First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 µm, and 80.7 × 3.9 µm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.

Effects of graphene oxide on PCR amplification for microbial community survey.

BACKGROUND: Graphene oxide (GO) has been suggested as an efficient assistant additive to eliminate non-specific amplification of the polymerase chain reaction (PCR). Although many studies have focused on exploring its molecular mechanism, the practice of GO on the quantitation of microbial community has not been implemented yet. In this study, GO was added in PCR system to explore the changes on removing typical amplification errors, such as chimera and mismatches on two kinds of mock communities (an evenly mixed and a staggered mock communities) and environmental samples. RESULTS: High-throughput sequencing of bacterial and fungal communities, based on 16S rRNA genes and internal transcribed spacers (ITS) respectively, showed that GO could significantly increase large segmental error (chimeric sequence) in PCR procedure while had no specific effect on point error (mismatched sequence). Besides, GO reduced the α-diversity of community, and changed the composition of fungal community more obviously than bacterial community. CONCLUSIONS: Our study provides the first quantitative data on microbial community level to prove the negative effect of GO, and also indicates that there may be a more complex interaction between GO and comprehensive DNA fragments in PCR process.

Down-regulation of RFWD3 inhibits cancer cells proliferation and migration in gastric carcinoma.

The E3 ligase RING finger and WD repeat domain 3 (RFWD3) can stabilize p53 in response to DNA damage, participate in replication checkpoint, and have an important role in multiple myeloma, testicular germ cell tumor and lung carcinogenesis. Its expression and molecular mechanisms have never been explored in gastric cancer. In present study, the RFWD3 was over-expressed in both AGS and HGC-27 gastric cancer cells are found. Knockdown of RFWD3 suppressed cells proliferation activity of gastric cancer cells. And further study showed down-regulation of RFWD3 promotes cell apoptosis, suppresses cell migration and invasion and blocks G2/M cell cycle progression, which may be related with AKT, ERK/P38 and Slug pathways. Collectively, the results of the present study showed that RFWD3 might be a candidate oncogene for gastric cell proliferation and may have an important role in gastric carcinogenesis.

The influence of low-temperature resistant lactic acid bacteria on the enhancement of quality and the microbial community in winter Jerusalem Artichoke (Helianthus tuberosus L.) silage on the Qinghai-Tibet Plateau.

Jerusalem Artichoke (Helianthus tuberosus L.), an emerging "food and fodder" economic crop on the Qinghai-Tibet Plateau. To tackle problems such as incomplete fermentation and nutrient loss occurring during the low-temperature ensilage of Jerusalem Artichokes in the plateau's winter, this study inoculated two strains of low-temperature resistant lactic acid bacteria, Lactobacillus plantarum (GN02) and Lactobacillus brevis (XN25), along with their mixed components, into Jerusalem Artichoke silage material. We investigated how low-temperature resistant lactic acid bacteria enhance the quality of low-temperature silage fermentation for Jerusalem Artichokes and clarify its mutual feedback effect with microorganisms. Results indicated that inoculating low-temperature resistant lactic acid bacteria significantly reduces the potential of hydrogen and water-soluble carbohydrates content of silage, while increasing lactic acid and acetic acid levels, reducing propionic acid, and preserving additional dry matter. Inoculating the L. plantarum group during fermentation lowers pH and propionic acid levels, increases lactic acid content, and maintains a dry matter content similar to the original material. Bacterial community diversity exhibited more pronounced changes than fungal diversity, with inoculation having a minor effect on fungal community diversity. Within the bacteria, Lactobacillus remains consistently abundant (>85%) in the inoculated L. plantarum group. At the fungal phylum and genus levels, no significant changes were observed following fermentation, and dominant fungal genera in all groups did not differ significantly from those in the raw material. L. plantarum exhibited a positive correlation with lactic acid and negative correlations with pH and propionic acid. In summary, the inoculation of L. plantarum GN02 facilitated the fermentation process, preserved an acidic silage environment, and ensured high fermentation quality; it is a suitable inoculant for low-temperature silage in the Qinghai-Tibet Plateau.

RACGAP1 drives proliferation, migration and invasion and suppresses autophagy of gastric cancer cells via inhibiting SIRT1/Mfn2.

Objective: Gastric cancer is the most frequent gastrointestinal malignancy with a poor prognosis. Rac GTPase activation protein 1 (RACGAP1) is a novel tumor promotor, whose detailed effect on gastric cancer remains to be further elucidated. Hence, this study identifies the action of RACGAP1 on gastric cancer and investigates the potential mechanism. Methods: RACGAP1 expression in gastric cancer was analyzed based on the data of The Cancer Genome Atlas (TCGA) database. Cell proliferation was measured by CCK-8 and colony formation assay. Cell migration and invasion were evaluated by transwell assay. Cell apoptosis was assessed by flow cytometry. Cell autophagy was evaluated via determining LC3. Results: RACGAP1 presented at high level in gastric cancer cells. Overexpressed RACGAP1 potentiated gastric cancer cell proliferation, migration, and invasion. Besides, silenced RACGAP1 induced cell apoptosis and autophagy. Furthermore, RACGAP1 suppressed the expression of SIRT1 and Mfn2. Conclusion: RACGAP1 was overexpressed in gastric cancer. RACGAP1 potentiated aggressive behaviors of gastric cancer, and suppressed cell apoptosis and autophagy via modulating SIRT1/Mfn2. RACGAP1 may be a valuable target in the treatment of gastric cancer.

[Effects of the Transformation from Natural Alpine Grassland to Mixed Artificial Grassland on the Characteristics of Soil Microbial Community].

This study aimed to analyze the effects of the transformation from natural alpine grassland (NAG) to mixed artificial grassland (MAG) on the characteristics of soil microbial community. We used Illumina Miseq high-throughput sequencing technology to investigate the soil microbial community of natural grassland and mixed artificial grassland. The results showed that plant diversity and the content of soil organic matter decreased significantly from NAG to MAG. In total, 29 and 11 phyla bacteria and fungi were detected, respectively. Compared with that in NAG, the Shannon indexes of the bacterial community increased significantly in MAG (from 9.51 to 9.89), whereas these differences were not significant between the NAG and MAG fungal community. The structure and composition of the soil microbial community showed significant differences between NAG and MAG. In addition, Mantel test results suggested that soil total organic matter, total nitrogen, and soil moisture were significantly correlated with variations in the bacterial community, and soil total organic matter and soil moisture were significantly correlated with variations in fungal community. The results of linear discriminant analysis (LEfSe) indicated that Atribacteria and Ascomycota microorganisms could be considered as the indicator groups for NAG, whereas Gemmata and Trichocomaceae microorganisms could be considered as the indicator groups for MAG. Tax4Fun2 results showed that the transition from NAG to MAG affected the utilization of different carbon sources by bacteria.

Grass-microbial inter-domain ecological networks associated with alpine grassland productivity.

Associations between grasses and soil microorganisms can strongly influence plant community structures. However, the associations between grass productivity and diversity and soil microbes, as well as the patterns of co-occurrence between grass and microbes remain unclear. Here, we surveyed grass productivity and diversity, determined soil physicochemical, and sequenced soil archaea, bacteria and fungi by metabarcoding technology at 16 alpine grasslands. Using the Distance-decay relationship, Inter-Domain Ecological Network (IDEN), and Mantel tests, we investigated the relationship between grass productivity, diversity and microbial diversity, and the patterns of co-occurrence between grass and microbial inter-domain network in alpine grassland. We found the archaea richness, bacteria richness and Shannon, and fungi α-diversity were significantly negatively correlation with grass diversity, but archaea and bacteria diversity were positively correlation with grass productivity. Moreover, an increase in microbial ß-diversity was observed along with increased discrepancy in grass diversity and productivity and soil variables. Variance partitioning analysis suggested that the contribution of grass productivity on microbial community was higher than that of soil variables and grass diversity, which implies that microbial community was more related to grass productivity. Inter-Domain Ecological Network showed that the grass species formed complex and stable ecological networks with some bacterial, archaeal, and fungal species, and the grass-fungal ecological networks showed the highest robustness, which indicated that soil fungi could better co-coexist with aboveground grass in alpine grasslands. Besides, the connectivity degrees of the grass-microbial network were significantly positively correlated with grass productivity, suggesting that the coexistence pattern of grasses and microbes had a positive feedback effect on the grass productivity. The results are important for establishing the regulatory mechanisms between plants and microorganisms in alpine grassland ecosystems.

Non-targeted metabolomics analysis reveals the mechanism of arbuscular mycorrhizal symbiosis regulating the cold-resistance of Elymus nutans.

Elymus nutans is a perennial grass of the Gramineae family. Due to its cold-resistance and nutrition deficiency tolerance, it has been applied to the ecological restoration of degraded alpine grassland on the Qinghai-Tibet Plateau. As an important symbiotic microorganism, arbuscular mycorrhizal fungi (AMF) have been proven to have great potential in promoting the growth and stress resistance of Gramineae grasses. However, the response mechanism of the AMF needs to be clarified. Therefore, in this study, Rhizophagus irregularis was used to explore the mechanism regulating cold resistance of E. nutans. Based on pot experiments and metabolomics, the effects of R. irregularis were investigated on the activities of antioxidant enzyme and metabolites in the roots of E. nutans under cold stress (15/10°C, 16/8 h, day/night). The results showed that lipids and lipid molecules are the highest proportion of metabolites, accounting for 14.26% of the total metabolites. The inoculation with R. irregularis had no significant effects on the activities of antioxidant enzyme in the roots of E. nutans at room temperature. However, it can significantly change the levels of some lipids and other metabolites in the roots. Under cold stress, the antioxidant enzyme activities and the levels of some metabolites in the roots of E. nutans were significantly changed. Meanwhile, most of these metabolites were enriched in the pathways related to plant metabolism. According to the correlation analysis, the activities of antioxidant enzyme were closely related to the levels of some metabolites, such as flavonoids and lipids. In conclusion, AMF may regulate the cold-resistance of Gramineae grasses by affecting plant metabolism, antioxidant enzyme activities and antioxidant-related metabolites like flavonoids and lipids. These results can provide some basis for studying the molecular mechanism of AMF regulating stress resistance of Gramineae grasses.

Grass-Legume Mixture with Rhizobium Inoculation Enhanced the Restoration Effects of Organic Fertilizer.

The establishment of artificial grassland is crucial in restoring degraded grassland and resolving the forage-livestock conflict, and the application of organic fertilizer and complementary seeding of grass-legume mixture are effective methods to enhance grass growth in practice. However, its mechanism behind the underground is largely unclear. Here, by utilizing organic fertilizer in the alpine region of the Qinghai-Tibet Plateau, this study assessed the potential of grass-legume mixtures with and without the inoculation of Rhizobium for the restoration of degraded grassland. The results demonstrated that the application of organic fertilizer can increase the forage yield and soil nutrient contents of degraded grassland, and they were 0.59 times and 0.28 times higher than that of the control check (CK), respectively. The community composition and structure of soil bacteria and fungi were also changed by applying organic fertilizer. Based on this, the grass-legume mixture inoculated with Rhizobium can further increase the contribution of organic fertilizer to soil nutrients and thus enhance the restoration effects for degraded artificial grassland. Moreover, the application of organic fertilizer significantly increased the colonization of gramineous plant by native mycorrhizal fungi, which was ~1.5-2.0 times higher than CK. This study offers a basis for the application of organic fertilizer and grass-legume mixture in the ecological restoration of degraded grassland.

Application of Organic Fertilizer Changes the Rhizosphere Microbial Communities of a Gramineous Grass on Qinghai-Tibet Plateau.

The effects of organic fertilizer application on the soil microbial community in grassland systems have been extensively studied. However, the effects of organic fertilizers on the structure of rhizosphere microbial communities are still limited. In this study, the diversity and composition of rhizosphere microbial communities of a gramineous grass Elymus nutans under organic fertilizer treatment were studied in an artificial pasture on Qinghai-Tibet Plateau. After a growing season, the application of organic fertilizer not only increased the height and biomass of Elymus nutans, but also changed the rhizosphere microbial compositions. In particular, organic fertilizer increased the diversity of rhizosphere bacterial community and inhibited the growth of pathogenic bacteria such as Acinetobacter, but the opposite trend was observed for the diversity of fungal community. The assembly process of fungal community was changed from a stochastic process to a deterministic process, indicating that selection was strengthened. Additionally, both the infection rate of arbuscular mycorrhizal fungi (AMF) toward host plants and the development of AMF-related structures were significantly increased after the application of organic fertilizer. Our study demonstrated that the addition of organic fertilizer to artificial pasture could improve the growth of grass through the alteration of the rhizosphere microbial communities. Organic fertilizer had a greater selectivity for the bacterial and the fungal communities that enhanced the niche filtration in this community, further benefiting the yield of forages.

Homogeneous Selection and Dispersal Limitation Dominate the Effect of Soil Strata Under Warming Condition.

Global warming is likely to affect the underground microbial communities in various ecosystems, but the response of soil microbial communities along a vertical depth profile to global warming has been elusive. Herein, we leveraged a warming field experiment in Qinghai-Tibet Plateau grassland and investigated the community structure of prokaryotes and fungi from the upper (0-15 cm) and lower (15-30 cm) strata under ambient and elevated temperature treatments. Three-years continual warming only significantly shifted the prokaryotic community within the upper strata and there was no significant effect observed for the fungal community. Additionally, under ambient temperature, there were significant differences between the two strata in both the prokaryotic and fungal communities, but under warming, this effect was alleviated. Next, the prokaryotic and fungal community assembly processes were measured by a phylogenetic-bin-based null approach (iCAMP). Though deterministic and stochastic processes dominated the assembly of prokaryotic and fungal communities, respectively, the deterministic processes were strengthened under warming for both communities. Specifically, the increased portion of homogeneous selection, contributing to a homogenous state, led to a smaller difference between prokaryotic communities of the two soil strata under warming. The smaller difference in the stochastic process component, i.e., dispersal limitation, contributed to the similar fungal community structures between the two strata under warming. This study deepens our understanding of warming effects on grassland microbial communities and gives greater insights on the underlying mechanisms for microbial assembly between upper and lower soil strata under warming scenarios.

A novel AVPR2 missense mutation in a Chinese boy with severe inherited nephrogenic diabetes insipidus.

Inherited nephrogenic diabetes insipidus (NDI) is characterized by renal resistance to arginine vasopressin (AVP). The most common cause is mutations in the AVP receptor 2 (AVPR2) gene at Xq28. Severe complications of NDI are rare but can occur after severe dehydration without treatment. A 7-year-old boy presented with short stature and severe intellectual disability other than polyuria and polydipsia. The karyotype was normal. Direct sequencing revealed a novel missense mutation c.506T > C (p.L169P) in AVPR2 in the patient. His mother was heterozygous for the mutation. The mutation was absent in 103 unrelated healthy males and predicted to be consistently pathogenic by several prediction methods, including Polyphen, SIFT, PMut, PhD-SNP, SNPs3D, PANTHER, and MEMPACK. Awareness of the primary signs of NDI, polyuria, and polydipsia would facilitate early diagnosis and treatment to prevent its severe complications. Also, molecular analysis will provide a rapid and definitive diagnosis and facilitate genetic counseling for family planning.

Meadow degradation increases spatial turnover rates of the fungal community through both niche selection and dispersal limitation.

The alpine meadow ecosystem, as the main ecosystem of the Qinghai-Tibet Plateau, has been heavily degraded over the past several decades due to overgrazing and climate change. Although soil microorganisms play key roles in the stability and succession of grassland ecosystems, their response to grassland degradation has not been investigated at spatial scale. Here, we systematically analyzed the spatial turnover rates of soil prokaryotic and fungal communities in degraded and undegraded meadows through distance-decay relationship (DDR) and species area relationship (SAR), as well as the community assembly mechanisms behind them. Although the composition and structure of both fungal and prokaryotic communities showed significant changes between undegraded and degraded meadows, steeper spatial turnover rates were only observed in fungi (Degraded Alpine Meadow ß = 0.0142, Undegraded Alpine Meadow ß = 0.0077, P < 0.05). Mantel tests indicated that edaphic variables and vegetation factors showed significant correlations to the ß diversity of fungal community only in degraded meadow, suggesting soil and vegetation heterogeneity both contributed to the variation of fungal community in that system. Correspondingly, a novel phylogenetic null model analysis demonstrated that environmental selection was enhanced in the fungal community assembly process during meadow degradation. Interestingly, dispersal limitation was also enhanced for the fungal community in the degraded meadow, and its relative contribution to other assembly process (i.e. selection and drift) showed a significant linear increase with spatial distance, suggesting that dispersal limitation played a greater role as distance increased. Our findings indicated the spatial scaling of the fungal community is altered during meadow degradation by both niche selection and dispersal limitation. This study provides a new perspective for the assessment of soil microbial responses to vegetation changes in alpine areas.

miR-574-3p inhibits proliferation and invasion in esophageal cancer by targeting FAM3C and MAPK1.

Esophageal cancer is considered as one of the leading malignancies. MicroRNA-574-3p (miR-574-3p) was used as a postoperative prognostic indicator in patients with esophageal squamous cell carcinoma. However, the underlying mechanism miR-574-3p involvement in esophageal cancer remains unclear. In this study, the expression of miR-574-3p was reduced in esophageal cancer tissues and cells. In vitro, miR-574-3p mimics and inhibitor were transfected into esophageal cancer cells (TE-1 and TE-8 cells) to up- or downregulating of miR-574-3p. miR-574-3p inhibited proliferation, migration and invasion, and promoted apoptosis in esophageal cancer cells. In addition, miR-574-3p was confirmed to target family with sequence similarity 3 member C (FAM3C) and mitogen-activated protein kinase 1 (MAPK1). miR-574-3p suppressed phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling via regulating FAM3C and MAPK1. In vivo, overexpression of miR-574-3p suppressed tumor growth in mice. Our findings indicated that miR-574-3p repressed proliferation and invasion of esophageal cancer via regulation of FAM3C and MAPK1, which provides a new biomarker for esophageal cancer treatment.

Culture conditions and nutrition requirements for the mycelial growth of Isaria farinosa (Hypocreales: Cordycipitaceae) and the altitude effect on its growth and metabolome.

Isaria farinosa is a pathogen of alpine Thitarodes larvae that are hosts for the Chinese medicinal fungus, Ophiocordyceps sinensis. A matrix analysis indicated that the optimal culture conditions for the mycelial growth of I. farinosa are a 50-mL liquid broth in a 250-mL flask at more than 100-rpm rotation and 15-25 °C. Illumination does not affect the mycelial growth. The optimal nutrition requirements are D-(+)-galactose and D-(-)-fructose as carbon resources and D-cysteine as well as yeast powder, peptone, and beef extract as nitrogen resources at a carbon-to-nitrogen ratio of 1:1 to 1:7. The mineral component and vitamins also significantly increase the mycelial growth of I. farinosa. Based on the optimal culture conditions and nutrition requirements for the mycelial growth of I. farinosa, the effects of altitude on mycelial growth and its metabolome were evaluated using quadrupole-time-of-flight/mass spectrometry, principal component analysis and partial least squares discriminant analysis. The altitude did not affect the mycelial production but significantly regulated its metabolome. The study presents a new approach to better select a method for producing more useful metabolites and highlights the necessity of establishing standards for culturing methods related to altitude to preserve fungal quality; additionally, the results indicate that the use of a fermenter may meet the demands of large-scale mycelial production.

Synthesis and biological evaluation of deoxy salacinols, the role of polar substituents in the side chain on the alpha-glucosidase inhibitory activity.

Three analogs (5, 6, and 7) lacking polar substituents in the side chain of a naturally occurring alpha-glucosidase inhibitor, salacinol (1a), were synthesized by the coupling reaction of a thiosugar, 1,4-dideoxy-1,4-epithio-D-arabinitol (3), with cyclic sulfates (8, 9, and 10), and their alpha-glucosidase inhibitory activities were examined. All these simpler analogs (5, 6, and 7) showed less inhibitory activity compared to 1a, and proved the importance of cooperative role of the polar substituents for the alpha-glucosidase inhibitory activity. A practical synthetic route to 3 starting from D-xylose is also described.