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The nematode-trapping fungi are ideal agents for controlling pathogenic nematodes. Arthrobotrys oligospora is a representative species of the same, producing traps for nematode predation. Here, three orthologous Ras GTPases (Ras2, Ras3, and Rheb) were characterized in A. oligospora . Our results indicate that they play pleiotropic roles in regulating the mycelial growth, conidiation, stress resistance, and pathogenicity of A. oligospora. Furthermore, deletion of Aoras2 and Aorheb significantly affected the mitochondrial activity, reactive oxygen species levels, lipid storage, and autophagy. Transcriptome analyses of Δ Aoras2 mutant revealed that many repressed genes were associated with signal transduction, energy production, and carbohydrate transport and metabolism. Moreover, metabolic profile analyses showed that AoRas2 and AoRheb affect the biosynthesis of secondary metabolites in A. oligospora . Collectively, these findings provide an in-depth insight into the essential roles of Ras GTPases in vegetative growth, development, and pathogenicity and highlight their importance in the lifestyle switch of the nematode-trapping fungi., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Aims/hypothesis: Glucagon-like peptide 1 receptor agonists (GLP-1 RA) such as exenatide are used as monotherapy and add-on therapy for maintaining glycaemic control in patients with type 2 diabetes mellitus. The current study investigated the safety and efficacy of once-weekly PB-119, a PEGylated exenatide injection, in treatment-naive patients with type 2 diabetes., Methods: In this Phase II, randomised, placebo-controlled, double-blind study, we randomly assigned treatment-naive Chinese patients with type 2 diabetes in a 1:1:1:1 ratio to receive subcutaneous placebo or one of three subcutaneous doses of PB-119 (75, 150, and 200 μg) for 12 weeks. The primary endpoint was the change in HbA 1c from baseline to week 12, and other endpoints were fasting plasma glucose, 2 h postprandial glucose (PPG), and proportion of patients with HbA 1c  < 53 mmol/mol (<7.0%) and ≤48 mmol/mol (≤6.5%) at 2, 4, 8 and 12 weeks of treatment. Safety was assessed in all patients who received at least one dose of study drug., Results: We randomly assigned 251 patients to one of the four treatment groups (n = 62 in placebo and 63 each in PB-119 75 μg, 150 μg and 200 μg groups). At the end of 12 weeks, mean differences in HbA 1c in the treatment groups were -7.76 mmol/mol (95% CI -9.23, -4.63, p < 0.001) (-0.72%, 95% CI -1.01, -0.43), -12.89 mmol/mol (95% CI -16.05, -9.72, p < 0.001) (-1.18%, 95% CI -1.47, -0.89) and -11.14 mmol/mol (95% CI -14.19, -7.97, p <0 .001) (-1.02%, 95% CI -1.30, -0.73) in the 75 μg, 150 μg and 200 μg PB-119 groups, respectively, compared with that in the placebo group after adjusting for baseline HbA 1c . Similar results were also observed for other efficacy endpoints across different time points. There was no incidence of treatment-emergent serious adverse event, severe hypoglycaemia or death., Conclusions/interpretation: All tested PB-119 doses had superior efficacy compared with placebo and were safe and well tolerated over 12 weeks in treatment-naive Chinese patients with type 2 diabetes., Trial Registration: ClinicalTrials.gov NCT03520972 FUNDING: The study was funded by National Major Scientific and Technological Special Project for Significant New Drugs Development and PegBio.

Understanding how fungi interact with other organisms has significant medical, environmental, and agricultural implications. Nematode-trapping fungi (NTF) can switch to pathogens by producing various trapping devices to capture nematodes. Here we perform comparative genomic analysis of the NTF with four representative trapping devices. Phylogenomic reconstruction of these NTF suggested an evolutionary trend of trapping device simplification in morphology. Interestingly, trapping device simplification was accompanied by expansion of gene families encoding adhesion proteins and their increasing adhesiveness on trap surfaces. Gene expression analysis revealed a consistent up-regulation of the adhesion genes during their lifestyle transition from saprophytic to nematophagous stages. Our results suggest that the expansion of adhesion genes in NTF genomes and consequential increase in trap surface adhesiveness are likely the key drivers of fungal adaptation in trapping nematodes, providing new insights into understanding mechanisms underlying infection and adaptation of pathogenic fungi., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chitin is one of the most important carbohydrates of the fungal cell wall, and is synthesized by chitin synthases. Chitin can be degraded by chitinases, which are important virulence factors in pathogenic fungi. Knowledge about the biosynthesis and degradation of chitin, and the enzymes responsible, has accumulated in recent years. In this review, we analyze the amino acid sequences of chitin synthases from several typical fungi. These enzymes can be divided into seven groups. While the different chitin synthases from a single fungus share a low degree of similarity, the same type of chitin synthase from different fungi shows high similarity. The number of chitinase genes in fungi display wide variation, from a single gene in Schizosaccharomyces pombe, to 36 genes in Trichoderma virens. Chitinases from different fungi can be divided into four groups. The functions of chitin synthases and chitinases in several typical fungi are summarized, and the crystal structures of chitinases and chitinase modification are also discussed.

Arthrobotrys oligospora is a typical nematode-trapping fungus. In this study, 37 transformants of A. oligospora were obtained by REMI (restriction enzyme mediated integration) method and phenotypic properties of nine transformants were analyzed. The nine transformants showed differences in growth, conidiation, trap formation, stress tolerance, and/or pathogenicity among each other and with those of the parental wild-type strain (WT). The insertional sites of the hph cassette were identified in transformants X5 and X13. In X5, the cassette was inserted in the non-coding region between AOL&#95;s00076g273 (76g273) and AOL&#95;s00076g274 (76g274) and the transcription of 76g274, but not 76g273, was enhanced in X5. 76g274p had two conserved domains and was predicted as a nucleoprotein, which we confirmed by its nuclear localization in Saccharomyces cerevisiae using the green fluorescent protein-fused 76g274p. The transcription of 76g274 was stimulated or inhibited by several environmental factors. The sporulation yields of 76g274-deficient mutants were decreased by 70%, and transcription of several sporulation-related genes was severely diminished compared to the WT during the conidiation. In summary, a method for screening mutants was established in A. oligospora and using the method, we identified a novel C 2 H 2 -type transcription factor that positively regulates the conidiation of A. oligospora.

Nicotine in tobacco is harmful to health and the environment, so there is an environmental requirement to remove nicotine from tobacco and tobacco wastes. In this study, the biotransformation of nicotine by Rhodococcus sp. Y22 was investigated, and three metabolites (NIC1, NIC4 and NIC5) were isolated by column separation, preparative TLC and solid plate's method, respectively. NIC1 was identified as 6-hydoxynicotine based on the results of NMR, MS, HPLC-UV and HRESIMS analysis; NIC4 was a novel compound and identified as 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one based on the results of NMR, MS and UV analysis; NIC5 was identified as nicotine blue based on the results of NMR and MS analysis. Meanwhile, two metabolites NIC2 and NIC3 were identified as 6-hydroxy-N-methylmyosmine and 6-hydroxypseudooxynicotine by HRESIMS analysis, respectively. According to these metabolites, the possible pathway of nicotine degradation by Rhodococcus sp. Y22 was proposed. The nicotine can be transformed to nicotine blue through two pathways (A and B), and 6-hydroxy-N-methylmyosmine is the key compound, which can be converted to 6-hydroxypseudooxynicotine (pathway A) and 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one (pathway B), respectively. Moreover, the encoding gene of nicotine dehydrogenase, ndh, was amplified from Rhodococcus sp. Y22, and its transcriptional level could be up-regulated obviously under nicotine induction. Our studies reported the key metabolites and possible biotransformation pathway of nicotine in Rhodococcus sp. Y22, and provided new insights into the microbial metabolism of nicotine.

Objective: To evaluate the efficacy and safety of alogliptin in Chinese patients with type 2 diabetes (T2DM)., Methods: This was a multicenter, randomized, double-blind, placebo-controlled phase III trial. A total of 491 subjects with T2DM were randomized in a 1:1 ratio to receive alogliptin (25 mg once daily) or placebo for 16 weeks. Among them, 181 were in the monotherapy group (group A), 186 were in the add-on to metformin group (group B), and 124 were in the add-on to pioglitazone group (group C)., Results: After 16 weeks of therapy, glycosylated hemoglobin A1c (HbA1c) levels decreased in both alogliptin and placebo groups. The mean changes in HbA1c for alogliptin and placebo were 1.00% and 0.43% (P<0.001), 0.91% and 0.23% (P<0.001), and 0.76% and 0.25% (P<0.001) in group A, B and C, respectively. Compared with placebo, alogliptin treatment led to a greater decrease in fasting plasma glucose (FPG) and a higher percentage of subjects who achieved HbA1c targets of ≤ 6.5% and ≤ 7.0%. The percentage of subjects who experienced all adverse events including hypoglycemia with alogliptin were comparable to those with placebo., Conclusions: Alogliptin 25 mg once daily reduced HbA1c and FPG, and increased a greater proportion of subjects achieving HbA1c goals of ≤6.5% and ≤7.0% compared with placebo when used as a monotherapy, add-on to metformin, or add-on to pioglitazone. The hypoglycemia rates and safety profiles with alogliptin were similar to those with placebo.

Objective: To explore the association of choroidal thickness variations in type 2 diabetes mellitus (T2DM) patients with diabetic retinopathy (DR) at different stages., Methods: A total of 161 patients with T2DM were included in this study, from October 2012 to June 2014. According to Early Treatment Diabetic Retinopathy Study (ETDRS) criteria, the patients were divided into 5 groups: non-DR without diabetic macular edema (DME) group(DR-/DME- group, 45 eyes), nonproliferative diabetic retinopathy (NPDR) without DME group(NPDR+/DME- group, 58 eyes), proliferative diabetic retinopathy (PDR) without DME group (PDR+/DME- group, 12 eyes), NPDR with DME group (NPDR+/DME+ group, 41 eyes), PDR with DME group (PDR+/DME+ group, 5 eyes). Meanwhile, 60 normal subjects were enrolled as the control group. All study subjects received optical coherence tomography enhanced depth imaging (EDI-OCT) examination to detect and compare subfoveal choroidal thickness at different stages of DR., Results: Mean SFCT was (271 ± 36), (270 ± 35), (262 ± 38), (244 ± 36), (229 ± 35) µm respectively in control, DR-/DME-, NPDR+/DME-, PDR+/DME-, NPDR+/DME+ groups. The SFCTs of PDR+/DME- and NPDR+/DME+ group were statistically lower than that of control group (P=0.004, P=0.001). The SFCT of PDR+/DME- group was lower than that of DR-/DME- group (P=0.003), and there was also a significant difference of SFCT between NPDR+/DME+ and NPDR+/DME- group (P=0.001). There was linear correlation between SFCT and the logMAR best-corrected visual acuity (r=0.397, P<0.01), but the SFCT was independent of diabetic duration, fasting blood glucose, HbA1c, axial length, diastolic blood pressure (DBP) and systolic blood pressure SBP (r=-0.024, 0.159, 0.089, 0.036, 0.143, 0.057, all P>0.05). There was no significant difference of SFCT among different DME types (F=0.071, P>0.05)., Conclusion: The SFCT decreased with increasing severity of DR. To monitor the SFCT in T2DM patients may be helpful to evaluate the severity of DR and provide a new treatment conception.

MicroRNAs (miRs) play important roles in initiation and progression of many pathologic processes. However, the roles of miRs in diabetic nephropathy remain unclear. This study was to determine whether miR-21 was involved in diabetic nephropathy and to explore the relationship between miR-21 and MMP9/TIMP1 expression in diabetic nephropathy. In situ hybridization studies showed that miR-21 was primarily localized and distributed in cortical glomerular and renal tubular cells in diabetic kk-ay kidney. Real-time quantitative RT-PCR demonstrated that the expression of miR-21 was significantly increased in kk-ay mice, compared with control C57BL mice. Interestingly, miR-21 expression positively correlated with urine albumin creatine ratio (ACR), TIMP1, collagen IV (ColIV), and fibronectin (FN); while negatively correlated with creatine clearance ratio (Ccr) and MMP-9 protein. Importantly, antagomir-21 not only ameliorated Ccr and ACR but also decreased TIMP1, ColIV, and FN proteins. In conclusion, our data demonstrate that miR-21 contributes to renal fibrosis by mediating MMP9/TIMP1 and that inhibition of miR-21 may be a novel target for diabetic nephropathy.

Microorganisms can attack and kill nematodes by diverse processes such as capturing, parasitizing, and producing toxins and enzymes. Extracellular enzymes, including serine proteases, chitinases, and collagenases are shown to be important virulence factors that can degrade the main chemical constituents of the nematode cuticle and eggshell. Here, we review the structure, function, regulation, and evolution of these nematicidal enzymes and provide insights into the mechanisms of microbial infections against nematodes. We discuss the practical applications of these nematicidal enzymes in agriculture and other areas.

Nematode-trapping fungi can secrete many extracellular hydrolytic enzymes such as serine proteases and chitinases to digest and penetrate nematode/egg-cuticles. However, little is known about the structure and function of chitinases in these fungi. In this study, 16 ORFs encoding putative chitinases, which all belong to glycoside hydrolase (GH) family 18, were identified from the Arthrobotrys oligospora genome. Bioinformatics analyses showed that these 16 putative chitinases differ in their functional domains, molecular weights and pI. Phylogenetic analysis grouped these A. oligospora chitinases into four clades: clades I, II, III and IV, respectively, including an A. oligospora-specific subclade (Clade IV-B) that contained high-molecular weight chitinases (≥100 kDa). Transcriptional analysis of A. oligospora chitinases suggested that the expression of most chitinases was repressed by carbon starvation, and all chitinases were up-regulated under nitrogen starvation. However, chitinase AO-190 was up-regulated under carbon and/or nitrogen starvation. Moreover, several chitinases (such as AO-59, AO-190 and AO-801) were up-regulated in the presence of chitinous substrates or a plant pathogenic fungus, indicating that they could play a role in biocontrol applications of A. oligospora. Our results provided a basis for further understanding the functions, diversities and evolutionary relationships between chitinase genes in nematode-trapping fungi.

Understanding the mechanisms of host-pathogen interaction can provide crucial information for successfully manipulating their relationships. Because of its genetic background and practical advantages over vertebrate model systems, the nematode Caenorhabditis elegans model has become an attractive host for studying microbial pathogenesis. Here we report a "Trojan horse" mechanism of bacterial pathogenesis against nematodes. We show that the bacterium Bacillus nematocida B16 lures nematodes by emitting potent volatile organic compounds that are much more attractive to worms than those from ordinary dietary bacteria. Seventeen B. nematocida-attractant volatile organic compounds are identified, and seven are individually confirmed to lure nematodes. Once the bacteria enter the intestine of nematodes, they secrete two proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. This Trojan horse pattern of bacterium-nematode interaction enriches our understanding of microbial pathogenesis.

Cuticle-degrading proteases are involved in the breakdown of cuticle/eggshells of nematodes or insects, a hard physical barrier against fungal infections. Understanding the 3-dimensional structures of these proteins can provide crucial information for improving the effectiveness of these fungi in biocontrol applications, e.g., by targeted protein engineering. However, the structures of these proteases remain unknown. Here, we report the structures of two cuticle-degrading proteases from two species of nematophagous fungi. The two structures were solved with X-ray crystallography to resolutions of 1.65 A (Ver112) and 2.1 A (PL646), respectively. Crystal structures of PL646 and Ver112 were found to be very similar to each other, and similar to that of proteinase K from another fungus Tritirachium album. Differences between the structures were found among residues of the substrate binding sites (S1 and S4). Experimental studies showed that the enzymes differed in hydrolytic activity to synthetic peptide substrates. Our analyses of the hydrophobic/hydrophilic and electrostatic features of these two proteins suggest that their surfaces likely play important roles during fungal infection against nematodes. The two crystal structures provide a solid basis for investigating the relationship between structure and function of cuticle-degrading proteases.

Four series (S, M, R, and W) of Alternaria longipes isolates were obtained based on consecutive induction with Dimethachlon (Dim) and ultraviolet irradiation. These isolates were then characterized according to their tolerance to Dim, sensitivity to osmotic stress, and phenotypic properties. All the induced Dim-resistant isolates showed a higher osmosensitivity than the parental strains, and the last generation was more resistant than the first generation in the M, R, and W series. In addition, the changes in the Dim resistance and osmotic sensitivity were not found to be directly correlated, and no distinct morphologic characteristics were found among the resistant and sensitive isolates, with the exception of the resistant isolate K-11. Thus, to investigate the molecular basis of the fungicide resistance, a group III two-component histidine kinase (HK) gene, AlHK1, was cloned from nineteen A. longipes isolates. AlHK1p was found to be comprised of a six 92- amino-acid repeat domain (AARD), HK domain, and response regulator domain, similar to the Os-1p from Neurospora crassa. A comparison of the nucleotide sequences of the AlHK1 gene from the Dim-sensitive and -resistant isolates revealed that all the resistant isolates contained a single-point mutation in the AARD of AlHK1p, with the exception of isolate K-11, where the AlHK1p contained a deletion of 107 amino acids. Moreover, the AlHK1p mutations in the isolates of each respective series involved the same amino acid substitution at the same site, although the resistance levels differed significantly in each series. Therefore, these findings suggested that a mutation in the AARD of AlHK1p was not the sole factor responsible for A. longipes resistance to dicarboximide fungicides.

Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchi1 was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchi1 ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchi1 encodes 426 amino acid residues and shares a high degree of similarity with other chitinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchi1 in response to carbon (5'-SYGGRG-3') and nitrogen (5'-GATA-3') were identified in the upstream of Crchi1. Expression of Crchi1 gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchi1 expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor.

Objective: We designed a multi-center, double-blind, randomized, parallel, with metformin controlled clinical trial to evaluate the efficacy and safety of low dose rosiglitazone combined with sulphonylurea therapy in type 2 diabetic patients who were inadequately controlled with sulphonylurea alone., Methods: Patients were treated with 4 mg rosiglitazone once daily plus sulphonylurea (test group) or 0.5 g metformin twice daily plus sulphonylurea (control group) for 12 weeks. The mean levels of HbA(1c), fasting and postprandial plasma glucose were recorded and compared between the two groups., Results: The mean levels of HbA(1c) decreased by 1.09% and 0.95% in the test group (n = 102) and control group (n = 96) respectively. Fasting and postprandial plasma glucose levels in the test group decreased by 25.0% and 35.6%, and in the control group, decreased by 17.7% and 23.8% as compared with the baseline (both P < 0.01). No liver damage was found., Conclusion: Combination treatment of rosiglitazone and sulphonylurea can effectively improve glycemic control in type 2 diabetic patients inadequately controlled with sulphonylurea alone.