitters. Endogenous H2 S plays a crucial role in the progression of cancer. However, the effect of endogenous H2 S on the development of nasopharyngeal carcinoma (NPC) is still unknown. In this study, aminooxyacetic acid (AOAA, an inhibitor of cystathionine-ß-synthase), dl-propargylglycine (PAG, an inhibitor of cystathionine-γ-lyase), and l-aspartic acid (l-Asp, an inhibitor of 3-mercaptopyruvate sulfurtransferase) were adopted to detect the role of endogenous H2 S in NPC growth. The results indicated that the combine (PAG + AOAA + l-Asp) group had higher inhibitory effect on the growth of NPC cells than the PAG, AOAA, and l-Asp groups. There were similar trends in the levels of apoptosis and reactive oxygen species (ROS). In addition, the combine group exhibited lower levels of phospho (p)-extracellular signal-regulated protein kinase but higher expressions of p-p38 and p-c-Jun N-terminal kinase than those in the AOAA, PAG, and l-Asp groups. Furthermore, the combine group exerted more potent inhibitory effect on NPC xenograft tumor growth without obvious toxicity. In summary, suppression of endogenous H2 S generation could dramatically inhibit NPC growth via the ROS/mitogen-activated protein kinase pathway. Endogenous H2 S may be a novel therapeutic target in human NPC cells. Effective inhibitors for H2 S-producing enzymes could be designed and developed for NPC treatment.

Infant-Type Hemispheric Glioma in a Chinese Girl: A Newly Defined Entity.

BACKGROUND: Infant-type hemispheric glioma is a newly defined entity in the updated 2021 WHO classification of tumors of the central nervous system. This lesion occurs in the cerebral hemispheres of newborns and infants and harbors molecular alterations in the NTRK family, ALK, ROS, or MET. Case report: A four-month-old female infant presented with a large space occupying lesion of the left cerebral hemisphere, whose histological manifestation was high-grade hemispheric infantile glioma. Tumor expressed panTRK, indicative of rearranged NTRK1, which was validated by next generation sequencing (NGS) as TPM3-NTRK1 fusion. There was homozygous deletion of CDKN2A/B, and there were ROS1, TLX3, FAT1, ABL1, MSH2, and PALB2 mutations. Conclusion: The additional genetic alterations in this case may expand the genotypic spectrum of this distinct cohort.

Synchronous Hepatoblastoma and Neuroblastoma in Two Chinese Infants.

Background: Commonly, pediatric solid tumors occur independently. Only two patients with synchronous hepatoblastoma (HBL) and neuroblastoma (NBL) have been reported. Case reports: Two Chinese infants presented with abdominal mass at 10 and 8 months. Computed tomography (CT) scans in both revealed hepatic masses with additional mediastinal or adrenal masses. Pathology confirmed synchronous HBLs in the liver and NBLs in the mediastinum and adrenal. Next generation sequencing (NGS) found no remarkable germline mutations. Both patients received gross total resections with chemotherapy before or after surgery. They were followed up for 36 and 8 months, and recovered well. Conclusion: These two cases of synchronous HBL and NBL tumors lacked significant genetic alterations.

Impact of the factors shaping gut microbiota on obesity.

Obesity is considered as a risk factor for chronic health diseases such as heart diseases, cancer and diabetes 2. Reduced physical activities, lifestyle, poor nutritional diet and genetics are among the risk factors associated with the development of obesity. In recent years, several studies have explored the link between the gut microbiome and the progression of diseases including obesity, with the shift in microbiome abundance and composition being the main focus. The alteration of gut microbiome composition affects both nutrients metabolism and specific gene expressions, thereby disturbing body physiology. Specifically, the abundance of fibre-metabolizing microbes is associated with weight loss and that of protein and fat-metabolizing bacteria with weight gain. Various internal and external factors such as genetics, maternal obesity, mode of delivery, breastfeeding, nutrition, antibiotic use and the chemical compounds present in the environment are known to interfere with the richness of the gut microbiota (GM), thus influencing weight gain/loss and ultimately the development of obesity. However, the effectiveness of each factor in potentiating the shift in microbes' abundance to result in significant changes that can lead to obesity is not yet clear. In this review, we will highlight the factors involved in shaping GM, their influence on obesity and possible interventions. Understanding the influence of these factors on the diversity of the GM and how to improve their effectiveness on disease conditions could be keys in the treatment of metabolic diseases.

The Potential of Hydrogen Sulfide Donors in Treating Cardiovascular Diseases.

Hydrogen sulfide (H2S) has long been considered as a toxic gas, but as research progressed, the idea has been updated and it has now been shown to have potent protective effects at reasonable concentrations. H2S is an endogenous gas signaling molecule in mammals and is produced by specific enzymes in different cell types. An increasing number of studies indicate that H2S plays an important role in cardiovascular homeostasis, and in most cases, H2S has been reported to be downregulated in cardiovascular diseases (CVDs). Similarly, in preclinical studies, H2S has been shown to prevent CVDs and improve heart function after heart failure. Recently, many H2S donors have been synthesized and tested in cellular and animal models. Moreover, numerous molecular mechanisms have been proposed to demonstrate the effects of these donors. In this review, we will provide an update on the role of H2S in cardiovascular activities and its involvement in pathological states, with a special focus on the roles of exogenous H2S in cardiac protection.

Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes.

BACKGROUND: Lipid expression is increased in the atrial myocytes of mitral regurgitation (MR) patients. This study aimed to investigate key regulatory genes and mechanisms of atrial lipotoxic myopathy in MR. METHODS: The HL-1 atrial myocytes were subjected to uniaxial cyclic stretching for eight hours. Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism were analyzed by PCR assay (168 genes). RESULTS: The stretched myocytes had significantly larger cell size and higher lipid expression than non-stretched myocytes (all p < 0.001). Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism in the myocytes were analyzed by PCR assay (168 genes). In comparison with their counterparts in non-stretched myocytes, seven genes in stretched monocytes (Idi1, Olr1, Nr1h4, Fabp2, Prkag3, Slc27a5, Fabp6) revealed differential upregulation with an altered fold change >1.5. Nine genes in stretched monocytes (Apoa4, Hmgcs2, Apol8, Srebf1, Acsm4, Fabp1, Acox2, Acsl6, Gk) revealed differential downregulation with an altered fold change <0.67. Canonical pathway analysis, using Ingenuity Pathway Analysis software, revealed that the only genes in the "superpathway of cholesterol biosynthesis" were Idi1 (upregulated) and Hmgcs2 (downregulated). The fraction of stretched myocytes expressing Nile red was significantly decreased by RNA interference of Idi1 (p < 0.05) and was significantly decreased by plasmid transfection of Hmgcs2 (p = 0.004). CONCLUSIONS: The Idi1 and Hmgcs2 genes have regulatory roles in atrial lipotoxic myopathy associated with atrial enlargement.

Synthetic Porcine Hepcidin Exhibits Different Roles in Escherichia coli and Salmonella Infections.

Hepcidin, an antimicrobial peptide, was discovered to integrate diverse signals from iron status and an infection threat and orchestrate a series of host-protective responses. Several studies have investigated the antimicrobial role of hepcidin, but the results have been controversial. Here, we aimed to examine the role of hepcidin in bacterial adherence and invasion in vitro We found that porcine hepcidin could decrease the amount of the extracellular pathogen enterotoxigenic Escherichia coli (ETEC) K88 that adhered to cells because it caused the aggregation of the bacteria. However, addition of hepcidin to macrophages infected with the intracellular pathogen Salmonella enterica serovar Typhimurium enhanced the intracellular growth of the pathogen through the degradation of ferroportin, an iron export protein, and then the sequestration of intracellular iron. Intracellular iron was unavailable by use of the iron chelator deferiprone (DFO), which reduced intracellular bacterial growth. These results demonstrate that hepcidin exhibits different functions in extracellular and intracellular bacterial infections, which suggests that different defense strategies should be taken to prevent bacterial infection.

Iron Reduces M1 Macrophage Polarization in RAW264.7 Macrophages Associated with Inhibition of STAT1.

Iron metabolism in inflammation has been mostly characterized in macrophages exposed to pathogens or inflammatory conditions. The aim of this study is to investigate the cross-regulatory interactions between M1 macrophage polarization and iron metabolism. Firstly, we characterized the transcription of genes related to iron homeostasis in M1 RAW264.7 macrophages stimulated by IFN-γ. The molecular signature of M1 macrophages showed high levels of iron storage (ferritin), a low level of iron export (ferroportin), and changes of iron regulators (hepcidin and transferrin receptors), which favour iron sequestration in the reticuloendothelial system and are benefit for inflammatory disorders. Then, we evaluated the effect of iron on M1 macrophage polarization. Iron significantly reduced mRNA levels of IL-6, IL-1ß, TNF-α, and iNOS produced by IFN-γ-polarized M1 macrophages. Immunofluorescence analysis showed that iron also reduced iNOS production. However, iron did not compromise but enhanced the ability of M1-polarized macrophages to phagocytose FITC-dextran. Moreover, we demonstrated that STAT1 inhibition was required for reduction of iNOS and M1-related cytokines production by the present of iron. Together, these findings indicated that iron decreased polarization of M1 macrophages and inhibited the production of the proinflammatory cytokines. The results expanded our knowledge about the role of iron in macrophage polarization.

Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology.

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.

Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.

Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human ß-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human ß-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii.

Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28ΔN), C-terminal (rVP28ΔC) and middle (rVP28ΔM). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1-27) and middle region (residues 35-95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV.

Expanding the phenotype and metabolic basis of ATP6AP2-congenital disorder of glycosylation in a Chinese patient with a novel variant c.185G>A (p.Gly62Glu).

Background: A rare X-linked hereditary condition known as ATP6AP2-congenital disorder of glycosylation (ATP6AP2-CDG) is caused by pathogenic variants in ATP6AP2, resulting in autophagic misregulation with reduced siganling of mammalian target of rapamycin (mTOR) that clinically presents with aberrant protein glycosylation, hepatosteatosis, immunodeficiency, cutis laxa, and psychomotor dysfunction. To date, only two missense mutations have been reported in three patients from two unrelated families. Methods: In order to extend the profiles of phenotype and genotype associated with ATP6AP2-CDG, we assessed the clinical history, whole exome sequencing (WES), and liver histology as well as immunohistochemistry in a Chinese patient, and performed quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and untargeted metabolomics in genetic exogenously constructed cells. Results: The 11-month-old Chinese boy presented with recurrent jaundice, cutis laxa, cirrhosis, growth retardation, coagulopathy, anemia, and cardiomegaly, and underwent liver transplantation. A novel mutation, c.185G>A (p.Gly62Glu), was identified in exon 3 of ATP6AP2. The expression of ATP6AP2 was observed to remain unchanged in the liver sample of the patient as well as in HEK293T cells harboring the p.Gly62Glu. This missense mutation was found to dysregulate autophagy and mTOR signaling. Moreover, metabolomics analysis revealed that the exogenously introduced Gly62Glu mutant resulted in the downregulation of numerous metabolites involved in lipid metabolism pathway. Conclusion: This study may enable a more detailed exploration of its precise pathogenesis and potential therapeutic interventions.

Neonatal sclerosing cholangitis with novel mutations in DCDC2 (doublecortin domain-containing protein 2) in Chinese children.

Background: Neonatal sclerosing cholangitis (NSC) is a rare and severe autosomal recessive inherited liver disease with mutations in DCDC2, commonly requiring liver transplantation (LT) for decompensated biliary cirrhosis in childhood. Methods: The information of four Chinese patients with NSC caused by mutations in DCDC2 from Children's Hospital of Fudan University were gathered. The four patients' clinicopathological and molecular features were summarized by clinical data, liver biopsy, immunohistochemical, and molecular genetic analysis. Results: All patients presented with jaundice, hepatosplenomegaly, hyperbilirubinemia and bile embolism, and high serum γ-glutamyl transferase activity (GGT). Liver biopsies revealed varying degrees of bile duct hyperplasia, portal-tract inflammation, and/or fibrosis. Whole-exome sequencing (WES) found novel heterozygous variants of c.1024-1G > T /p.? and c.544G > A /p. Gly182Arg in the DCDC2. Conclusion: This study expands the genetic spectrum of DCDC2 in NSC.

Hydrogen sulfide functions as a micro-modulator bound at the copper active site of Cu/Zn-SOD to regulate the catalytic activity of the enzyme.

The present study examines whether there is a mechanism beyond the current concept of post-translational modifications to regulate the function of a protein. A small gas molecule, hydrogen sulfide (H2S), was found to bind at active-site copper of Cu/Zn-SOD using a series of methods including radiolabeled binding assay, X-ray absorption near-edge structure (XANES), and crystallography. Such an H2S binding enhanced the electrostatic forces to guide the negatively charged substrate superoxide radicals to the catalytic copper ion, changed the geometry and energy of the frontier molecular orbitals of the active site, and subsequently facilitated the transfer of an electron from the superoxide radical to the catalytic copper ion and the breakage of the copper-His61 bridge. The physiological relevance of such an H2S effect was also examined in both in vitro and in vivo models where the cardioprotective effects of H2S were dependent on Cu/Zn-SOD.

Evidence for cell apoptosis suppressing white spot syndrome virus replication in Procambarus clarkii at high temperature.

In shrimp, higher water temperatures (~32°C) can suppress the ability of white spot syndrome virus (WSSV) to replicate and cause mortality, but the mechanisms remain unclear. To investigate whether cell apoptosis might be involved, a Tdt-mediated dUTP nick-end label (TUNEL) method was used to assess levels of chromosomal DNA fragmentation in hepatopancreas and gill cells of Procambarus clarkii crayfish infected with WSSV and maintained at either 32 ± 1°C or 24 ± 1°C. Based on relative cell numbers with yellow-green colored TUNEL-positive nuclei, the apoptotic index was elevated in WSSV-infected crayfish maintained at 32°C. In gill tissue sections examined by transmission electron microscope, cells with nuclei displaying apoptotic bodies or marginated, condensed and fragmented chromatin without concurrent cell cytoplasm damage were also more prevalent. Flow cytometry sorting of annexin-stained cells showed apoptosis to be most prevalent in granular haemocytes, and assays for caspase-3 activity showed it to be most elevated in hepatopancreas tissue. Despite these indicators of cell apoptosis but consistent with WSSV replication being restricted at elevated temperatures, no increases in transcription of the viral anti-apoptosis genes ORF390 and ORF222 were detected by RT-PCR in shrimp maintained at 32°C, possibly due to the elevated levels of cellular apoptosis.

TMEM199-Congenital Disorder of Glycosylation With Novel Phenotype and Genotype in a Chinese Boy.

Background: TMEM199-congenital disorder of glycosylation (TMEM199-CDG) is a rare autosomal recessive inherited disease characterized by chronically elevated serum transaminase, decreased serum ceruloplasmin, steatosis and/or fibrosis, TMEM199 mutation, reduced level of TMEM199 protein, and abnormal protein glycosylation. Methods: The information of a Chinese patient with TMEM199-CDG in the Children's Hospital of Fudan University was reviewed. The patient's clinical, pathological, and molecular features were obtained by clinical data study, liver biopsy, immunohistochemistry, and molecular genetic analysis. Results: A 4-year-old Chinese boy presented with hypertransaminasemia, hypercholesterolemia, elevated alkaline phosphatase, decreased serum ceruloplasmin and serum copper level, and coagulopathy since birth. To the best of our knowledge, novel findings included strabismus, cirrhosis by liver biopsy, reduced expression of TMEM199 by immunohistochemistry, and a frameshift variant of c.128delA/p.Lys43Argfs*25 in the TMEM199 gene. Conclusion: This case added to the phenotypic and genotypic spectrum of TMEM199-CDG.

A Case Series of Pediatric Intestinal Ganglioneuromatosis With Novel Phenotypic and Genotypic Profile.

Introduction: Intestinal ganglioneuromatosis (IGN) is a rare condition with enteric involvement. Herein, we report a case series of pediatric IGN with a novel phenotypic and genotypic profile. Methods: The clinical presentation, histopathology, immunochemistry, molecular features, treatment, and prognosis of 3 cases of IGN were assessed. Results: The cases involved 3 boys with an age range of 1 year and 4 months to 8 years, mimicking juvenile polyps or pseudomembranous enteritis. One patient carried a novel germline mutation in RTEL1 (c.296C > T/p.Pro99Leu) along with variants in F11 (c.1489C > T/p.Arg497Xaa), NBAS (c.1514delC/p.Pro505Hisfs*15), and FECH (c.315-48T > C/splicing), who died due to intractable inflammation. The other two patients underwent recurrence without significant signs of systemic syndrome or malignant progression. Conclusion: This case series added to the phenotypic and genotypic spectrum of pediatric IGN, which requires the accumulation of more cases and research for in-depth understanding.

Role of Hydrogen Sulfide in Oral Disease.

Oral diseases are among the most common human diseases yet less studied. These diseases affect both the physical, mental, and social health of the patients resulting in poor quality of life. They affect all ages, although severe stages are mostly observed in older individuals. Poor oral hygiene, genetics, and environmental factors contribute enormously to the development and progression of these diseases. Although there are available treatment options for these diseases, the recurrence of the diseases hinders their efficiency. Oral volatile sulfur compounds (VSCs) are highly produced in oral cavity as a result of bacteria activities. Together with bacteria components such as lipopolysaccharides, VSCs participate in the progression of oral diseases by regulating cellular activities and interfering with the immune response. Hydrogen sulfide (H2S) is a gaseous neurotransmitter primarily produced endogenously and is involved in the regulation of cellular activities. The gas is also among the VSCs produced by oral bacteria. In numerous diseases, H2S have been reported to have dual effects depending on the cell, concentration, and donor used. In oral diseases, high production and subsequent utilization of this gas have been reported. Also, this high production is associated with the progression of oral diseases. In this review, we will discuss the production of H2S in oral cavity, its interaction with cellular activities, and most importantly its role in oral diseases.

Molecular Evolution of Attachment Glycoprotein (G) and Fusion Protein (F) Genes of Respiratory Syncytial Virus ON1 and BA9 Strains in Xiamen, China.

Monitoring viral transmission and analyzing the genetic diversity of a virus are imperative to better understand its evolutionary history and the mechanism driving its evolution and spread. Especially, effective monitoring of key antigenic mutations and immune escape variants caused by these mutations has great scientific importance. Thus, to further understand the molecular evolutionary dynamics of respiratory syncytial virus (RSV) circulating in China, we analyzed nasopharyngeal swab specimens derived from hospitalized children ≤5 years old with acute respiratory tract infections (ARIs) in Xiamen during 2016 to 2019. We found that infants under 6 months of age (52.0%) were the main population with RSV infection. The prevalent pattern "BBAA" of RSV was observed during the epidemic seasons. RSV ON1 and BA9 genotypes were the dominant circulating strains in Xiamen. Interestingly, we observed four Xiamen-specific amino acid substitution combinations in the G protein and several amino acid mutations primarily occurring at antigenic sites Ø and V in the F protein. Our analyses suggest that introduction of new viruses and local evolution are shaping the diversification of RSV strains in Xiamen. This study provides new insights on the evolution and spread of the ON1 and BA9 genotypes at local and global scales. IMPORTANCE Monitoring the amino acid diversity of the RSV G and F genes helps us to find the novel genotypes, key antigenic mutations affecting antigenicity, or neutralizing antibody-resistant variants produced by natural evolution. In this study, we analyzed the molecular evolution of G and F genes from RSV strains circulating in Xiamen, China. These data provide new insights on local and global transmission and could inform the development of control measures for RSV infections.