Histone Methyltransferase SsDim5 Regulates Fungal Virulence through H3K9 Trimethylation in Sclerotinia sclerotiorum.

Histone post-translational modification is one of the main mechanisms of epigenetic regulation, which plays a crucial role in the control of gene expression and various biological processes. However, whether or not it affects fungal virulence in Sclerotinia sclerotiorum is not clear. In this study, we identified and cloned the histone methyltransferase Defective in methylation 5 (Dim5) in S. sclerotiorum, which encodes a protein containing a typical SET domain. SsDim5 was found to be dynamically expressed during infection. Knockout experiment demonstrated that deletion of SsDim5 reduced the virulence in Ssdim5-1/Ssdim5-2 mutant strains, accompanied by a significant decrease in H3K9 trimethylation levels. Transcriptomic analysis further revealed the downregulation of genes associated with mycotoxins biosynthesis in SsDim5 deletion mutants. Additionally, the absence of SsDim5 affected the fungus's response to oxidative and osmotic, as well as cellular integrity. Together, our results indicate that the H3K9 methyltransferase SsDim5 is essential for H3K9 trimethylation, regulating fungal virulence throug mycotoxins biosynthesis, and the response to environmental stresses in S. sclerotiorum.

Effect and Safety of Herbal Medicine Foot Baths in Patients with Diabetic Peripheral Neuropathy: A Multicenter Double-Blind Randomized Controlled Trial.

OBJECTIVE: To evaluate the effect and safety of foot baths with Tangbi Waixi Decoction (TW) in treating patients with diabetic peripheral neuropathy (DPN). METHODS: It is a multicenter double-blinded randomized controlled trial. Participants with DPN were recruited between November 18, 2016 and May 30, 2018 from 8 hospitals in China. All patients received basic treatments for glycemic management. Patients received foot baths with TW herbal granules either 66.9 g (intervention group) or 6.69 g (control group) for 30 min once a day for 2 weeks and followed by a 2-week rest, as a therapeutic course. If the Toronto Clinical Scoring System total score (TCSS-TS) ⩾6 points, the patients received a total of 3 therapeutic courses (for 12 weeks) and were followed up for 12 weeks. The primary outcome was change in TCSS-TS score at 12 and 24 weeks. Secondary outcomes included changes in bilateral motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) of the median and common peroneal nerve. Safety was also assessed. RESULTS: Totally 632 patients were enrolled, and 317 and 315 were randomized to the intervention and control groups, respectively. After the 12-week intervention, patients in both groups showed significant declines in TCSSTS scores, and significant increases in MNCV and SNCV of the median and common peroneal nerves compared with pre-treatment (P<0.05). The reduction of TCSS-TS score at 12 weeks and the increase of SNCV of median nerve at 24 weeks in the control group were greater than those in the intervention group (P<0.05). The number of adverse events did not differ significantly between groups (P>0.05), and no serious adverse event was related with treatment. CONCLUSION: Treatment of TW foot baths was safe and significantly benefitted patients with DPN. A low dose of TW appeared to be more effective than a high dose. (Registry No. ChiCTR-IOR-16009331).

SsCak1 Regulates Growth and Pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating fungal pathogen that causes severe crop losses worldwide. It is of vital importance to understand its pathogenic mechanism for disease control. Through a forward genetic screen combined with next-generation sequencing, a putative protein kinase, SsCak1, was found to be involved in the growth and pathogenicity of S. sclerotiorum. Knockout and complementation experiments confirmed that deletions in SsCak1 caused defects in mycelium and sclerotia development, as well as appressoria formation and host penetration, leading to complete loss of virulence. These findings suggest that SsCak1 is essential for the growth, development, and pathogenicity of S. sclerotiorum. Therefore, SsCak1 could serve as a potential target for the control of S. sclerotiorum infection through host-induced gene silencing (HIGS), which could increase crop resistance to the pathogen.

Regulation of Grain Chalkiness and Starch Metabolism by FLO2 Interaction Factor 3, a bHLH Transcription Factor in Oryza sativa.

Chalkiness is a key determinant that directly affects the appearance and cooking quality of rice grains. Previously, Floury endosperm 2 (FLO2) was reported to be involved in the formation of rice chalkiness; however, its regulation mechanism is still unclear. Here, FLO2 interaction factor 3 (OsFIF3), a bHLH transcription factor, was identified and analyzed in Oryza sativa. A significant increase in chalkiness was observed in OsFIF3-overexpressed grains, coupled with a round, hollow filling of starch granules and reduced grain weight. OsFIF3 is evolutionarily conserved in monocotyledons, but variable in dicotyledons. Subcellular localization revealed the predominant localization of OsFIF3 in the nucleus. The DAP-seq (DNA affinity purification sequencing) results showed that OsFIF3 could affect the transcriptional accumulation of ß-amylase 1, α-amylase isozyme 2A-like, pectinesterase 11, ß-glucosidase 28 like, pectinesterase, sucrose transport protein 1 (SUT1), and FLO2 through the binding of the CACGTG motif on their promoters. Moreover, FLO2 and SUT1 with abundant OsFIF3 binding signals showed significant expression reduction in OsFIF3 overexpression lines, further confirming OsFIF3's role in starch metabolism regulation and energy material allocation. Taken together, these findings show that the overexpression of OsFIF3 inhibits the expression of FLO2 and SUT1, thereby increasing grain chalkiness and affecting grain weight.

mRNA Turnover Protein 4 Is Vital for Fungal Pathogenicity and Response to Oxidative Stress in Sclerotinia sclerotiorum.

Ribosome assembly factors have been extensively studied in yeast, and their abnormalities may affect the assembly process of ribosomes and cause severe damage to cells. However, it is not clear whether mRNA turnover protein 4 (MRT4) functions in the fungal growth and pathogenicity in Sclerotinia sclerotiorum. Here, we identified the nucleus-located gene SsMRT4 using reverse genetics, and found that knockdown of SsMRT4 resulted in retard mycelia growth and complete loss of pathogenicity. Furthermore, mrt4 knockdown mutants showed almost no appressorium formation and oxalic acid production comparing to the wild-type and complementary strains. In addition, the abilities to ROS elimination and resistance to oxidative and osmotic stresses were also seriously compromised in mrt4 mutants. Overall, our study clarified the role of SsMRT4 in S. sclerotiorum, providing new insights into ribosome assembly in regulating pathogenicity and resistance to environmental stresses of fungi.

Genome-Wide Identification of GYF-Domain Encoding Genes in Three Brassica Species and Their Expression Responding to Sclerotinia sclerotiorum in Brassica napus.

GYF (glycine-tyrosine-phenylalanine)-domain-containing proteins, which were reported to participate in many aspects of biological processes in yeast and animals, are highly conserved adaptor proteins existing in almost all eukaryotes. Our previous study revealed that GYF protein MUSE11/EXA1 is involved in nucleotide-binding leucine-rich repeat (NLR) receptor-mediated defense in Arabidopsis thaliana. However, the GYF-domain encoding homologous genes are still not clear in other plants. Here, we performed genome-wide identification of GYF-domain encoding genes (GYFs) from Brassica napus and its parental species, Brassica rapa and Brassica oleracea. As a result, 26 GYFs of B. napus (BnaGYFs), 11 GYFs of B. rapa (BraGYFs), and 14 GYFs of B. oleracea (BolGYFs) together with 10 A. thaliana (AtGYFs) were identified, respectively. We, then, conducted gene structure, motif, cis-acting elements, duplication, chromosome localization, and phylogenetic analysis of these genes. Gene structure analysis indicated the diversity of the exon numbers of these genes. We found that the defense and stress responsiveness element existed in 23 genes and also identified 10 motifs in these GYF proteins. Chromosome localization exhibited a similar distribution of BnaGYFs with BraGYFs or BolGYFs in their respective genomes. The phylogenetic and gene collinearity analysis showed the evolutionary conservation of GYFs among B. napus and its parental species as well as Arabidopsis. These 61 identified GYF domain proteins can be classified into seven groups according to their sequence similarity. Expression of BnaGYFs induced by Sclerotinia sclerotiorum provided five highly upregulated genes and five highly downregulated genes, which might be candidates for further research of plant-fungal interaction in B. napus.

Benefits of herbal formulae containing Poria cocos (Fuling) for type 2 diabetes mellitus: A systematic review and meta-analysis.

BACKGROUND: Poria cocos (Schw.) Wolf or Fuling is one of the top 10 most frequently prescribed herbs in China for the treatment of type 2 diabetes mellitus (T2DM). OBJECTIVE: The purpose of this systematic review is to determine the additional benefit of Fuling formulae use in addition to hypoglycaemic agents for T2DM in randomised clinical trials. METHODS: English (5) and Chinese (4) medical databases were searched from their inception to August 2021. RCTs that included Fuling in herbal formulae for T2DM were included. Risk of bias were assessed using the Cochrane Collaboration's procedures. Stata software (13.0) was used for data analysis. RESULTS: Seventy-three RCTs (6,489 participants) with herbal formulae containing Fuling were included. Most studies were at risk of bias and strength of the evidence were low to moderate. Meta-analysis findings showed that the addition of formulae containing Fuling to hypoglycaemic agent-treatments could benefit people with T2DM by reducing fasting blood glucose (MD -0.82 [-0.93, -0.71]; I2 = 79.6%, P = 0.00), 2-hour postprandial blood glucose (MD-1.15 [-1.31, -0.98], I2 = 80%, P = 0.00) and haemoglobin A1c (MD-0.64 [-0.75, -0.53], I2 = 84.7%, P = 0.00). Adverse events were also significantly lower in the integrative group than in the hypoglycaemic alone group (RR 0.99 [0.93, 1.06], P = 0.87). CONCLUSION: Evidence from this study supports the use of Fuling formulae combined with hypoglycaemic agents for T2DM. The combined therapies appear to be well tolerated. TRAIL REGISTRATION: This review is registered with the PROSPERO international prospective register of systematic reviews (CRD42020214635).

A Single Amino Acid Substitution in RFC4 Leads to Endoduplication and Compromised Resistance to DNA Damage in Arabidopsis thaliana.

Replication factor C (RFC) is a heteropentameric ATPase associated with the diverse cellular activities (AAA+ATPase) protein complex, which is composed of one large subunit, known as RFC1, and four small subunits, RFC2/3/4/5. Among them, RFC1 and RFC3 were previously reported to mediate genomic stability and resistance to pathogens in Arabidopsis. Here, we generated a viable rfc4e (rfc4-1/RFC4G54E) mutant with a single amino acid substitution by site-directed mutagenesis. Three of six positive T2 mutants with the same amino acid substitution, but different insertion loci, were sequenced to identify homozygotes, and the three homozygote mutants showed dwarfism, early flowering, and a partially sterile phenotype. RNA sequencing revealed that genes related to DNA repair and replication were highly upregulated. Moreover, the frequency of DNA lesions was found to be increased in rfc4e mutants. Consistent with this, the rfc4e mutants were very sensitive to DSB-inducing genotoxic agents. In addition, the G54E amino acid substitution in AtRFC4 delayed cell cycle progression and led to endoduplication. Overall, our study provides evidence supporting the notion that RFC4 plays an important role in resistance to genotoxicity and cell proliferation by regulating DNA damage repair in Arabidopsis thaliana.

SsNEP2 Contributes to the Virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious soilborne fungal pathogen that causes serious economic losses globally. The necrosis and ethylene-inducible peptide 1 (NEP1)-like proteins (NLPs) were previously shown to play an important role in pathogenicity in fungal and oomycete pathogens. Here, we generated S. sclerotiorum necrosis and ethylene-inducible peptide 2 (SsNEP2) deletion mutant through homologous recombination and found that SsNEP2 contributes to the virulence of S. sclerotiorum without affecting the development of mycelia, the formation of appressoria, or the secretion of oxalic acid. Although knocking out SsNEP2 did not affect fungal sensitivity to oxidative stress, it did lead to decreased accumulation of reactive oxygen species (ROS) in S. sclerotiorum. Furthermore, Ssnlp24SsNEP2 peptide derived from SsNEP2 triggered host mitogen-activated protein kinase (MAPK) activation, increased defense marker gene expression, and enhanced resistance to Hyaloperonospora arabidopsidis Noco2. Taken together, our data suggest that SsNEP2 is involved in fungal virulence by affecting ROS levels in S. sclerotiorum. It can serve as a pathogen-associated molecular pattern (PAMP) and trigger host pattern triggered immunity to promote the necrotrophic lifestyle of S. sclerotiorum.

Urine proteomics identifies biomarkers for diabetic kidney disease at different stages.

BACKGROUND: Type 2 diabetic kidney disease is the most common cause of chronic kidney diseases (CKD) and end-stage renal diseases (ESRD). Although kidney biopsy is considered as the 'gold standard' for diabetic kidney disease (DKD) diagnosis, it is an invasive procedure, and the diagnosis can be influenced by sampling bias and personal judgement. It is desirable to establish a non-invasive procedure that can complement kidney biopsy in diagnosis and tracking the DKD progress. METHODS: In this cross-sectional study, we collected 252 urine samples, including 134 uncomplicated diabetes, 65 DKD, 40 CKD without diabetes and 13 follow-up diabetic samples, and analyzed the urine proteomes with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We built logistic regression models to distinguish uncomplicated diabetes, DKD and other CKDs. RESULTS: We quantified 559 ± 202 gene products (GPs) (Mean ± SD) on a single sample and 2946 GPs in total. Based on logistic regression models, DKD patients could be differentiated from the uncomplicated diabetic patients with 2 urinary proteins (AUC = 0.928), and the stage 3 (DKD3) and stage 4 (DKD4) DKD patients with 3 urinary proteins (AUC = 0.949). These results were validated in an independent dataset. Finally, a 4-protein classifier identified putative pre-DKD3 patients, who showed DKD3 proteomic features but were not diagnosed by clinical standards. Follow-up studies on 11 patients indicated that 2 putative pre-DKD patients have progressed to DKD3. CONCLUSIONS: Our study demonstrated the potential for urinary proteomics as a noninvasive method for DKD diagnosis and identifying high-risk patients for progression monitoring.

Additional Benefit of Chinese Medicine Formulae Including Dioscoreae rhizome (Shanyao) for Diabetes Mellitus: Current State of Evidence.

Background: Chinese medicine has been used to treat diabetes symptoms for thousands of years. Dioscoreae rhizome or Shanyao is a Chinese medicinal herb that is routinely used in the treatment of diabetes mellitus (DM). Objective: The purpose of this study is to evaluate the evidence of the added benefits and safety of herbal formulae containing Shanyao in clinical studies and the possible mechanisms of Shanyao in the prevention and treatment of DM in experimental studies. Methods: We searched nine databases for randomized controlled trials (RCTs) that included Shanyao in the formulae in the treatment of type 2 DM. Furthermore, experimental studies on the prevention and treatment of DM by Shanyao in English- and Chinese-language databases were identified. Results: Fifty-three moderate quality RCTs with herbal formulae containing Shanyao were identified. Results from meta-analysis indicated that Shanyao alone or formulae containing Shanyao in addition to conventional treatments could benefit people with type 2 DM in lowering blood glucose, blood lipids and reducing insulin resistance. Moreover, adverse events were significantly lower in the CHM plus conventional group than those in the conventional group. Shanyao may exert the benefit through various mechanisms including inhibition of α-glucosidase and DPP-IV activity, increase of endogenous GLP-1 and immune regulating activities. Conclusion: Evidence from this review suggested that there appeared to be added clinical benefits associated with the use of Shanyao for DM, whether as a food supplement or as a CHM combined with hypoglycemic agents with a good safety profile.

Exenatide and Renal Outcomes in Patients with Type 2 Diabetes and Diabetic Kidney Disease.

BACKGROUND: Cardiovascular outcomes in clinical trials with type 2 diabetes mellitus (T2DM) patients have shown that glucagon-like peptide-1 receptor agonist can have a beneficial effect on the kidney. This trial aimed to assess the effects of exenatide on renal outcomes in patients with T2DM and diabetic kidney disease (DKD). METHODS: We performed a randomized parallel study encompassing 4 general hospitals. T2DM patients with an estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2 and macroalbuminuria, defined as 24-h urinary albumin excretion rate (UAER) >0.3 g/24 h were randomized 1:1 to receive exenatide twice daily plus insulin glargine (intervention group) or insulin lispro plus glargine (control group) for 24 weeks. The primary outcome was the UAER percentage change from the baseline after 24 weeks of intervention. The rates of hypoglycemia, adverse events (AEs), and change in eGFR during the follow-up were measured as safety outcomes. RESULTS: Between March 2016 and April 2019, 92 patients were randomized and took at least 1 dose of the study drug. The mean age of the participants was 56 years. At baseline, the median UAER was 1,512.0 mg/24 h and mean eGFR was 70.4 mL/min/1.73 m2. After 24 weeks of treatment, the UAER percentage change was significantly lower in the intervention group than in the control group (p = 0.0255). Moreover, the body weight declined by 1.3 kg in the intervention group (the difference between the 2 groups was 2.7 kg, p = 0.0001). Compared to the control group, a lower frequency of hypoglycemia and more gastrointestinal AEs were observed in the intervention group. CONCLUSION: Exenatide plus insulin glargine treatment for 24 weeks resulted in a reduction of albuminuria in T2DM patients with DKD.

Herbal medicine foot bath for the treatment of diabetic peripheral neuropathy: protocol for a randomized, double-blind and controlled trial.

BACKGROUND: As a common complication of diabetes, the incidence of diabetic peripheral neuropathy (DPN) is 60-70% worldwide. DPN is a major risk factor for diabetic foot, which may lead to foot ulceration and even amputation. The treatment of DPN remains challenging. Our preliminary study demonstrated that the external application of Tangbi Waixi (TW) decoction to the lower extremities relieved clinical symptoms and improved nerve conduction velocity in DPN patients. The aim of this study was to verify the efficacy of TW among DPN patients and evaluate the herb mixture's safety using rigorous methodological designs. METHODS/DESIGN: This study is a multicenter, double-blind, randomized controlled trial. A total of 640 DPN patients will be recruited and randomized to receive a foot bath with either the TW decoction or control drug. Participants will be assessed at baseline and 12 and 24 weeks after treatment. The primary outcome was the change of the Toronto Clinical Scoring System (TCSS). Secondary outcomes were nerve conduction velocity, blood glucose, blood lipids, serum inflammatory cytokines, and the European Quality of Life Five-Dimension Scale (EQ-5D) and TCM symptom scores. DISCUSSION: This multicenter, prospective, randomized clinical trial will provide valuable data regarding the efficacy and safety of foot bath treatment with TW decoction. Positive results would provide a novel treatment regimen for DPN patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-IOR-16009331 . Registered on 8 October 2016.

[Effect of tangtong recipe on the proliferation of cultured iliac arterial smooth muscle cells of diabetic rats in vitro].

OBJECTIVE: To explore the inhibition of Tangtong Recipe (TTR) on proliferation of vascular smooth muscle cells (VSMCs) in rat model of diabetes mellitus using seropharmacological and modern molecular biological technologies. METHODS: The drug-serum of TTR was prepared by feeding pure-breed New Zealand rabbits at various doses (18 mL/kg, 13 mL/kg, and 8 mL/kg) of TTR, and used to intervene the VSMCs get from the iliac artery of diabetic model rats cultured by tissue block plantation. The impact of drug-serum on VSMCs proliferation was estimated through measuring tritiated thymine deoxy-ribonucleotide (3H-TDR) incorporation using liquid-scintillation detector, and the cell cycle was detected using flow cytometry. RESULTS: The 3H-TDR incorporation in cells after intervention with various doses of TTR drug-serum were reduced significantly, in a concentration-dependent manner, to the level lower than that in the normal serum control group (P < 0.01). And the percentage of S and G2/M phase cells in the drug-serum treated groups also were markedly lower than that in the normal control group (P < 0.01). CONCLUSION: Drug-serum of TTR could inhibit the proliferation of VSMC of diabetic rat in vitro in a concentration-dependent manner.

[Effects of Jiangtang Bushen Recipe on serum C-reactive protein, tumor necrosis factor-alpha and interleukin-6 in patients with type 2 diabetes mellitus].

UNLABELLED: OBJECTIVE To observe the effect of Jiangtang Bushen Recipe (JBR) on inflammatory cytokines in patients with type 2 diabetes mellitus (T2DM), and explore its therapeutic mechanism. METHODS Sixty-four patients with T2DM were randomly divided into the treated group (n = 34) and the control group (n = 30). Education course, dietary treatment and conventional hypoglycemic agent were given to both groups, and JBR, mainly composed of Cibot Rhizome, Radix Dipsaci, Glossy Privet Fruit; Ecliptae Herba, Radicis Lycii, Radix Astragali, Rehmannia Dride Rhizome, etc., was given additionally to patients in the treated group, one dose a day for 4 weeks by boiled water and taking in two times. Fasting blood glucose (FBG), fasting insulin (FINS), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and C-reactive protein (CRP) were measured, and insulin sensitive index (ISI) and clinical TCM symptom score were calculated before and after treatment. RESULTS: After treatment, the clinical symptom score dropped in both groups (P < 0.01), and it was lower in the treated group than that in the control group (P < 0.01). At the same time, serum levels of FINS, CRP, TNF-alpha and IL-6 decreased and ISI increased significantly, the effect showed in the treated group was also superior to that in the control group (P < 0.05). CONCLUSION: JBR, which could tonify Pi and Shen, nourish Yin and clear away heat, can improve insulin resistance and alleviate clinical symptoms of T2DM patients, the mechanism may be related with its actions in regulating the production of inflammatory cytokines and inhibiting inflammatory reac-