Virulence Variability and Genetic Diversity in Blumeria graminis f. sp. hordei in Southeastern and Southwestern China.

Powdery mildew is a key airborne foliar disease of barley in southeastern and southwestern China. Barley varieties usually partially or wholly lose resistance to the pathogen Blumeria graminis (DC.) f. sp. hordei 3 to 5 years after release due to the frequent acquirements of new virulences in the pathogen population. However, no B. graminis f. sp. hordei virulence detection has been carried out in the recent decade and, thus, no information is available on the present virulence components and major pathotypes in epidemic regions. Twenty-one near-isogenic lines of Pallas were selected to detect B. graminis f. sp. hordei virulence variation, with 97 pathotypes identified from the isolates collected from 2015 to 2019. The virulence complexities ranged from 1 to 12, with 1.5 isolates on average assigned per pathotype, suggesting a natural trait of high pathotype diversity and low virulence complexity in the Chinese B. graminis f. sp. hordei populations. Eleven high-virulence pathotypes were detected in the traditional barley-growing regions in Yunnan and Zhejiang. Six virulent pathotypes to resistance gene mlo-5 were detected only in the two traditional epidemic regions, with a virulence frequency (VF) of 4.8% (7 of 147). Compared with the results from a decade ago, VFs for resistance alleles Mla3, mlo-5, Mla6 + Mla14, Mla7 + Mlk, Mlg + MlCP, and Mla13 + MlRu3 + MlaRu4 increased from 0 to 0.7 to 25.8%. Isolates from Yunnan and Zhejiang had similar virulence profiles, which differed from those identified in Tibet. In addition, genetic diversities differed in the isolate groups collected from Tibet, Yunnan, and Zhejiang.

Identification and molecular mapping of YrBm for adult plan resistance to stripe rust in Chinese wheat landrace Baimangmai.

KEY MESSAGE: A new adult plan resistance gene YrBm for potentially durable resistance to stripe rust was mapped on wheat chromosome arm 4BL in landrace Baimangmai. SSR markers closely flanking YrBm were developed and validated for use in marker-assisted selection. The wheat stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst) frequently acquires new virulences and rapidly adapts to environmental stress. New virulences in Pst populations can cause previously resistant varieties to become susceptible. If those varieties were widely grown, consequent epidemics can lead to yield losses. Identification and deployment of genes for durable resistance are preferred method for disease control. The Chinese winter wheat landrace Baimangmai showed a high level of adult plant resistance (APR) to stripe rust in a germplasm evaluation trial at Langfang in Hebei province in 2006 and has continued to confer high resistance over the following 15 years in field nurseries in Hebei, Sichuan and Gansu. A recombinant inbred line population of 200 F10 lines developed from a cross of Baimangmai and a susceptible genotype segregated for APR at a single locus on chromosome 4BL; the resistance allele was designated YrBm. Allelism tests of known Yr genes on chromosome 4B and unique closely flanking marker alleles Xgpw7272189 and Xwmc652164 among a panel of Chinese wheat varieties indicated that YrBm was located at a new locus. Moreover, those markers can be used for marker-assisted selection in breeding for stripe rust resistance.

Endothelial Mitochondrial Preprotein Translocase Tomm7-Rac1 Signaling Axis Dominates Cerebrovascular Network Homeostasis.

Objective- Mitochondria are the important yet most underutilized target for cardio-cerebrovascular function integrity and disorders. The Tom (translocases of outer membrane) complex are the critical determinant of mitochondrial homeostasis for making organs acclimate physiological and pathological insults; however, their roles in the vascular system remain unknown. Approach and Results- A combination of studies in the vascular-specific transgenic zebrafish and genetically engineered mice was conducted. Vascular casting and imaging, endothelial angiogenesis, and mitochondrial protein import were performed to dissect potential mechanisms. A loss-of-function genetic screening in zebrafish identified that selective inactivation of the tomm7 (translocase of outer mitochondrial membrane 7) gene, which encodes a small subunit of the Tom complex, specially impaired cerebrovascular network formation. Ablation of the ortholog Tomm7 in mice recapitulated cerebrovascular abnormalities. Restoration of the cerebrovascular anomaly by an endothelial-specific transgenesis of tomm7 further indicated a defect in endothelial function. Mechanistically, Tomm7 deficit in endothelial cells induced an increased import of Rac1 (Ras-related C3 botulinum toxin substrate 1) protein into mitochondria and facilitated the mitochondrial Rac1-coupled redox signaling, which incurred angiogenic impairment that underlies cerebrovascular network malformation. Conclusions- Tomm7 drives brain angiogenesis and cerebrovascular network formation through modulating mitochondrial Rac1 signaling within the endothelium.

Low-density lipoprotein receptor-related protein 6 regulates alternative pre-mRNA splicing.

Low-density lipoprotein receptor-related protein 6 (LRP6) serves as a Wnt coreceptor. Although Wnt/LRP6 signalling is best known for the ß-catenin-dependent regulation of target genes in tissue development and homeostasis, emerging evidence demonstrates the biological aspects of LRP6 beyond a Wnt coreceptor. Whether LRP6 modulates tissue development in a Wnt/ß-catenin signalling-independent manner remains unknown. Using a model of striated muscle development, we observed that LRP6 was almost undetectable in proliferating myoblasts, whereas its expression gradually increased in the nucleus of myodifferentiating cells. During myodifferentiation, LRP6 modulated the muscle-specific splicing of integrin-ß1D and consequent myotube maturation independently of the ß-catenin-dependent Wnt signalling. Furthermore, we identified that the carboxy-terminal serine-rich region in LRP6 bond to the adenine-rich sequence within alternative exon D (AED) of integrin-ß1 pre-mRNA, and therefore, elicited AED inclusion when the spliceosome was recruited to the splice site. The interaction of LRP6 with the adenine-rich sequence was sufficient to overcome AED exclusion by a splicing repressor, polypyrimidine tract binding protein-1. Besides the integrin-ß1, deep RNA sequencing in different types of cells revealed that the LRP6-mediated splicing regulation was widespread. Thus, our findings implicate LRP6 as a potential regulator for alternative pre-mRNA splicing.

Vasodilator-stimulated phosphoprotein-guided Clopidogrel maintenance therapy reduces cardiovascular events in atrial fibrillation patients requiring anticoagulation therapy and scheduled for percutaneous coronary intervention: a prospective cohort study.

BACKGROUND: In a previous study, we found that titrating clopidogrel maintenance doses (MDs) according to vasodilator-stimulated phosphoprotein (VASP) monitoring minimised the rate of major adverse cardiovascular and cerebral events (MACCE) after percutaneous coronary intervention (PCI) without increasing bleeding in patients with high on-treatment platelet reaction to clopidogrel. This study aimed to investigate whether VASP-guided clopidogrel MD could reduce thromboembolism and bleeding in atrial fibrillation (AF) patients requiring anticoagulation and scheduled for PCI. METHODS: AF patients scheduled for PCI were recruited between July 2014 and July 2016. These patients were allocated into VASP-guided (n = 250) and control (n = 253) groups depending on the clopidogrel MD profile. In the VASP-guided group, clopidogrel MD was titrated by the platelet reactivity index (PRI), whereas in the control group, clopidogrel MD was fixed at 75 mg per day. The primary endpoint was MACCE and secondary endpoints were thrombolysis in myocardial infarction (TIMI) major and minor bleeding 1 year after PCI. RESULTS: Five hundred and three patients were included in the present study, with 1-year data available for 95.6% patients. The average CHA2DS2-VASc score of the whole population was 3.7 ± 0.7 and the average HAS-BLED score was 3.2 ± 0.4. MACCE was less in the VASP-guided group than in the control group (2.5% vs. 5.0%, P = 0.02). The incidence of major bleeding was comparable between both groups (3.0% vs. 2.8%, P = 0.72) and minor bleeding was higher in the VASP-guided group than in the control group (15.3% vs. 9.7%, P = 0.03). Kaplan-Meier analysis indicated that there was no difference in survival between both groups (log-rank test, P = 0.68). CONCLUSIONS: In AF patients requiring anticoagulation and scheduled for PCI, VASP-guided antiplatelet therapy reduced major cardiovascular and cerebral adverse events, accompanied by increased minor bleeding events. TRIAL REGISTRATION: The present study was retrospectively registered in the Chinese Clinical Trial Registry, A Primary Registry of the International Clinical Trial Registry Platform, World Health Organisation (Registration no: ChiCTR-IOR-17013854 ). The registered date was December 11, 2117.

Interactions of Methylenetetrahydrofolate Reductase Gene Polymorphisms, Folate, and Homocysteine on Blood Pressure in a Chinese Hypertensive Population.

BACKGROUND: High blood pressure is related to cardiovascular diseases. We aimed to explore the interactions of methylenetetrahydrofolate reductase (MTHTR) gene C677T and A1298C mutations and folate/homocysteine (Hcy) status on blood pressure in a Chinese hypertensive population. METHODS: The clinical data in the present study derived from a previous trial (NCT00520247). Genotypes in Hcy pathway enzymes were detected by PCR-RFLP methods. Supine blood pressure was measured with a mercury sphygmomanometer. Serum Hcy was measured by high-performance liquid chromatography, and serum folate was measured by chemiluminescent immunoassay. RESULTS: This study showed that hyperhomocysteinemia independently elevated diastolic blood pressure (DBP) (ß (SE): 2.02 (0.85), p = 0.018). Furthermore, individuals with high Hcy and MTHFR1298AC + CC genotypes showed higher DBP than the normal Hcy and 1298AA carriers (ß (SE): 1.81 (0.54), p = 0.001). This correlation was verified by the trend test (p = 0.003). However, polymorphisms of MTHFR C677T, MTR A2756G or MTRR A66G do not affect baseline blood pressure level. CONCLUSIONS: The present study demonstrated that the MTHFR A1298C mutation accompanied by hyperhomocysteinemia jointly elevated DBP. Further studies are necessary to confirm the role of these genotypes and Hcy on blood pressure in a larger population.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli.

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

The reporting quality of randomized controlled trials in Chinese herbal medicine (CHM) formulas for diabetes based on the consort statement and its extension for CHM formulas.

Background: This study aimed to assess the overall reporting quality of randomized controlled trials (RCTs) in Chinese herbal medicine (CHM) formulas for patients with diabetes, and to identify factors associated with better reporting quality. Methods: Four databases including PubMed, Embase, Cochrane Library and Web of Science were systematically searched from their inception to December 2022. The reporting quality was assessed based on the Consolidated Standards of Reporting Trials (CONSORT) statement and its CHM formula extension. The overall CONSORT and its CHM formula extension scores were calculated and expressed as proportions separately. We also analyzed the pre-specified study characteristics and performed exploratory regressions to determine their associations with the reporting quality. Results: Seventy-two RCTs were included. Overall reporting quality (mean adherence) were 53.56% and 45.71% on the CONSORT statement and its CHM formula extension, respectively. The strongest associations with reporting quality based on the CONSORT statement were multiple centers and larger author numbers. Compliance with the CHM formula extension, particularly regarding the disclosure of the targeted traditional Chinese medicine (TCM) pattern (s), was generally insufficient. Conclusion: The reporting quality of RCTs in CHM formulas for diabetes remains unsatisfactory, and the adherence to the CHM formula extension is even poorer. In order to ensure transparent and standardized reporting of RCTs, it is essential to advocate for or even mandate adherence of the CONSORT statement and its CHM formula extension when reporting trials in CHM formulas for diabetes by both authors and editors.

CoFe Alloy-Coupled Mo2C Wrapped by Nitrogen-Doped Carbon as Highly Active Electrocatalysts for Oxygen Reduction/Evolution Reactions.

Molybdenum carbide (Mo2C) with a Pt-like d-band electron structure exhibits certain activities for oxygen reduction and evolution reactions (ORR/OER) in alkaline solutions, but it is questioned due to its poor OER stability. Combining Mo2C with transition metals alloy is a feasible way to stabilize its electrochemical activity. Herein, CoFe-Prussian blue analogues are used as a precursor to compound with graphitic carbon nitride and Mo6+ to synthesize FeCo alloy and Mo2C co-encapsulated N-doped carbon (NG-CoFe/Mo2C). The morphology of NG-CoFe/Mo2C (800 °C) shows that CoFe/Mo2C heterojunctions are well wrapped by N-doped graphitic carbon. Carbon coating not only inhibits growth and agglomeration of Mo2C/CoFe, but also enhances corrosion resistance of NG-CoFe/Mo2C. NG-CoFe/Mo2C (800 °C) exhibits an excellent half-wave potential (E1/2 = 0.880 V) for ORR. It also obtains a lower OER overpotential (325 mV) than RuO2 due to the formation of active species (CoOOH/ß-FeOOH, as indicated by in-situ X-ray diffraction tests). E1/2 shifts only 6 mV after 5000 ORR cycles, while overpotential for OER increases only 19 mV after 1000 cycles. ORR/OER performances of NG-CoFe/Mo2C (800 °C) are close to or better than those of many recently reported catalysts. It provides an interfacial engineering strategy to enhance the intrinsic activity and stability of carbides modified by transition-metals alloy for oxygen electrocatalysis.

MALAT1 modulated FOXP3 ubiquitination then affected GINS1 transcription and drived NSCLC proliferation.

An increasing number of studies have shown that long-noncoding RNAs (lncRNAs) are involved in the post-translational modifications (PTMs) of protein in a variety of tumors. However, little is known about the exact regulation mechanism of lncRNAs in regulating PTMs in non-small-cell lung carcinoma (NSCLC) proliferation. Metastasis-associated lung adenocarcinoma transcript1 (MALAT1) and GINS complex subunit 1(GINS1) both were upregulated and promoted proliferation progression in NSCLC. In this study, the clinicopathologic significance of MALAT1 and GINS1 in NSCLC was investigated, a positive correlation in their expression was found. The silencing of MALAT1 decreased GINS1 expression and inhibited NSCLC proliferation in vitro and in vivo. The upregulation of GINS1 reversed NSCLC proliferation inhibited by MALAT1 knockdown. FOXP3 (forkhead box protein 3) was identified as the critical transcription factor for GINS1 transcription. In addition, MALAT1 could stabilize FOXP3 by binding to zinc finger (ZF) domain and leucine zipper (LZ) domain of FOXP3. Interestingly, these two domains were also interaction domains for FOXP3 binding with E3 ligase STUB1 (STIP1 homology and U-box containing protein 1). In this way, MALAT1 masked the protein-interacting domain, and inhibited FOXP3 ubiquitination by STUB1. Together, our results identified a novel regulatory axis of MALAT1-FOXP3-GINS1, and demonstrated that MALAT1 played an important modulatory role in PTM of FOXP3 which affects GINS1 transcription and drives proliferation character in NSCLC.

Abnormalities occurring during female gametophyte development result in the diversity of abnormal embryo sacs and leads to abnormal fertilization in indica/japonica hybrids in rice.

Embryo sac abortion is one of the major reasons for sterility in indica/japonica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indica/japonica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.

Establishment and Evaluation of a Simple Size-Selective Method for Exosome Enrichment and Purification.

Procedures for enrichment, isolation and purification of exosomes from complex biological samples are difficult, tedious, non-standardized, and require bulky instrumentation such as ultracentrifugation (UC). In this article, a simple method for isolating exosomes. Size-Selective Method (SSM) was established based on commercially available materials, and the UC and ExoQuick-TC kits (EQkit) methods were compared in terms of morphology, particle size, quantity, Western Blot (WB), and extraction time. Results showed that all three different exosome separation methods could obtain circular membranous vesicles, with a diameter of 30-110 nm. There were more non-exosome components in the samples extracted by SSM, such as large microvesicles, with a lower purity. UC obtained a large number of exosomes with a higher purity, but it required an ultracentrifuge, costed much time and had low yield. Both EQkit and SSM were easy to operate, but EQkit tended to aggregate exosomes and consume much time. WB results showed that exosomes extracted by all the three methods expressed CD63 protein. The SSM had the highest CD63 protein content and at the same protein concentration. The above evidences showed that SSM was fast and had high recovery, low cost and high protein concentration, but had more non-exosome protein components, which can be a choice for exosome separation.

Comparison of gross tumor volume of primary oesophageal cancer based on contrast-enhanced three-dimensional, four-dimensional, and cone beam computed tomography.

BACKGROUND: To explore motion information included in 3DCT, 4DCT and CBCT by comparing volumetric and positional differences of GTV. RESULTS: Independent of tumor location, significant differences were observed among volumes [IGTV10 > (IGTVCBCT or IGTVMIP) > (GTV3D or GTV4D50)]. The underestimations or overestimations between IGTV10 and IGTVCBCT were larger than those between IGTV10 and IGTVMIP (p < 0.001-0.011; p < 0.001-0.023). For upper oesophageal tumors, GTV4D50/IGTVCBCT negatively correlated with motion vector (r = -0.756, p = 0.011). In AP direction, the centroid coordinates of IGTVCBCT differed from GTV3D, GTV4D50, IGTVMIP and IGTV10 (p = 0.006, 0.013, 0.038, and 0.010). For middle oesophageal tumors, IGTV10/IGTVCBCT positively correlated with motion vector (r = 0.695, p = 0.006). The centroid coordinates of IGTVCBCT differed from those of IGTV10 (p = 0.046) in AP direction. For distal oesophageal tumors, the centroid coordinates of IGTVCBCT showed significant differences to those of IGTVMIP (p = 0.042) in LR direction. For both middle and distal tumors, the degrees of associations of IGTV10 outside IGTVCBCT significantly correlated with the motion vector (r = 0.540, p = 0.046; r = 0.678, p = 0.031). MATERIALS AND METHODS: Thirty-four oesophageal cancer patients underwent 3DCT, 4DCT and CBCT. GTV3D, GTV4D50, internal GTVMIP (IGTVMIP) and IGTVCBCT were delineated on 3DCT, 4DCT50, 4DCTMIP and CBCT. GTVs from 10 respiratory phases were combined to produce GTV10. Differences in volume, position for different targets, correlation between volume ratio and motion vector were evaluated. The motion vector was the spatial moving of the target centroid position. CONCLUSIONS: IGTVCBCT encompasses more motion information than GTV3D and GTV4D50 for upper oesophageal tumors, but slightly less than IGTV10 for middle and distal oesophageal tumors. IGTVCBCT incorporated similar motion information to IGTVMIP. However, motion information encompassed in CBCT and MIP cannot replace each other.

Analysis on genetic diversification and heterosis in autotetraploid rice.

Polyploidization has played an important role in plant evolution and is a pathway for plants to increase genetic diversification and to get higher heterosis comparing with that of diploid does. This study was undertaken to assess the genetic variation and relationships among 40 autotetraploid rice genotypes and their counterpart diploid cultivars with 99 SSR markers screened from published rice genome. The 99 SSR markers detected polymorphism among autotetraploid genotypes and revealed a total of 291 alleles with an average of 2.949 alleles per locus. Autotetraploid lines showed higher genetic diversity and significant variation in agronomic traits than diploid cultivars. Phylogenetic analysis revealed that most of autotetraploid lines were genetically different from their diploid parents, and inter-subspecific hybrids were prepared on the basis of genetic distance between parents. Inter-subspecific autotetraploid hybrids showed a higher and positive heterobeltiosis and competitive heterosis than diploid hybrids, especially for grain yield. Genetic distance appeared not to predict heterosis in diploid rice for all traits; however, it showed a significant correlation with grain yield, grain length and grain length to width ratio in autotetraploid rice. This extensive research on autotetraploid heterosis and genetic diversity will be useful for the development of autotetraploid rice hybrids.