First Report of Neopestalotiopsis clavispora Causing Fruit Brown Spot of Pomegranate in China.

Pomegranate (Punica granatum) is an important fruit crop for therapeutic and food applications. In June 2022, brown spots were observed on the fruit surface of pomegranate cultivar named Guangyan in Mengzi (23°20'6''N,103°25'5''E), Yunnan, China. The early spots appeared as circular or irregular lesions, measuring 1~1.5 mm in diameter. They were light brown with a clear boundary between disease and healthy tissues. Over time, these spots developed into polygonal lesions covering the entire fruit surface. Eventually, the diseased fruits decayed, and more than 50% of fruits were infected in pomegranate orchards. The tissues from the interface between health and disease were cut down, immersed in 75% ethanol for 15 s, then 5% NaOCl disinfecting for 2 min, washed three times with sterile water, and the PDA cultured at 26 °C in an incubator under dark conditions. Twenty-five samples were collected for pathogen isolation, ten fungal isolates were obtained by single spore germination, and these isolates had similar morphological characters. The colonies were white with 81 mm diameter at 7 days of incubation, containing undulate edges with dense aerial mycelium. After 14 days, the black conidiomata formed superficially, gathering into black droplets. Conidiogenous cells were hyaline, short, and filiform. Conidia were fusiform, straight or slightly curved, and comprised five cells, 24.12 to 34.53 (x̄=29.78) µm × 4.21 to 12.15 (x̄=8.68) µm (n=50). The three median cells were 13.13 to 25.22 µm (x̄=18.54), dark brown, whose septa and periclinal walls were darker than the other two cells. The apical cells showed two to four appendages, 12.31 to 29.15 (x̄=21.56) µm. Only a single appendage was found on the basal cell, 2.34 to 7.16 µm. Based on morphological features, these isolates were identified as Neopestalotiopsis clavispora (Maharachchikumbura et al., 2012, 2014). Molecular identification of isolate YNSL-3 was performed by amplification and sequencing of ITS4/ITS5, BT2A/ BT2B and EF1-728F/EF-2, respectively (White et al. 1990, Glass et al.1995, Carbone et al. 1999, O'Donnell et al. 1998). These base sequences were deposited in GenBank with accession numbers OQ891378 (ITS), OR088917 (Tef) and OR513439(Tub), respectively. BLAST searches of the sequences revealed 100% (478/478 bp), 100% (484/484 bp), and 94.67% (426/450 bp) homology with those of N. clavispora NM16311a from GenBank (LC209216, LC209220, and LC209221), respectively. Phylogenetic analysis (IQ-TREE) by maximum likelihood method showed that the isolate YNSL-3 was clustered with N. clavispora. The pathogenicity was tested with the isolate of YNSL-3, YNSL-5 and YNSL-8 by detached assay. The fruit surface of pomegranate cultivar Guangyan was wounded with a sterilized needle. The mycelial blocks (5mm2) of isolates cultured on PDA for 7 days were attached to the points of inoculation. Controls were inoculated with sterile PDA agar. All inoculated fruits were maintained in a growth chamber at 26°C with 75% relative humidity. The test was performed thrice. The brown lesions were observed after 7 days, whereas the controls showed no symptoms. The same pathogens reisolated were identical to the original isolates based on morphological characterization and molecular analyses. N. clavispora could cause different diseases in many plants (Rajashekara et al. 2023, Loredana et al. 2020). To our knowledge, this is the first report of fruit brown spot on Punica granatum caused by N. clavispora in China. This finding will help improve management strategies against the fruit brown spots on P. granatum in China.

Dimension-shifting multifunctional biocompatible nanocomposites.

A facile method to prepare dimension-shifting biocompatible multifunctional nanocomposites is described. The design is based on magnetic - and electrostatic - induced transitions from the dispersed state to the assembled state of zero-dimensional nanoparticles, resulting in dimension conversion.

A new antifungal and cytotoxic C-methylated flavone glycoside from Picea neoveitchii.

A new C-methylated flavone glycoside, 5,7-dihydroxy-3-methoxy-6-C-methylflavone 8,4'-di-O-ß-D-glucopyranoside (1), was isolated from the twigs and leaves of Picea neoveitchii Mast, together with eight known compounds, 5,7,8,4'-tetrahydroxy-3-methoxy-6-methylflavone 8-O-ß-D-glucopyranoside (2) kaempferol 3,4'-di-O-ß-D-glucopyranoside (3), apigenin 7-O-ß-D-glucopyranoside (4), tiliroside (5), massonianoside B (6), umbeliferone 7-O-ß-D-glucopyranoside (7), dihydroconiferin (8) and gleditschiaside A (9). Their structures were elucidated on the basis of analyses of spectroscopic data. Compound 1 showed moderate antifungal activity against tested plant pathogens (Pyricularia grisea (Cooke) Sacc., Sclerotium rocfsii Sacc. and Alternaria mali Roberts), however, compounds 2 and 5 had obvious inhibitory effect against S. rocfsii and A. mali, respectively. Compounds 1, 2, 3 and 9 also exhibited potent cytotoxicity against Spodoptera litura Fabricius cells.

Anthriscifolcine A, a C18-diterpenoid alkaloid.

The title compound, C(26)H(39)NO(7), which was isolated from Delphinium anthriscifolium var. majus, has a lycoctonine carbon skeleton containing four six-membered rings (A, B, D and E) and three five-membered rings (C, F and G). Rings A, B and E adopt chair conformation, while ring D adopts a boat conformation. Rings C and F adopt envelope conformations.

A deep investigation into the adipogenesis mechanism: profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/beta-catenin signaling pathway.

BACKGROUND: MicroRNAs (miRNAs) are a large class of tiny non-coding RNAs (approximately 22-24 nt) that regulate diverse biological processes at the posttranscriptional level by controlling mRNA stability or translation. As a molecular switch, the canonical Wnt/beta-catenin signaling pathway should be suppressed during the adipogenesis; However, activation of this pathway leads to the inhibition of lipid depots formation. The aim of our studies was to identify miRNAs that might be involved in adipogenesis by modulating WNT signaling pathway. Here we established two types of cell model, activation and repression of WNT signaling, and investigated the expression profile of microRNAs using microarray assay. RESULTS: The high throughput microarray data revealed 18 miRNAs that might promote adipogenesis by repressing WNT signaling: miR-210, miR-148a, miR-194, miR-322 etc. Meanwhile, we also identified 29 miRNAs that might have negative effect on adipogenesis by activating WNT signaling: miR-344, miR-27 and miR-181 etc. The targets of these miRNAs were also analysed by bioinformatics. To validate the predicted targets and the potential functions of these identified miRNAs, the mimics of miR-210 were transfected into 3T3-L1 cells and enlarged cells with distinct lipid droplets were observed; Meanwhile, transfection with the inhibitor of miR-210 could markedly decrease differentiation-specific factors at the transcription level, which suggested the specific role of miR-210 in promoting adipogenesis. Tcf7l2, the predicted target of miR-210, is a transcription factor triggering the downstream responsive genes of WNT signaling, was blocked at transcription level. Furthermore, the activity of luciferase reporter bearing Tcf7l2 mRNA 3' UTR was decreased after co-transfection with miR-210 in HEK-293FT cells. Last but not least, the protein expression level of beta-catenin was increased in the lithium (LiCl) treated 3T3-L1 cells after transfection with miR-210. These findings suggested that miR-210 could promote adipogenesis by repressing WNT signaling through targeting Tcf7l2. CONCLUSIONS: The results suggest the presence of miRNAs in two cell models, providing insights into WNT pathway-specific miRNAs that can be further characterized for their potential roles in adipogenesis. To our knowledge, present study represents the first attempt to unveil the profile of miRNAs involved in adipogenesis by modulating WNT signaling pathway, which contributed to deeper investigation of the mechanism of adipogenesis.

Characterization and transcriptional regulation analysis of the porcine TNFAIP8L2 gene.

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a new member of the tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) family that functions as an important factor in the maintenance of immune homeostasis. In this study, we cloned the cDNA sequences and analyzed the genomic structure of porcine TNFAIP8L2. RH mapping using the IMpRH panel showed that this gene was closely linked to microsatellite marker SW512 in pig chromosome 4. Subcellular localization analysis showed GFP-TNFAIP8L2 fusion protein distributed in nucleus as well as cytoplasm including mitochondria and endoplasmic reticulum. Real-time PCR analysis revealed that porcine TNFAIP8L2 was more highly expressed in spleen than other tissues. To understand its characterization of transcriptional regulation, we cloned approximately 2 kb of 5'-regulatory region upstream to the porcine TNFAIP8L2 translational start site and generated sequential deletion constructs evaluated in dual-luciferase reporter assay. The results demonstrated that its core promoter is 435 base pairs (bp) upstream to the transcription initiation site. Then, site-directed mutation experiment combined with electrophoretic mobility shift assay (EMSA) indicated that M-CAT binding factor (MCBF) and activator protein 1 (AP-1) were important transcription factors for porcine TNFAIP8L2. These findings provide an important basis for further understanding of porcine TNFAIP8L2 regulation and function in swine.

Development and validation of nomograms to accurately predict risk of recurrence for patients with laryngeal squamous cell carcinoma: Cohort study.

BACKGROUND: Recurrence is still major obstacle to long-term survival in laryngeal squamous cell carcinoma (LSCC). We aimed to establish and validate a nomogram to precisely predict recurrence probability in patients with LSCC. METHODS: A total of 283 consecutive patients with LSCC received curative-intend surgery between 2011 and 2014 at were enrolled in this study. Subsequently, 283 LSCC patients were randomly assigned to a training cohort (N = 171) and a validation cohort (N = 112) in a 3:2 ratio. According to the results of multivariable Cox regression analysis in the training cohort, we developed a nomogram. The predictive accuracy and discriminative ability of the nomogram were evaluated by calibration curve and concordance index (C-index), and compared with TNM stage system by C-index, receiver operating characteristic (ROC) analysis. Decision curve analysis (DCA) was performed to estimate clinical value of our nomogram. RESULTS: Six independent factors rooted in multivariable analysis of the training cohort to predict recurrence were age, tumor site, smoking, alcohol, N stage and hemoglobin, which were all integrated into the nomogram. The calibration curve for the probability of recurrence presented that the nomogram-based predictions were in good correspondence with actual observations. The C-index of the nomogram was 0.81 (0.75-0.88), and the area under curve (AUC) of nomogram in predicting recurrence free survival (RFS) was 0.894, which were significantly better than traditional TNM stage. Decision curve analysis further affirmed that our nomogram had a larger net benefit than TNM stage. The results were confirmed in the validation cohort. CONCLUSION: A risk prediction nomogram for patients with LSCC, incorporating readily assessable clinicopathologic variables, generates more accurate estimations of the recurrence probability when compared TNM stage alone, but still needs additional data before being used in clinical implications.

Initial surgical versus non-surgical treatments for advanced hypopharyngeal cancer: A meta-analysis with trial sequential analysis.

BACKGROUND: There is no consensus on whether initial surgical or non-surgical treatments should be the standard treatment pattern for advanced hypopharyngeal cancer. The aim of the meta-analysis was systematically and quantitatively compare the relative efficacy between initial surgical and non-surgical therapies in patients with advanced-stage hypopharyngeal carcinoma. METHODS: A comprehensive search was performed in PubMed, the ISI Web of Knowledge, the Cochrane Library, and Embase databases from inception to April 10, 2019. Citation screening, data abstraction and quality assessment were performed in duplicate. Meta-analysis with trial sequential analysis (TSA) was used to assess the primary and secondary outcomes. Besides, we used the Grading of Recommendations Assessment Development and Evaluation (GRADE) to evaluate the certainty of the body of evidence. RESULTS: A total of 17 trials was appraised with 2539 patients that complied with inclusion and exclusion criterion. Pooled analyses indicated patients receiving primary surgical and non-surgical therapy did not significantly differ in overall survival (OS) (relative risk [RR] = 1.04, 95% confidence interval [CI] = 0.94 to 1.15), with TSA inconclusive. Additionally, patients treated with primary surgical experienced an increased disease free survival (DFS) probability compared with non-surgical treatment (RR 1.20, 95% CI = 1.05 to1.37), while TSA is inconclusive. Notably, non-surgical management did have a beneficial efficacy on larynx preservation (RR 0.48, 95% CI = 0.33 to 0.70), and TSA also provided conclusive evidence. GRADE indicated the level of evidence was low or very low for primary or secondary outcomes. CONCLUSION: The results of our meta-analysis indicated when compared to surgical treatments, non-surgical therapy for patients with advanced hypopharyngeal carcinoma appears to have equivalent efficacy, and it offers an opportunity to preserve laryngeal function. Due to inconclusive evidence by TSA, further investigation with large randomized clinical trials (RCTs) using modern approaches should be undertaken to verify the results of this meta-analysis. TRIAL REGISTRATION: PROSPERO registration number: CRD42018118563. Registered on December 19, 2018.

Is oval window transport a royal gate for nanoparticle delivery to vestibule in the inner ear?

Drug delivery to the inner ear by nanomedicine strategies has emerged as an effective therapeutic approach for the management of inner ear diseases including hearing and balance disorders. It is well accepted that substance enters the perilymph from the middle ear through the round window membrane (RWM), but the passage through the oval window (OW) has long been neglected. Up to now, researchers still know little about the pathway via which nanoparticles (NPs) enter the inner ear or how they reach the inner ear following local applications. Herein, we engineered fluorescence traceable chitosan (CS) NPs, investigated the NP distribution within cochlear and vestibular organs, and assessed the availability of RWM and OW pathways to NP transport. Intriguingly, there were high levels of CS NPs in vestibular hair cells, dark cells and supporting cells, but negligible ones in cochlear hair cells and epithelial cells after intratympanic administration. However, the NPs were visualized in two cell models, L929 and HEI-OC1 cell lines, and in the hair cells of cochlear explants after co-incubation in vitro. These combined studies implied that CS NPs might enter the vestibule directly through the OW and then preferentially accumulated in the cells of vestibular organs. Thus, in vivo studies were carried out and clearly revealed that CS NPs entered the inner ear through both the RWM and OW, but the latter played a governing role in delivering NPs to the vestibule with vivid fluorescence signals in the thin bone of the stapes footplate. Overall, these findings firstly suggested that the OW, as a royal gate, afforded a convenient access to facilitate CS NPs transport into inner ear, casting a new light on future clinical applications of NPs in the effective treatment of vestibular disorders by minimizing the risk of hearing loss associated with cochlear hair cell pathology.

Silencing of microRNA-708 promotes cell growth and epithelial-to-mesenchymal transition by activating the SPHK2/AKT/ß-catenin pathway in glioma.

Aberrant microRNA-708 (miR-708) expression is frequently reported in cancer studies; however, its role in glioma has not been examined in detail. We investigated miR-708 function in glioma and revealed that miR-708 expression was significantly down-regulated in glioma tissues and cell lines. Restoration of miR-708 inhibited glioma cell growth and invasion both in vitro and in vivo. The oncogene SPHK2 (sphingosine kinase 2) was identified as a downstream target of miR-708 using luciferase and western blot assays. miR-708 inhibited AKT/ß-catenin signaling, which is activated by SPHK2. In addition, we revealed that miR-708 was transcriptionally repressed by EZH2 (enhancer of zeste homolog 2)-induced histone H3 lysine 27 trimethylation and promoter methylation. In summary, our findings revealed that miR-708 is a glioma tumor suppressor and suggest that miR-708 is a potential therapeutic target for glioma patients.

Heterojunctions of silver-iron oxide on graphene for laser-coupled oxygen reduction reactions.

We report a two-step hybridization of N-doped graphene and Ag-decorated Fe2O3 hematite to realize a balanced oxygen adsorption/desorption equilibrium and a laser-coupled ORR (LORR). The stable plateau currents with n values of 3.9 in a wide potential range (0.2-0.7 V) and 7.5% peroxide inhibition of the LORR are found to be directly associated with the Ag/Fe2O3 heterojunction, where interactions of semiconductor band gap excitation and plasmonic resonance-induced hot electrons are proposed to occur.

General Solvent-dependent Strategy toward Enhanced Oxygen Reduction Reaction in Graphene/Metal Oxide Nanohybrids: Effects of Nitrogen-containing Solvent.

A general solvent-dependent protocol directly influencing the oxygen reduction reaction (ORR) in metal oxide/graphene nanohybrids has been demonstrated. We conducted the two-step synthesis of cobalt oxide/N-doped graphene nanohybrids (CNG) with solvents of water, ethanol, and dimethylformamide (DMF), representing tree typical categories of aqueous, polar organic, and organic N-containing solvents commonly adopted for graphene nanocomposites preparation. The superior ORR performance of the DMF-hybrids can be attributed to the high nitrogen-doping, aggregation-free hybridization, and unique graphene porous structures. As DMF is the more effective N-source, the spectroscopic results support a catalytic nitrogenation potentially mediated by cobalt-DMF coordination complexes. The wide-distribution of porosity (covering micro-, meso-, to macro-pore) and micron-void assembly of graphene may further enhance the diffusion kinetics for ORR. As the results, CNG by DMF-synthesis exhibits the high ORR activities close to Pt/C (i.e. only 8 mV difference of half-wave potential with electron transfer number of 3.96) with the better durability in the alkaline condition. Additional graphene hybrids comprised of iron and manganese oxides also show the superior ORR activities by DMF-synthesis, confirming the general solvent-dependent protocol to achieve enhanced ORR activities.

Chemical constituents of the aerial part of Derris elliptica.

A new coumaronochromone, 6,4'-dihydroxy-7,5' -dimethoxy-coumaronochromone (1), together with eleven known flavonoids (2-12) were isolated from the ethanol extract of the aerial part of Derris elliptica. Their structures were elucidated on the basis of spectroscopic analysis. Compounds 2, 4, 7, 8 and 9 exhibited moderate insecticidal activities against larvae of Aedes albopictus. All compounds showed strong cytotoxic activities against Spodoptera litura (SL) and Trichoplusia ni BTI-Tn-5B1-4 (Hi-5) cells comparison to positive control of rotenone.

Chemical constituents of Picea neoveitchii.

Four flavonoids, 5,7,4'-trihydroxy-3,8,-dimethoxy-6-C-methylflavone (1), 5,8,4'-trihydroxy-3,7-dimethoxy-6-C-methylflavone (2), 7-methoxy-6-C-methylkaempferol (3) and kaempferol-7-O-(2″-E-p-coumaroyl)-α-l-arabinofuranoside (4), together with 15 known compounds, were isolated from the twigs and leaves of Picea neoveitchii Mast. Their structures were elucidated on the basis of analyses of spectroscopic data. Compound 4 showed strong anti-fungal activity against Fusarium oxysporum whereas compounds 1-4 were all active against Rhizoctonia solani.

Transcription factor Sp1 is essential for the regulation of the porcine caveolin-1 gene.

Caveolin-1 (CAV-1) is a key structural component of caveolae that regulates cholesterol. Employing transgenic techniques to regulate the cholesterol content of pork through CAV-1 is hindered by our lack of knowledge about its regulation. To investigate the regulatory mechanism of porcine CAV-1, a DNA segment containing the 5'-flanking region of CAV-1 was isolated from porcine genomic DNA and sequenced. The luciferase reporter assay detected five cis-acting elements for efficient expression of the CAV-1 gene at the region spanning nucleotides -213 to -20 with serially deleted 5'-flanking sequences and site-directed mutants, -123 to -114 was the core promoter. The electrophoretic mobility shift assay demonstrated potential binding of Sp1 protein to this core promoter. The purpose of this study is to systematically elucidate the transcriptional regulation mechanism of porcine CAV-1 and to contribute to the investigation of the interaction between CAV-1 and cholesterol.

Characterization and transcriptional regulation analysis of the porcine PAQR6 gene.

Progestin and adipoQ receptor family member VI (PAQR6) is a new member of the Progestin and adipoQ receptors (PAQRs) family that functions as an important factor in the nongenomic actions of rapid steroid response. Considering the important role of PAQR6 in these nongenomic actions, we determined porcine PAQR6 expression and regulation. We cloned the cDNA of porcine PAQR6 and analyzed its genomic structure. Subcellular localization analysis showed that PAQR6-green fluorescent protein fusion protein was distributed in cytoplasm. Spatial expression patterns analysis revealed that porcine PAQR6 was more highly expressed in small intestinal than in other tissues. To investigate the regulatory mechanism of porcine PAQR6, we cloned approximately 1.5 kb of porcine PAQR6 5'-regulatory region and generated sequential deletion constructs and evaluated their activity in a dual-luciferase reporter assay. The results suggested that the core promoter region of this gene is located in the first exon region from +90 to +241. Site-directed mutagenesis indicated that adiponectin receptor protein 1, E2F transcription factor 1, and Sp1 transcription factor might be important transcription factors for porcine PAQR6. This study is the first attempt to elucidate the transcriptional regulation of porcine PAQR6, which established a foundation for further study and contributed to the deeper investigation of the regulation and function of PAQR6.

[Comparison of internal fixation treatment of tibia intercondylar eminence fracture between absorbable screw and metallic screw].

OBJECTIVE: To compare advantage and disadvantage of internal fixation method for tibia intercondylar eminence fracture between absorbable screw and metallic screw. METHODS: From 1996 to 2002, 200 patients with fracture of tibia intercondylar eminence were divided into group A (with absorbable screw, n = 120) and group B (with metallic screw, n = 80). And the biological compatibility, biomechanics, bone union and complications were compared between two groups. RESULTS: There were no obvious differences in operating time and circumstance of recovery position between two groups. Group A was obviously better than group B in biological compatibility, biomechanics, bone union, joint function recovery. The average healing time of group A was three months, that of group B was three and half months. The good rates of joint function in two groups were 98.0% and 95.0% respectively. The occurrence rates of wound arthritis were 1.7% and 3.7%. There was significant difference between them (P < 0.01). CONCLUSION: Absorbable screw is a more ideal material of internal fixation to treat tibia intercondylar eminence fracture.