Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Methods We designed,developed,and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection.The precision of the liquid transfer and temperature control was tested.A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction(RT-PCR).The entire process,from SARS-CoV-2 nucleic acid extraction to amplification,was evaluated. Results The precision of the syringe transfer volume was 19.2±1.9 μL(set value was 20),32.2±1.6(set value was 30),and 57.2±3.5(set value was 60).Temperature control in the amplification tube was measured at 60.0±0.0 ℃(set value was 60)and 95.1±0.2 ℃(set value was 95)respectively.SARS-Cov-2 nucleic acid extraction yield through the device was 7.10×106 copies/mL,while a commercial kit yielded 2.98×106 copies/mL.The mean time to complete the entire assay,from SARS-CoV-2 nucleic acid extraction to amplification detection,was 36 min and 45 s.The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test(POCT).

Objective:To investigate the clinical features and characteristics of gene mutation of patients with Hereditary spherocytosis(HS) and report novel mutations found in this work.Method:We reviewed the literatures for the clinical and genetic features of the 35 patients (24 boys and 11 girls, median age 7.8 years) with HS admitted to Wuhan Children′s Hospital from June 2017 to January 2023. Analyze clinical manifestations the blood routine, and biochemical laboratory results, and use Next-generation sequencing (NGS) to detect pathogenic mutation. Mann-Whitney U testing method was used to analyze the hematological parameters between different genotypes. Results:All of the patients showed clinical manifestations of anemia; 33 children had jaundice, 28 children had splenomegaly, and 11 children had gallstones. Gene mutations were detected in all 35 patients. 17 patients carried ANK1 variants, 14 patients harbored SPTB variants, 1 patient carried SPTA1variants, 1 patient carried SLC4A1 variants, and 2 patients harbored both ANK1 and SPTB variants. A total of 39 heterozygous mutations were found, among which 27 were pathogenic, 6 was likely pathogenic and 6 were of unknown significance according to the ACMG guideline; Among 39 mutations identified, 31 were novel. There were no significant differences in peripheral blood cell parameters and hemolysis indicators between the ANK1 mutant group and the SPTB group (all P>0.05). Conclusion:SPTB and ANK1 mutations are the most common mutations in HS pathogenic genes among patients in Hubei, China, and there was no significant difference in phenotype between ANK1 and SPTB genotype. We found 31 unreported novel variants and expanded the spectrum of variation in this type of disease .

Objective:To investigate the mechanism of Candida albicans Int1 in regulating septin organization. Methods:A series of full-length and truncated fragments of Int1 were constructed and fused with green fluorescent protein (GFP). The intracellular localization of the fusion proteins was observed under a fluorescence microscope. The region in Int1 that was required for bud neck localization was identified. Full-length and fragments of Int1 were overexpressed in the yeast Saccharomyces cerevisiae and the changes in cell growth, cell morphology and septin organization were investigated to determine the functional region in Int1 that mediated the interaction with septin. Moreover, the co-localization of the region and septin was analyzed. Results:The full-length Int1 consisted of 1 661 amino acid residues. A middle region of 209 amino acid residues, Int1-M4 (739-947 aa), that could be localized at the bud neck during both small and large bud periods was identified. Overexpression of Int1-M4 led to significant growth defects, elongated bud and disorganized septin. In the cells with elongated bud, Int1-M4 and septin with abnormal structures could be co-localized.Conclusions:Int1-M4 (739-947 aa), the middle region of Int1 containing 209 amino acid residues, mediated the bud neck localization and the interaction with septin, playing an important role in regulating septin organization.

Objective:To investigate the efficacy of Qingruxiao granules combined with tamoxifen in the treatment of breast hyperplasia and its effect on serum hypoxia-inducible factor-alpha (HIF-α), angiopoietin-2 (Ang-2) and prolactin (PRL) levels. Methods:Ninety-eight patients with breast hyperplasia admitted to Xi'an No.3 Hospital from June 2020 to January 2022 were retrospectively included in this study. They were divided into control and observation groups ( n = 49/group) according to different treatments. The control group was treated with tamoxifen alone. The observation group was treated with Qingruxiao granules combined with tamoxifen. Clinical efficacy, symptom score, ultrasound parameters (glandular layer thickness, longest diameter of mass, maximum diameter of hypoechoic area, inner diameter of lactating tube), endocrine hormone levels (estradiol, progesterone, and prolactin), HIF-α, and Ang-2 pre- and post-treatment, as well as the incidence of adverse reactions were compared between the two groups. Results:Total response rate in the observation group was significantly higher than that in the control group [93.88% (4/49) vs. 77.55%, χ2 = 5.33, P < 0.05). After treatment, breast mass score, breast pain, systemic accompanying symptom, and nipple discharge in the observation group were (1.34 ± 0.29) points, (1.02 ± 0.36) points, (0.68 ± 0.17) points, (0.97 ± 0.15) points, respectively, which were significantly lower than (1.57 ± 0.23) points, (1.45 ± 0.41) points, (0.95 ± 0.26) points, and (1.28 ± 0.26) points, respectively, in the control group ( t = 4.35, 5.52, 6.08, 7.23, all P < 0.001). The glandular layer thickness, the longest diameter of mass, the maximum diameter of hypoechoic area, and the inner diameter of lactating duct in the observation group were (9.45 ± 1.67) mm, (11.46 ± 3.68) mm, (14.37 ± 4.22) mm, and (1.23 ± 0.39) mm, respectively, which were significantly lower than (11.26 ± 2.51) mm, (16.33 ± 4.01) mm, (19.87 ± 5.01) mm, (1.54 ± 0.48) mm in the control group ( t = 4.20, 2.26, 5.88, 3.51, all P < 0.001). Serum estradiol and prolactin levels in the observation group were (122.35 ± 29.76) ng/L and (205.64 ± 36.42) IU/L, respectively, which were significantly lower than (139.76 ± 30.48) ng/L and (251.49 ± 41.87) IU/L in the control group ( t = 2.86, 5.78, both P < 0.05). Serum progesterone level in the observation group was (9.22 ± 1.57) μg/L, which was significantly higher than (7.18 ± 1.21) μg/L in the control group ( t = -7.20, P < 0.05). Serum HIF-α and Ang-2 levels in the observation group were (0.15 ± 0.05) ng/L and (0.98 ± 0.11) ng/L, respectively, which were significantly lower than (0.24 ± 0.07) ng/L and (1.49 ± 0.22) ng/L in the control group ( t = 7.32, 14.51, both P < 0.001). There was no significant difference in the incidence of adverse reactions between the two groups ( P > 0.05). Conclusion:Qingruxiao granules combined with tamoxifen can effectively improve clinical symptoms, reduce tumor size, regulate endocrine hormone levels, decrease the expression of angiogenic factors in patients with breast hyperplasia, and is highly safe.

Objective:To investigate the clinical features and characteristics of gene mutation of patients with neurodevelopmental disorder caused by CTNNB1 gene. Method:Genetic mutation analysis of the patients were obtained by using the whole exome sequencing and Sanger sequencing. We reviewed the literatures for the clinical and genetic features of CTNNB1 related neurodevelopmental disorder. Results:Six inpatients, three boys and three girls, who came for speech impairment motor delay were included in this study. The average age for the patients was 17.8±11.1 months. The main clinical manifestations of the patients were craniofacial dysmorphism, microcephaly, hypertonia or spasm, speech impairment motor delay, esotropia and valgus. WES showed that 6 patients carried de novo mutations of CTNNB1 gene, which were c.1057delA, c.1493_1494insA, c.418_424del, c.1985_1988del, c.1420C>T and c.1550T>C. No abnormality was found in the patients′ parents. Conclusions:The clinical manifestation of CTNNB1 related neurodevelopmental disorder involves multiple systems. We found five unreported variants and expanded the variation spectrum of the CTNNB1 gene.

Objective:To summarize the electrophysiological characteristics of neuronal intranuclear inclusion disease (NIID) and explore the value of electrophysiological examination in NIID auxiliary diagnosis.Methods:Twenty NIID patients diagnosed by pathological biopsy and genetic confirmation (15 were symptomatic, 5 were asymptomatic), admitted to Department of Neurology, Affiliated Hospital of Xuzhou Medical University from February 2020 to June 2022 were chosen. Peripheral motor/sensory nerve conduction, needle electromyography, F wave, repetitive electrical stimulation, skin sympathetic reflex (SSR), and tremor were analyzed. Peripheral nerve conduction and SSR parameters were compared between 15 patients with symptomatic NIID (symptomatic NIID group) and 11 age- and gender-matched normal control subjects (control group).Results:(1) All 15 patients with symptomatic NIID were with abnormal electrophysiological findings: 14 patients had abnormal peripheral nerve conduction, including 14 with slowed motor nerve conduction velocity (MCV), 4 with reduced composite muscle action potential (cMAP) wave amplitude, 12 with slowed sensory nerve conduction velocity (SCV), and 3 with reduced sensory nerve action potential (sNAP) wave amplitude, and overall slowed nerve conduction velocity and relatively preserved wave amplitude were noted; 4 patients had neurogenic lesions by needle electromyography; 13 patients had prolonged F-wave latency at varied degrees; 12 showed abnormal SSR; 4 exhibited synchronous tremor from 4.0 to 7.5 Hz. (2) In 5 patients with asymptomatic NIID, 3 had abnormal peripheral nerve conduction, including 3 with slowed MCV, 2 with slowed SCV, and 1 with reduced sNAP wave amplitude; 3 showed abnormal SSR. (3) Significant differences in MCV and SCV, some cMAP and sNAP amplitudes, and SSR latency and amplitude were noted in nerves of the upper and lower extremities between the symptomatic NIID group and control group ( P<0.05). Conclusion:Peripheral nerve damages are common in patients with NIID, especially myelin damage and autonomic nerve injury, and some patients may have electrophysiological abnormalities before clinical symptoms; therefore, peripheral nerve conduction and SSR can be recommended as auxiliary screening tools for NIID.

The clinical data of 2 patients with LIG4 syndrome who presented to Wuhan Children′s Hospital from May 2020 to December 2021 were retrospectively analyzed and genetically analyzed. Both patients were male, aged from 5 months to 3 years. The clinical presentations were scattered rash and repeated infections (bacterial infection, EB virus, cytomegalovirus, etc). Laboratory tests showed that the neutrophil and lymphocyte counts decreased. Immunoassay revealed a significant is CD4+T, CD8+T, CD19+B lymphocytes and NK. By Whole exome sequencing, we found 2 inherited mutations inherited in the LIG4 gene (c.833G>T and c.1271_1275delAAAGA) from patient1, and another 2 inherited mutations (c.980T>G, c.1271_1275del) from patient 2. In this study, we found two new variants of LIG4 gene and expanded the mutation spectrum of this gene.

The patient in this study was a 3 years 8 month old boy. The patient presented with facial dysmorphism including wide eye distance and flat nose. The major clinical manifestations were poor response, backward language and motor development; and his fingers cannot be bent. Moreover, the patient′s hands were also uncoordinated. In addition the patient suffered from congenital myopia and nystagmus; and the teeth were fall off easily. The abnormal reproductive system was characterized by small penis and small testicle. No obvious abnormality was found in liver and kidney function and serum immunoglobulin level through laboratory biochemical test. The results of the spine X-Ray examination indicates scoliosis. Results from brain MRI showed cerebellar dysplasia. Compound heterozygous variants in COG5 gene (c.1039C>T and c.928+3A>G), each inheranted from his parents were found in this patient by high-throughput sequencing and Sanger sequencing. After a clear diagnosis, the patient received language rehabilitation training and motor rehabilitation training. In this study, we found two new variants in COG5 gene and increased the mutation spectrum of this gene.

Objective:This study retrospectively analyzed an outbreak of dengue fever in Puyang of Henan province in 2019, in order to find the sources of infection.Methods:Dengue virus IgM/IgG and NS1 antigen were tested by colloidal gold method. E gene was amplified by PCR. MegaX was used for sequences alignment to construct evolutionary distance trees.Results:After clinical and laboratory confirmation, there were 81 cases of dengue fever, 17 of which were imported case who were local farmers and worked in Combadia and Thailand, and 64 of which were indigenous cases. The E gene alignment results showed that the pathogen of this epidemic was Vietnamese 1 and highly homologous with the Vietnamese strain. After the local outbreak, dengue virus E gene developed a nucleotide site mutation which can be steadily transmission.Conclusion:The dengue fever outbreak in Puyang was a local outbreak caused by dengue virus type 1, which was associated with imported cases. Gene sequencing showed that the imported pathogen had a relatively stable and transmissible nucleotide mutation after the local epidemic.

Objective:This study retrospectively analyzed an outbreak of dengue fever in Puyang of Henan province in 2019, in order to find the sources of infection.Methods:Dengue virus IgM/IgG and NS1 antigen were tested by colloidal gold method. E gene was amplified by PCR. MegaX was used for sequences alignment to construct evolutionary distance trees.Results:After clinical and laboratory confirmation, there were 81 cases of dengue fever, 17 of which were imported case who were local farmers and worked in Combadia and Thailand, and 64 of which were indigenous cases. The E gene alignment results showed that the pathogen of this epidemic was Vietnamese 1 and highly homologous with the Vietnamese strain. After the local outbreak, dengue virus E gene developed a nucleotide site mutation which can be steadily transmission.Conclusion:The dengue fever outbreak in Puyang was a local outbreak caused by dengue virus type 1, which was associated with imported cases. Gene sequencing showed that the imported pathogen had a relatively stable and transmissible nucleotide mutation after the local epidemic.