The causal relationship between gut microbiota and type 2 diabetes: a two-sample Mendelian randomized study.

Background: Type 2 diabetes mellitus (T2DM) is a commonly observed metabolic anomaly globally, and as of the present time, there's no recognized solution. There is an increasing body of evidence from numerous observational studies indicating a significant correlation between gut flora and metabolic disease progression, particularly in relation to T2DM. Despite this, the direct impact of gut microbiota on T2DM isn't fully understood yet. Methods: The summary statistical figures for intestinal microbiota were sourced from the MiBioGen consortium, while the summary statistical data for T2DM were gathered from the Genome-Wide Association Studies (GWAS) database. These datasets were used to execute a two-sample Mendelian randomization (MR) investigation. The Inverse Variance Weighted (IVW), Maximum Likelihood, MR-Egger, Weighted Median, and Weighted Models strategies were employed to assess the impact of gut microbiota on T2DM. Findings were primarily obtained using the IVW technique. Techniques like MR-Egger were employed to identify the occurrence of horizontal pleiotropy among instrumental variables. Meanwhile, Cochran's Q statistical measures were utilized to assess the variability or heterogeneity within these instrumental variables. Results: The outcomes from the IVW analysis demonstrated that the genus Alistipes (OR = 0.998, 95% confidence interval: 0.996-1.000, and P = 0.038), genus Allisonella (OR = 0.998, 95% confidence interval: 0.997-0.999, P = 0.033), genus Flavonifractor (OR = 0.995, 95% confidence interval: 0.993-0.998, P = 3.78 × 10-3), and genus Haemophilus (OR = 0.995, 95% confidence interval: 0.993-0.998, P = 8.08 × 10-3) all acted as defense elements against type 2 diabetes. Family Clostridiaceae1 (OR = 1.003, 95% confidence interval: 1.001-1.005, P = 0.012), family Coriobacteriaceae (OR = 1.0025, 95% confidence interval: 1.000-1.005, P = 0.043), genus Actinomyces (OR = 1.003,95% confidence interval: 1.001-1.005, P = 4.38 × 10-3), genus Candidatus Soleaferrea (OR = 1.001,95% confidence interval: 1.000-1.002 P = 0.012) were risk factors for type 2 diabetes. False Discovery Rate correction was performed with finding that genus.Allisonella, genus.Alistipes, family Coriobacteriaceaeand T2DM no longer displayed a significant causal association. In addition, no significant heterogeneity or horizontal pleiotropy was found for instrumental variable. Conclusion: This MR study relies on genetic variation tools to confirm the causal effect of genus Flavonifractor, genus Haemophilus, family Clostridiaceae1, genus Actinomyces and genus Candidatus Soleaferrea on T2DM in the gut microbiome, providing new directions and strategies for the treatment and early screening of T2DM, which carries significant clinical relevance. To develop new biomarkers and better understand targeted prevention strategies for T2DM, further comprehensive investigations are required into the protective and detrimental mechanisms exerted by these five genera against T2DM.

[Identification and pedigree analysis for an A(W)37B subtype due to c.940A>G variant of ABO gene].

OBJECTIVE: To delineate the serological and molecular profiles of a patient with A(w)37B subtype. METHODS: The ABO bloodtypes of the proband, his wife and daughter were determined with a standard serological method. Their ABO genotypes were determined by sequence-specific primer polymerase chain reaction (PCR-SSP). All exons of the ABO gene were directly sequenced. Exons 6 and 7 of the ABO gene were further analyzed by cloning and sequencing. RESULTS: The red blood cells of the proband showed a weak B phenotype. His serum sample contained weak reactive anti-A antibody, which was defined as A(w)B blood group based on the serological characteristics. The A and B alleles were detected by blood group genotyping. Gene cloning and sequencing have identified a characteristic c.940A>G variant (ABO*AW.37) in exon 7 of the ABO gene, which resulted in substitution of Lysine by Glutamate at position 314. The proband's daughter has inherited the ABO*AW.37 allele. CONCLUSION: The c.940A>G variant in exon 7 of the ABO gene probably underlay the decreased activity of GTA transferase and resulted in the Aw37 phenotype.

[Study of a Chinese pedigree carrying a novel variant of α-1, 3-N-acetyl galactosaminyl transferase gene].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree with a novel ABO subtype. METHODS: The proband and his family members were subjected to serological analysis, and their genotypes were determined by fluorescence PCR and direct sequencing of the coding regions of the ABO gene. Exons 6 to 7 of the ABO gene were also subjected to clone sequencing for haplotype analysis. RESULTS: The proband was determined as an AxB subtype. By fluorescence PCR, he was typed as A/B. Clone sequencing has revealed a insertional mutation c.797_798 insT in exon 7 of the ABO gene, which yielded a novel allele. Pedigree analysis confirmed that the novel ABO*A1.02 allele carried by the proband and his sister was inherited from their father. The c.797_798insT mutation has been submitted to GenBank with an accession number of MK125137. CONCLUSION: The c.797_798insT mutation of exon 7 of the ABO gene probably has led to weakened expression of A antigen.

[Whole exome sequencing and pedigree analysis for a case with an ABw03 subtype].

OBJECTIVE: To explore the molecular basis for a blood donor with an ABO subtype. METHODS: The proband and his family members were subjected to serological analysis. Their genotypes were determined by real-time PCR and sequencing of the coding regions of ABO gene. RESULTS: The proband was determined as an ABw subtype. By sequencing analysis, the proband was typed as A102/BW03. Compared with ABO*B.01, the proband was found to harbor a 721C>T variant (ABO*BW.03 allele) in exon 7 of the ABO gene, which caused substitution of Arginine at position 241 by Tryptophan resulting in a ABW phenotype. The blood type of the proband's sister was similar to that of the proband. The maternal serological pattern was B type, and the result of sequencing suggested that the genotype fit with B101/Bw03. CONCLUSION: The 721C>T in the exon 7 of the ABO glycosyltransferase gene probably underlies the Bw03 phenotype. The ABO*Bw.03 variant of the proband and his sister were inherited from their mother.

Prognostic Factors and Treatment of Spinal Astrocytomas: A Multi-institutional Cohort Analysis.

STUDY DESIGN: Retrospective cohort analysis of patients with spinal astrocytoma from multi-institutional data and the literature. OBJECTIVE: To determine the prognostic factors, treatment, and survival of patients. SUMMARY OF BACKGROUND DATA: Our current understanding of the epidemiology, prognosis, and optimal treatment of spinal astrocytoma is limited. The literature is confined to case reports or small institutional case series. METHOD: Patient demographics, tumor characteristics, treatments, and outcomes were extracted. Univariate Kaplan-Meier survival analysis was performed to identify prognostic factors followed by multivariate Cox proportional hazard analysis. Wilcoxon signed-rank test was performed on pre- and postoperational functional status as measured by McCormick score. RESULTS: Ninety-four patients from four institutions and 339 patients from the literature were included. For the multi-institutional cohort, WHO grade IV tumors had shorter progression-free survival (PFS) than those of lower grades, whereas gross total resection (GTR) (hazard ratio [HR]: 0.41, 95% confidence interval [CI]: 0.14-1.27, P = 0.124) trended toward longer PFS when compared to subtotal resection (STR). Age 18 years or older, paresthesia as a presenting symptom, and higher WHO grade were associated with shorter overall survival (OS), whereas thoracic tumor location when compared to cervical tumor location, biopsy when compared to STR, and radiotherapy (HR: 0.42, 95% CI: 0.20-0.88, P = 0.022) were associated with longer OS. For the literature cohort, GTR (HR 0.43, 95% CI: 0.24-0.77, P = 0.005) was associated with longer PFS when compared to STR, whereas higher WHO grade was associated with shorter PFS. Higher WHO grade and recurrence/progression were associated with shorter OS. Postoperative McCormick score was significantly higher than preoperative score (P < 0.001), but subgroup analysis of the change in McCormick score by extent of resection revealed no differences among groups (P = 0.551). CONCLUSION: In patients with spinal astrocytomas, GTR likely resulted in longer PFS when compared to STR. Adjuvant radiotherapy appears to be effective in improving survival outcomes for high-grade tumors. LEVEL OF EVIDENCE: 4.

Impact of "a" determinant mutations on detection of hepatitis B surface antigen (HBsAg) in HBV strains from Chinese patients with occult hepatitis B.

This study was designed to detect mutations that occur within the "a" determinant in the S gene of the hepatitis B virus (HBV) in patients with occult hepatitis B (OHB), and to analyze the influence of these mutations on expression and reactivity of the hepatitis B surface antigen (HBsAg). Twenty-three certified OHB samples were compared to 32 HBsAg positive samples from patients with chronic hepatitis B. The median HBV DNA levels in the OHB group were significantly lower than those in the control group (P < 0.0001). Mutations within the "a" determinant were analyzed by gene amplification and sequencing. This revealed mixed infections in which clones within a sample displayed either different mutations or mutations in association with clones that exhibited wild type amino acid patterns. Sequencing analysis also showed a significant difference between the proportions of amino acid mutations observed in the OHB and control groups. Seven recombinant S (rS) proteins with corresponding OHB mutations and three wild type alleles were expressed and purified in the Pichia pastoris expression system to preserve conformational attributes, and their reactivity analyzed using six commercial HBsAg assays. The OHB sera were HBsAg nonreactive while the rS proteins with corresponding OHB mutations were universally reactive. Thus, we postulate that the reduced binding affinity between mutated HBsAg and its antibody may not be as important in defining OHB as is the effect of specific mutations in the preS/S region of the genome that affect the synthesis and secretion of the S protein and/or the virion.

Prognostic Factors in Patients With Spinal Chordoma: An Integrative Analysis of 682 Patients.

BACKGROUND: The low incidence of spinal chordoma precludes a prospective study of prognostic factors with a large patient cohort. OBJECTIVE: To perform a comprehensive integrative analysis on the prognostic factors, treatment, and outcomes of patients with spinal chordoma using data from 2 institutions and the literature. METHODS: Appropriate studies were identified per search criteria. The local database was retrospectively searched to include a similar patient cohort. RESULTS: Overall, 108 studies from the literature and 30 patients from our local institution were identified, resulting in a total of 682 patients. The median age was 57 years old and 35.2% were female. The median follow-up was 46 months (range: 1-408). The median progression-free survival (PFS) and overall survival (OS) were 72 months and 115 months, respectively. Significant prognostic factors for PFS on multivariate analysis included age (pediatric vs adult, hazard ratio [HR]: 2.00-14.36), tumor location (mobile spine vs sacral spine, HR: 0.31-0.87), pathology (differentiated vs classic, HR: 2.48-10.90),and chemotherapy (HR:1.11-3.85). Significant prognostic factors for OS on multivariate analysis included age (geriatric vs adult, HR: 1.52-3.45 and pediatric vs adult, HR: 1.73-9.36), bladder or bowel dysfunction (HR: 1.27-5.43), pathology (dedifferentiated vs classic, HR: 2.38-11.09), recurrence or progression (HR: 1.72-4.48), and metastases (HR: 1.11-2.47). CONCLUSION: In patients with spinal chordoma, young age, location in sacral spine, dedifferentiated pathology, and chemotherapy were negative predictors of PFS, while young and old age, bladder or bowel dysfunction at presentation, dedifferentiated pathology, recurrence or progression, and metastases portended a worse OS.

Study of novel pure culture HBCD-1, effectively degrading Hexabromocyclododecane, isolated from an anaerobic reactor.

In this study, two pure strains, named HBCD-1 and HBCD-2, were isolated from a continuous anaerobic reactor over 300-days acclimation, which processed high capability of biodegrading Hexabromocyclododecane. Both of the two strains degraded HBCD diastereomers in different extents, especially strain HBCD-1, which interestingly degraded α-HBCD effectively. All of the degrading results were well fitted with the first-order kinetics model. By morphological observation and 16S rRNA gene sequence analysis, the strain HBCD-1 showed highest similarity with Achromobacter sp. Under the optimal culturing conditions of 30°C, pH 7 and the initial HBCD concentration of 500µg/L, the biodegradation rate of HBCD-1 reached 90% after 8days treatment. Moreover, during the biodegradation process by HBCD-1 strain, the concentration of bromide ion was lower than the theoretical value. Finally, 4 metabolites were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as well as a biodegradation pathway was proposed.

Molecular analysis of the hepatitis B virus presurface and surface gene in patients from eastern China with occult hepatitis B.

This study was designed to detect and analyze mutations that occur within the presurface and surface (pre-S/S) gene of HBV in patients with occult hepatitis B, and determine their relationship to that disorder. Among 254 HBsAg negative samples of blood collected in eastern China, 183 were positive for anti-HBc alone, 61 were positive for anti-HBe alone, and 10 samples were positive for HBeAg. Within this group, 15 samples were found to be HBV DNA positive by real-time PCR and were designated Group I. A control group of 28 HBsAg positive samples were chosen at random from patients with chronic hepatitis B and designated Group II. The HBV pre-S/S gene was amplified by PCR and subjected to sequencing analysis. Occult hepatitis B was found in 1.6% of the patients with anti-HBc alone and in 3.3% of those with anti-HBe alone. Occult hepatitis B also was found in all HBsAg negative but HBeAg positive samples. Sequencing analysis showed a significant correlation between point mutations within the "a" determinant and occult hepatitis B (P < 0.0001), and a close relationship between pre-S deletion mutations and occult hepatitis B (P = 0.06). There were unique amino acid mutations at the G145 position other than G145R. The HBV DNA levels in patients with occult hepatitis B were significantly lower than those found in the control group. The "a" determinant mutations and pre-S deletions may play important roles in occult hepatitis B by affecting the expression, synthesis and secretion of the S protein and by impeding viral release and replication.

[Occult HBV infection in patients with anti-HBc positive alone].

OBJECTIVE: This study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection. METHODS: Sera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive. RESULTS: DNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples. CONCLUSIONS: Occult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.

PreS deletion mutations of hepatitis B virus in chronically infected patients with simultaneous seropositivity for hepatitis-B surface antigen and anti-HBS antibodies.

Hepatitis B surface antigen (HBsAg) and anti-HBs antibodies (anti-HBs) may coexist in certain chronic hepatitis B (CHB) patients. This study was designed to further explore the relationship between this coexistence and hepatitis B Virus (HBV) preS deletions. Sera of 28 patients carrying both HBsAg and anti-HBs (Group I) and those of another 28 HBsAg positive but anti-HBs negative patients (Group II) were collected from CHB patients. Direct sequencing of polymerase chain reaction products or sequencing of clones was applied to both groups to determine sequences of HBV preS and S genes. Genotyping of the S gene indicated that all sampled HBVs were either Genosubtype Ba or Genosubtype Ce. Seven samples in Group I harbored HBV preS deletion mutations. Three of the seven samples showed large deletion mutations in 3' terminus of preS1 and co-existence of the mutant type and the full-length wild type, and the remaining four samples showed deletion mutations in 5' terminus of preS2. All mutant strains were found to be genosubtype Ce. Only two samples in Group I showed G145R/A mutation. Only one sample in Group II contained preS deletion mutation. It is therefore concluded that HBV preS deletion mutations are likely to be related to the coexistence of HBsAg and anti-HBs in CHB patients (P-value = 0.024). Some immune reactions may select for the preS deletion in CHB patients with anti-HBs, the possible marker for immune selection.

[A new method of processing quantitative PCR data].

Today standard PCR can't satisfy the need of biotechnique development and clinical research any more. After numerous dynamic research, PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700. But the error of this technique is too great to satisfy the need of biotechnique development and clinical research. A better quantitative PCR technique is needed. The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system. This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions, and can reflect the accumulating rule of PCR product molecule accurately. Accurate quantitative PCR analysis can be made use this function relation. Accumulated PCR product quantity can be obtained from initial template number. Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used. For an example, when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1,000,000, the quantitative result accuracy will be more than 99%. The difference of result error is distinct using same condition,same instrument but different analysis method. Moreover,if the PCR quantitative analysis system is used to process data, it will get result 80 times of accuracy than using CT method.