First report of leaf spot on Gleditsia sinensis caused by Colletotrichum gloeosporioides in China.

Gleditsia sinensis Lam (Lamarck et al., 1788) is an endemic species widely distributed in China. In Sep. 2022, leaf spot symptoms were observed on G. sinensis in Xuhui district (31◦9'16''N, 121◦26'36''E), Shanghai, China, with an incidence rate of 55% in the examination of 9 trees. The leaves showed typical symptoms of anthracnose with irregular gray-brown spots and sunken areas. For isolation, 5 × 5 mm sections were cut from the lesion edge of 20 infected leaves collected from 2 trees. The surface of the sections was sterilized by immersion in 75% ethanol for 30 s, followed by 5% NaClO for 1 min, rinsed three times with sterile water, and dried on sterile filter paper. These sections were placed on PDA plates incubated at 25°C in darkness. Eighteen isolates with similar colony morphology were obtained and purified by single spore culturing. Two isolates (YKY2301, 2302) from separate trees were further tested. On the 6th day, the colonies had a diameter of 7.6 to 8.4 cm and appeared white to gray-white with aerial hyphae. The colony's central part exhibited an orange hue due to the conidia accumulation, while the undersides displayed an orange-yellow color. The hyphae were hyaline and smooth, with septa and branches, and the conidia were cylindrical with blunt to slightly rounded ends, measuring 13.1 to 18.8 (average 15.9) µm× 4.0 to 6.6 (average 5.4) µm (n=184). From conidia germinated on glass slides, the appressoria measured 5.5 to 6.3 µm ×4.9 to 5.1 µm (n=50) and were nearly spherical or elliptical in shape. These characteristics matched those of the Colletotrichum gloeosporioides species complex (Cannon et al., 2012; Weir et al., 2012). For molecular identification, the genomic DNA was extracted using a modified CTAB method (Luo et al., 2012). Gene fragments including ITS (PP125667, PP125668), GAPDH (PP153428, PP153429), ACT (PP153424, PP153425), TUB2(PP153917, PP190256), and ApMAT (PP153426, PP153427) were obtained by PCR using universal primers (Huang et al., 2022) and sequenced. The sequences exhibited 98.19% to 99.82% identity with the corresponding gene of the type strain C. gloeosporioides IMI356878 (JX010152, JX010056, JX009531, JX010445, JQ807843) in NCBI BLAST. A multilocus Maximum likelihood phylogenetic tree was constructed based on concatenated the five genes by PhyloSuite. It showed that YKY2301, 2302 were on the same branch with C. gloeosporioides. Based on these results, the isolates were identified as C. gloeosporioides. Pathogenicity tests were conducted by mycelial and conidia inoculation. 5 mm mycelial or blank agar plugs were inoculated onto the leaves of 2 healthy trees in a garden (25 to 30 °C), with and without wounds made by toothpick pricking (n≥3 per group). All mycelial inoculated leaves showed leaf spots on the 6th day. Three healthy 2-year-old seedlings were inoculated with either conidia (108 conidia/ml) or water by leaf spray, and maintained in a climate chamber (27 °C, 80% humidity). Inoculated seedlings showed necrotic leaf spots on day 14, and wilted within 3 weeks. The controls in all tests remained asymptomatic. The pathogen has been re-isolated and confirmed by sequencing, thus fulfilling Koch's postulates. This is the first report of leaf spots caused by C. gloeosporioides on G. sinensis in the world. As illustrated by the example of legume pod infection (Gerusa et al., 2019), it poses a potential threat to the fruits of G. sinensis, despite currently only affecting their ornamental value. This report provides basic information for future research.

MicroRNA-142-3p, a novel target of tumor suppressor menin, inhibits osteosarcoma cell proliferation by down-regulation of FASN.

Menin, encoded by MEN1 gene, has been viewed as a tumor suppressor in several types of tumors, such as insulinoma, parathyroid tumor, and adrenocortical and lung carcinoma. However, its expression and molecular regulation mechanism in osteosarcoma has not been elucidated. Here, our results show menin expression is significantly down-regulated in osteosarcoma tissues, compared with adjacent normal tissues. Besides, we report that MicroRNA-142-3p as a novel target of menin. Up-regulation of MicroRNA-142-3p by menin overexpression inhibits cell proliferation in U2OS and MG63 cells. At the molecular level, MicroRNA-142-3p inhibits the protein expression of FASN through binding to its 3'-untranslated region. Therefore, we elucidate a novel regulation pathway in osteosarcoma cells and suggest a potential therapeutic approach for the tumor therapy.

MicroRNA-32 inhibits osteosarcoma cell proliferation and invasion by targeting Sox9.

Increasing reports suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for human cancers, including osteosarcoma. Previous studies have shown that miR-32 was dysregulated in breast and endometrial cancer. However, its biological roles in osteosarcoma remain unclear. In the current study, we found that miR-32 was significantly down-regulated in osteosarcoma tissues, compared with the adjacent normal tissues. In vitro studies further demonstrated that miR-32 mimics were able to suppress, while its antisense oligos promoted cell proliferation in Saos-2 and U2OS cells. At the molecular level, our data further revealed that expression of Sox9 was negatively regulated by miR-32. Therefore, our results identify an important role for miR-32 in the osteosarcoma through regulating Sox9 expression.

MicroRNA-126 inhibits osteosarcoma cells proliferation by targeting Sirt1.

Numerous studies have recently suggested that miRNAs contribute to the development of various types of human cancer as well as to their proliferation and metastasis. The aim of this study was to investigate the functional significance of miR-126 and to identify its possible target genes in osteosarcoma (OS) cells. Here, we found that expression level of miR-126 was reduced in osteosarcoma cells in comparison with the adjacent normal tissues. The enforced expression of miR-126 was able to inhibit cell proliferation in U2OS and MG63 cells, while miR-126 antisense oligonucleotides (antisense miR-126) promoted cell proliferation. At the molecular level, our results further revealed that expression of Sirt1, a member of histone deacetylase, was negatively regulated by miR-126. Therefore, the data reported here demonstrate that miR-126 is an important regulator in osteosarcoma, which will contribute to better understanding of the important misregulated miRNAs in osteosarcoma cells.

Clinical application of SARS-CoV-2 antibody detection and monoclonal antibody therapies against COVID-19.

The purpose of this study was to investigate the clinical application of severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) specific antibody detection and anti-SARS-CoV-2 specific monoclonal antibodies (mAbs) in the treatment of coronavirus infectious disease 2019 (COVID-19). The dynamic changes of SARS-CoV-2 specific antibodies during COVID-19 were studied. Immunoglobulin M (IgM) appeared earlier and lasted for a short time, while immunoglobulin G (IgG) appeared later and lasted longer. IgM tests can be used for early diagnosis of COVID-19, and IgG tests can be used for late diagnosis of COVID-19 and identification of asymptomatic infected persons. The combination of antibody testing and nucleic acid testing, which complement each other, can improve the diagnosis rate of COVID-19. Monoclonal anti-SARS-CoV-2 specific antibodies can be used to treat hospitalized severe and critically ill patients and non-hospitalized mild to moderate COVID-19 patients. COVID-19 convalescent plasma, highly concentrated immunoglobulin, and anti-SARS-CoV-2 specific mAbs are examples of anti-SARS-CoV-2 antibody products. Due to the continuous emergence of mutated strains of the novel coronavirus, especially omicron, its immune escape ability and infectivity are enhanced, making the effects of authorized products reduced or invalid. Therefore, the optimal application of anti-SARS-CoV-2 antibody products (especially anti-SARS-CoV-2 specific mAbs) is more effective in the treatment of COVID-19 and more conducive to patient recovery.

Multiple synostoses syndrome is due to a missense mutation in exon 2 of FGF9 gene.

Fibroblast growth factors (FGFs) play diverse roles in several developmental processes. Mutations leading to deregulated FGF signaling can cause human skeletal dysplasias and cancer.(1,2) Here we report a missense mutation (Ser99Asp) in exon 2 of FGF9 in 12 patients with multiple synostoses syndrome (SYNS) in a large Chinese family. In vitro studies demonstrate that FGF9(S99N) is expressed and secreted as efficiently as wild-type FGF9 in transfected cells. However, FGF9(S99N) induces compromised chondrocyte proliferation and differentiation, which is accompanied by enhanced osteogenic differentiation and matrix mineralization of bone marrow-derived mesenchymal stem cells (BMSCs). Biochemical analysis reveals that S99N mutation in FGF9 leads to significantly impaired FGF signaling, as evidenced by diminished activity of Erk1/2 pathway and decreased beta-catenin and c-Myc expression when compared with wild-type FGF9. Importantly, the binding of FGF9(S99N) to its receptor is severely impaired although the dimerization ability of mutant FGF9 itself or with wild-type FGF9 is not detectably affected, providing a basis for the defective FGFR signaling. Collectively, our data demonstrate a previously uncharacterized mutation in FGF9 as one of the causes of SYNS, implicating an important role of FGF9 in normal joint development.

[Efficacy of catheter-based renal denervation in mongrel neurogenic hypertensive dogs].

OBJECTIVE: Hypertension is the most common risk factor for cardiovascular diseases and stroke. Renal sympathetic hyperactivity is associated with hypertension. The aim of this study was to explore the efficacy of renal denervation for hypertension. METHODS: Eighteen mongrel neurogenic hypertensive dogs were divided into intervention [mean arterial pressure: (150.6 ± 18.8) mm Hg (1 mm Hg = 0.133 kPa) plus renal sympathetic denervation by percutaneous catheter-based radiofrequency, n = 10] and control [mean arterial pressure (147.4 ± 13.2) mm Hg, n = 8] group. Mean arterial pressure before and at 2, 4, 6 and 10 weeks after procedure was invasively measured. Renin activity (PRA), angiotensin II (AngII), aldosterone (Ald), and creatinine (Cr) were detected at 2, 6 and 10 weeks after procedure. RESULTS: Mean arterial pressure remained unchanged in control group. In intervention group, mean arterial pressure (MAP) decreased to (130.4 ± 14.1) mm Hg, (136.2 ± 17.1) mm Hg, (128.7 ± 14.7) mm Hg and (126.1 ± 12.7) mm Hg respectively at 2, 4, 6, and 10 weeks after procedure. Meanwhile, the level of PRA, AngII, Ald significantly reduced post procedure compared with pre-procedural level (P < 0.05) and the Cr level remained unchanged post procedure (P > 0.05). CONCLUSION: Sympathetic nervous system plays an important role in the progression of hypertension. Catheter-based renal denervation results in substantial and sustained blood-pressure reduction in this model.

Epidemiological Characteristics of COVID-19 Outbreak in Yangzhou, China, 2021.

Objective: Epidemiological characteristics of COVID-19 outbreak in Yangzhou city caused by the highly contagious Delta variant strain of SARS-CoV-2 virus were investigated in this retrospective descriptive study to provide prevention and control guidelines for outbreaks in the future. Methods: All the epidemiological data used in this study were collected manually from the official website of the Yangzhou Municipal Health Committee from 28 July to 26 August 2021, and then were analyzed systematically and statistically in this study. Results: A total of 570 COVID-19 cases were reported during the short-term outbreak in Yangzhou City. The ages of infected individuals ranged from 1 to 90 years with the average age at 49.47 ± 22.69 years. As for gender distributions, the ratio of male- to-female patients was 1:1.36 (242:328). Geographic analysis showed that 377 patients (66.1%) were in Hanjiang District while 188 patients (33.0%) were in Guangling District. Clinical diagnosis showed that 175 people (30.7%) had mild symptoms, 385 people were in moderate conditions (67.5%), and 10 people were in severe situations (1.8%). Significant age differences were found among the three groups (P < 0.001). However, no significant difference was identified in terms of gender ratio (P > 0.05). Based on the transmission chain formed by 6 generations of infected persons with a clear transmission relationship, the age showed a gradually decreasing trend, while the median time of diagnosis in 2 adjacent generations was 3 days. In addition, the estimated basic reproduction number R 0 of the Delta variant was 3.3651 by the classical Susceptible, Infectious, and/or Recovered (SIR) model. Conclusion: The Delta variant of SARS-CoV-2 was highly infectious and has obvious clustering characteristics during the Yangzhou outbreak in China.

Mutations in a beta-tubulin confer resistance of Gibberella zeae to benzimidazole fungicides.

ABSTRACT Wheat head blight caused by Gibberella zeae (anamorph: Fusarium graminearum) is a threat to food safety in China because of mycotoxin contamination of the harvested grain, the frequent occurrence of the disease, and the failure of chemical control in some areas due to benzimidazole resistance in the pathogen population. The molecular resistance mechanism, however, of G. zeae to benzimidazole fungicides (especially carbendazim; active ingredient: methyl benzimidazol-2-yl carbamate [MBC]) is poorly understood. DNA sequences of a beta-tubulin gene (beta(2)tub) (GenBank access number FG06611.1) in G. zeae were analyzed. Mutations in beta(2)tub in moderately resistant strains (MBC(MR)) included TTT (Phe)-->TAT (Tyr) at codon 167 or TTC (Phe)-->TAC (Tyr) at codon 200. A highly resistant strain (MBC(HR)) had two point mutations, one at codon 73, CAG (Gln)-->CGG (Arg), and the other at codon 198, GAG (Glu)-->CTG (Leu). To confirm that mutations in the beta(2)tub confer resistance to benzimidazole fungicides, the entire beta(2)tub locus was deleted from MBC(MR) and MBC(HR) strains of G. zeae. The resulting Deltabeta(2)tub mutants from both MBC(MR) and MBC(HR) strains grew normally on MBC-free potato dextrose agar medium and were supersensitive to MBC. Complementation of the Deltabeta(2)tub mutants by transformation with a copy of the intact beta(2)tub locus from their parent strains exhibited less resistance than the original strains, and complementation of the Deltabeta(2)tub mutants by transformation with a copy of the intact beta(2)tub locus from sensitive strains restored MBC sensitivity. The results indicated that the mutations in the beta(2)tub gene conferred resistance of G. zeae to benzimidazole fungicides and this gene can be used as a genetic marker in G. zeae.

[Association of tryptophan hydroxylase gene A218C and serotonin transporter gene polymorphism with essential hypertension in Chinese northern Han population].

OBJECTIVE: To investigate the relationship between tryptophan hydroxylase (TPH) gene A218C in intron 7 and 5-hydroxytryptamine transporter (5-HTT) gene variable number tandem repeat (VNTR) in intron 2 and gene-linked polymorphic region (LPR) deletion/insertion polymorphism and essential hypertension (EH) in Chinese northern Han population. METHODS: A total of 280 EH patients and 200 normotensive controls were genotyped using polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: There were no significant differences in the frequencies of the genotypes and alleles of TPH gene A218C and 5-HTTVNTR between EH patents and controls (all P > 0.05). The genotype frequencies of SS, LS and LL in the 5-HTTLPR polymorphism was 68%, 29% and 3% in EH patients, 53%, 37% and 10% in the controls respectively (P < 0.01). The frequencies of allele S and L of the 5-HTTLPR were 82% and 18% in EH patients, 72% and 28% in the controls respectively (P < 0.01). Compared with the carriers of L allele (LS + LL), the EH risk was significantly higher in the SS homozygote (OR = 1.90, 95%CI = 1.31 - 2.77, P = 0.001). After adjustment of age, gender, body mass index, blood lipids, fasting blood glucose and blood uric acid level, the binary logistic regression analysis demonstrated that SS genotype in the 5-HTTLPR polymorphism was significantly related to occurrence of EH (OR = 1.47, 95%CI = 1.06 - 2.04, P = 0.021). CONCLUSIONS: The SS genotype of the 5-HTTLPR might be a susceptible gene to EH, while the TPH gene A218C and 5-HTTVNTR polymorphism is not associated with EH in Chinese northern Han population.

[CT-guided percutaneous removal of osteoid osteoma].

OBJECTIVE: To evaluate the methods of CT-guided percutaneous removal of osteoid osteoma. METHODS: From February 2003 to December 2003, there were 11 patients, at a mean age of 19.3 years old (from 14 to 32 years old). Seven cases were male, and 4 cases were female. Diagnosis was supported by the complete clinical and imaging file. Eleven cases with osteoid osteomas were treated with CT-guided percutaneous excision. The location involved were femoral neck (6 cases), acetabulum (4 cases), humeral neck (1 case). At first, CT scanning was done in the whole nidus with thin slices (2 mm thick), and the CT scan slice passing through the center of the nidus was selected. Then the whole nidus was removed by trephine along the guidewire which was inserted into the bone up to the nidus and was protected by the trocar. Finally complete lesion resection was checked with CT. RESULTS: Nine cases had a final pathological diagnosis of osteoid osteomas. Complete pain relief was obtained in these patients after the day of operation. There were no complications, no recurrence and patients remained symptom free at follow-up of 8-18 months (mean, 15.2 months). CONCLUSION: The methods of CT guided percutaneous removal of osteoid osteoma is a minimally invasive technique that represents the efficacy and safety.

Repair of radius defect with bone-morphogenetic-protein loaded hydroxyapatite/collagen-poly(L-lactic acid) composite.

OBJECTIVE: To explore the method to repair bone defect with bone-morphogenetic-protein loaded hydroxyapatite/collagen-poly(L-lactic acid) composite. METHODS: 18 adult beagle dogs were randomly divided into 3 groups. In Group A, bone-morphogenetic-protein (BMP) loaded hydroxyapatite/collagen-poly(L-lactic acid) (HAC-PLA) scaffold was implanted in a 2 cm diaphyseal defect in the radius. In Group B, unloaded pure HAC-PLA scaffold was implanted in the defects. No material was implanted in Group C (control group). The dogs were sacrificed 6 months postoperatively. Features of biocompatibility, biodegradability and osteoinduction were evaluated with histological, radiological examinations and bone mineral density (BMD) measurements. RESULTS: In Group A, the radius defect healed after the treatment with BMP loaded HAC-PLA. BMD at the site of the defect was higher than that of the contralateral radius. Fibrous union developed in the animals of the control group. CONCLUSIONS: BMP not only promotes osteogenesis but also accelerates degradation of the biomaterials. Optimized design parameters of a three-dimensional porous biomaterial would give full scope to the role of BMP as an osteoinductive growth factor.

Pattern of instent neointimal formation compared to native atherosclerosis in the coronary bifurcation lesions: volumetric intravascular ultrasound analysis.

BACKGROUND: No clinical study has systematically analyzed and compared circumferential neointimal and plaque distribution of stent neointimal proliferation and in native atherosclerotic plaques. This study aimed to investigate and compare the pattern of instent neointimal formation and native atherosclerosis in the coronary bifurcation lesions by volumetric analysis using systematic intravascular ultrasound (IVUS). METHODS: We examined bifurcation lesions in native coronary artery (plaque group, n = 102) and stented bifurcations at 9-month follow-up (neointima group, n = 51) using volumetric IVUS analysis of both the main vessel (MV) and side branch (SB). Three 5-mm segments were analyzed; the proximal MV (MVp), distal MV (MVd) and SB ostium (SBo). For each segment, volumetric analysis was performed in each of four quadrants (divided according to the branch takeoff and the geometric center of the lumen); carinal, epicardial, abcarinal, and myocardial. The eccentricity index was defined as the ratio of the abcarinal plaque (or neointimal) volume to the carinal plaque (or neointimal) volume. RESULTS: The plaque distribution differed significantly between the four quadrants, with the largest in the abcarinal quadrant, followed by the myocardial, epicardial, and carinal quadrants. The distribution of neointima was similar in the MV, but the four quadrants in the SB did not differ significantly. The eccentricity indices of both the MVd (P < 0.001) and SBo (P = 0.001) were significantly higher for the plaque group than the neointima group. CONCLUSIONS: The distribution of neointimal proliferation seems to have a similar pattern to that of atherosclerotic plaque in native coronary arteries, particularly in the main vessel, but the trend is less prominent.