Identification and Fungicide Screening of Fungal Species Associated with Walnut Anthracnose in Shaanxi and Liaoning Provinces, China.

Walnut is cultivated around the world for its precious woody nut and edible oil. Recently, walnut infected by Colletotrichum spp. resulted in a great yield and quality loss. In August and September 2014, walnut fruits with anthracnose were sampled from two commercial orchards in Shaanxi and Liaoning provinces, and five representative isolates were used in this study. To identify the pathogen properly, four genes per region (internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, actin, and chitin synthase) were sequenced and used in phylogenetic studies. Based on multilocus phylogenetic analysis, five isolates clustered with Colletotrichum fioriniae, including its ex-type, with 100% bootstrap support. The results of multilocus phylogenetic analyses, morphology, and pathogenicity confirmed that C. fioriniae was one of the walnut anthracnose pathogens in China. All 13 fungicides tested inhibited mycelial growth and spore germination. Flusilazole, fluazinam, prochloraz, and pyraclostrobin showed the strongest suppressive effects on the mycelial growth than the others, the average EC50 values ranged from 0.09 to 0.40 µg/ml, and there was not any significant difference (P < 0.05). Pyraclostrobin, thiram, and azoxystrobin were the most effective fungicides on spore germination (P < 0.05), and the EC50 values ranged from 0.01 to 0.44 µg/ml. Pyraclostrobin, azoxystrobin, fluazinam, flusilazole, mancozeb, thiram, and prochloraz exhibited a good control effect on walnut anthracnose caused by C. fioriniae, and preventive activities were greater than curative activities. Pyraclostrobin at 250 a.i. µg/ml and fluazinam at 500 a.i. µg/ml provided the highest preventive and curative efficacy, and the values ranged from 81.3 to 82.2% and from 72.9 to 73.6%, respectively. As a consequence, mancozeb and thiram could be used at the preinfection stage, and pyraclostrobin, azoxystrobin, flusilazole, fluazinam, and prochloraz could be used at the early stage for effective prevention and control of walnut anthracnose caused by C. fioriniae. The results will provide more significant instructions for controlling the disease effectively in northern China.

Complexity of influences on atrazine phytoremediation of coexisting graphene oxide in water: Mitigating its phytotoxicity while decreasing plant removal contribution.

Phytoremediation is an efficient technology for the removal of herbicide atrazine (ATZ) contamination in water bodies, but its ability to reduce ATZ under combined pollution remains unclear, especially ATZ co-existing with the emerging pollutant graphene oxide (GO) that may have potential effects on ATZ fate, plants and microbes. Herein, we investigated the phytoremediation potential of an emergent plant (Iris pseudacorus) for ATZ and the response of bacteria in a hydroponic system with and without GO. The results showed that plants enhanced ATZ dissipation in water with the increased removal rate by a factor of 1.7-4.0. GO restricted ATZ uptake by plants, but favored ATZ bioconcentration in cell walls. The plant contributed most to changes in the bacterial communities, decreasing the alpha diversity, while enriching the functional categories involving in amino acid and carbohydrate metabolisms. These findings indicated that I. pseudacorus can be employed as an effective candidate of phytoremediation for ATZ co-existing with GO at environmentally relevant concentrations, tending to recruit bacteria with plant stress tolerance and growth-promotion activities more than with ATZ degradation activities; GO exerted a mitigating effect on ATZ stress improving the barrier function of cell walls, but decreased the contribution of plants to ATZ removal.

Transcriptome analysis of anaerobic glycolysis effects on Jurkat T cell proliferation.

Introduction: To explore the effects of anaerobic glycolysis on Jurkat T cell proliferation and clarify the possible mechanism via transcriptomic analysis. Material and methods: The monocarboxylate transporter 1 inhibitor AZD3965 was used to target and block the transmembrane transport of lactate, thereby inhibiting anaerobic glycolysis in Jurkat T cells. Then, genes with differential expression between treated and untreated cells were detected by transcriptomic analysis, and constructs were generated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses as well as protein-protein interaction (PPI) network analysis were performed to explore the potential mechanism. Results: Inhibition of anaerobic glycolysis reduced Jurkat T-cell proliferation. RNA sequencing identified 1723 transcripts that were differentially expressed, including 1460 upregulated genes and 263 downregulated genes. GO functional enrichment analysis showed that the differentially expressed genes were mainly involved in the biological processes of response to unfolded protein, response to topologically incorrect protein, and protein folding. KEGG pathway analysis of differentially expressed genes or hub genes from the PPI network analysis revealed enrichment in the estrogen signaling and PI3K-Akt pathways. Conclusions: Anaerobic glycolysis contributes to the regulation of Jurkat T-cell proliferation. The underlying mechanism may involve the estrogen signaling pathway or PI3K-Akt signaling pathway as well as protein metabolism.

Salt Restriction and Angiotensin-Converting Enzyme Inhibitor Improve the Responsiveness of the Small Artery in Salt-Sensitive Hypertension.

For salt-sensitive hypertension (SSH), salt restriction and angiotensin-converting enzyme (ACE) inhibitors are essential treatments, but their effect on the function of resistance arteries is unclear. Here, we present an intravital study to detect the effect of salt restriction and ACE inhibitors on the function of the mesenteric small artery (MSA) in SSH. Dahl salt-sensitive rats were randomized into the following groups: ACE inhibitor gavage, salt restriction, ACE inhibitor combined with salt restriction, and high-salt diet. After a 12-week intervention, the mesenteric vessels maintained their perfusion in vivo, and the changes in the diameter and blood perfusion of the MSAs to norepinephrine (NE) and acetylcholine (ACh) were detected. Switching from a high-salt diet to a low-salt diet (i.e., salt restriction) attenuated the vasoconstriction of the MSAs to NE and promoted the vasodilatation to ACh, while ACE inhibitor improved the vasodilatation more obviously. Pathologically, changes in local ACE, AT1R, and eNOS expression were involved in these processes induced by a high-salt diet. Our study suggests that salt restriction and ACE inhibitor treatment improve high salt intake-induced MSA dysfunction in SSH, and salt restriction is a feasible and effective treatment. Our findings may provide a scientific basis for the treatment of hypertension.

RTP4, a Biomarker Associated with Diagnosing Pulmonary Tuberculosis and Pan-Cancer Analysis.

Background: Pulmonary tuberculosis (PTB) is a global epidemic of infectious disease; the purpose of our study was to explore new potential biomarkers for the diagnosis of pulmonary tuberculosis and to use the biomarkers for further pan-cancer analysis. Methods: Four microarray gene expression sets were downloaded from the GEO public databases and conducted for further analysis. Healthy control (HC) samples and samples of pulmonary tuberculosis (PTB) were calculated with enrichment scores in folate biosynthesis pathways. The scores acted as a new phenotype combined with clinical information (control or PTB) for subsequent analysis. Weight gene coexpression network analysis (WGCNA) was used to seek the modules mostly related to PTB and folate biosynthesis in training sets. Twenty-nine coexistence genes were screened by intersecting the genes in the green-yellow module of GSE28623 and the brown module of GSE83456. We used the protein-protein interaction network analysis to narrow the gene range to search for hub genes. Then, we downloaded the unified and standardized pan-cancer data set from the UCSC database for correlations between biomarkers and prognosis and tumor stage differences. Results: Eventually, RTP4 was selected as a biomarker. To verify the reliability of this biomarker, an area under the ROC (AUC) was calculated in gene sets (GSE28623, GSE83456, and GSE34608). Lastly, to explore the difference in RTP4 expression before and after antituberculosis treatment, the GSE31348 gene set was enrolled to compare the expressions in weeks 0 and 26. The results showed significant differences between these two time points (p < 0.001). RTP4 was significantly upregulated in the pulmonary tuberculosis group compared to the healthy control group in three gene sets and downregulated after antituberculosis therapy in one gene set. These results suggest that RTP4 can be used as a potential biomarker in diagnosing tuberculosis. The results of pan-cancer analysis showed that high expression of RTP4 in 4 tumor types was positively correlated with poor prognosis and high expression of RTP4 in 6 tumor types was negatively correlated with poor prognosis. We found significant differences in the expression of the RTP4 gene at different stages in 5 types of tumors. Conclusion: RTP4 might be a new potential biomarker for diagnosing pulmonary tuberculosis.

Reduced 2,4-dienoyl-CoA reductase 1 is served as an unfavorable biomarker and is related to immune infiltration in cervical cancer.

BACKGROUND: Worldwide, cervical cancer (CC) remains the most prevalent malignancy of the female reproductive system, posing a threat to women's life and health, and increasing the medical and economic burden on society. Therefore, the search for tumor biomarkers for CC remains an important research direction. Immunotherapy has significantly improved patient outcomes, and genes related to tumor immune infiltration have been clinically relevant and highly reproducible biomarkers that affect the prognosis and response to treatment of CC. 2,4-dienoyl-CoA reductase 1 (DECR1) was considered to be an oncogene in a previous study, but relationship between DECR1 and immune infiltration was not mentioned. Our study aimed to reveal the clinical value of DECR1 in CC and to investigate its relationship with immune infiltration. METHODS: Human Protein Atlas was used to identify the localization of DECR1. The Ualcan database, TCGA, and IHC were used to assess the prognostic value of DECR1. GSEA was used to assess the possible signaling pathways of DECR1 in CC. The TIMER database was applied to reveal the relevance between DECR1 and immune infiltration. GEPIA was conducted to detect the co-relationship among DECR1, immune markers, and typical molecules of apoptosis. RESULTS: DECR1 was mainly distributed in the cytoplasm and overlapped with the endoplasmic reticulum. DECR1 was downregulated in CC compared to adjacent tissue. Survival analysis showed that patients with lower expression of DECR1 have a worse prognosis in CC. GSEA suggested that DECR1 was closely related to apoptosis signaling. TIMER showed that DECR1 was positively correlated with CD8+ T cell and CD4+ T cell but not with B cell in CC. CONCLUSION: DECR1 may be a potential cancer suppressor in CC and may be involved in apoptotic pathways and associated with immune infiltration.

First report of jujube anthracnose caused by Colletotrichum siamense in China.

Jujube (Zizyphus jujuba Mill.), a native small deciduous tree of China, is widely cultivated in China, Korea, India, Japan, Europe, and the United States (Chen et al. 2020). The fruit have been commonly consumed as healthy food supplements and traditional Chinese medicine for over 2000 years (Li et al. 2007). In August 2019, anthracnose-like leaf spot symptoms were observed on jujube plants in Xiaomenya Village, Jinan City, Shandong Province, China (36°27'39″N, 117°3'13″E), with over 30% leaf disease incidence. The spots were circular, sunken, brown in the center and with dark brown edges. As the spots enlarged and coalesced, it resulted in leaf perforation and early defoliation. Sometimes acervuli were observed on the lesions (Fig. S1a, b). To identify the causal agent, 20 diseased leaves were sampled, the margins of the lesions were cut into pieces (5 × 5 mm), sterilized and cultured following the protocol described previously (Wan et al. 2020) at 25 ℃ for 5 days. Twelve monospore isolates showing identical colony morphology were obtained. Three representative isolates, JNZG11, JNZG311, JNZG313, were used for further study. When grown on PDA the colony color was initially white and then turned pale-gray to gray in 5-day-old cultures. On the reverse, colonies were brown-black with an orange pigmentation near the center. Aerial mycelium was cottony, dense, white to pale-gray. Conidia were hyaline, 1-celled, smooth-walled, subcylindrical, oblong, attenuated with slightly rounded ends, (11.1-) 12.7-13.3 (-17.8) ×(-4.4) 5.2-5.5 (-6.3) µm (n=50). Appressoria were dark-brown, oval or irregular, (7.3-) 8.6-9.2 (-9.8) ×(-5.1) 5.8-6.9 (-7.0) µm (n=50) (Fig. S1c-g). The morphology resembled those of Colletotrichum gloeosporioides species complex (Cannon et al. 2012). For accurate identification, the sequences of the ribosomal internal transcribed spacer (ITS), actin (ACT), ß-tub2 (TUB2), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) of the 3 isolates were sequenced (Weir et al. 2012), and deposited into GenBank (Accession Nos. see Table 1). The six loci (ITS, GAPDH, ACT, CHS-1, CAL, and TUB2) were concatenated and the aligned sequences (1904 bp) were 99.7% homologous to ex-type C. siamense ICMP18578. The sequences of 38 Colletotrichum species (44 isolates) were downloaded from GenBank for phylogenetic analyses. In the maximum likelihood phylogenetic tree generated, the highest log likelihood was -8798.90 and the three isolates were all in the C. siamense clade (bootstrap support 94 %) (Fig. S2). To complete Koch's postulates, 60 healthy, mature jujube leaves on 12 branches (5 leaves per branch) (variety 'Zhongqiuhong') were inoculated with 20 µL of spore suspension (106 conidia/mL) or sterile water as a control. The branches were placed in sterile beakers containing a small amount of sterile water sealed with plastic wrap and maintained at 28 °C, 12 h light/dark. Five days after inoculation, all treated leaves showed the typical anthracnose symptom, similar to that observed in the field (Fig. S1h). The same fungus was re-isolated from the margins of the lesions using the aforementioned methods. Whereas no fungus were isolated from the controls. Previously, C. siamense has been reported to infect Z. mauritiana in China (Shu et al. 2020). To our knowledge, this is the first report of C. siamense causing anthracnose on Z. jujuba in China. This finding provides crucial information for the effective management of this disease.

Defining key metabolic roles in osmotic adjustment and ROS homeostasis in the recretohalophyte Karelinia caspia under salt stress.

The recretohalophyte Karelinia caspia is of forage and medical value and can remediate saline soils. We here assess the contribution of primary/secondary metabolism to osmotic adjustment and ROS homeostasis in Karelinia caspia under salt stress using multi-omic approaches. Computerized phenomic assessments, tests for cellular osmotic changes and lipid peroxidation indicated that salt treatment had no detectable physical effect on K. caspia. Metabolomic analysis indicated that amino acids, saccharides, organic acids, polyamine, phenolic acids, and vitamins accumulated significantly with salt treatment. Transcriptomic assessment identified differentially expressed genes closely linked to the changes in above primary/secondary metabolites under salt stress. In particular, shifts in carbohydrate metabolism (TCA cycle, starch and sucrose metabolism, glycolysis) as well as arginine and proline metabolism were observed to maintain a low osmotic potential. Chlorogenic acid/vitamin E biosynthesis was also enhanced, which would aid in ROS scavenging in the response of K. caspia to salt. Overall, our findings define key changes in primary/secondary metabolism that are coordinated to modulate the osmotic balance and ROS homeostasis to contribute to the salt tolerance of K. caspia.

First report of Colletotrichum siamense causing leaf spot on Manglietia decidua in the world.

Manglietia decidua Q. Y. Zheng is a deciduous broad-leaved plant and native to Jiangxi province, China. It is cultivated for its timber and urban landscaping (Xiong et al., 2014). In September 2019, a new foliar disease was observed on approx. 25% of 121 M. decidua trees in Jiangxi Agricultural University (N28°45'56″, E115°50'21″), Nanchang city, Jiangxi Province, China. The disease site belongs to the subtropical monsoon humid climate, with rainfall (1,600-1,700 mm) and red soil region. Initially, infection appeared on the leaf margins or tips as water-soaked, irregular lesions, then expanded to the center, developed into large black-brown, irregular necrotic lesions. Finally, the lesions fall off the leaves. To identify the pathogen, 15 diseased leaves were collected from 5 trees (3 leaves per tree) randomly. Small pieces (5 × 5 mm) cut from the lesion margins were surfaced sterilized (70% ethanol for 30 s, 3% NaOCl for 1 min, rinsed 3 times with sterile water), and placed on potato dextrose agar (PDA) at 25 °C. Among the isolated fungi, Colletotrichum-like colonies were about 91%, and 18 monoconidial isolates were obtained. Isolates HML-1, HML-4, and HML-7 were selected and preserved for further studies. Colonies on PDA were white, cottony, and grayish-white on the reverse side. Setae absent. Acervuli were brown, circular. Conidiophores were clear, septate, non-branching or branching at the base, conidiogenous cells were enteroblastic, phialidic, colorless, cylindrical, ampulliform. Conidia were elliptical, single-celled, straight, hyaline, and measured 13.3-17.9 × 4.3-5.7 µm (14.8 ± 1.2 × 4.8 ± 0.4 µm, n = 100). Appressoria were oval to irregular, dark brown, and ranged from 5.3-9.1 × 4.4-6.3 µm (7.2 ± 0.3 × 5.1 ± 0.2 µm, n=100). Morphological characteristics matched the description of Colletotrichum gloeosporioides sensu lato (Weir et al. 2012). The internal transcribed spacer regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS-1), calmodulin (CAL), and beta-tubulin 2 (TUB2) were sequenced (Weir et al., 2012), and deposited in GenBank (ITS: OL757565-OL757567; ACT: OL627398-OL627400; CHS-1: OL757358-OL757360; GAPDH: OL757361-OL757363; CAL: OL757355-OL757357; TUB2: OL757364-OL757366). Six loci were concatenated, and the aligned sequences (2056 bp) were 99.9%, 99.8% homologous to C. siamense ICMP 18574 and ex-type ICMP18578, respectively. In the maximum-likelihood phylogenetic tree, the highest log likehihood was -9259.74, and 3 isolates were in the C. siamense clade. Based on the phylogeny and morphology, 3 isolates were identified as C. siamense. The pathogenicity of 3 isolates was tested on 12 M. decidua plants (variety: Yi lin ke) grown in the field. Healthy leaves were wounded slightly with a needle (Φ=0.5 mm) and inoculated with 10 µL of spore suspension (106 conidia/mL). Controls were treated with ddH2O (Si et al. 2021). All the treated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiments were repeated twice. Within 9 days, all the inoculated points showed similar symptoms to those observed in the field, whereas controls were asymptomatic. The same isolate was re-isolated from the lesions, whereas no fungus was isolated from control leaves. Manglietia decidua is an ancient and endangered plant, threatened with southern blight (Sclerotium rolfsii) (Yi et al. 2021a), root rot (Calonectria ilicicola) (Yi et al. 2021b). This is the first report of the newly emerging disease caused by C. siamense in the world. The potential threat should be evaluated for conservation in the future. This study provided crucial information for epidemiological studies and appropriate control strategies.

First report of Colletotrichum fioriniae causing leaf spot on Schima superba in the world.

Schima superba Gardn. et Champ. is a subtropical evergreen tree species naturally distributed mainly in China, Japan, and Vietnam. It is primarily planted for its timber and urban landscaping in China (Ni, 1996). In September 2018, leaves necrotic spots were observed on S. superba in Jiangxi Forest Breeding Center (28°57'19.52" N, 115°39'21.32" E), Jiangxi Province, China. The disease incidence was about 30%. Initially, spots were circular to semicircular, grayish-brown in the center with dark brown margin, then expanded and eventually collapsed into sunken necrotic lesions. To identify the agent, diseased leaves were collected randomly. Pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol (30 s), 3% NaOCl (60 s), and rinsed 3 times in sterile water. These pieces were put on potato dextrose agar (PDA) and cultured at 25 °C. Pure cultures were obtained by monosporic isolation, and 3 isolates (MH-1, MH-2, MH-3) were used for morphological studies and phylogenetic analyses. On PDA, colonies were initially white, cottony, then became pinkish to deep-pink at the center and pink on the reverse. Conidia were fusiform with acute ends, smooth-walled, hyaline, 13.7-18.5 × 4.6-6.1 µm (16.4 ± 1.3× 5.3 ± 0.6 µm, n = 100). Conidiophores were colorless to pale brown, smooth, septate. Conidiogenous cells were colorless to pale brown, smooth, cylindrical to ampulliform. The morphological characteristics fit the descriptions of Colletotrichum acutatum J. H. Simmonds sensu lato (Damm et al., 2012). For accurate identification, genomic DNA of 3 isolates was extracted, and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin 2 (TUB2), and chitin synthase (CHS-1) were amplified and sequenced using the corresponding primers (Weir et al., 2012). The sequences were deposited in GenBank (ITS: MZ325946, MZ325947, MW584318; ACT: MZ399375, MZ419566, MW661171; CHS-1: MZ399376, MZ419567, MW661172; MZ399377, GAPDH: MZ419568, MW661173; TUB2: MZ399378, MZ419569, MW661174). Five loci were concatenated, and the aligned sequences (1528bp) were 99.89% homologous to ex-type C. fioriniae (Marcelino & Gouli) R. G. Shivas & Y. P. Tan CBS128517. Phylogenetic analysis using the maximum likelihood showed that 3 isolates were clustered in C. fioriniae clade with 100% bootstrap support. Based on the multi-locus phylogeny and morphology, 3 isolates were identified as C. fioriniae. Pathogenicity tests were performed on 36 seedlings of S. superba (2-year-old). The leaves were wounded slightly and inoculated with a drop of spore suspension (106 conidia/mL). The sterile water was used as controls. All the tested leaves were covered with black plastic bags to keep them moist for 2 days. All seedlings were placed in the greenhouse (25 °C, 12 h light/dark) for 10 days, and all inoculated leaves had typical symptoms. The controls were asymptomatic. The same fungus was reisolated from the lesions, fulfilling Koch's postulates. Colletotrichum fioriniae was described as a new species from the C. acutatum s. l. (Shivas et al., 2009), and it was an important plant pathogen, such as Pyrus spp. (Pavlovic et al., 2019), Morus alba L. (Xue et al., 2019), and so on. This is the first report of the newly emerging disease of S. superba caused by C. fioriniae in the world, and its potential threat should be evaluated in the future. This study provided crucial information for epidemiologic studies and appropriate control strategies.

Effects of Graphene Oxide on Atrazine Phytotoxicity Effects of Graphene Oxide on Photosynthetic Response of Iris Pseudacorus to Atrazine Stress and Accumulation of Atrazine in the Plant.

To evaluate combined effects of co-existed pesticides and nanomaterials on aquatic plants, the toxicity of herbicide atrazine (ATZ) on Iris pseudacorus in the presence and absence of Graphene oxide (GO) was investigated using chlorophyll a fluorescence transients. Results showed that GO reduced ATZ accumulation in plant. ATZ or ATZ combined with GO mainly blocked electron transport beyond QA at PSII as indicated by the sharp rise of the J-step level of fluorescence rise kinetics. The pronounced increase in Fm and the loss of I-step were observed when ATZ was at 2.0 mg·L- 1 implying the damage on the oxygen evolution complex and PSI. GO at environmentally relevant concentration did not exhibit significant photosynthetic inhibitory effects on I. pseudacorus. GO at 1.0 mg·L- 1 promoted photosynthesis of I. pseudacorus under ATZ stress at 2.0 mg·L- 1. These result indicated that the presence of GO alleviated the photosynthesis inhibition by ATZ at high levels.

On the Quantization of AB Phase in Nonlinear Systems.

Self-intersecting energy band structures in momentum space can be induced by nonlinearity at the mean-field level, with the so-called nonlinear Dirac cones as one intriguing consequence. Using the Qi-Wu-Zhang model plus power law nonlinearity, we systematically study in this paper the Aharonov-Bohm (AB) phase associated with an adiabatic process in the momentum space, with two adiabatic paths circling around one nonlinear Dirac cone. Interestingly, for and only for Kerr nonlinearity, the AB phase experiences a jump of π at the critical nonlinearity at which the Dirac cone appears and disappears (thus yielding π-quantization of the AB phase so long as the nonlinear Dirac cone exists), whereas for all other powers of nonlinearity, the AB phase always changes continuously with the nonlinear strength. Our results may be useful for experimental measurement of power-law nonlinearity and shall motivate further fundamental interest in aspects of geometric phase and adiabatic following in nonlinear systems.

First report of leaf spot disease caused by Colletotrichum siamense on Cornus hongkongensis (Hemsl.) in China.

Cornus hongkongensis (Hemsl.) is an excellent ornamental tree species in China and elsewhere. In 2019, C. hongkongensis anthracnose was firstly observed at the campus of Jiangxi Agricultural University (JXAU) (28°45'56″N, 115°50'21″E), then found in parks, Nanchang, China. In early August, the disease appeared and lasted until the leaves dropped (November). The disease incidence was above 60%, and the diseased leaf rate was above 70%. The lesions mostly appeared along the leaf edges. Some small round to irregular lesions also developed in other parts of the leaves. These diseased leaves had circular or irregularly shaped spots with gray-white color in the center and dark brown on the edge of the lesions. Later, the lesions became necrotic and shriveled. As the disease progressed, the spots coalesced so that affected leaves appeared blighted (Supplementary Figure 1 A-C). To identify the pathogen, leaves with typical symptoms from the campus of JXAU were collected and small pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 1 min in 3% NaOCl, and then rinsed with sterile distilled water three times. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 25 °C under a 12-h light/dark cycle (3000 lx). Pure cultures were obtained from individual conidia by single spore isolates. For studies of microscopic morphology, a representative isolate JX-S4 was subcultured on PDA. The colony of JX-S4 was white and turning gray and light gray on the reverse side, producing dark-green pigmentation near the center (Supplementary Figure 1 D). The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and 16.9 ± 1.6 × 6.0 ± 0.6 µm (n = 50) in size. Appressoria were one-celled, pale brown, thick-walled, ellipsoidal, and measured 8.7 ± 1.7 × 6.4 ± 0.8 µm (n = 50) (Supplementary Figure 1 E, F). The morphological characteristics of JX-S4 matched those of the Colletotrichum siamense species (Weir et al. 2012). For accurate identification, the internal transcribed spacer (ITS) and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-I), beta-tubulin 2 (TUB2), and calmodulin (CAL) were respectively amplified with primers ITS1/ITS4, GDF/GDR, CHS-79F/CHS-345R, ßt2a/ßt2b, and CL1/CL2. The sequences were deposited in GenBank (Accession nos. MT587807, MT628710, MT628709, MT628711, and MT628708). Phylogenetic analysis was calculated with concatenated sequences (ITS, GAPDH, CHS-I, CAL, and TUB2) using MEGA 7. In the maximum likelihood phylogenetic tree, Isolate JX-S4 was clustered with C. siamense with 93% bootstrap support (Supplementary Figure 2). Based on the morphological characteristics and phylogenetic analysis, JX-S4 was identified as C. siamense. Pathogenicity test of JX-S4 was verified on 45 attached healthy leaves from three C. hongkongensis plants (10-year-old) at the campus of JXAU inoculated with mycelial plugs (φ=5 mm) from the culture edge (6-day-old) on PDA. And an additional 45 healthy leaves were inoculated with PDA plugs as controls. The leaves were wounded with a red-hot needle (φ=0.5 mm). All treatment and control leaves were wrapped up with black plastic bags to keep them moist for 2 days. The pathogenicity tests were repeated twice. Within 7 days, all the inoculated leaves developed the lesions, which were similar to those observed in the field. Control leaves were asymptomatic (Supplementary Figure 1 G, H). The same fungus was re-isolated from the symptomatic tissues, fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing C. hongkongensis anthracnose. This finding provides crucial information for managing this disease. For example, when diagnosing Cornus anthracnose, C. siamense needs to be looked out for and appropriate control measures implemented.

Circular RNA YAP1 acts as the sponge of microRNA-21-5p to secure HK-2 cells from ischaemia/reperfusion-induced injury.

Circular RNA YAP1 (circYAP1) was reported to participate in progression of gastric cancer. However, the role of circYAP1 in acute kidney injury (AKI) remains obscure. We attempted to examine the effects of circYAP1 on ischaemia/reperfusion-stimulated renal injury. AKI model was established by treating HK-2 cells in ischaemia/reperfusion (I/R) environment. CircYAP1 expression in blood of AKI patients and I/R-treated HK-2 cells was evaluated via RT-qPCR. CCK-8, flow cytometry, ELISA and ROS assay were executed to test the impact of circYAP1 on cell viability, apoptosis, inflammatory cytokines and ROS generation. Bioinformatic analysis was executed to explore miRNA targets. The relativity between circYAP1 and miR-21-5p was verified by RT-qPCR and luciferase assay. The functions of miR-21-5p in I/R-triggered injury were reassessed. PI3K/AKT/mTOR pathway was detected by Western blot. Down-regulated circYAP1 was observed in AKI blood samples and I/R-treated HK-2 cells. CircYAP1 overexpression expedited cell growth and weakened secretion of inflammatory factors and ROS generation in I/R-disposed cells. Besides, we found circYAP1 could sponge to miR-21-5p. Interestingly, miR-21-5p overexpression overturned the repressive effects of circYAP1 on cell injury. Moreover, PI3K/AKT/mTOR pathway was activated by circYAP1 via inhibiting miR-21-5p. We demonstrated that circYAP1 activated PI3K/AKT/mTOR pathway and secured HK-2 cells from I/R injury via sponging miR-21-5p.

The circular RNA-NRIP1 plays oncogenic roles by targeting microRNA-505 in the renal carcinoma cell lines.

We explored the roles and regulatory mechanisms of the circular RNA (circRNA) nuclear receptor-interacting protein 1 (NRIP1; circNRIP1) in ACHN and CAKI-1 cells. ACHN and CAKI-1 cells were transfected with small-interfering-circNRIP1 (si-circNRIP1) and microRNA-505 (miR-505) inhibitor or the corresponding controls. Cell viability was detected with the Cell Counting Kit-8. The protein expression levels of Bcl-2, Bax, cleaved-caspase-3, matrix metalloproteinase (MMP)-2, MMP-9, adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), protein kinase B (AKT), phosphatidylinositol 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) were individually determined via Western blot. Quantitative reverse transcription polymerase chain reaction was used to examine the expressions of circNRIP1 and miR-505 both in tumor cells and tissues. The apoptotic rate, the colony numbers, and the migration rate were separately determined by the Annexin V-fluorescein isothiocyanate/propidium iodide and flow cytometer, colony formation assay, and migration assay. We found that circNRIP1 was overexpressed in tumor tissue but miR-505 was overproduced. Silencing circZNF292 induced inhibition of cell viability, colony formation, and migration, as well as the activity of AMPK and PI3K/AKT/mTOR cascades but enhancement of apoptosis. si-circNRIP1 stimulated the upregulation of miR-505, whose silence abolished the effects of si-circNRIP1 on these elements mentioned above. In conclusion, the circNRIP1 played oncogenic roles in the ACHN and the CAKI-1 cell lines by targeting miR-505 via stimulating AMPK and PI3K/AKT/mTOR cascades.

Identification, Virulence and Fungicide Sensitivity of Colletotrichum gloeosporioides s.s. Responsible for Walnut Anthracnose Disease in China.

Walnut (Juglans regia L.) is an economically important woody nut and edible oil tree all over the world. However, walnut production is limited by walnut anthracnose, which is a disastrous disease that causes significant yield losses. Studying the etiology of anthracnose on walnut and the pathogens' virulence and sensitivities to fungicides would be beneficial for effective control. This study was conducted to identify the pathogen of walnut anthracnose and reveal the population diversity of pathogens through virulence, sensitivities to fungicides, and genetic variation. A total of 13 single-spore Colletotrichum isolates were collected from walnut anthracnose-diseased fruits and leaves from 13 walnut commercial orchards in Henan, Hubei, Shandong, and Shaanxi provinces in China. The isolates were identified as Colletotrichum gloeosporioides sensu stricto (s.s.) according to multilocus phylogenetic analyses (internal transcribed spacer, actin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase), morphological as well as cultural characters, and pathogenicity. When the same walnut tissue was inoculated with different isolates, the disease lesion size was different. The results showed that the virulence of all isolates was considerably different, and the differences were not correlated with geographic origins. The virulence to walnut leaves and fruits inoculated with the same isolate was significantly different. Based on the virulence to walnut leaves and fruits, the 13 isolates were divided into three groups. Virulence of 69.2% of the isolates to walnut fruits was higher than that to leaves; 15.4% of isolates had no difference in pathogenicity, and the virulence to walnut leaves was higher for 15.4% of isolates. Tebuconazole, difenoconazole, flusilazole, and carbendazim inhibited the growth of fungal mycelia, and the concentration for 50% of maximal effect (EC50) values were 0.4 to 20.5, 0.6 to 2.6, 0.2 to 1.6, and 0.002 to 0.2 µg/ml, respectively, with average values of 6.5 ± 6.9, 1.5 ± 0.6, 0.9 ± 0.4, and 0.1 ± 0.05 µg/ml, respectively. All isolates were more sensitive to difenoconazole, flusilazole, and carbendazim than tebuconazole (P < 0.01). Isolate sensitivities to the same fungicide were different. Isolates SL-31 and TS-09 were the least sensitive to carbendazim and tebuconazole, respectively, and the resistance ratios were 87.3 and 51.6, respectively. Sensitivities to difenoconazole and flusilazole were largely consistent among all isolates, and the resistance ratios were from 1 to 4.6 and from 1 to 7, respectively. Therefore, difenoconazole and flusilazole could be chosen for disease control. The differences of pathogenicity and fungicide sensitivity were not correlated with geographic regions. These results indicated that there was high intraspecific diversity of populations in C. gloeosporioides s.s. that caused walnut anthracnose. For effective management, the targeted control strategy should be implemented based on the different geographic regions.

High Sodium Intake Impairs Small Artery Vasoreactivity in vivo in Dahl Salt-Sensitive Rats.

The effects of high sodium intake on the functionality of resistance arteries have been repeatedly studied in vitro, but no study has focused on salt-sensitive hypertension in vivo. We studied the in vivo reactivity of mesenteric small arteries (MSAs) to vasoactive agents in Dahl salt-sensitive (DS) rats with various sodium diets. Twenty-four male DS rats were randomized into 3 groups: LS (0.3% NaCl diet), NS (0.6% NaCl diet), and HS (8% NaCl diet). After a 12-week intervention, the diameter changes of the MSAs after noradrenaline (NA) and acetylcholine (ACh) exposure were detected by a microscope, and changes in blood perfusion through the MSAs were measured by full-field laser perfusion imaging. HS enhanced the constrictive response of the MSAs to NA and attenuated the relaxing response to ACh. Low sodium intake reduced the response of the MSAs to NA and promoted ACh-induced vasodilatation. HS also aggravated NA-induced blood perfusion reduction and impaired ACh-induced hyperperfusion of the MSAs. Pathologically, HS was associated with arteriolar structural damage and fibrosis of the MSAs. We conclude that sodium intake affects the responsiveness of the MSAs to vasoactive agents in DS rats and might play important roles in modulating blood pressure in hypertensive individuals.

Monocarboxylate transporter 1 and monocarboxylate transporter 4 in cancer-endothelial co-culturing microenvironments promote proliferation, migration, and invasion of renal cancer cells.

BACKGROUND: The Warburg effect demonstrates the importance of glycolysis in the development of primary and metastatic cancers. We aimed to explore the role of monocarboxylate transporter 1 (MCT1) and MCT4, two essential transporters of lactate, in renal cancer progression during cancer-endothelial cell co-culturing. METHODS: Renal cancer cells (786-O) and human vascular endothelial cells (HUVECs) were single-cultured or co-cultured in transwell membranes in the presence or absence of a MCT-1/MCT-4 specific blocker, 7ACC1. Cell proliferation was evaluated with the CCK-8 kit, while cell migration, after a scratch and invasion in transwell chambers, was evaluated under a microscope. Real-time qPCR and western blot were employed to determine the mRNA and protein levels of MCT1 and MCT4, respectively. The concentration of lactic acid in the culture medium was quantified with an l-Lactic Acid Assay Kit. RESULTS: 786-O cells and HUVECs in the co-culturing mode exhibited significantly enhanced proliferation and migration ability, compared with the cells in the single-culturing mode. The expression of MCT1 and MCT4 was increased in both 786-O cells and HUVECs in the co-culturing mode. Co-culturing promoted the invasive ability of 786-O cells, and markedly increased extracellular lactate. Treatments with 7ACC1 attenuated cell proliferation, migration, and invasion, and down-regulated the levels of MCT1/MCT4 expression and extracellular lactate. CONCLUSIONS: The Warburg effect accompanied with high MCT1/MCT4 expression in the cancer-endothelial microenvironments contributed significantly to renal cancer progression, which sheds new light on targeting MCT1/MCT4 and glycolytic metabolism in order to effectively treat patients with renal cancers.

Long Non-Coding RNA DUXAP8 Enhances Renal Cell Carcinoma Progression via Downregulating miR-126.

BACKGROUND Renal cell carcinoma (RCC) is one of the common malignant tumors in the urinary system, which endangers human health for a long time. The past decade, the molecular biology of renal cell carcinoma has made considerable progress, so that we have a more profound understanding of renal cell carcinoma. Molecular biological mechanism of renal cell carcinoma remains to be explored. Evidence indicates that long non-coding RNAs (lncRNAs) may be important players in human cancer progression, including RCC. In this study, we found that a newly discovered pseudogene-derived lncRNA named DUXAP8, a 2107-bp RNA, was remarkably upregulated in RCC. MATERIAL AND METHODS Expression of lncRNA DUXAP8 was determined by a qRT-PCR assay in RCC tissues. The proliferation and invasion of RCC cell were measured by a cell proliferation assay and a Transwell invasion assay. Expression of miR-126 was detected by real-time PCR. Interactions between lncRNA DUXAP8 and miR-126 were measured by a luciferase reporter assay and an RNA-pull down assay. In vivo experiments were used to detect tumor formation. RESULTS Together, our study not only identifies lncRNA DUXAP8 as a negative regulator of renal cancer with potential clinical value, but also reveals a regulatory mechanism by long non-coding RNAs to control tumor development. CONCLUSIONS Results from this study provide evidence that lncRNA DUXAP8 enhances renal cell carcinoma progression via downregulating of miR-126, which offers a new approach for the treatment of RCC.