Objective·To achieve the rapid detection of Staphylococcus aureus using nanopore Cas9-targeted sequencing(nCATS)technology,and simultaneously perform staphylococcal protein A(spa)typing and staphylococcal cassette chromosome mec(SCCmec)typing.Methods·The spa gene and SCCmec gene elements were selected as two regions of interest(ROIs)for targeted sequencing.Four types of CRISPR RNAs(crRNAs)were designed to form Cas9 ribonucleoproteins(RNPs)to cleave sequences flanking the two ROIs.For each crRNA,a 42 bp synthetic target DNA was designed.Appropriate crRNAs were screened,the cleavage system was optimized,and both the cleavage reaction time and Cas9 RNP synthesis temperature were determined based on the results of Cas9 RNP cleavage efficiency testing.Genomic DNA was extracted from a methicillin-resistant Staphylococcus aureus(MRSA)strain,and the ends flanking the cleaved ROIs were dephosphorylated and dA-tailed.Sequencing adapters were ligated,and sequencing was performed using a nanopore platform.The quality scores of the sequencing data were analyzed,and the obtained nucleic acid sequences were compared with those in the mecA,spa and SCCmec databases.Based on the comparison results,the presence of Staphylococcus aureus,MRSA or not,and spa and SCCmec types were determined.Results·Two sets of crRNAs were designed.Based on grayscale analysis of electrophoresis results,the set with higher cleavage efficiency was selected for further experiments.Optimization showed that a 1∶1 ratio of Cas9 RNP to target DNA,a 15 min cleavage reaction time,and a Cas9 RNP synthesis temperature of 25℃yielded a cleavage efficiency of 87.41%.nCATS sequencing quality scores ranged between 15(Q15)and 20(Q20),indicating an approximate sequencing accuracy of 99%.Sequence comparisons with the mecA,spa and SCCmec databases revealed that the strain's spa type was t2 and its SCCmec type was Ⅱ.These results were consistent with those obtained by PCR amplification sequencing and multiplex PCR.Conclusion·The nCATS technique enables rapid detection of Staphylococcus aureus,while simultaneously providing spa and SCCmec typing information.

Objective·To achieve the rapid detection of Staphylococcus aureus using nanopore Cas9-targeted sequencing(nCATS)technology,and simultaneously perform staphylococcal protein A(spa)typing and staphylococcal cassette chromosome mec(SCCmec)typing.Methods·The spa gene and SCCmec gene elements were selected as two regions of interest(ROIs)for targeted sequencing.Four types of CRISPR RNAs(crRNAs)were designed to form Cas9 ribonucleoproteins(RNPs)to cleave sequences flanking the two ROIs.For each crRNA,a 42 bp synthetic target DNA was designed.Appropriate crRNAs were screened,the cleavage system was optimized,and both the cleavage reaction time and Cas9 RNP synthesis temperature were determined based on the results of Cas9 RNP cleavage efficiency testing.Genomic DNA was extracted from a methicillin-resistant Staphylococcus aureus(MRSA)strain,and the ends flanking the cleaved ROIs were dephosphorylated and dA-tailed.Sequencing adapters were ligated,and sequencing was performed using a nanopore platform.The quality scores of the sequencing data were analyzed,and the obtained nucleic acid sequences were compared with those in the mecA,spa and SCCmec databases.Based on the comparison results,the presence of Staphylococcus aureus,MRSA or not,and spa and SCCmec types were determined.Results·Two sets of crRNAs were designed.Based on grayscale analysis of electrophoresis results,the set with higher cleavage efficiency was selected for further experiments.Optimization showed that a 1∶1 ratio of Cas9 RNP to target DNA,a 15 min cleavage reaction time,and a Cas9 RNP synthesis temperature of 25℃yielded a cleavage efficiency of 87.41%.nCATS sequencing quality scores ranged between 15(Q15)and 20(Q20),indicating an approximate sequencing accuracy of 99%.Sequence comparisons with the mecA,spa and SCCmec databases revealed that the strain's spa type was t2 and its SCCmec type was Ⅱ.These results were consistent with those obtained by PCR amplification sequencing and multiplex PCR.Conclusion·The nCATS technique enables rapid detection of Staphylococcus aureus,while simultaneously providing spa and SCCmec typing information.

Aim To investigate the effect of dipeptidyl peptidase-4 inhibitor(DPP-4i)on serum creatinine(Cr)in patients with type 2 diabetes mellitus(T2DM).Methods A systematic search was performed across data-bases of PubMed,Embase,Cochrane Library and Web of Science,and randomized controlled trials(RCT)of DPP-4i therapy for regulating Cr in T2DM patients was included.A fixed-effect or random-effect model was used for data fitting,heterogeneity was quantitatively evaluated according to the index of I2,and sensitivity analysis and publication bias testing were performed by using the standard methods.Results After searching the database through the system,12 RCTs were included,with a total of 2 276 participants.Due to the potential heterogeneity,a random effect model was used for data fitting.DPP-4i treatment could mildly increase Cr levels in T2DM patients(WMD:0.15 mg/L,95％CI:0.03～0.27,I=1 8％,P=0.02),and the results showed statistical differences.According to sensitivity testing,the results of Meta-analysis were relatively reliable.No publication bias was observed according to Begg's and Egger's tests.Conclusions The use of DPP-4i for hypoglycemic treatment in T2DM patients may result in mild elevation of blood Cr lev-els.Further multicenter studies with larger samples are needed in the future to explore the clinical significance of DPP-4i treatment induced changes in Cr levels.

Objective To explore the mechanism underlying ITGB1-induced drug resistance in gastric cancer based on the circRNA-miRNA-ITGB1 regulatory network.Methods Tumor tissue samples were collected from 21 patients with gastric cancer.The ITGB1 gene expression levels were determined using real-time fluorescent quantitative polymerase chain reaction,and circRNA sequencing was performed to compare the differences in circRNAs between patients with low and high ITGB1 expression.BGC-823 cells were trans-fected with si-circ_0027189,si-miR-455,or si-NC and divided into the si-circ_0027189,si-miR-455,or si-NC groups,respectively.The circ_0027189,miR-455,and ITGB1 expression levels in each group and the sensitivity to oxaliplatin were measured.Results The circRNA regulatory network showed that circ_0027189 regulated ITGB1 expression through miR-455.Compared to the si-NC group,the si-circ_0027189 group exhibited decreased expression levels of circ_0027189 and ITGB1,increased expression levels of miR-455,and reduced sensitivity to oxaliplatin.In contrast,the si-miR-455 group showed decreased expression levels of miR-455,increased expression levels of ITGB1,and enhanced sensitivity to oxaliplatin compared to the si-NC group.Conclusion circ_0027189 can increase ITGB1 expression levels by targeting miR-455 expression,ultimately increasing drug resistance in gastric cancer cells.

Objective:To summarize the clinical manifestations, gene variations, diagnosis and treatment of 3 cases with SLC35A2 variations characterized by congenital glycosylation disorder Ⅱm (CDG Ⅱm). Methods:A total of 3 patients admitted to the Department of Pediatrics of Xiangya Hospital of Central South University in China from 2018 to 2020 were examined in detail. The studies till January 2022 were searched with key words of "congenital disorders of glycosylation Ⅱm", " SLC35A2" and "CDG Ⅱm" in both English and Chinese in the databases of China National Knowledge Infrast Ructure (CNKI), Wanfang, Online Mendelian Inheritance in Man and PubMed, and the clinical manifestations, genetic variation, treatments and prognosis of patients with SLC35A2 mutation were summarized. Results:The patients all presented with intractable infantile spasm and global developmental delay, onset in infancy. A variety of antiepileptic treatments had temporary and partial efficacy. Otherwise, proband 2 and 3 presented with abnormal glutamic-pyruvic transaminase and increased platelets. Funduscopy showed dysplasia of the retinal pigment epithelium in both eyes, and they both received D-galactose treatment. A total of 22 relevant case reports, including 99 patients, were collected. The 99 patients all were heterozygous mutations, and a total of 75 different variation sites were reported. The clinical manifestations were characterized by global developmental delay or mental retardation ( n=89), epileptic seizure ( n=75), hypotonia ( n=57), facial deformity ( n=57), skeletal abnormality ( n=50), visual impairment ( n=42), elevated glutamic-pyruvic transaminase ( n=31), gastrointestinal symptoms ( n=28), skin deformity ( n=26), microcephaly ( n=23) and congenital heart disease ( n=12). Craniocerebral magnetic resonance imaging may be normal in the early stage. With age, magnetic resonance imaging may show abnormal white matter signals, brain atrophy, dysplasia of corpus callosum, delayed myelination, enlargement of lateral ventricle, brain stem atrophy and so on. Studies have shown that galactose treatment may be effective. Conclusions:SLC35A2 variants lead to CDG Ⅱm, whose clinical manifestations mainly include epileptic encephalopathy and global developmental delay. Multiple antiepileptic therapies can temporarily or partially control seizures, while oral galactose may improve the clinical symptoms, showing its prospect as a dietary therapy.

A 25-year-old female patient took 4 Rupi Sanjie capsules orally (0.55 g/capsule) thrice daily and Xiangshao granules 1 bag (4 g/bag) orally thrice daily by herself for breast nodule. After 2 months, the patient developed yellow urine, skin itching, and yellowish skin and sclera. Laboratory tests showed alanine aminotransferase (ALT) 1 334 U/L, aspartate aminotransferase (AST) 717 U/L, alkaline phosphatase (ALP) 164 U/L, total bilirubin (TBil) 88 μmol/L, and γ-glutamyltransferase (γ-GT) 127 U/L. Liver injury caused by Rupi Sanjie capsules and Xiangshao granules were considered. The 2 drugs were stopped and the treatments of liver protection and cholagogic were given. Three weeks later, her symptoms above-mentioned disappeared and the laboratory tests showed ALT 106 U/L, AST 88 U/L, ALP 88 U/L, TBil 13 μmol/L, and γ-GT 32 U/L. At 1 month of follow-up, the patient′s liver function returned to normal. The patient′s liver injury might be related to the component of bupleurum in Rupi Sanjie capsules and Xiangshao granules and the component of prunella vulgaris in Rupi Sanjie capsules.

A 25-year-old female patient took 4 Rupi Sanjie capsules orally (0.55 g/capsule) thrice daily and Xiangshao granules 1 bag (4 g/bag) orally thrice daily by herself for breast nodule. After 2 months, the patient developed yellow urine, skin itching, and yellowish skin and sclera. Laboratory tests showed alanine aminotransferase (ALT) 1 334 U/L, aspartate aminotransferase (AST) 717 U/L, alkaline phosphatase (ALP) 164 U/L, total bilirubin (TBil) 88 μmol/L, and γ-glutamyltransferase (γ-GT) 127 U/L. Liver injury caused by Rupi Sanjie capsules and Xiangshao granules were considered. The 2 drugs were stopped and the treatments of liver protection and cholagogic were given. Three weeks later, her symptoms above-mentioned disappeared and the laboratory tests showed ALT 106 U/L, AST 88 U/L, ALP 88 U/L, TBil 13 μmol/L, and γ-GT 32 U/L. At 1 month of follow-up, the patient′s liver function returned to normal. The patient′s liver injury might be related to the component of bupleurum in Rupi Sanjie capsules and Xiangshao granules and the component of prunella vulgaris in Rupi Sanjie capsules.

Objective:To investigate the clinical efficacy of anterior approach combined with blocking plates and screws in the management of acetabular fracture involving the quadrilateral area.Methods:A retrospective case series analysis was performed for 16 patients with acetabular fracture involving the quadrilateral area admitted to First and Second Affiliated Hospital of Guangxi Medical University from January 2017 to January 2019. There were 12 males and 4 females，with the age of 21-66 years［（45.3±10.6）years］. According to Letournel-Judet classification，there were 9 patients with bi-column fracture，6 with anterior and posterior traverse fracture and 1 with anterior column fracture. A total of 9 patients were operated via the ilioinguinal approach and 7 via the lateral-rectus approach. Reduction and fixation of the pelvis and acetabulum were performed，using 3.5 mm cortical bone screws or plates to block the internal displacement of fracture in the quadrilateral body. The incision length，operation time and intraoperative blood loss were recorded. The quality of fracture reduction was assessed according to the Matta reduction criteria at postoperative 2 days and hip function by the modified Merle D'Aubigne-Postel score at postoperative 3 months and 12 months. Postoperative complications were observed.Results:All patients were followed up for 13-24 months［（16.1±2.9）months］. The ilioinguinal approach and lateral-rectus approach showed surgical incision of 12-26 cm［（18.6±4.0）cm］and 8-15 cm［（10.7±2.3）cm］，respectively. The operation time was 107-215 minutes［（159.2±27.8）minutes］and the intraoperative blood loss was 200-2，300 ml［（853.1±489.7）ml］. According to Matta reduction criteria，the results were excellent in 9 patients and good in 7. Three months after operation，the modified Merle D'Aubigne-Postel score was 11-18 points［（15.2±2.2）points］，which showed the results were excellent in 4 patients，good in 7，fair in 4 and poor in 1，with the excellent and good rate of 69%. Twelve months after operation，the modified Merle D'Aubigne-Postel score was 13-18 points［（16.9±1.4）points］，which showed the results were excellent in 7 patients，good in 8 and fair in 1，with the excellent and good rate of 94%. The liquefaction of post-surgical incision was seen in a patient，bladder injury in a patient，lateral femoral cutaneous nerve injury in a patient，and heterotopic ossification in a patient. There was no loosening or breakage of the internal fixation.Conclusion:For acetabular fracture involving the quadrilateral area，anterior approach combined with blocking plates and screws can prevent the displacement of quadrilateral fracture and attain satisfactory reductiongood hip function recovery and few complications.

Objective:To analyze the clinical manifestation, laboratory examination, treatment and prognosis of congenital factor Ⅺ (FⅪ) deficiency.Methods:The clinical data of 80 patients with congenital FⅪ deficiency in our hospital from September 2006 to October 2020 were analyzed retrospectively.Results:Among the 80 patients, there were 33 males (41.3%) and 47 females (58.8%) , with a median age of 32 (2-66) years. Twenty-eight cases (35.0%) had bleeding events, including 11 cases of spontaneous bleeding (13.8%) , 9 cases of ecchymosis or bleeding after skin trauma (11.3%) , 9 cases of postoperative bleeding (11.3%) . Among the female patients, there were 11 cases of menorrhagia (23.4%) and 1 case of bleeding after vaginal delivery (2.1%) . Laboratory examination were characterized by prolonged activated partial thromboplastin time (APTT) , normal prothrombin time (PT) , and decreased FⅪ activity (FⅪ∶C) . Nine patients (11.3%) were tested for FⅪ gene (F11) with 11 mutations. Twenty-seven patients (33.8%) received fresh frozen plasma (FFP) treatment, 15 patients (18.8%) were received for prophylaxis with no bleeding occurred during and after operation.Conclusion:Most patients with congenital FⅪ deficiency have no or mild bleeding symptoms. There was no significant correlation between FⅪ∶C and the severity of bleeding symptoms, and there was a well consistency between FⅪ∶C and F11 homozygous or heterozygous mutation type. Prophylactic infusion of FFP can effectively reduce the risk of operative bleeding.

Objective:To analyze the clinical phenotype and molecular pathogenesis of nine patients with hereditary factor Ⅴ (FⅤ) deficiency.Methods:Nine patients with hereditary FⅤ deficiency who were admitted to the Institute of Hematology and Blood Diseases Hospital from April 1999 to September 2019 were analyzed. The activated partial thromboplastin time, prothrombin time, and FⅤ procoagulant activity (FⅤ∶C) were measured for phenotypic diagnosis. High-throughput sequencing was employed for the F5 gene mutation screening, Sanger sequencing was adopted to confirm candidate variants and parental carrying status, Swiss-model was used for three-dimensional structure analysis, and ClustalX v.2.1 was used for homologous analysis.Results:The FⅤ∶C of the nine patients ranged from 0.1 to 10.6. Among them, eight had a hemorrhage history, with kin/mucosal bleeding as the most common symptom (three cases, 37.5%) , whereas one case had no bleeding symptom. There were five homozygotes and four compound heterozygotes. A total of 12 pathogenic or likely pathogenic mutations were detected, of which c.6100C>A/p.Pro2034Thr, c.6575T>C/p.Phe2192Ser, c.1600_1601delinsTG/p. Gln534*, c.4713C>A/p.Tyr1571*, and c.952+5G>C were reported for the first time.Conclusion:The newly discovered gene mutations enriched the F5 gene mutation spectrum associated with hereditary FⅤ deficiency. High-throughput sequencing could be an effective method to detect F5 gene mutations.