Targeted inhibition of gut bacterial ß-glucuronidases by octyl gallate alleviates mycophenolate mofetil-induced gastrointestinal toxicity.

Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20 mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20 mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.

YAP1 as a Novel Negative Biomarker of Immune Checkpoint Inhibitors for EGFR-Mutant Non-Small-Cell Lung Cancer.

Background: Immune checkpoint inhibitors (ICIs) have become a standard care in non-small-cell lung cancer (NSCLC). However, its application to epidermal growth factor receptor (EGFR)-mutant NSCLC patients is confronted with drug resistance. This study aimed to clarify the potential role of Yes1-associated transcriptional regulator (YAP1) in ICIs treatment for EGFR-mutant NSCLC population. Methods: All the clinical data of NSCLC were downloaded from Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) for GSE11969 and GSE72094. Based on YAP1 expression, all the NSCLC patients including the EGFR-mutant and EGFR-wildtype (WT) patients were divided into two groups, YAP1_High and YAP1_Low. Using cBioPortal, genetic alterations were analyzed for identification of immunogenicity in EGFR-mutant NSCLC. MR analysis was used to analyze the hub gene of EGFR. The infiltration of immune cells and the expression of the identified tumor-associated antigens were identified with TIMER. By graph learning-based dimensionality reduction analysis, the immune landscape was visualized. Moreover, survival analysis was performed to verify the predictive value of YAP1 in ICIs treatment for EGFR-mutant NSCLC population using Ren's research data (NCT03513666). Results: YAP1 was a poor prognostic factor of EGFR-mutant NSCLC population rather than lung adenocarcinoma (LUAD) patients. MR analysis revealed that the EGFR gene regulated YAP1 expression. YAP1 was identified as a hub gene closely associated with immunosuppressive microenvironment and poor prognosis in EGFR-mutant NSCLC population in TCGA LUAD. Tumors with YAP1_High showed an immune-"cold" and immunosuppressive phenotype, whereas those with YAP1_Low demonstrated an immune-"hot" and immunoactive phenotype. More importantly, it was verified that YAP1_High subpopulation had a significantly shorter progression-free survival (PFS) and overall survival (OS) after ICIs treatment in EGFR-mutant NSCLC patients in the clinical trial. Conclusions: YAP1 mediates immunosuppressive microenvironment and poor prognosis in EGFR-mutant NSCLC population. YAP1 is a novel negative biomarker of ICIs treatment in EGFR-mutant NSCLC population. Clinical Trials. This trial is registered with NCT03513666.

[Olecranon osteotomy approach for Dubberley type â ¢ coronal fractures of the distal humerus].

OBJECTIVE: To retrospectively analyze the clinical efficacy of olecranon osteotomy approach in the treatment of Dubberley type Ⅲ coronal fractures of the distal humerus and summarize the treatment experience. METHODS: From January 2016 to June 2020, 17 patients (5 males and 12 females) with Dubberley type Ⅲ coronal fractures of the distal humerus were treated by olecranon osteotomy approach. The age ranged from 37 to78 years old with an average of (58.5±12.9) years old. According to Dubberley classification, there were 5 cases of type Ⅲ A and 12 cases of type Ⅲ B. The curative effect was evaluated using the Borberg-Morrey elbow function score. The flexion, extension and rotation range of motion of the elbow joint, complications and postoperative imaging evaluation were recorded. RESULTS: All the 17 patients got bony union. The follow-up time ranged from 12 to 33 months with an average of (15.6±5.6) months. There was 1 case of ischemic necrosis of capitulum humeri, 2 cases of traumatic arthritis and 1 case of heterotopic ossification, 1 case of malunion of fracture. The range of motion was (114.80±19.50) °. The Broberg-Morrey score was 85.3±8.2, excellent in 5 cases, good in 9 cases, fair in 3 cases and poor in 0 case. CONCLUSION: Through olecranon osteotomy approach, the articular surface of distal humerus could be fully exposed, and the operation is convenient. Anatomical reduction and rigid fixation of the articular surface of distal humerus are the key factors for the succesful outcome.

[Ulna osteotomy and joint capsule release and tight for old Monteggia's fracture in children].

OBJECTIVE: To explore the experience and effect of surgical treatment in old Monteggia fracture in children. METHODS: From January 2013 to December 2017, 32 cases of old Monteggia's fracture were treated including 18 males and 14 females with an average age of(5.3±1.2) years old ranging from 2 to 9 years old. No symptoms of radial nerve injury were found. The preoperative symptoms of the patients were the pain and deformity of the elbow joint, the flexion and extension and the limited forearm rotation. The X-ray showed the union of the ulna or the "arched sign", the dislocation of the radial head or the subluxation of the head. The posterior incision of the ulna ridge was performed in the operation, and the long oblique osteotomy was performed at the most obvious point of the ulna angle deformity. Then the Boyd incision was used to expose the humeral and radial joint and the upper ulnar radial joint. The scar tissue in the joint was cleaned and the radial head was repositioned. On the premise of maintaining the stability of the elbow joint, the ulna osteotomy was treated with plate and screw internal fixation. RESULTS: All 32 cases were followed up for 12 to 24 months with an average of 14.8 months, of which 1 case had incision infection. There were no pain symptoms of elbow and wrist in 32 patients after operation, 29 patients with elbow joint flexion and extension (130±5)°/0°, forearm pronation and supination 90°/(85±5)°; 2 patients with elbow flexion and extension(119°/8°, 121°/7°), forearm pronation and supination (90°/75°, 85°/60°); 1 patient with elbow flexion and extension 90°/10°, forearm pronation and supination 80°/60°. According to Mackay criteria, the result was excellent in 29 cases, good in 2 cases, medium in 1 case. CONCLUSIONS: Ulna osteotomy, elbow posterior capsular release, anterior capsule contraction is a effective method in the treatment of old Monteggia's fracture in children.

[Surgical treatment for 14 patients with old fractures of humeral lateral condyle in children].

OBJECTIVE: To study clinical effects of operation for the treatment of old fractures of the humerus lateral condyle in children. METHODS: From January 2012 to January 2014 in our department, 14 children of old humeral lateral condyle fractures were treated with operation. Ten cases were male, 4 cases were female; age from 2 to 12 years old, average 5.8 years old. The initial diagnosis was type IIfracture according to the Milch ciassification, the loss of treatment in 11 cases, conservative treatment in 3 cases of nonunion after fracture displacement. Two cases had mild cubitus valgus deformity; 10 cases had elbow disorders, and the motion range was limited from 15° to 60°; 6 cases had pain in activity. The time from injury to operation was 32 to 176 days(62 days on average) in 14 cases, the 14 cases were treated with open reduction and internal fixation. According to the Modified An-Morrey elbow function assessment criteria after surgery for curative effect. RESULTS: Fourteen cases were followed up for 1 to 3 years, average 1.8 years. No nonunion, malunion, aseptic necrosis of the epiphysis, cubitus varus or valgus occurred. Five cases had mild protrusion deformity of external condyle, 3 cases still had mild dysfunction. The time of clinical bone union was 4 to 8 weeks in X-ray films. Five cases had bony spur formation, 3 cases had signs of early closure of epiphysis; 2 cases had a increasing volume of humeral lateral condyle; and 2 cases appeared tail deformity. Modified An-Morrey score averaged(95.2±3.6) points, 13 excellent, 1 good. CONCLUSIONS: For the old fracture of humeral lateral condyle, operation can effectively restore the appearance and function of elbow joint, and the short-term curative effect is satisfactory, but the long-term effect needs further observation.

Odor fingerprinting of Listeria monocytogenes recognized by SPME-GC-MS and E-nose.

Microorganisms can produce species-specific microbial volatile organic compounds (MVOCs), or odor compounds, which can be characterized by odor fingerprinting. The objective of this study was to characterize the odor fingerprint of Listeria monocytogenes. Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and electronic nose (E-nose) were used to recognize the MVOCs of L. monocytogenes in pure culture medium. The main MVOCs of L. monocytogenes were identified by SPME-GC-MS analysis as alcohols, aldehydes, ketones, alkanes, and heterocyclics, among which the relative peak area of one compound, 3-hydroxy-2-butanone, increased along with the growth of L. monocytogenes. The odor fingerprint of L. monocytogenes at different growth stages could be clearly discriminated by E-nose. In addition, E-nose signals had a very good linear relationship with the concentration of this bacterium (R(2) = 0.9937). Our study may help to establish the analysis of the odor fingerprint of microorganisms as a potential routine method in microbiology.

A chimeric dengue virus vaccine using Japanese encephalitis virus vaccine strain SA14-14-2 as backbone is immunogenic and protective against either parental virus in mice and nonhuman primates.

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.

[Analysis of full-length gene sequence of rabies vaccine virus aG strain].

To sequence and analyze the full-length gene sequence of rabies vaccine virus aG strain. The full-length gene sequence of aG strain was amplified by RT-PCR by 8 fragments,each PCR product was cloned into vector pGEM-T respectively, sequenced and assemblied; The 5' leader sequence was sequenced with method of 5' RACE. The homology between aG and other rabies vaccine virus was analyzed by using DNAstar and Mega4. 0 software. aG strain was 11 925nt(GenBank accession number: JN234411) in length and belonged to the genotype I . The Bioinformatics revealed that the homology showed disparation form different rabies vaccine virus. the full-length gene sequence of rabies vaccine virus aG strain provided a support for perfecting the standard for quality control of virus strains for production of rabies vaccine for human use in China.

[Progress in the research of phenotype and genotype of Japanese encephalitis virus in China].

Japanese encephalitis virus(JEV)is one of the leading cause of viral encephalitis in Asia. The phenotypic and genotypic characteristics of isolated virus strains are reviewed in this paper. Studies on the biological characteristics of the isolates showed that different isolates existed apparent differences in virus plaque morphology, neuroinvasive pathogenicity in mice, protective antigenicity and hemagglutination property. In China, only genotype III JEV strains were isolated before 1977. But since 1977, both genotype I and I JEV strains were isolated and the genotype I virus, which was isolated from mosquitoes mostly, has become the dominant strain. Study on the genomic sequence indicated that there was only a few amino acid difference (< or = 43%) between the two genotype isolates. Comparison between both genotype isolates and widely used live vaccine strain SA14-14-2 revealed that there were only < or = 3% amino acid differences, most of which were the SA14-14-2 unique attenuating sites. These results indicate that the SA14-14-2 live vaccine is able to protect people against infection of the both genotype I and Ill JEV strains.

A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation.

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a -1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.

[Studies on the biological and genetic characteristics of a highly neurovirulent Japanese encephalitis virus strain SA4].

The biological and genetic characteristics of a highly neurovirulent JE virus strain SA4 were studied. Mice were inoculated intracerebrally with strain SA4 and SA14, and observed for 14 days, respectively. On different days, mice brains were harvested for titrations of the virus content in the brains. Full-length genome of SA4 was sequenced and compared with SA14 as well as other JE virus strains in the world. The results indicated that the mice inoculated by SA4 induced sickness and death more rapidly (24 hours faster) than those induced by the SA14. The virus titers in the brains of mice infected with SA4 were 0.5-1.0 lg PFU/mL higher than that infected with SA14. The sequence comparison indicated that the nucleotide and amino acid homology between SA4 and the other 21 JE strains were 84.6%-99.0% and 95.2%-99.7% respectively. Comparison with strain SA14 revealed that there were 17 amino acid differences between the two strains, of which 5 were in the E protein region. The results demonstrate that strain SA4 is a highly neurovirulent strain. The substitutions of the 17 amino acids in the SA4 strain can be the molecular basis for the biological characteristics of high neurovirulence.

[Study on the compliance and economic cost of rabies vaccination].

OBJECTIVE: To learn the compliance status regarding completing full rabies vaccination schedule, and to analyse the economic cost. METHODS: Interviewing patients who visited doctor at Emergency Department, the People's Hospital, Beijing University from June 2007 to January 2009 on vaccination compliance of Essen regimens. The economic cost of Essen regimen and Zagreb regimen were estimated using average vaccination cost, average release amount by National Institue for Control of Pharmaceutical and Biological Products. RESULTS: In total, 3440 patients were interviewed. The proportion completing full vaccine schedule was 33.3%, 77.1% and 78.0% for exposure category I, II and III respectively. The compliance was high for first 3 doses, it reduced significantly after the third dose. The individual medical costs was 694 RMB for Essen regimen, and 482.2 RMB for Zagreb regimen. The estimated annual medical costs was 9.709 billion RMB for Essen regimen, and 6.746 billion RMB for Zagreb regimen nationaly. CONCLUSIONS: The Zagreb regimen should be recommended in order to simplify the vaccine administration, to increase compliance and to reduce rabies vaccination cost.

[Analysis of full-length gene sequence of a rabies vaccine strain CTN-1 for human use in China].

CTN-1 is one of the rabies vaccine strains for human use in China, but there has been no report on the full-length gene sequence of CTN-1. In this study, the full-length gene of CTN-1 was amplified by RT-PCR, each PCR product was cloned into T vector and then sequenced, assemblied and compared with other vaccine strains as well as the wild Chinese rabies isolates. The phylogenetic tree of G gene was constructed and the genetic homology was analyzed. The results revealed that CTN-1 was 11 925nt (GenBank accession number: FJ959397)in length and belonged to the genotype I. The full-length nucleotide homologies among CTN-1 and other rabies virus strains were between 81.5%-93.4%, of which the lowest 81.5% was between CTN-1 strain and bat isolate SHBRV, and the highest 93.4% was between CTN-1 and Chinese isolate HN10. The phylogenetic analysis revealed that the majority of Chinese isolates could be grouped into the same clade with the CTN-1 strain, but aG and some vaccine strains from abroad such as Flury, PM, PV, ERA, RC-HL and a few Chinese strains were grouped in another clade. Comparsion of the G protein genes also showed that the homologies among CTN-1 and most of the Chinese isolates were higher than that of the other vaccine strains to those Chinese strains. Therefore, it suggests that the CTN-1 strain is more suitable and rational to be used for the production of rabies inactivated vaccine in China than the others.

[Comparison of viremia formation between guinea-pigs infected with wild and attenuated (SA14-14-2) Japanese encephalitis viruses].

OBJECTIVE: To study the viremia formation in guinea-pigs infected with wild type and attenuated Japanese encephalitis virus (JEV). METHODS: Guniea pigs were inoculated intraperitoneally with different wild JEV strains and the attenuated vaccine strain and its parent virulent strain. Viremia was detected on different days following virus inoculation. RESULTS: All the guinea-pigs inoculated with the wild JEV strains induced different levels of viremia (1.00-3.40 Lg pfu) on the 1st and 3rd day post inoculation. Using a virus titer of 10(4) pfu for inoculation, the animals inoculated with the SA14 parent strain induced relatively high viremia (10(2.4)-10(3.4) pfu), however no viremia coulds be detected on any tested days. CONCLUSION: The degree of viremia in guinea pigs can be used as a new method to evaluate the attenuation of JEV.

[Completed sequences analysis on the Chinese attenuated yellow fever 17D vaccine strain and the WHO standard yellow fever 17D vaccine strain].

OBJECTIVE: To compare the molecular characteristics of the Chinese attenuated yellow fever 17D vaccine strain and the WHO reference yellow fever 17D vaccine strain. METHODS: The primers were designed according to the published nucleotide sequences of YFV 17D strains in GenBank. Total RNA of was extracted by the Trizol and reverse transcripted. The each fragments of the YFV genome were amplified by PCR and sequenced subsequently. The fragments of the 5' and 3' end of the two strains were cloned into the pGEM T-easy vector and then sequenced. RESULTS: The nucleotide acid and amino acid sequences of the homology to both strains were 99% with each other. No obvious nulceotide changes were found in the sequences of the entire genome of each 17D strains. Moreover, there was no obvious changes in the E protein genes. But the E173 of YF17D Tiantan, associted with the virulence, had mutantions. And the two live attenuated yellow fever 17D vaccine strains fell to the same lineage by the phylogenetic analysis. CONCLUSION: The results indicated that the two attenuated yellow fever 17D vaccine viruses accumulates mutations at a very low frequency and the genomes were relative stable.

[Duplication of an animal model of myocardial ischemia with blood stasis syndrome in mini-swines].

OBJECTIVE: To establish an animal model of myocardial ischemia with blood stasis syndrome in mini-swines. METHODS: An animal model of myocardial ischemia was established in mini-swines by oppressing the coronary artery through the expansion of inner layer of Ameroid constrictor and the Ameroid constrictor was implanted into the distal end of the initial part of the first branch of interventricular septum of ramus descendens anterior arteriae coronariae sinistrae. Dynamic observation of behavior changes, general health status and changes of hemorheological parameters in the mini-swines were made after operation. RESULTS: The coronary angiography showed that the stenosis rate in ischemic group was more than 75% four weeks after operation. Compared with before operation and sham-operated group, there were great changes of behavior, general health status, tongue color and hemorheological parameters in ischemic group (P<0.05). CONCLUSION: The animal model of myocardial ischemia with blood stasis syndrome in mini-swines was established successfully 4 weeks after operation. The pathological process in the animal model is similar to that in the patients with chronic myocardial ischemia. So this model can be adopted in the research of myocardial ischemia with blood stasis syndrome.

[Study on the phenotypic characteristics of Japanese encephalitis virus strains isolated from different years].

In order to reveal the phenotypic characteristics of 17 JE virus strains isolated from different years, plaque sizes, mice neurovirulence and mice neuroinvasiveness of the isolates were studied and compared. BHK21 cell monolayers were used for testing the plaque sizes. The virus neurovirulence was tested in 9-11g mice inoculated intracerebrally and the virus neuroinvasiveness was tested in 9-11g and 14-16g by subcutaneous inoculation. Results showed that all the viruses produced clear plaques on the BHK21 cell monolayers with different sizes and all the virus strains appeared high neurovirulence in the mice with higher than lg8. 0/0.03 mL virus titers, while no apparent difference among them. The neuroinvasiveness (subcutaneous virulence) tested in the 9-11g mice had shown a little difference, but when tested in the 12-14 g mice,the difference was apparent. The results demonstrated that JEV in nature were highly neurovirulent with no apparent difference. However the neuroinvasiveness of the JEV in nature was greatly different, which didn't relate to the years of isolation and genotypes, but most of the viruses isolated from patients showed higher neuroinvasiveness.