First report of Nigrospora chinensis causing dragon fruit (Selenicereus monacanthus) stem spot in China.

Dragon fruit (Selenicereus monacanthus), renowned for its economic value and dual utility in both culinary and medicinal applications, is predominantly cultivated in China. In July 2023, a stem spot disease was found on dragon fruit ("Zi honglong" cultivar) plants with 37% incidence, in Huajiang Town (N25°40', E105°39'), Guanling County, Anshun City, Guizhou Province. The symptoms appeared as yellow spots surrounded by watery stains, then the spots expanded to suborbicular, which finally led stem to wither. Twelve symptomatic stem samples were collected in a 1.3-hectare plantation and cut into small pieces (5 mm × 5 mm), sterilized the surface with 75% ethanol for 30 seconds, washed 3 times with disinfected distilled water, moved to potato dextrose agar (PDA) medium, and incubated at 28°C for 5 days. Once the mycelium had developed, they were transferred to another PDA medium and cultured at 28°C for a period of 3-5 days. Totally fifteen identical strains were isolated, their colonies were white and round in shape; hyphae were smooth, hyaline; conidia were globose or subglobose, smooth, aseptate, 5.8-11.9 × 4.2-10.6 µm (av. = 8.8 × 7.4 µm, n = 30), light brown in early stage and gradually turning black over time; sterile cells were terminal on hyphae, pale to dark brown, uhceiform or oval, 17.5-21.6 × 4.6-8.7 µm (av. = 19.4 × 5.8 µm, n = 30). The morphologic characteristics of the isolates matched Nigrospora chinensis described by Wang et al. (2017). The PCR amplification was carried out by 3 primers ITS1/ITS4 (Vilgalys et al. 1990; White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone et al. 1999) and BT2A/BT2B (Glass et al. 1995) belonged to the internal transcribed spacer (ITS), translation elongation factor-1 (TEF1) and ß-tubulin (TUB2) gene loci, respectively. The sequences of a representative strain (GUCC 524) had 99.38% (ITS: 483/486 bp, PP391347 vs KX985970), 99.79% (TEF1: 482/483 bp, PP400678 vs KY019427) and 99.73% (TUB2: 372/373 bp, PP400677 vs KY019497) identities with those of N. chinensis (strain LC 3085). The phylogenetic tree constructed by three gene combinations showed that GUCC 524 was significantly clustered with N. chinensis. Ten 6-month-old dragon fruit ("Zi honglong" cultivar) seedlings were engrafted with 10 µL conidial suspension (1×105 conidia/mL), packaged with sealed film, two of them were inoculated with sterile distilled water as controls, and placed in a greenhouse at 28℃ for 10 days, inoculated plants showed yellow spots analogous to field symptoms, no symptoms were found in control plants. This experiment was repeated three times. Morphological character and molecular identification based on 3 gene loci of the strains isolated from the inoculated stems, were consistent with those of the original isolated strains. Therefore, based on morphological identification, phylogenetic analysis and pathogenicity test, the pathogen was identified as N. chinensis. Our study firstly reported N. chinensis as a pathogen causing stem spot disease on dragon fruit. N. chinensis is an important agent resulting for economic losses, previously reported on Camellia sinensis (Wang et al. 2017), Saccharum officinarum (Raza et al. 2019), Aucuba japonica (Qin et al. 2021). This report establishes a pivotal reference point for the progression of scientific strategies in preventing and controlling this disease associated with N. chinensis.

Metformin combined with glucose starvation synergistically suppress triple-negative breast cancer by enhanced unfolded protein response.

Metformin (MET) is a well-documented drug used in the treatment of type II diabetes. Recent studies have revealed its potential anti-tumor effects in various types of cancer. However, the dosage of MET required to exhibit anti-tumor activity is considerably higher than the clinically recommended dosage. In this study, we investigated the synergistical anti-tumor effect of glucose deprivation and MET in MDA-MB-231 cells, which represents a triple-negative breast cancer subtype (TNBC). Our findings demonstrate that glucose deprivation significantly enhances the anti-tumor activity of MET by reducing cell proliferation and increasing cell apoptosis. RNA-seq was performed to identify the key molecules involved in this process. Our results indicate that unfolded protein response of endoplasmic reticulum (UPRER) was significantly activated upon glucose starvation combining with MET compared to glucose starvation alone. Notably, the combined treatment significantly activated UPRER signaling pathway through ATF4/ATF3/CHOP axis, due to enhanced UPRER stress. In conclusion, our study suggests that the synergistic effects of MET and glucose deprivation suppress cell proliferation in TNBC by activating pro-apoptotic molecules through UPRER stress. These findings have potential implications for the anti-tumor application of MET in TNBC.

First Report of Leaf Spot Disease Associated with Faba Bean (Vicia faba) Caused by Pestalotiopsis rosea in China.

Faba bean (Vicia faba L.) is a cool-season legume crop, planted worldwide as an essential source of protein-rich foods, vegetables, and animal feeds. In China, the total cultivated area of faba bean in 2019 was 839,618 square hectometers (hm2) and the production was 1,740,945 tons (t) (Ji et al. 2022). In May 2021, a leaf spot disease on faba bean plants with about 80% disease incidence was observed in a 0.3 ha commercial field located at Zunyi City (27°31'43.80″ N, 106°23'34.27″ E), Guizhou Province, China. The leaves of the early affected plants appeared circular dark brown spots, which then rapidly develop into large irregular shaped lesions if conditions remain favorable. Severe infection can result in extensive defoliation of plants and lesions on pods. Symptomatic leaves were collected and cut into small pieces, surface sterilized with 75% ethanol for 30 s followed by 2% NaClO for 1 min, rinsed with sterile distilled water three times, and incubated on PDA plates amended with streptomycin sulfate (0.5 mg/L) at 25°C for 2-4 days. Two purified cultures were obtained through single-spore culture. Colonies on PDA attaining 62 mm diam after 2 weeks, white or pale red, edge undulate, with dense aerial mycelium on the surface, fruiting bodies black to reddish brown. Conidia fusoid to ellipsoid, 4-septate, straight to slightly curved, 18.5-22 × 6-7 µm (av. = 20 × 7 µm, n = 30); basal cell obconic, hyaline, 3.5-5 µm long, with a single appendage, 2.5-6 µm; three median cells doliiform, verruculose, olivaceous with slightly red (second cell from base 4-5 µm long; third cell 4.5-5.5 µm long; fourth cell 3-5.5 µm long); apical cell conical, hyaline, 2.5-4 µm long, with 1-3 tubular appendages, 13-22.5 µm long. The morphological characters of our studied specimens fit well with Pestalotiopsis rosea (Maharachchikumbura et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region, partial ß-tubulin (tub2) and translation elongation factor 1-alpha (tef1-α) genes were amplified and sequenced using primer pairs ITS5/ITS4 (White et al. 1990), T1/Bt-2b (Glass and Donaldson 1995) and EF1-728F/EF-2 (Carbone and Kohn 1999). The DNA sequences of two isolates GUCC 195257 (OP364052, OP391714, OP391713) and GUCC 195258 (OP364053, OP391716, OP391715) were deposited in GenBank. The BLAST searches revealed that these sequences had 99% (537/539 bp), 100% (453/453 bp), 99% (591/593 bp), 99% (537/539 bp), 100% (453/453 bp) and 99% (574/576 bp) nucleotide identity to the ex-type strain of P. rosea (JX399005, JX399036, JX399069), respectively. In addition, multi-locus phylogenetic analysis showed that both isolates clustered with P. rosea with full statistical support. The phylogenetic relationship of Pestalotiopsis species supported the identification of our isolates as P. rosea. In the pathogenicity test, the leaves of ten healthy 2-week-old faba bean plants were spray inoculated with a conidial suspension (1 × 106 conidia/ml) of the two isolates. Another set of five plants that were sprayed with sterilized distilled water served as the controls. Treated plants were kept at 25°C in a greenhouse with a photoperiod of 12 h and 70% relative humidity. After one week, all inoculated leaves showed symptoms similar to those of the infected faba bean observed in the field, whereas controls were symptomless. The pathogenicity test was performed twice with similar results. The fungus was reisolated from the inoculated plants and identified as P. rosea by morphological and molecular evidence, thus confirming Koch's postulates. To our knowledge, this is the first report of P. rosea causing leaf spot on faba bean in the world. Pestalotiopsis species are well-known phytopathogens that can cause a variety of diseases, including leaf spots, chlorosis, and various postharvest (Wang et al. 2019). The results of this study not only contribute to accurately identify this disease in the fields of faba bean production, but also provide an important reference for developing specific control measures.

First Report of Leaf Spot on Zanthoxylum bungeanum Caused by Nigrospora sphaerica in China.

Zanthoxylum bungeanum Maxim., a deciduous shrub in Zanthoxylum genus of the Rutaceae family, has not only highly economical values as condiment and medicine, but also significantly ecological values in soil and water conservation. In March 2023, a typical leaf spot disease on Z. bungeanum (Variety "Xiao Qingjiao") was observed in the field with an area of 26.68 ha with 35% incidence and 25.4% disease intensity in Zhenfeng County (25°38'57.60″ N, 105°64'98.64″ E, 1,156 m), Guizhou Province, China. The symptom leaves showed as irregularly shaped necrotic lesions, brown to dark brown with black margin. 30 samples with typical symptoms were collected and cut into 0.5 cm × 0.5 cm pieces. Their surfaces were disinfected with 1.5% NaClO for 2 min followed by 75% ethanol for 35 s, rinsed three times with sterile distilled water, finally incubated on PDA plates at 27°C. A total of 36 isolates were obtained through single-spore cultivation. The colonies on PDA were fluffy with abundant aerial mycelia and covered the whole plates (diameter 90 mm) in 7 days. Conidia were brown to black, single-celled, smooth, spherical or oblate, 12.0-17.0 × 12.5-18.5 µm (av. = 14.5 × 15.5 µm, n = 50) and grew on a colorless transparent vesicle at the apical cell of conidiophores. The morphological characteristics were similar with N. sphaerica (Wang et al. 2017). The 5.8S DNA (ITS), translation elongation factor 1-alpha (TEF1-α) and ß-tubulin (TUB2) genes were amplified with primers ITS4/ITS5, EF1-728F/EF2, and BT2A/BT2B, respectively (White et al. 1990; Carbone and Kohn 1999, O'Donnell et al. 1998; Glass and Donaldson 1995). The ITS, TEF1-α and TUB2 sequences of two randomly selected isolates, GUCC 21-187 and GUCC 21-235, had > 99% nucleotide identities (ITS: 99.60% (504/506 bp, OR646539) and 99.61% (506/508 bp, OR640300); TEF: 100% (470/470 bp, OR654285) and 100.00% (471/471 bp, OR654286); TUB: 100.00% (408/408 bp, OR661269) and 99.52% (411/413 bp, OR661270), respectively) with those sequences of N. sphaerica (LC 7294) in GenBank (KX985932, KY019397 and KY019602, respectively). The phylogenetic tree based on sequences of ITS, TEF1-α and TUB2 indicated that GUCC 21-187 and GUCC 21-235 were most closely related to N. sphaerica (LC 7294), supported with 100%/100%/1 bootstraps. Based on morphological characteristics and molecular datasets analyses, the isolates were identified as N. sphaerica. 10 healthy 2-years-old Z. bungeanum plants were sprayed with conidial suspensions (1 × 106 conidia/mL) of the isolates and the other 5ere sprayed with sterile water as the controls, all the treated plants were cultivated in a glasshouse at 25°C under 85% relative humidity. Typical leaf spot symptoms appeared on inoculated Z. bungeanum plants after 8 days, while the control plants remained asymptomatic. N. sphaerica was re-isolated from the lesions of inoculated plants and identified by morphological and molecular identification. Pathogenicity test was performed three times with analogous results, fulfilling Koch's postulates. N. sphaerica had been reported as a common pathogen on a variety of plants including sugarcane, kiwifruit and blueberry (Cui et al. 2018; Chen et al. 2016; Wright et al. 2008). To our knowledge, this is the first report of leaf spot disease caused by N. sphaerica on Z. bungeanum in China. Our report would be helpful to Z. bungeanum growers to recognize this leaf spot disease, and corresponding measures could be taken to minimize or avoid the economic losses caused by it.

Characteristics and risk factors for renal recovery after acute kidney injury in critically ill patients in cohorts of elderly and non-elderly: a multicenter retrospective cohort study.

BACKGROUND: The purpose of this study was to explore the risk factors for renal nonrecovery among elderly and nonelderly patients with acute kidney injury (AKI) in critically ill patients. METHODS: A multicenter retrospective cohort of 583 critically ill patients with AKI was examined. We found the best cutoff value for predicting renal recovery by age was 63 years old through logistic regression. All patients were divided into two cohorts, age <63 and age ≥63-years old; on the basis of renal recovery at 30 days after AKI, the two patient cohorts were further divided into a renal recovery group and a renal nonrecovery group. Multivariate logistic regression was used to analyze the risk factors affecting renal recovery in the two cohorts. RESULTS: The 30-day renal recovery rate of patients aged <63 years was 70.0% (198/283), multivariate analysis showed that the independent risk factors affecting renal nonrecovery in age <63 years old included AKI stage, blood lactate level and hemoglobin level. The 30-day renal recovery rate of patients aged ≥63 years was 28.7% (86/300), multivariate analysis showed that the independent risk factors for renal nonrecovery in age ≥63-years old included diabetes mellitus, surgery with general anesthesia, AKI stage, APACHE II score, eGFR, and hemoglobin level. CONCLUSIONS: The renal nonrecovery after AKI in critically ill patients in patients aged ≥63 years was more strongly affected by multiple risk factors, such as diabetes mellitus, surgery with general anesthesia, eGFR, and APACHE II score, in addition to hemoglobin and AKI stage.

27-gauge microincision vitrectomy surgery compared with 25-gauge microincision vitrectomy surgery on wound closure and need for wound suture and other postoperative parameters in the treatment of vitreoretinal disease: A meta-analysis.

We performed a meta-analysis to evaluate the effect of 27-gauge microincision vitrectomy surgery compared with 25-gauge microincision vitrectomy surgery on wound closure and the need for wound suture and other postoperative parameters in the treatment of vitreoretinal disease. A systematic literature search up to June 2022 was performed and 1264 subjects with the vitreoretinal disease at the baseline of the studies; 562 of them were using the 27-gauge microincision vitrectomy surgery, and 722 were using 25-gauge microincision vitrectomy surgery. Odds ratio (OR), and mean difference (MD) with 95% confidence intervals (CIs) were calculated to assess the effect of 27-gauge microincision vitrectomy surgery compared with 25-gauge microincision vitrectomy surgery on wound closure and the need for wound suture and other postoperative parameters in the treatment of vitreoretinal disease using the dichotomous, and contentious methods with a random or fixed-effect model. The 27-gauge microincision vitrectomy surgery subjects had a significantly lower intraoperative and postoperative wound complication (OR, 6.66; 95% CI, 0.46-0.95, P = .02), and wound suture number (OR, 0.38; 95% CI, 0.20-0.71, P = .002), and best corrected visual acuity (MD, -0.03; 95% CI, -0.05 to -0.001, P = .02) compared with 25-gauge microincision vitrectomy surgery in subjects with vitreoretinal disease. However, 27-gauge microincision vitrectomy surgery subjects had no significant difference in the wound closure time (MD, -8.45; 95% CI, -23.44 to 6.55, P = .27), operation time (MD, 0.85; 95% CI, -1.17 to 2.86, P = .41), intraocular pressure at postoperative day 1 (MD, 0.42; 95% CI, -1.45-2.28, P = .66), primary anatomical success rate (OR, 0.83; 95% CI, 0.42-1.63, P = .58), and central macular thickness (MD, 1.81; 95% CI, -21.76 to 25.37, P = .88) compared to 25-gauge microincision vitrectomy surgery in subjects with vitreoretinal disease. The 27-gauge microincision vitrectomy surgery subjects had a significantly lower intraoperative and postoperative wound complication, wound suture number, and best corrected visual acuity, and no significant difference in the wound closure time, operation time, intraocular pressure at postoperative day 1, primary anatomical success rate, and central macular thickness compared to 25-gauge microincision vitrectomy surgery in subjects with vitreoretinal disease. The analysis of outcomes should be with caution because of the low sample size of 12 out of 15 studies in the meta-analysis and a low number of studies in certain comparisons.

Reelin Improves Cognition and Extends the Lifespan of Mutant Ndel1 Mice with Postnatal CA1 Hippocampus Deterioration.

The glycoprotein Reelin maintains neuronal positioning and regulates neuronal plasticity in the adult brain. Reelin deficiency has been associated with neurological diseases. We recently showed that Reelin is depleted in mice with a targeted disruption of the Ndel1 gene in forebrain postnatal excitatory neurons (Ndel1 conditional knockout (CKO)). Ndel1 CKO mice exhibit fragmented microtubules in CA1 pyramidal neurons, profound deterioration of the CA1 hippocampus and a shortened lifespan (~10 weeks). Here we report that Ndel1 CKO mice (of both sexes) experience spatial learning and memory deficits that are associated with deregulation of neuronal cell adhesion, plasticity and neurotransmission genes, as assessed by genome-wide transcriptome analysis of the hippocampus. Importantly, a single injection of Reelin protein in the hippocampus of Ndel1 CKO mice improves spatial learning and memory function and this is correlated with reduced intrinsic hyperexcitability of CA1 pyramidal neurons, and normalized gene deregulation in the hippocampus. Strikingly, when treated with Reelin, Ndel1 CKO animals that die from an epileptic phenotype, live twice as long as nontreated, or vehicle-treated CKO animals. Thus, Reelin confers striking beneficial effects in the CA1 hippocampus, and at both behavioral and organismal levels.

Optimal Extraction Study of Gastrodin-Type Components from Gastrodia Elata Tubers by Response Surface Design with Integrated Phytochemical and Bioactivity Evaluation.

Gastrodia elata tuber (GET) is a popular traditional Chinese medicines (TCMs). In this study, response surface methodology (RSM) with a Box⁻Behnken design (BBD) was performed to optimize the extraction parameters of gastrodin-type components (gastrodin, gastrodigenin, parishin A, parishin B, parishin C and parishin E). Different from the conventional studies that merely focused on the contents of phytochemical, we gave consideration to both quantitative analysis of the above six components by HPLC and representative bioactivities of GET, including antioxidation and protection of human umbilical vein endothelial cells (HUVEC). Four independent variables (ethanol concentration, liquid-material ratio, soaking time and extraction time) were investigated with the integrated evaluation index of phytochemical contents. With the validation experiments, the optimal extraction parameters were as follows: ethanol concentration of 41%, liquid⁻solid ratio of 28.58 mL/g, soaking time of 23.91 h and extraction time of 46.60 min. Under the optimum conditions, the actual standardized comprehensive score was 1.8134 ± 0.0110, which was in accordance with the predicted score of 1.8100. This firstly established method was proved to be feasible and reliable to optimize the extraction parameters of the bioactive components from GET. Furthermore, it provides some reference for the quality control and extraction optimization of TCMs.

Ndel1 and Reelin Maintain Postnatal CA1 Hippocampus Integrity.

UNLABELLED: How the integrity of laminar structures in the postnatal brain is maintained impacts neuronal functions. Ndel1, the mammalian homolog of NuDE from the filamentous fungus Aspergillus nidulans, is an atypical microtubule (MT)-associated protein that was initially investigated in the contexts of neurogenesis and neuronal migration. Constitutive knock-out mice for Ndel1 are embryonic lethal, thereby necessitating the creation a conditional knock-out to probe the roles of Ndel1 in postnatal brains. Here we report that CA1 pyramidal neurons from mice postnatally lacking Ndel1 (Ndel1 conditional knock-out) exhibit fragmented MTs, dendritic/synaptic pathologies, are intrinsically hyperexcitable and undergo dispersion independently of neuronal migration defect. Secondary to the pyramidal cell changes is the decreased inhibitory drive onto pyramidal cells from interneurons. Levels of the glycoprotein Reelin that regulates MTs, neuronal plasticity, and cell compaction are significantly reduced in hippocampus of mutant mice. Strikingly, a single injection of Reelin into the hippocampus of Ndel1 conditional knock-out mice ameliorates ultrastructural, cellular, morphological, and anatomical CA1 defects. Thus, Ndel1 and Reelin contribute to maintain postnatal CA1 integrity. SIGNIFICANCE STATEMENT: The significance of this study rests in the elucidation of a role for Nde1l and Reelin in postnatal CA1 integrity using a new conditional knock-out mouse model for the cytoskeletal protein Ndel1, one that circumvents the defects associated with neuronal migration and embryonic lethality. Our study serves as a basis for understanding the mechanisms underlying postnatal hippocampal maintenance and function, and the significance of decreased levels of Ndel1 and Reelin observed in patients with neurological disorders.

Influence of brewing conditions on taste components in Fuding white tea infusions.

BACKGROUND: White tea has received increasing attention of late as a result of its sweet taste and health benefits. During the brewing of white tea, many factors may affect the nutritional and sensory quality of the resulting infusions. The present study aimed to investigate the effect of various infusion conditions on the taste components of Fuding white tea, including infusion time, ratio of tea and water, number of brewing steps, and temperature. RESULTS: Brewing conditions had a strong effect on the taste compound profile and sensory characteristics. The catechin, caffeine, theanine and free amino acid contents generally increased with increasing infusion time and temperature. Conditions comprising an infusion time of 7 min, a brewing temperature of 100 °C, a tea and water ratio of 1:30 or 1:40, and a second brewing step, respectively, were shown to obtain the highest contents of most compounds. Regarding tea sensory evaluation, conditions comprising an infusion time of 3 min, a brewing temperature of 100 °C, a tea and water ratio of 1:50, and a first brewing step, resulted in the highest sensory score for comprehensive behavior of color, aroma and taste. CONCLUSION: The results of the present study reveal differences in the contents of various taste compounds, including catechins, caffeine, theanine and free amino acids, with respect to different brewing conditions, and sensory scores also varied with brewing conditions. © 2016 Society of Chemical Industry.

Fungal Systematics and Evolution: FUSE 3.

The present study introduces seven new species, one new combination, one new variety and several interesting taxonomical notes and/or geographical records. Most of the new taxa are Ascomycetes, but the study also includes a new variety of a Basidiomycete. Novel species include Gyromitra khanspurensis (Discinaceae, Pezizales, Pezizomycetes) from Pakistan growing near Cedrus deoadara and Paramyrothecium guiyangense and Paramyrothecium verruridum (Stachybotriaceae, Hypocreales, Sordariomycetes) both isolated from soil in China. New species from South Africa are Sclerostagonospora elegiae on culm litter of Elegia equisetacea, Sclerostagonospora fusiformis on culm litter of Thamnochortus spicigerus, Sclerostagonospora pinguis on culm litter of Cannomois virgata and Sclerostagonospora sulcata on culm litter of Ischyrolepis subverticellata (Phaeosphaeriaceae, Pleosporales, Dothideomycetes). Hapalocystis berkeleyi var. kickxii with its basionym Hypoxylon kickxii is shown to be a taxon on species level and thus recombined as Hapalocystis kickxii (Sydowiellaceae, Diaporthales, Sordariomycetes), and it is lecto- and epitypified. The new variety Pluteus romellii var. luteoalbus (Pluteaceae, Agaricales, Agaricomycetes) growing on a mossy fallen stem of a deciduous tree is described from Czech Republic. Cortinarius scaurocaninus (Cortinariaceae, Agaricales, Agaricomycetes) is new for Austria, Humicola grisea (Chaetomiaceae, Sordariales, Sordariomycetes) is an interesting new record for Chile. Two taxa are reported as new for Turkey: the lichenicolous fungus Opegrapha parasitica (Opegraphaceae, Arthoniales, Arthoniomycetes) growing partly immersed in the thallus of Aspicilia and the lichen Rinodina zwackhiana (Physciaceae, Teloschistales, Lecanoromycetes) from calcareous rock. Finally, Xerula strigosa (Physalacriaceae, Agaricales, Agaricomycetes), described from China, is confirmed to be present also in Pakistan.

Effect of water quality on the main components in Fuding white tea infusions.

The aim of this study was to investigate the effect of water quality on the main components in Fuding white tea infusions, including catechins, caffeine, theanine and free amino acids. Pure, tap and spring water were tested, and water quality was found to have a distinct effect on the main compounds extracted. Pure water, which was weakly acidic and low in dissolved ions, achieved the highest catechin content, whereas caffeine and theanine, and amino acids, were higher in infusions made with spring and tap water, respectively. Sensory evaluation was performed to evaluate infusion colour, taste and aroma, and sensory quality was similarly influenced by water type, due primarily to differences in dissolved ions. Pure water was more suitable for brewing white tea with superior colour, aroma and taste.

Opposite roles of myocardin and atrogin-1 in L6 myoblast differentiation.

L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low-concentration of serum, but the underlying mechanism is not well understood. The role of atrogin-1, an E3 ligase with well-characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin-1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up-regulation of atrogin-1 and down-regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, over-expression of atrogin-1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co-expression of myocardin with atrogin-1 inhibited atrogin-1-induced myogenin expression. Over-expression of atrogin-1 decreased myocardin protein level, albeit without affecting its mRNA level. Small-interfering RNA-mediated knockdown of atrogin-1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin-1 without involving Foxo1. Taken together, our results have demonstrated that atrogin-1 plays a positive role in skeletal muscle differentiation through down-regulation of myocardin.

Induction of microRNA-1 by myocardin in smooth muscle cells inhibits cell proliferation.

OBJECTIVE: Myocardin is a cardiac- and smooth muscle-specific transcription co-factor that potently activates the expression of downstream target genes. Previously, we demonstrated that overexpression of myocardin inhibited the proliferation of smooth muscle cells (SMCs). Recently, myocardin was reported to induce the expression of microRNA-1 (miR-1) in cardiomyocytes. In this study, we investigated whether myocardin induces miR-1 expression to mediate its inhibitory effects on SMC proliferation. METHODS AND RESULTS: Using tetracycline-regulated expression (T-REx) inducible system expressing myocardin in human vascular SMCs, we found that overexpression of myocardin resulted in significant induction of miR-1 expression and inhibition of SMC proliferation, which was reversed by miR-1 inhibitors. Consistently, introduction of miR-1 into SMCs inhibited their proliferation. We isolated spindle-shaped and epithelioid human SMCs and demonstrated that spindle-shaped SMCs were more differentiated and less proliferative. Correspondingly, spindle-shaped SMCs had significantly higher expression levels of both myocardin and miR-1 than epithelioid SMCs. We identified Pim-1, a serine/threonine kinase, as a target gene for miR-1 in SMCs. Western blot and luciferase reporter assays further confirmed that miR-1 targeted Pim-1 directly. Furthermore, neointimal lesions of mouse carotid arteries displayed downregulation of myocardin and miR-1 with upregulation of Pim-1. CONCLUSIONS: Our data demonstrate that miR-1 participates in myocardin-dependent of SMC proliferation inhibition.

Screening for broad-spectrum antimicrobial endophytes from Rosa roxburghii and multi-omic analyses of biosynthetic capacity.

Plants with certain medicinal values are a good source for isolating function-specific endophytes. Rosa roxburghii Tratt. has been reported to be a botanical source of antimicrobial compounds, which may represent a promising candidate for screening endophytic fungi with antimicrobial potential. In this study, 54 endophytes were isolated and molecularly identified from R. roxburghii. The preliminary screening using the plate confrontation method resulted in 15 different endophytic strains showing at least one strong inhibition or three or more moderate inhibition against the 12 tested strains. Further re-screening experiments based on the disc diffusion method demonstrated that Epicoccum latusicollum HGUP191049 and Setophoma terrestris HGUP190028 had excellent antagonistic activity. The minimum inhibitory concentration (MIC) test for extracellular metabolites finally indicated that HGUP191049 had lower MIC values and a broader antimicrobial spectrum, compared to HGUP190028. Genomic, non-target metabolomic, and comparative genomic studies were performed to understand the biosynthetic capacity of the screened-out endophytic fungus. Genome sequencing and annotation of HGUP191049 revealed a size of 33.24 megabase pairs (Mbp), with 24 biosynthetic gene clusters (BGCs), where the putative antimicrobial compounds, oxyjavanicin, patulin and squalestatin S1 were encoded by three different BGCs, respectively. In addition, the non-targeted metabolic results demonstrated that the strain contained approximately 120 antimicrobial secondary metabolites and was structurally diverse. Finally, comparative genomics revealed differences in pathogenicity, virulence, and carbohydrate-active enzymes in the genome of Epicoccum spp. Moreover, the results of the comparative analyses presumed that Epicoccum is a promising source of antimicrobial terpenes, while oxyjavanicin and squalestatin S1 are antimicrobial compounds shared by the genus. In conclusion, R. roxburghii and the endophytic HGUP191049 isolated from it are promising sources of broad-spectrum antimicrobial agents.

Proteasomal degradation of myocardin is required for its transcriptional activity in vascular smooth muscle cells.

Myocardin is a transcriptional co-activator of serum response factor (SRF) and can be degraded through ubiquitin-proteasome system. Our preliminary studies unexpectedly revealed that accumulation of myocardin in response to proteasome inhibition by MG132 or lactacystin resulted in decrease of transcriptional activity of myocardin as indicated by reduced expression of SMC contractile marker genes (SM α-actin, SM22, and calponin) and muscle-enriched microRNAs (miR-143/145 and miR-1/133a), and reduced contractility of human vascular smooth muscle cells (SMCs) embedded in collagen gel lattices, suggesting that myocardin degradation is required for its transcriptional activity. Further studies using chromatin immunoprecipitation assay revealed that proteasome inhibition, although increased the occupancy of myocardin and SRF on the promoter of SM α-actin gene, abolished myocardin-dependent recruitment of RNA polymerase II. We further examined the degradation of myocardin in epithelioid and spindle-shaped SMCs and revealed that myocardin in more differentiated spindle-shaped SMCs was more quickly degraded and had shorter half-life than in epithelioid SMCs. In neointimal lesions, we found that stabilization of myocardin protein was companied by downregulation of transcripts of ubiquitin and proteasome subunits, further illustrating the mechanism underlying reduction of myocardin transcriptional activity. In summary, our results have suggested that proteasomal degradation of myocardin is required for its transcriptional activity.

Multigene Phylogeny, Diversity and Antimicrobial Potential of Endophytic Sordariomycetes From Rosa roxburghii.

Rosa roxburghii Tratt. is widely applied in food, cosmetics, and traditional medicine, and has been demonstrated to possess diverse bioactivities. Plant endophytic fungi are important microbial resources with great potential for application in many fields. They not only establish mutualistic symbiosis with host plants but also produce a variety of bioactive compounds. Therefore, in the present study, endophytic fungi were isolated from R. roxburghii, the diversity and antimicrobial activities were evaluated. As a result, 242 strains of endophytic Sordariomycetes were successfully isolated. Multigene phylogenetic analyses showed that these isolates included eight orders, 19 families, 33 genera. The dominant genera were Diaporthe (31.4%), Fusarium (14.4%), Chaetomium (7.9%), Dactylonectria (7.0%), Graphium (4.5%), Colletotrichum (4.1%), and Clonostachys (4.1%). For different tissues of R. roxburghii, alpha diversity analysis revealed that the diversity of fungal communities decreased in the order of root, fruit, stem, flower, leaf, and seed, and Clonostachys and Dactylonectria exhibited obvious tissue specificity. Meanwhile, functional annotation of 33 genera indicated that some fungi have multitrophic lifestyles combining endophytic, pathogenic, and saprophytic behavior. Additionally, antimicrobial activities of endophytic Sordariomycetes against Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Pyricularia oryzae, Rhizoctonia solani, Fusarium oxysporum, Pseudomonas syringae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa were screened. Dual culture test assays showed that there were 40 different endophytic species with strong inhibition of at least one or moderate inhibition of two or more against the 12 tested strains. The results from the filter paper diffusion method suggested that extracellular metabolites may be more advantageous than intracellular metabolites in the development of antimicrobial agents. Eleven isolates with good activities were screened. In particular, Hypomontagnella monticulosa HGUP194009 and Nigrospora sphaerica HGUP191020 have shown promise in both broad-spectrum and intensity. Finally, some fungi that commonly cause disease have been observed to have beneficial biological activities as endophytic fungi. In conclusion, this study showed the species composition, alpha diversity, and lifestyle diversity of endophytic Sordariomycetes from R. roxburghii and demonstrated these isolates are potential sources for exploring antimicrobial agents.

Behavioral Deficits in Mice with Postnatal Disruption of Ndel1 in Forebrain Excitatory Neurons: Implications for Epilepsy and Neuropsychiatric Disorders.

Dysfunction of nuclear distribution element-like 1 (Ndel1) is associated with schizophrenia, a neuropsychiatric disorder characterized by cognitive impairment and with seizures as comorbidity. The levels of Ndel1 are also altered in human and models with epilepsy, a chronic condition whose hallmark feature is the occurrence of spontaneous recurrent seizures and is typically associated with comorbid conditions including learning and memory deficits, anxiety, and depression. In this study, we analyzed the behaviors of mice postnatally deficient for Ndel1 in forebrain excitatory neurons (Ndel1 CKO) that exhibit spatial learning and memory deficits, seizures, and shortened lifespan. Ndel1 CKO mice underperformed in species-specific tasks, that is, the nest building, open field, Y maze, forced swim, and dry cylinder tasks. We surveyed the expression and/or activity of a dozen molecules related to Ndel1 functions and found changes that may contribute to the abnormal behaviors. Finally, we tested the impact of Reelin glycoprotein that shows protective effects in the hippocampus of Ndel1 CKO, on the performance of the mutant animals in the nest building task. Our study highlights the importance of Ndel1 in the manifestation of species-specific animal behaviors that may be relevant to our understanding of the clinical conditions shared between neuropsychiatric disorders and epilepsy.

Ascodesmisrosicola sp. nov. and Talaromycesrosarhiza sp. nov., two endophytes from Rosaroxburghii in China.

BACKGROUND: Rosaroxburghii Tratt., a deciduous shrub of the family Rosaceae, is usually used as food and medicinal materials and also cultivated as an ornamental. Plant endophytic fungi are a large class of microbial resources not fully researched, with great potential applications. Two strains of Ascodesmis and Talaromyces were isolated during a survey of biodiversity on endophytic fungi of R.roxburghii in China. Multigene phylogenetic analyses showed that each of the two fungi formed a distinct lineage and separated from known congeneric species and they are proposed as two novel taxa. NEW INFORMATION: Ascodesmisrosicola sp. nov. usually has one or two conspicuous simple or branched ridges extending to the majority of the ascospore surface and remarkably small asci, distinguishing it from the previously-described species in the genus Ascodesmis. Talaromycesrosarhiza sp. nov., of the section Talaromyces, is closely related to T.francoae. It differs from the latter by having both monoverticillate and biverticillate conidiophores, while those of T.francoae are biverticillate. Both novel endophytes are illustrated and described.