[Establishment and validation of nomogram prediction model for complicated acute appendicitis].

Objective: To establish and internally validate a nomogram model for predicting complicated acute appendicitis (CA). Methods: The clinical data from 663 acute appendicitis patients from the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine from October 2015 to October 2022 were retrospectively analyzed. There were 411 males and 252 females, aged (M (IQR)) 41 (22) years (range: 18 to 84 years). There were 516 cases of CA and 147 cases of uncomplicated acute appendicitis. The minimum absolute contraction and selection operator regression model was used to screen the potential relative factors of CA, and the screened factors were included in the Logistic regression model for multivariate analysis. Software R was used to establish a preoperative CA nomogram prediction model, the receiver operating characteristic curve of the model was drawn, and the value of area under the curve (AUC) was compared to evaluate its identification ability, and the Bootstrap method was used for internal verification. Results: The elderly (age≥60 years) (OR=2.428, 95%CI: 1.295 to 4.549), abdominal pain time (every rise of 1 hour) (OR=1.089, 95%CI: 1.072 to 1.107), high fever (body temperature≥39 ℃) (OR=1.122, 95%CI: 1.078 to 1.168), total bilirubin (every rise of 1 µmol/L) (OR=2.629, 95%CI: 1.227 to 5.635) were independent relative factors of CA (all P<0.05). The AUC of this model was 0.935 (95%CI: 0.915 to 0.956). After internal verification using the Bootstrap method, the model still had a high discrimination ability (AUC=0.933), and the predicted CA curve was still in good agreement with the actual clinical CA curve. Conclusion: The clinical prediction model based on the elderly (age≥60 years), prolonged abdominal pain time, high fever (body temperature≥39 ℃), and increased total bilirubin can help clinicians effectively identify CA.

Raspberry ketone glucoside suppresses melanin synthesis through IL6/JAK1/STAT3 signal pathway.

OBJECTIVE: This study aimed to evaluate the anti-melanogenic activity of raspberry ketone glucoside (RKG) and further explore the specific molecular mechanisms by which RKG affects melanogenesis. MATERIALS AND METHODS: The B16F10 cells model, the mushroom tyrosinase model and the zebrafish model were used to assess the whitening activity of RKG. We subsequently identified possible pathways related to RKG inhibition of melanogenesis by RNA-seq analysis and qRT-PCR on the zebrafish model, and further explored the effects of key genes on the pathway on the melanogenic effect of RKG by using related pathway inhibitors and Tg [mpeg: EGFP] transgenic zebrafish line. RESULTS: RKG could noticeably inhibit melanogenesis in B16F10 cells in vitro and on zebrafish in vivo. The RNA-Seq analysis and the qRT-PCR in zebrafish embryos indicated that the inhibition of melanogenesis by RKG could be achieved by activating JAK1/STAT3 signal pathway and inhibiting the expression levels of the MITFa, TYR, TYRP1a genes directly associated with melanogenesis. The inhibitor tests revealed that the inhibitory effect of the RKG on melanogenesis was restored by the IL6, JAK1/2, and STAT3 inhibitors, specifically STAT3 inhibitor. We further examine the relationship between the JAK1/STAT3 signal pathway and the MITFa. The achieved results indicate that the RKG could activate the zebrafish macrophages via the JAK1, but the inhibition of macrophage activation by loganin did not affect the anti-pigmentation effect of the RKG. CONCLUSIONS: RKG showed remarkable whitening activity on both B16F10 cells in vitro and zebrafish model in vivo. Furthermore, RKG could inhibit melanogenesis by activating the IL6/JAK1/STAT3 pathway, inhibiting the transcriptional activity of MITFa, and its downstream expression levels of the TYR and TYRP1a genes.

[Phenotype-genotype analysis of the autosomal recessive hereditary hearing loss caused by OTOA variations].

Objective: To analyze the phenotypic-genotypic characteristics of hereditary deafness caused by OTOA gene variations. Methods: Family histories, clinical phenotypes and gene variations of six pedigrees were analyzed, which were diagnosed with hearing loss caused by OTOA gene variations at the PLA General Hospital from September 2015 to January 2022. The sequence variations were verified by Sanger sequencing and the copy number variations were validated by multiplex ligation-dependent probe amplification (MLPA) in the family members. Results: The hearing loss phenotype caused by OTOA variations ranged from mild to moderate in the low frequencies, and from moderate to severe in the high frequencies in the probands, which came from six sporadic pedigrees, among which a proband was diagnosed as congenital deafness and five were diagnosed as postlingual deafness. One proband carried homozygous variations and five probands carried compound heterozygous variations in OTOA gene. Nine pathogenic variations (six copy number variations, two deletion variations and one missense variation) and two variations with uncertain significance in OTOA were identified in total, including six copy number variations and five single nucleotide variants, and three of the five single nucleotide variants were firstly reported [c.1265G>T(p.Gly422Val),c.1534delG(p.Ala513Leufs*11) and c.3292C>T(p.Gln1098fs*)]. Conclusions: OTOA gene variations can lead to autosomal recessive nonsyndromic hearing loss. In this study, the hearing loss caused by OTOA defects mostly presents as bilateral, symmetrical, and postlingual, and that of a few presents as congenital. The pathogenic variations of OTOA gene are mainly copy number variations followed by deletion variations and missense variations.

[Analysis of COL1A1 gene variation and clinical prevention and treatment in patients with Van der Hoeve syndrome].

Objective: To investigate the clinical phenotype, treatment and prevention of Van der Hoeve syndrome, and analyze the variation characteristics of its related gene COL1A1. Methods: Hearing and sequencing data of syndromic deafness patients who had undergone genetic testing for deafness at the Chinese People's Liberation Army General Hospital since January 2008 to October 2020 were retrospectively reviewed. The variation of the COL1A1 gene and return visits to traceable patients and families were summarized, the disease progress and clinical treatment effects were analyzed, and the prevention strategies were discussed. Results: A total of 7 patients with COL1A1 gene mutation underwent clinical intervention. The mutation sites were c.1342A>T (p.Lys448*), c.124C>T (p.Gln42*), c.249insG(p.Ala84*), c.668insC(p.Gly224*), c.2829+1G>C, c.1081C>T (p.Arg361*), c.1792C>T (p.Arg598*), of which c.1081C>T and c.1792C>T had been previously reported, and the remaining 5 were novo mutations that have not been reported. All the 7 probands underwent stapes implantation and received genetic counseling and prevention guidance. Conclusions: Van der Hoeve syndrome belongs to osteogenesis imperfecta type Ⅰ. The disease has high penetrance. Timely surgical intervention for hearing loss can improve the life quality in patients. Accurate genetic counseling and preimplantation genetic diagnosis can achieve the primary prevention for the disease.

[Surgical strategy of minimally invasive cochlear implantation].

Cochlear implantation is currently the most effective treatment for patients with severe-to-profound sensorineural hearing loss. How to achieve minimally invasive treatment, preserve the residual hearing, and further improve curative effect and reduce surgical complications is the goal of cochlear implantation practice. This article introduces the minimally invasive cochlear implantation technique in terms of the idea of minimally invasive operation, the advantages of electric acoustic stimulation, the key points of electrode implantation technique, the design of surgical incision, and the precise processing of perioperative period. This technique not only has the merits of less operative damage and better hearing and speech rehabilitation after surgery, but also reserves favorable structures and function for the future application of gene therapy and hair cell regeneration technique. Therefore, it is strongly recommended for further promotion in clinical practice.

[Phenotype and genotype analysis of recessive hereditary moderate sensorineural hearing loss caused by new mutations in OTOGL gene].

Objective: To perform the phenotype and genetic analysis on two families with moderate sensorineural hearing impairment and determine the cause of deafness. Methods: The phenotype and genetic analysis was performed on the two hearing impairment pedigrees coming to Chinese PLA General Hospital from January 2014 to August 2020. DNA samples of the proband from family 1 and the parents from family 2 were collected and tested through next generation sequencing on all deafness genes, and Sanger sequencing was performed to verify the mutation sites. The reported pathogenic variants of the otogelin-like (OTOGL) gene, the autosomal recessive inherited deafness genes that cause moderate sensorineural hearing loss and the clinical manifestations of the deafness genes that have the similar expression location as the OTOGL gene were summarized and analyzed. Results: The pathogenic variants in the families were compound heterozygous variants in the OTOGL gene c.2773C>T/c.2826C>G (p.Arg925*/p.Tyr942*) and c.4455G>A/c.875C>G (Trp1485*/p.Ser292*), respectively. c.2773C>T was an already reported pathogenic variant causing hearing impairment in the literature, while c.2826C>G, c.4455G>A and c.875C>G were novel reported variant sites. The above four variants were classified as pathogenic variants according to the variant interpretation standards and guideline of the Amercian College of Medical Genetics and Genomics. Conclusions: Pathogenic variants in OTOGL gene is an important genetic factor leading to moderate sensorineural hearing loss. The newly discovered variant sites c.2826C>G, c.4455G>A and c.875C>G enrich the variant spectrum of OTOGL gene. The results of the current study provide a basis for genetic counseling of the related families and a new target for the treatment of hereditary hearing loss in the future.

Effect of TNF-alpha on endometrial glucose transporter-4 expression in patients with polycystic ovary syndrome through nuclear factor-kappa B signaling pathway activation.

The present study aimed to investigate the possible mechanism of action of tumor necrosis factor-alpha (TNF-α) in endometrial glucose transporter-4 (GLUT-4) expression regulation by detecting the expression levels of endometrial inflammatory factors and GLUT-4 in patients with polycystic ovary syndrome (PCOS). A total of 140 patients were included in this study and divided into four groups: the PCOS group, the obesity + PCOS group, the normal group, and the obesity group (n = 35 each). The general clinical data of all patients were collected, and their expression levels of TNF-α, nuclear factor kappa B p65 (NF-κBp65), and GLUT-4 in the endometrium were tested via immunohistochemistry. Endometrial stromal cells were cultured in vitro and treated with TNF-α or pyrrolidine dithiocarbamate (PDTC) + TNF-α, and the expression levels of NF-κBp65, phospho-NF-κBp65 (p-NF-κBp65), and GLUT-4 were tested using Western blotting (WB) before and after treatment. As a result, we got: 1) Compared with the normal group, the abundance of the protein for TNF-α and NF-κBp65 in the endometrium of the patients with PCOS was elevated, while the expression level of GLUT-4 was decreased; the difference was statistically different (P < 0.05). The comparison between the obesity + PCOS group and the PCOS group yielded the same results. 2) According to the WB results, compared with the normal group, the abundance of the protein for endometrial GLUT-4 was decreased in the PCOS group, and the expression levels of p-NF-κBp65 and NF-κBp65 were increased in the obesity + PCOS group; the differences were statistically different (P < 0.05). The addition of TNF-α could decrease the abundance of the protein for GLUT-4 and increase the abundance of the protein for p-NF-κBp65. After treatment with PDTC + TNF-α, the abundance of the protein for p-NF-κBp65 decreased and GLUT-4 increased compared with the TNF-α group; these values were close to those of the control group. We concluded that the abundance of the proteins for local inflammatory factors in the endometrial cells of patients with PCOS was increased, indicating that TNF-α could affect the expression of endometrial GLUT-4 in such patients by activating the p-NF-κBp65 signaling pathway.

[Case report and diagnosis of Noonan syndrome with multiple lentigines with deafness as its main clinical feature].

Summary Noonan syndrome with multiple lentigines（NSML） is a disorder with syndromic hearing loss. Abnormalities of other systems in NSML have received increasing attention, but hearing loss is rarely concerned. And due to the incomplete phenotype, some patients with NSML maybe missed or maybe confused with other syndromic deafness such as Waardenburg syndrome. Our study will familiarize more otolaryngologists with Leopard syndrome. A 5-year-old boy with bilateral sensorineural hearing loss and numerous symmetrically distributed dark brown macules that had good effect of cochlear implantation was collected in this study. And his father had bilateral sensorineural hearing loss and numerous symmetrically distributed dark brown macules. Waardenburg syndrome was initially diagnosed by clinical phenotype and its molecular etiology was confirmed by gene diagnosis. Waardenburg syndrome-related deafness genes and 131 known deafness genes were not identified by second-generation sequencing. Whole-exon sequencing was performed for 4 individuals in the family and the results were confirmed by Sanger sequencing. This study confirmed the diagnosis by identifying a disease-causing mutation in the PTPN11 gene, which was a heterozygous missense mutation at p. Tyr279Cys（c. 836A>G）. The mutation co-segregated with hearing loss in the family. Our results demonstrated that hearing loss in this family was caused by heterozygous mutations in PTPN11. These cases will familiarize more otolaryngologists with NSML, and they emphasize the importance of considering NSML as a possible cause of hearing problems.

[One novel pathologic variation in KMT2D cause Kabuki syndrome with hearing loss as the main phenotype and related research on types of deafness].

Objective:To make the molecular diagnosis of a patient complaining hearing loss and with specific facial features, developmental delay, vertebral dysplasia, hypotonia and other suspected phenotypes of Kabuki make-up syndrome（KS）; to investigate the characteristics and main phenotypes of KS. Method:①Whole-exome sequencing and bioinformatics analysis were performed for proband and her parents. ②Literatures describing the clinical features of KS patients with clear molecular diagnosis from the period of Aug 2010 to Mar 2019 were collected from databases of PubMed and CNKI. Result:①The proband carries the c. 15777insT variant（p. Pro5260fs*10） in KMT2D gene. The variant causes the termination codon to appear prematurely. KMT2D c. 15777insT was classified as PVS1+PS1+PM2 according to the ACMG variation interpretation standard, which is a disease-causing mutation. The c. 15777insT was first reported as a pathogenic mutation of KS. ②77 peer-reviewed publications on KS were analysed including 462 patients with KS. The main findings were intellectual disability（305 cases）, congenital heart defects（227 cases）, hypotonia（184 cases）, short fingers（147 cases）, short stature（144 cases）, cleft palate（139 cases）, hearing loss（101 cases） and developmental delay（99 cases）. Of the 101 patients with hearing loss, 11 were confirmed to have conductive hearing loss（1 with recurrent otitis media）, 3 with mixed hearing loss, 12 with sensorineural deafness（1 with recurrence otitis media） and 75 patients with unidentified types of deafness（28 with recurrent otitis media）. Conclusion:KS involves defects of a wide range of organs, with each organ showing different severity of symptoms, which is easily misdiagnosed from the phenotypes. We suggest the diagnosis on hearing loss in KS patients should be strengthened. KMT2D and KDM6A are two pathogenic genes that have been identified for KS. With the increase of age, its typical clinical phenotypes become more and more obvious. When there is only atypical suspected KS symptoms in the early neonatal period, relevant genetic test should be performed as soon as possible to achieve early diagnosis and intervention.

[PTPN11 and the deafness].

Summary PTPN11 gene encodes tyrosine phosphatase SHP-2 which locates on chromosome 12（12q24.1）, expresses in most embryonic and adult tissues, and plays pivotal roles in cell proliferation, differentiation, survival and cell death. SHP-2 apparently participates in signaling events downstream of RAS-MAPK and JAK/STAT. Diseases related to PTPN11 gene mutations include the Noonan syndrome（NS） and the NS with Multiple Lentigines（NSML）. Both NS and NSML contain the phenotypes of deafness, craniofacial anomalies, short stature, congenital heart defects, skin disorders, ophthalmologic abnormalities and cancer predisposition.

[Histocompatibility of an original Chinese-made mid-urethral sling in a rabbit model].

Objective: To evaluate the histocompatibility of an original Chinese-made mid-urethral sling (Repelvica mid-urethral sling). Methods: In total 10 female New Zealand white rabbits were implanted with Repelvica mid-urethral sling or tension-free vaginal tape-obturator tape (TVT-O; Gynecare). Both brands of sling were implanted under deep fascia of the abdominal wall and in the space between vagina and bladder. All animal groups were sacrificed at set time intervals (4 weeks and 12 weeks), and the abdominal and vaginal slings were harvested for histological evaluation. Results: All slings appeared to be well incorporated into the abdominal wall and anterior vaginal wall. All specimens showed a thin, loose, fibrous interface between the synthetic graft and abdominal wall or vaginal wall, along with mild inflammatory reaction from 4 weeks to 12 weeks. Abdominal grafts of Repelvica mid-urethral sling and TVT-O induced comparable tissue reaction (histological score 10.5 versus 10.5 at 4 weeks, 10.0 versus 9.5 at 12 weeks; both P>0.05). Vaginal grafts of Repelvica mid-urethral sling had lower histological score than TVT-O (histological score 6.0 versus 12.0 at 4 weeks, 8.5 versus 12.5 at 12 weeks), however the differences were not statistically significant (both P>0.05). Conclusions: Chinese-made Repelvica mid-urethral sling exhibits good histocompatibility. Vaginal graft of Repelvica mid-urethral sling evoks minor tissue reaction, which could be attributed to its lightweighted property and favored its clinical application.

A Multiple-Cell Microenvironment in a 3-Dimensional System Enhances Direct Cellular Reprogramming Into Hepatic Organoids.

OBJECTIVES: The difficulty in proliferation and availability and the rapid loss functions of primary human hepatocytes highlight the need to develop an alternative, preferably renewable source of human induced hepatocytes in regenerative medicine. Liver organoids generated on a multiple-cell microenvironment in a 3-dimensional (3D) system can provide a highly efficient solution to this issue. METHODS: Human hepatocytes were induced from fibroblasts by the lentiviral expression of FOXA3, HNF1A, and HNF4A. Together with these induced hepatocytes, human umbilical vein endothelial cells and mesenchymal stem cells in a 3D system were used to produce liver organoids. Liver-related gene and protein expression of liver organoids and induced hepatocytes were tested using a 2-dimensional (2D) system. RESULTS: Liver organoids notably increased the expression of hepatic transcription factors, marker genes, transporter genes, and liver metabolism enzyme genes, while it decreased the specific gene expression of fibroblasts. Liver organoids expressed comparable liver-specific proteins, such as ALB, AAT, and HNF4A in the 3D system. CONCLUSION: Direct reprogramming in multiple-cell microenvironments in 3D systems is more controllable and efficient than cell reprogramming in 2D systems. Liver organoids have the potential for use in disease modeling, pharmaceutical applications, and cellular transplantation.

Mutations of transglutaminase-1 in Chinese patients with autosomal recessive congenital ichthyosis: a case report with clinical and genetic analysis of Chinese cases reported in literature.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of diseases of keratinization, characterized primarily by abnormal skin scaling over the whole body surface. Recently, ARCI has been designated to include the major forms of lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) and harlequin ichthyosis. The first two conditions are the most common major clinical subtypes, and both are caused principally by mutations in the transglutaminase 1 gene, TGM1, although other genes may be responsible in some cases. AIM: To identify the genetic mutations underlying LI in a Chinese family with LI, and to review all the known TGM1 mutations in Chinese patients with ARCI. METHODS: The proband had the severe classic LI phenotype, and was a member of a four-generation family with close blood relationships. We sequenced the DNA of the patients and close relatives. We also reviewed 13 Chinese patients with ARCI from 8 reported families, comprising 10 patients with LI, 2 with CIE and 1 with bathing suit ichthyosis. RESULTS: We characterized 14 different TGM1 mutations. Six of these were reported in other ethnic groups initially and later in Chinese patients, while the remaining eight were first described in Chinese patients. Of the latter, five have been reported only in Chinese patients, while the remaining three have also been reported in other ethnic groups. CONCLUSION: This study expands the current spectrum on TGM1 mutation and increases the knowledge of TGM1 mutation characteristics.

Sequence characterization, polymorphism, and tissue expression profile of an effector immediate-early gene: activity-regulated cytoskeletal associated protein gene (Arc/Arg3.1) in swamp and river buffalo.

The activity-regulated cytoskeletal associated protein (Arc/Arg3.1) has been implicated in experience-dependent synaptic plasticity and memory formation. However, information regarding its coding gene in buffalo remains scarce. In this study, the full-length of Arc/Arg3.1 was isolated and characterized (accession No. JX491649) and genetic variations of six river buffalo and eight swamp buffalo were investigated. A tissue expression profile was obtained using semi-quantitative reverse transcription-polymerase chain reaction. The coding region sequence of Arc/Arg3.1 contained 1191 nucleotides encoding a putative protein of 396 amino acids with a theoretical isoelectric point (pI) and molecular weight (Mw) of 5.4 and 45.2 kDa, respectively. Four polymorphisms (c.63T>C, c.228T>C, c.558G>A, and c.625G>C) were found in buffalo; however, only substitution c.625G>C was non-synonymous, leading to an amino acid change from Val to Leu at the 209th position of the Arc/Arg3.1 protein sequence. Bioinformatics analysis revealed that this substitution had no significant effect on Arc/Arg3.1 function (subPSEC = -1.4039, Pdeleterious = 0.1685), which indicated that Arc/Arg3.1 was highly conserved and functionally important in buffalo. Phylogenetic analysis revealed that the gene is closely related to that of Bos taurus and Bos grunniens. The gene was moderately expressed in the hypophysis and the placenta; it was weakly expressed in the kidney, milk, mammary gland, cerebrum, lung, heart, rumen, fat, and uterus; and it was almost silent in the muscle, liver, and skin. These findings will provide further insights into the structure and function of the immediate-early gene in buffalo.