Erythropoietin inhibits apoptosis of retinal ganglion cells induced by high glucose through JNK signaling pathway.

The aim of this study was to investigate the effect of erythropoietin (EPO) on the apoptosis of retinal ganglion cells (RGCs) induced by high glucose and its mechanism. Rat primary RGCs were extracted to establish high glucose-induced apoptosis models using a 30 mM high-glucose medium. Then flow cytometry, cell counting kit-8 (CCK-8) assay and Western blotting assay were performed to detect the effects of high-, medium- and low-dose EPO on the apoptosis of RGCs induced by high glucose. Next, the molecular mechanism by which EPO suppressed the high glucose-induced apoptosis of RGCs was explored via gene array assay and bioinformatics analysis. The results and mechanism of bioinformatics analysis were verified by Western blotting assay. Finally, the small interfering ribonucleic acid (siRNA) experiment was applied to knock down tyrosine-protein phosphatase non-receptor type 1 (PTPN1) and PTPN11 to verify their roles in the inhibition of EPO on the apoptosis of RGCs triggered by high glucose. Flow cytometry-Annexin V/propidium iodide (PI) staining and CCK-8 assay confirmed that the high-, medium- and low-dose EPO inhibited the apoptosis of RGCs induced by high glucose in a dose-dependent manner (P<0.05). Subsequently, Western blotting assay results manifested that the high-, medium- and low-dose EPO reduced the expression levels of apoptosis-related proteins active-cysteinyl aspartate specific proteinase 3 (Caspase 3) and active- Caspase 9 in a dose-dependent manner (P<0.05). Moreover, according to gene array assay and bioinformatics analysis results, the c-Jun N-terminal kinase (JNK) signaling pathway, PTPN1 and PTPN11 might exert crucial effects in the inhibition of EPO on the apoptosis of RGCs induced by high glucose. Western blotting assay results also demonstrated that, compared with the high-glucose treatment, the high-dose EPO treatment decreased the protein expression level of phosphorylated (p)-JNK1/JNK but increased the protein expression levels of PTPN1 and PTPN11 (P<0.05). Moreover, flow cytometry-Annexin V/PI staining and CCK-8 assay results revealed that in EPO-treated cells, knocking down PTPN1 and PTPN11 significantly reversed the protective effect of EPO against high glucose-induced retinal ganglion cell apoptosis (P<0.05). Lastly, Western blotting assay illustrated that knocking down PTPN1 and PTPN11 significantly abolished the inhibition of high-dose EPO on the JNK signaling pathway. EPO may suppress the JNK signaling pathway by raising the expression levels of PTPN1 and PTPN11, so as to inhibit the apoptosis of RGCs triggered by high glucose.

Integrated analysis of the RNA-Seq data of liver hepatocellular carcinoma.

The present study aimed to explore the genetic changes involved in the liver hepatocellular carcinoma (HCC) development. The RNA-Seq data of 212 HCC tissue samples and 50 normal tissue samples were downloaded using TCGA-Assembler. A total of 4 subgroups were obtained, and 4167, 6279, 5379, and 2548 DEGs were screened in group 1, group 2, group 3, and group 4, respectively. Enrichment analysis found that cell cycle, metabolism, and translation related terms were the most significantly changed functions and pathways. There were 454 genes (1114 pairs), 803 genes (722 pairs), and 788 genes (724 pairs), separately interacted in the condition specific PPI network of group 1, 2, 3, and 4, with MMP2, ATNXN1, F2, and HDAC1 as the hub genes. What's more, using these genes, total 7, 20, 198, and 1 subtype related miRNAs; 35, 50, 47, and 17 subtype related TFs; 1, 1, 0, and 2 subtype related drugs were screened in group 1, 2, 3, and 4, respectively. The integrated biological analysis on RNA-Seq data provided substantial of bio-molecular related to the HCC development. miR-147b, SP1, and Riboflavin were the subtype-related regulator/drug for HCC. The study about the big data of HCC RNA-Seq data reveals the intrinsic gene expression pattern of the tumor, which provides a novel perspective to understand the heterogeneity of pathogenesis in HCC tumorigenesis.

First-principles calculation of the effects of Li-doping on the structure and piezoelectricity of (K0.5Na0.5)NbO3 lead-free ceramics.

The crystal structures of the lead-free piezoelectric ceramics (K0.5Na0.5)NbO3 and (K0.5Na0.5)0.94Li0.06NbO3 prepared by a solid-state method were investigated using first-principles calculations. The calculated values of piezoelectricity were in good agreement with the experimental data. We found that the primary contribution to piezoelectricity in this material comes from the hybridization of the O 2p and Nb 4d orbitals, which causes a change in the Nb-O bond length and the distortion of the Nb-O octahedral structure. Analysis of the band structure and the total density of states revealed that Li-doped (K0.5Na0.5)NbO3 enhances hybridization of the O 2p and Nb 4d orbitals. This hybridization enhancement further reduces the Nb-O1 bond length and enhances the distortion of the Nb-O octahedron along the [001] direction, which may be the main reason for the improvement of the piezoelectric properties. In addition, the piezoelectric coefficients are calculated here, which show the same trend as the experimental results.

[Role of autophagy in acute liver failure induced by D-galactosamine/lipopolysaccharide in mice].

Objective: To investigate the expression and role of autophagy in the progression of acute liver failure (ALF) using the mouse model of ALF induced by D-galactosamine/LPS (D-GalN/LPS). Methods: The C57BL/6 mice were used, and intraperitoneal injection of D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was performed to establish the mouse model of ALF. The mice were divided into control group and 2-, 4-, and 6-hour D-GalN/LPS-induced ALF model groups. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function, and the pathological changes in liver tissue were observed to evaluate the status of liver injury. Quantitative real-time PCR was used to measure the expression of autophagy-related genes, Western blot was used to measure the expression of autophagy-related proteins in liver tissue, and a fluorescence microscope was used to observe the expression of autophagosome in the progression of liver failure. A one-way ANOVA was used for comparison of means of multiple samples between any two groups (LSD-t test for data with homogeneity of variance and Games-Howell method for data with heterogeneity of variance).P< 0.05 was considered statistically significant. Results: The ALF model groups showed gradual liver impairment over the time of D-GalN/LPS stimulation. There were significant increases in ALT and AST after 4 hours; the pathological injury of liver tissue gradually aggravated over the time of D-GalN/LPS stimulation and fulfilled the criteria for ALF at 6 hours. The mRNA and protein expression of autophagy-related genes (ATG-7, ATG-5, Beclin-1, Lamp-1, and LC3a) increased in the early and medium stages of ALF (2 and 4 hours) and decreased after ALF progressed to liver failure (6 hours). As was observed via the fluorescence microscope, the 4-hour D-GalN/LPS-induced ALF model group showed the highest expression of autophagosome. Conclusion: The expression of autophagy gradually increases in the early and medium stages of ALF and decreases when ALF progresses to liver failure. Therefore, autophagy plays an important role in the pathogenesis of ALF.

[Role of glycogen synthase kinase-3ß in development of severe hepatitis liver failure caused by hepatitis B virus].

OBJECTIVE: To investigate the role of glycogen synthase kinase-3ß (GSK3ß) in the development of severe hepatitis liver failure (SHLF) caused by the hepatitis B virus. METHODS: Twelve patients with chronic hepatitis B (CHB) (CHB group), 12 patients with SHLF caused by hepatitis B virus (SHLF group), and 8 normal subjects (control group), who were admitted to Beijing You'an Hospital from January 2009 to December 2011, were included in this study. Their liver tissues were collected to do some clinical examinations. The GSK3ß activity in the liver tissue was detected with a GSK3ß activity assay kit. Western blot was used to determine the expression of p-GSK3, total GSK3, and -actin. The paraffin sections of liver tissue were prepared for immunofluorescence assay. All data were expressed as mean±standard deviation, and comparison between groups was made by least significant difference t test. P < 0.05 was considered statistically significant. RESULTS: Western blot results showed that compared with the control group, the CHB group had a higher level of p-GSK3ß and the SHLF group had a significantly lower level of p-GSK3ß (P = 0.0342). The immunofluorescence assay results showed that the SHLF group had a significantly lower level of p-GSK3ß than the control group. GSK3ß activity assay results showed that compared with the control group, the CHB group had a significantly lower GSK3ß activity and the SHLF group had a significantly higher GSK3ß activity (P = 0.0289), which were consistent with the results of Western blot and immunofluorescence assay. CONCLUSION: GSK3 is activated in the development of SHLF, so it is an important signaling molecule in the pathogenesis of SHLF. Inhibiting its activity may play a role in the prevention and treatment of SHLF.

Gradual electroforming and memristive switching in Pt/CuO(x)/Si/Pt systems.

We report a memristive switching effect in Pt/CuOx/Si/Pt devices prepared by the rf sputtering technique at room temperature. Differently from other Cu-based metal filament switching systems, a gradual electroforming process, marked by a gradual increase of the device resistance and a gradual decrease of the device capacitance, was observed in the current-voltage and capacitance characteristics. After the gradual electroforming, the devices show a uniform memristive switching behavior. By Auger electron spectroscopy analysis, a model based on the thickness change of the SiOx layer at the CuOx/Si interface and Cu ion migration is proposed for the gradual electroforming and uniform memristive switching, respectively. This work should be meaningful for the preparation of forming-free and homogeneous memristive devices.

Changes in quantity and spectroscopic properties of water-extractable organic matter during soil aquifer treatment.

The aim of this study was to identify qualitative and quantitative changes in the character of water-extractable organic matter (WEOM) in soils as a consequence of soil aquifer treatment (SAT). Soil samples were obtained from a soil-column system with a 2-year operation, and divided into seven layers from top to bottom: CS1 (0-12.5 cm), CS2 (12.5-25 cm), CS3 (25-50 cm), CS4 (50-75 cm), CS5 (75-100 cm), CS6 (100-125 cm) and CS7 (125-150 cm). A sample of the original soil used to pack the columns was also analysed to determine the effects of SAT. Following 2 years of SAT operation, both soil organic carbon and water-extractable organic carbon were shown to accumulate in the top soil layer (0-12.5 cm), and to decrease in soil layers deeper than 12.5 cm. The WEOM in the top soil layer was characterized by low aromaticity index (AI), low emission humification index (HIX) and low fluorescence efficiency index (F(eff)). On the other hand, the WEOM in soil layers deeper than 12.5 cm had increased values of HIX and F(eff), as well as decreased AI values relative to the original soil before SAT. In all soil layers, the percentage of hydrophobic and transphilic fractions decreased, while that of the hydrophilic fraction increased, as a result of SAT. The production of the amide-2 functional groups was observed in the top soil layer. SAT operation also led to the enrichment of hydrocarbon and amide-1 functional groups, as well as the depletion of oxygen-containing functional groups in soil layers deeper than 12.5 cm.

[Non-muscle myosin heavy chain 9 gene-related disorders with thrombocytopenia: report of two pedigrees and literature review].

Objective: To summarize the clinical characteristics and gene variants of 2 pedigrees of non-muscle myosin heavy chain 9 related diseases (MYH9-RD) in children. Methods: The basic information, clinical features, gene variants and laboratory tests of MYH9-RD patients from 2 pedigrees confirmed in the First Affiliated Hospital of Zhengzhou University in November 2021 and July 2022 were analyzed retrospectively. "Non-muscle myosin heavy chain 9 related disease" "MYH9" and "children" were used as key words to search at Pubmed database, CNKI and Wanfang database up to February 2023. The MYH9-RD gene variant spectrum and clinical data were analyzed and summarized. Results: Proband 1 (male, 11 years old) sought medical attention due to epistaxis, the eldest sister and second sister of proband 1 only showed excessive menstrual bleeding, the skin and mucous membrane of the their mother were prone to ecchymosis after bumping, the uncle of proband 1 had kidney damage, and the maternal grandmother and maternal great-grandmother of proband 1 had a history of cataracts. There were 7 cases of phenotypic abnormalities in this pedigree. High-throughput sequencing showed that the proband 1 MYH9 gene had c.279C>G (p.N93K) missense variant, and family verification analysis showed that the variant was inherited from the mother. A total of 4 patients including proband 1 and family members were diagnosed with MYH9-RD. The proband 2 (female, 1 year old) sought medical attention duo to fever and cough, and the father's physical examination revealed thrombocytopenia. There were 2 cases of phenotypic abnormalities in this pedigree. High-throughput sequencing showed that there was a c.4270G>A (p.D1424N) missense variant in the proband 2 MYH9 gene, and family verification analysis showed that the variant was inherited from the father. A total of 2 patients including proband 2 and his father were diagnosed with MYH9-RD. A total of 99 articles were retrieved, including 32 domestic literatures and 67 foreign literatures. The MYH9-RD cases totaled 149 pedigrees and 197 sporadic patients, including 2 pedigrees in our study. There were 101 cases with complete clinical data, including 62 sporadic cases and 39 pedigrees. There were 56 males and 45 females, with an average age of 6.9 years old. The main clinical manifestations were thrombocytopenia, skin ecchymosis, and epistaxis. Most patients didn't receive special treatment after diagnosis. Six English literatures related to MYH9-RD caused by c.279C>G mutation in MYH9 gene were retrieved. Italy reported the highest number of cases (3 cases). Twelve literatures related to MYH9-RD caused by c.4270G>A mutation in MYH9 gene were retrieved. China reported the highest number of cases (9 cases). Conclusions: The clinical manifestations of patients in the MYH9-RD pedigrees varied greatly. MYH9 gene c.279C>G and c.4270G>A mutations are the cause of MYH9-RD.

Association of CD14 G(-1145)A and C(-159)T polymorphisms with reduced risk for tuberculosis in a Chinese Han population.

Although the role of CD14 in mediating signals from Toll-like receptors to recognize Mycobacterium tuberculosis is known, how polymorphisms in this gene affect the susceptibility to develop tuberculosis are still not clear. We examined whether single nucleotide polymorphisms at positions -1145 and -159 in the promoter region of the CD14 gene are associated with tuberculosis in a Chinese Han population in a case-control study of 432 Chinese patients with tuberculosis and 404 ethnically matched healthy controls. Genotyping was performed to identify polymorphisms of the CD14 gene by PCR-DNA sequencing. Both the frequency of allele T in the C(-159)T polymorphism (odds ratio (OR) = 1.4; 95% confidence interval (95%CI) = 1.148-1.708) and allele G in the G(-1145)A polymorphism (OR = 1.512; 95%CI = 1.236- 1.849) were significantly more frequent in cases than in controls. The frequencies of genotypes CC and CT in the C(-159)T polymorphism, as well as the frequencies of genotypes AA and AG, were lower in cases than in controls. Based on our results, we conclude that G(-1145)A and C(-159)T polymorphisms of CD14 are associated with decreased risk for the development of tuberculosis in the Chinese Han population.

[Analysis of 8 cases of asparaginase related cerebral venous sinus thrombosis in children].

Objective: To summarize the clinical features, treatment and prognosis of asparaginase (ASP) related cerebral venous sinus thrombosis (CVST). Methods: Clinical profiles including age, sex, first symptoms, coagulation function, imaging findings, ASP type, treatment and prognosis of eight acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) children with ASP related CVST at the Department of Pediatrics, First Affiliated Hospital of Zhengzhou University from November 2016 to October 2021 were analyzed retrospectively. Results: Eight CVST children were all male, including 6 ALL and 2 LBL, with the onset age ranged from 5 to 15 years, 6 cases occurred in the stage of first induction remission, and the initial symptom were mainly epileptic seizures (7 cases). Magnetic resonance imaging combined magnetic resonance venography (MRV) showed the most common site of venous sinus enlargement was superior sagittal sinus (8 cases). Secondary cerebral hemorrhage was found in 5 cases. D-dimer elevated on the day of onset in all cases. Three patients were treated with intravascular mechanical thrombectomy and thrombolysis combined with anticoagulant therapy, 3 patients were treated with continuous anticoagulant therapy only, 2 patients were not treated with anticoagulant therapy. MRV follow-up for 3 months showed that the thrombi in patients were almost completely absorbed except in 2 patients who were not treated with anticoagulant therapy. Thrombolysis combined with anticoagulant therapy was the fastest way for thrombosis absorption. Among 8 patients, 1 died of early recurrence of ALL, and 7 patients accepted further asparaginase and no CVST recurrence or progression was found. There were no sequelae of nervous system except 1 patient with left upper limb muscle strength impairment. Conclusions: ASP related CVST is more common in older male children and the prognosis is good. ASP related CVST occurred mostly in the stage of first induction remission, and most initial manifestation is epileptic seizure. The superior sagittal region is a common site of thrombus, magnetic resonance imaging combined with MRV is helpful for accurately diagnosis. Timely anticoagulant treatment can improve the prognosis, and mechanical thrombectomy and thrombolysis can quickly recanalize the vessel.

[Clinical characteristics and prognosis of 83 children with newly treated hepatoblastoma].

Objective: To investigate the clinical features, survival and prognostic risk factors of children with hepatoblastoma (HB). Methods: Clinical data of 83 children with newly treated HB at the Department of Hematology and Oncology, Children's Hospital, the First Affiliated Hospital of Zhengzhou University from January 2012 to October 2019 were analyzed retrospectively. The sex, age, first clinical manifestations, pretreatment extent of disease (PRETEXT) stages, pathological types, initial alpha-fetoprotein (AFP), treatment methods and treatment outcome of all patients were summarized. The children diagnosed before 2018 were treated with "Wuhan Protocol", and those who diagnosed after 2018 were treated with the "Expert Consensus for Multidisciplinary Management of Hepatoblastoma"(CCCG-HB-2016) protocol. Kaplan-Meier survival analysis was used to calculate the survival rate, Log-Rank test was used in univariate analysis, and the Cox regression model was used in multivariate prognosis analysis. Results: Among 83 cases, there were 51 males and 32 females. The age of onset was 25.2 (9.0, 34.0) months old, and 64 cases (77%) were under 3 years old. The most common first clinical manifestation was abdominal mass in 45 cases (54%). There were 8 cases of PRETEXT stage Ⅰ, 43 cases of stage Ⅱ, 20 cases of stage Ⅲ and 12 cases of stage Ⅳ. During the follow-up period of 40 (17, 63) months, the 1-year overall survival (OS) rate and event-free survival (EFS) rate were (84±4) % and (79±5) %, respectively, and 5-year OS rate and EFS rate were (78±5) % and (76±5) %, respectively. Fifty-five cases were treated with "Wuhan Protocol", and the 5-year OS and EFS rate were (73±6) % and (71±6) %, respectively. Twenty-eight cases were treated with CCCG-HB-2016 protocol, and the 5-year OS and EFS rate were (88±7) % and (82±9) %, respectively. Multivariate COX regression analysis showed that AFP did not turn negative after 3 courses of postoperative chemotherapy (HR=9.228, 95%CI 1.017-83.692) and PRETEXT stage Ⅳ (HR=6.587, 95%CI 1.687-25.723) were independent risk factors affecting the prognosis of children with HB. Conclusions: The "Wuhan Protocol" and CCCG-HB-2016 protocol were effective in the treatment of children with HB. AFP did not turn negative after 3 courses of postoperative chemotherapy and PRETEXT stage Ⅳ were independent risk factors affecting the prognosis of children with HB.

Analysis results of 579 cases of genomic copy number variation sequencing of pregnant women in prenatal diagnosis.

OBJECTIVE: This study explored the usefulness of genomic copy number variation sequencing (CNV-Seq) in the prenatal diagnosis of pregnant women. PATIENTS AND METHODS: Based on prenatal diagnostic indications, CNV-Seq analysis was done in the samples from the 579 pregnant women of the 7 subgroups that included advanced maternal age (group A), high risk noninvasive prenatal test (NIPT) (group B), high risk Down's (Group C), abnormal ultrasound findings (Group D), adverse pregnancy history (Group E), chromosome abnormalities in couples (Group F), and the mixed group (Group G). RESULTS: A total of 57 (9.84%) cases have abnormal CNV-Seq results. Among them, 21 cases were aneuploid chromosomal number abnormalities (3.63%, 21/579), and 36 cases were CNV abnormalities (6.22%, 36/579), including 7 cases of pathogenic copy number alteration (pCNA) (1.21%, 7/579) and 29 cases variants of uncertain significance (VUS) (5.01%, 29/579). The total detection rates of abnormal CNV-Seq in Group G and Group B were 20.27% (15/74) and 15.91% (14/88), which were significantly higher than those in other groups (p < 0.05). Among 36 cases of abnormal CNV-Seq, 7 cases were chromosome fragment deletion or duplication, which were pathogenic CNV, and some rare chromosomal diseases were detected. CONCLUSIONS: Patients with a high risk of NIPT or multiple indications of prenatal diagnosis are highly suspected of chromosomal diseases. CNV-Seq is a useful tool for detecting chromosome abnormalities for prenatal diagnosis of pregnant women more accurately and provides more comprehensive information for prenatal diagnosis to reduce birth defects.

The influence of a bisphosphonate on bone generation determined using a chick-femur model.

AIM: To determine the direct influence of a bisphosphonate (pamidronate) delivered by one of two carriers, on bone generation in chick-femurs supported by chick egg chorio-allantoic membranes. METHODOLOGY: Twenty chick femurs freshly harvested from fertilized eggs were randomly allocated to two groups: (i) Affi-Gel blue bead carrier (n=10); and (ii) hydroxyapatite bead carrier (n=10). The femurs in each group were further randomly divided into control (n=4) and experimental (n=6) subgroups. Carriers charged with PBS solution or 0.1 M pamidronate were delivered into the bone marrow of each femur at its mid-portion through a needle puncture. Each femur was then grafted onto the chorio-allantoic membrane of a chick egg and incubated for 7 days. Each experimental and control subgroup femur yielded four histological sections at the puncture site, constituting the test and inter-bone controls. In addition, two histological sections were also obtained from 400 to 450 µm away from each end of the experimental puncture site to act as the intra-bone controls. Bone generation was quantified and the ratio of cross-sectional area of bone marrow to circumference of bone (outcome measure) was determined using a software package, Image-Pro(®) Plus. The data were analysed using Mann-Whitney tests and Wilcoxon signed rank tests. RESULTS: The outcome ratio in the test group was significantly (P<0.001) smaller than both the inter-bone and intra-bone control groups. There was evidence of increased bone formation directly over the pamidronate-charged carriers. CONCLUSIONS: The test model established that pamidronate had a positive effect on bone generation over a period of 7 days, regardless of the carrier type.

Effect of ultrasonic and alkaline pretreatment on sludge degradation and electricity generation by microbial fuel cell.

Both ultrasonic and alkaline pretreatment of excess sewage sludge were investigated to enhance organic degradation and electricity generation from sludge by the subsequent microbial fuel cell (MFC). The ultrasonic pretreatment showed that the degree of sludge disintegration was directly related to the energy input, ultrasonic density and duration. Alkaline pretreatment demonstrated that more soluble organic matters were released from the sludge with more NaOH dose and longer reaction time, and the degree of sludge disintegration within 30 min accounted for 45-76% of that for 24 h. When ultrasonic and alkaline pretreatment were combined, the released chemical oxygen demand (COD) was higher than those with ultrasonic or alkaline pretreatment alone. Ultrasonic and alkaline (pH=11) pretreatment could enhance electricity generation from sludge by the subsequent MFC, resulting in more degradation of total COD (TCOD) and volatile solids (VS). Slight change in power output from the MFC was observed due to the higher soluble chemical oxygen demand (SCOD) in the pretreated sludge. By using the combined ultrasonic and alkaline pretreatment of sludge, the removal efficiencies of TCOD and VS were increased from 27.1% to 61.0% and 35.2% to 62.9% in comparison with raw sludge, respectively, and the power output in MFC was slightly increased from 10.3 W/m(3) to 12.5 W/m(3).

Utility of the lateral arm free flap in oral cavity reconstruction: a single-centre experience with Chinese patients.

The lateral arm flap (LAF) may offer an alternative option for oral cavity repair. Twenty-five Chinese patients with oral cavity defects were reconstructed with a LAF. The anatomical characteristics of the flap, the donor site complications, and the functional and aesthetic assessments of recipient site were reviewed. The overall flap survival was 96.0% (24/25patients). The average pedicle length was 7.07±1.09cm when it was cut off at the insert of the deltoid, with an average arterial diameter of 1.30±0.37mm and vein diameter of 2.06±0.48mm. The average flap length was 7.06±1.01cm, and the average flap breadth was 5.28±0.66cm, with the average flap size ranging from 18 to 42cm2. One to three reliable perforators supplied the flap, with the proximal, middle, and distal perforators being located at 9.9±1.1cm, 8.6±1.4cm, and 5.7±1.2cm from the lateral epicondyle, respectively. The donor defect was closed primarily and healed uneventfully. A longitudinal scar was the most common morbidity of the donor site. The function and shape of the reconstructed tissues were well restored. The LAF provides a reliable choice for reconstructing medium-sized oral cavity defects, with minimal donor-site morbidity and ideally functional and aesthetic rehabilitation of the recipient site.

Induction of diabetes in rhesus monkeys and establishment of insulin administration strategy.

OBJECTIVES: The diabetic rhesus monkey seems to be a useful model for preclinical investigations of islet transplantation and new drug treatments for type 1 diabetes mellitus (T1DM). Information is limited regarding a standard technique to induce and assess diabetes in rhesus monkeys as well as the strategy to apply insulin administration. Herein, we have established and characterized a model of diabetic rhesus macaques. METHODS: Four monkeys were divided into 2 groups of 2 each: group 1, total pancreatectomy; and group 2, partial pancreatectomy (75%) with low-dose streptozotocin (STZ) administration. Pancreatic function was measured using intravenous glucose tolerance tests before the operation. Spiral computed tomography (CT) scans of the pancreas were obtained before and after pancreatectomy. Fasting blood glucose and postprandial blood glucose levels were monitored twice daily using blood samples from the fingers or toes. Various types and doses of insulin were administered twice daily. We performed regular assessments of hematological and serum biochemical parameters, insulin, and C-peptide. RESULTS: Both total pancreatectomy and partial pancreatectomy (75%) with STZ administration induced T1DM in rhesus monkeys; there was interindividual variation in the STZ dose. Excluding C-peptide and insulin, the hematological and serum biochemical parameters did not differ significantly from normal values postoperatively. The various insulin treatment strategies are achieved stable blood glucose (BG) levels. CONCLUSIONS: STZ injection after partial pancreatectomy may be a safe, reproducible method to induce T1DM. Porcine insulin administration was a safe, economical method to control BG levels in a diabetic rhesus monkey before islet transplantation.

[Review on the etiology and complications of hand, foot and mouth disease, using data from the national sentinel surveillance program, in China, 2015-2016].

Objective: To understand the characteristics relating to the etiology and complications of hand, foot and mouth disease (HFMD) based on data from the pilot National Sentinel Surveillance (NSS) program so as to explore the feasibility, advantages and disadvantages of the NSS. Methods: Data were extracted from the NSS system, conducted in 11 provinces of China from November 2015 to October 2016. Characteristics regarding the etiology, complications of HFMD and factors related to the positive rates of HFMD specimens were analyzed under the logistic regression method by SPSS 20.0 software. Results: A total of 4 783 specimens were collected, including 3 390 from mild, 1 390 from severe and 3 from death cases. The overall positive rate was 81.43% (3 895/4 783). Other enteroviruses (non EV71/Cox A16 enteroviruses) appeared the major serotype (52.68%, 1 482/2 813) for mild infection of the disease while EV71 was for the severe cases (65.31%, 706/1 081). The serotype spectrum revealed by the pilot NSS was almost identical with the existing surveillance system. Other enteroviruses tended to infect younger children (χ(2)=130.17, P<0.001) than EV71 and Cox A16, in China. The multivariate logistic regression results showed that higher positive rate was associated with specimens which were collected from males, at children' hospitals, in peak seasons, timely and in stools. The positive rates presented downwarding trends with the extension of the onset-sampling interval (χ(2)=14.47, P<0.001 in stool specimen; χ(2)=31.99, P<0.001 in throat swab; χ(2)=24.26, P<0.001 in anal swab). Aseptic meningitis, non-brainstem encephalitis and brainstem encephalitis appeared the top three complications of both EV71-associated and other enteroviruses-associated severe HFMD cases. Conclusions: Factors as gender, season/place/timeliness of specimen collection, and types of hospital all appeared independently influenced the positive rates. NSS seemed feasible to be used as an alternative or supplement tool to the existing surveillance program in China.

Mesenchymal stem cells from Chinese Guizhou minipig by hTERT gene transfection.

INTRODUCTION: To provide standard implemental cells for biomedical research, an immortalized mesenchymal stem cell (MSC) lineage was established from the bone marrow of Chinese Guizhou minipigs. We investigated the characteristics of the MSC transfected with hTERT. METHODS: MSCs isolated from bone marrow (BM) were steadily transfected using a plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT) at population doubling (PD) time of 3. The expression of hTERT was analyzed by reverse transcriptase polymerase chain reaction; the morphology of the cells, by phase contrast microscope; cell growth curve, by MTS assay, and phenotype, flow cytometry and immunofluorescence. Karyotype and osteogenic differentiation of transfected cells were compared with normal MSCs. RESULTS: After transfection with the hTERT gene, the MSCs showed vigorous proliferation activity undergoing more than 60 PDs. Expression of the hTERT gene was detected in these cells. Transfected MSCs showed a prolonged life span, maintaining similar morphology and karyotype compared with normal MSCs. The apoptotic rate of transfected MSCs at PD60 was far below that of the normal MSCs at PD15. More than 99% of transfected MSCs were positive for stem cell markers, including SH-2, SH-3, SB-21, and CD29, and negative for CD34, CD45, and SLA-II. Transfected MSCs possessed the ability to differentiate into osteogenic as same as that of normal MSCs. CONCLUSION: The hTERT gene-induced immortalization prolonged the life span of MSCs, maintaining the overall properties identical to the original cells, findings that will be useful for basic research.

Immortalization of mesenchymal stem cells from bone marrow of rhesus monkey by transfection with human telomerase reverse transcriptase gene.

INTRODUCTION: Our previous experiments indicated that bone marrow mesenchymal stem cells of rhesus monkey (RhBMSCs) have a low proliferative ability with a finite life span, which will hamper their application in biomedical research. Establishing an immortalized RhBMSC lineage might solve the problem. METHODS: RhBMSCs isolated from the bone marrow of rhesus monkeys using density gradient centrifugation were purified using adherence separation. Then, the cells were steadily transfected by plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT). We analyzed expression of hTERT, proliferation, phenotype (SH-2, SH-3, SB-10, CD29, CD34, CD45, and HLA-DR), differentiation toward osteogenic lineage, karyotype, and tumorigenesis of transfected cells. RESULTS: After transfection, the RhBMSCs proliferated vigorously, undergoing more than 50 population doublings (PDs). Apoptotic rate of transfected RhBMSCs at PD40 was only 4.5%, versus untransfected RhBMSCs at PD15, which was more than 33.5%. Compared with normal RhBMSC, the life span of transfected RhBMSCs was prolonged, retaining similar morphology, karyotype, and potential to differentiate into an osteogenic lineage. More than 99% of transfected RhBMSCs were positive for stem cell markers, including SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR. Furthermore, the transfected cell line was benign in nude mice tumor formation. CONCLUSION: Our results demonstrated that hTERT gene had been transfected into RhBMSCs. The transfected RhBMSCs proliferated vigorously. Phenotype, differentiation, and karyotype of transfected RhBMSC showed no significant difference from untransfected cells. The transfected RhBMSCs are a potential cell source for transplantation as well as tissue engineering.

Short and long peptidoglycan recognition proteins (PGRPs) in zebrafish, with findings of multiple PGRP homologs in teleost fish.

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (tn: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response.