OsGSTU34, a Bz2-like anthocyanin-related glutathione transferase transporter, is essential for rice (Oryza sativa L.) organs coloration.

Anthocyanins are a flavonoid compound known as one of the most important chromogenic substances. They play several functions, including health promotion and sustaining plants during adverse conditions. They are synthesized at the endoplasmic reticulum and sequestered in the vacuole. In this work, we generated knock-out lines of OsGSTU34, a glutathione transporter's tau gene family, with no transgene line and off-target through CRISPR/Cas9 mutagenesis and highlighted the loss of pigmentation in rice flowers, leaves, stems, shoots, and caryopsis. The anthocyanin quantification in the wild-type BLWT and mutant line BLG34-8 caryopsis showed that cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G) were almost undetectable in the mutant line. A tandem mass tag (TMT) labeling proteomic analysis was conducted to elucidate the proteomic changes in the BLWT and BLG34-8. The result revealed that 1175 proteins were altered, including 408 that were down-regulated and 767 that were upregulated. The accumulation of the OsGSTU34-related protein (Q8L576), along with several anthocyanin-related proteins, was down-regulated. The enrichment analysis showed that the down-regulated proteins were enriched in different pathways, among which the phenylpropanoid biosynthesis pathway, flavonoid biosynthesis metabolites, and anthocyanin biosynthesis pathway. Protein interaction network prediction revealed that glutathione-S-transferase (Q8L576) was connected to the proteins involved in the flavonoid and anthocyanin biosynthesis pathways, such as flavanone 3-dioxygenase 1 (Q7XM21), leucoanthocyanidin dioxygenase 1 (Q93VC3), 4-coumarate-CoA ligase 2 (Q42982), phenylalanine ammonia-lyase (P14717), chalcone synthase 1 (Q2R3A1), and 4-coumarate-CoA ligase 5 (Q6ZAC1). However, the expression of the most important anthocyanin biosynthesis gene was not altered, suggesting that only the transport mechanism was affected. Our findings highlight new insight into the anthocyanin pigmentation in black rice and provide new perspectives for future research.

Nationwide survey of physicians' familiarity and awareness of diabetes guidelines in China: a cross-sectional study.

OBJECTIVE: This study aims to investigate physicians' familiarity and awareness of four diabetes guidelines and their practice of the recommendations outlined in these guidelines. DESIGN: A cross-sectional study. SETTING: An online questionnaire survey was conducted among physicians affiliated with the Specialist Committee for Primary Diabetes Care of China Association of Chinese Medicine, using the snowball sampling method to ensure a broader representation of physicians. PARTICIPANTS: 1150 physicians from 192 cities across 30 provinces in China provided complete data. RESULTS: Tertiary care hospital physicians (TCPs) exhibited the highest familiarity with the Guideline for the Prevention and Treatment of Type 2 Diabetes Mellitus in China (91.3%), followed by the National Guidelines for the Prevention and Control of Diabetes in Primary Care (76.8%), the Standards of Medical Care in Diabetes (72.2%) and the Guidelines for Prevention and Treatment of Diabetes in Chinese Medicine (63.8%). Primary care practitioners (PCPs) exhibited familiarity with these four guidelines at about 50% or less. Self-reported reference to modern diabetes guidelines by physicians is more frequent than traditional Chinese medicine (TCM) diabetes guidelines, with rates at 73.2% and 33.8%, respectively. Approximately 90% of physicians provided instructions on self-monitoring of blood glucose to their patients with diabetes. Less than one-third of physicians referred patients to a specialised nutritionist. In terms of health education management, TCPs reported having a diabetes health management team at the rate of 75.7%, followed by secondary care hospital physicians at 57.0% and PCPs at 27.5%. Furthermore, approximately 40% of physicians did not fully grasp hypoglycaemia characteristics. CONCLUSIONS: Familiarity and awareness of the screening guidelines varied among physicians in different hospital settings. Importantly, significant discrepancies were observed between physicians' awareness and their self-reported reference to modern medicine guidelines and TCM guidelines. It is essential to consistently provide education and training on diabetes management for all physicians, particularly PCPs.

Improvement of resistance to rice blast and bacterial leaf streak by CRISPR/Cas9-mediated mutagenesis of Pi21 and OsSULTR3;6 in rice (Oryza sativa L.).

Rice (Oryza sativa L.) is a staple food in many countries around the world, particularly in China. The production of rice is seriously affected by the bacterial leaf streak and rice blast, which can reduce rice yield or even cause it to fail to be harvested. In this study, susceptible material 58B was edited by CRISPR/Cas9, targeting a target of the Pi21 gene and a target of the effector-binding element (EBE) of the OsSULTR3;6 gene, and the mutants 58b were obtained by Agrobacterium-mediated method. The editing efficiency of the two targets in the T0 generation was higher than 90.09%, the homozygous mutants were successfully selected in the T0 generation, and the homozygous mutation rate of each target was higher than 26.67%. The expression of the edited pi21 and EBE of Ossultr3;6 was significantly reduced, and the expression of defense responsive genes was significantly upregulated after infected with rice blast. The lesion areas of rice blast and bacterial leaf streak were significantly reduced in 58b, and the resistance of both was effectively improved. Furthermore, the gene editing events did not affect the agronomic traits of rice. In this study, the resistance of 58b to rice blast and bacterial leaf streak was improved simultaneously. This study provides a reference for using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) to accelerate the improvement of rice varieties and the development of new materials for rice breeding.

Detection of QTLs Regulating Six Agronomic Traits of Rice Based on Chromosome Segment Substitution Lines of Common Wild Rice (Oryza rufipogon Griff.) and Mapping of qPH1.1 and qLMC6.1.

Wild rice is a primary source of genes that can be utilized to generate rice cultivars with advantageous traits. Chromosome segment substitution lines (CSSLs) are consisting of a set of consecutive and overlapping donor chromosome segments in a recipient's genetic background. CSSLs are an ideal genetic population for mapping quantitative traits loci (QTLs). In this study, 59 CSSLs from the common wild rice (Oryza rufipogon Griff.) accession DP15 under the indica rice cultivar (O. sativa L. ssp. indica) variety 93-11 background were constructed through multiple backcrosses and marker-assisted selection (MAS). Through high-throughput whole genome re-sequencing (WGRS) of parental lines, 12,565 mapped InDels were identified and designed for polymorphic molecular markers. The 59 CSSLs library covered 91.72% of the genome of common wild rice accession DP15. The DP15-CSSLs displayed variation in six economic traits including grain length (GL), grain width (GW), thousand-grain weight (TGW), grain length-width ratio (GLWR), plant height (PH), and leaf margin color (LMC), which were finally attributed to 22 QTLs. A homozygous CSSL line and a purple leave margin CSSL line were selected to construct two secondary genetic populations for the QTLs mapping. Thus, the PH-controlling QTL qPH1.1 was mapped to a region of 4.31-Mb on chromosome 1, and the LMC-controlling QTL qLMC6.1 was mapped to a region of 370-kb on chromosome 6. Taken together, these identified novel QTLs/genes from common wild rice can potentially promote theoretical knowledge and genetic applications to rice breeders worldwide.

Clinical features and effectiveness of Chinese medicine in patients with COVID-19 from overseas: A retrospective study in Xiamen, China.

COVID-19, referred to as new coronary pneumonia, is an acute infectious disease caused by a new type of coronavirus SARS-CoV-2. To evaluate the effect of integrated Chinese medicine and Western medicine in patients with COVID-19 from overseas. Data were collected from 178 COVID-19 patients overseas at First Affiliated Hospital of Xiamen University from April 1, 2021 to July 31, 2021. These patients received therapy of integrated Chinese medicine and western medicine. Demographic data and clinical characteristics were extracted and analyzed. In addition, the prescription which induced less length of PCR positive days and hospitalization days than the median value was obtained. The top 4 frequently used Chinese medicine and virus-related genes were analyzed by network pharmacology and bioinformatics analysis. According to the chest computed tomography (CT) measurement, abnormal lung findings were observed in 145 subjects. The median length of positive PCR/hospitalization days was 7/7 days for asymptomatic subjects, 14/24 days for mild subjects, 10/15 days for moderate subjects, and 14/20 days for severe subjects. The most frequently used Chinese medicine were Scutellaria baicalensis (Huangqin), Glycyrrhiza uralensis (Gancao), Bupleurum chinense (Chaihu), and Pinellia ternata (Banxia). The putative active ingredients were baicalin, stigmasterol, sigmoidin-B, cubebin, and troxerutin. ACE, SARS-CoV-2 3CL, SARS-CoV-2 Spike, SARS-CoV-2 ORF7a, and caspase-6 showed good binding properties to active ingredients. In conclusion, the clinical results showed that integrated Chinese medicine and Western medicine are effective in treating COVID-19 patients from overseas. Based on the clinical outcomes, the putative ingredients from Chinese medicine and the potential targets of SARS-CoV-2 were provided, which could provide a reference for the clinical application of Chinese medicine in treating COVID-19 worldwide.

Comparative transcriptome-wide identification and differential expression of genes and lncRNAs in rice near-isogenic line (KW-Bph36-NIL) in response to BPH feeding.

Brown planthopper (BPH) is the most devastating pest of rice in Asia, causing substantial yield losses and has become a challenging task to be controlled under field conditions. Although extensive measures have been taken over the past decades, which resulted in the evolution of new resistant BPH strains. Therefore, besides other possible approaches, equipping host plants with resistant genes is the most effective and environment-friendly technique for BPH control. Here, we systematically analyzed transcriptome changes in the susceptible rice variety Kangwenqingzhan (KW) and the resistant near-isogenic line (NIL) KW-Bph36-NIL, through RNA-seq, depicting the differential expression profiles of mRNAs and long non-coding RNAs (lncRNAs) in rice before and after BPH feeding. We observed a proportion of genes (1.48%) and (2.74%) were altered in KW and NIL, respectively, indicating different responses of rice strains against BPH feeding. Nevertheless, we characterized 384 differentially expressed long non-coding RNAs (DELs) that can be impacted by the two strains by alternatively changing the expression patterns of the respective coding genes, suggesting their certain involvement in response to BPH feeding. In BPH invasion, KW and NIL responded differently by modifying the synthesis, storage, and transformation of intracellular substances, adjusting the nutrient accumulation and utilization inside and outside the cells. In addition, NIL expressed stronger resistance by acutely up-regulating genes and other transcription factors related to stress resistance and plant immunity. Altogether, our study elaborates valuable insights into the genome-wide DEGs and DELs expression profiles of rice under BPH invasion by high throughput sequencing and further suggests that NILs can be utilized in BPH resistance breeding programs in developing high-resistance rice lines.

Three new 3-formyl-2-arylbenzofurans from Itea yunnanensis and their anti-hepatocellular carcinoma effects.

Five 3-formyl-2-arylbenzofuran derivatives, including three new compounds (1-3) and two known analogues (4-5), were identified from the 95% EtOH extract of Itea yunnanensis. Extensive spectroscopic analyses were performed for the structure elucidation of all new benzofurans, and single-crystal X-ray diffraction analyses were further employed for the structure verification of iteafuranals C (1) and D (2). In MTT assay, iteafuranal E (3) and iteafuranal A (4) displayed significant growth inhibition effect on SK-Hep-1 cells with IC50 values of 5.365 µM and 6.013 µM, respectively. The colony formation assay of 3 and 4 further confirmed their remarkable inhibitory effect on cell growth. Preliminary mechanism study demonstrated that 3 remarkably down-regulated the phosphorylation level of ERK, which suggested 3 could inhibit cell growth and induce apoptosis of SK-Hep-1 cells by blocking RAS/RAF/MEK/ERK signaling pathway. This study highlighted the potential of 3-fomyl-2-benzofuran derivatives as novel lead compounds to treat Hepatocellular carcinoma.[Formula: see text].

CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.

Precise Editing of the OsPYL9 Gene by RNA-Guided Cas9 Nuclease Confers Enhanced Drought Tolerance and Grain Yield in Rice (Oryza sativa L.) by Regulating Circadian Rhythm and Abiotic Stress Responsive Proteins.

Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants lacking any off-targets and T-DNA were screened based on site-specific sequencing and used for morpho-physiological, molecular, and proteomic analysis. Mutant lines appear to accumulate higher ABA, antioxidant activities, chlorophyll content, leaf cuticular wax, and survival rate, whereas a lower malondialdehyde level, stomatal conductance, transpiration rate, and vascular bundles occur under stress conditions. Proteomic analysis found a total of 324 differentially expressed proteins (DEPs), out of which 184 and 140 were up and downregulated, respectively. The OsPYL9 mutants showed an increase in grain yield under both drought and well watered field conditions. Most of the DEPs related to circadian clock rhythm, drought response, and reactive oxygen species were upregulated in the mutant plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were only involved in circadian rhythm and Gene Ontology (GO) analysis showed that most of the DEPs were involved in response to abiotic stimulus, and abscisic acid-activated signaling pathways. Protein GIGANTEA, Adagio-like, and Pseudo-response regulator proteins showed higher interaction in protein-protein interaction (PPI) network. Thus, the overall results showed that CRISPR/Cas9-generated OsPYL9 mutants have potential to improve both drought tolerance and the yield of rice. Furthermore, global proteome analysis provides new potential biomarkers and understandings of the molecular mechanism of rice drought tolerance.

Development of Chromosome Segment Substitution Lines (CSSLs) Derived from Guangxi Wild Rice (Oryza rufipogon Griff.) under Rice (Oryza sativa L.) Background and the Identification of QTLs for Plant Architecture, Agronomic Traits and Cold Tolerance.

Common wild rice contains valuable resources of novel alleles for rice improvement. It is well known that genetic populations provide the basis for a wide range of genetic and genomic studies. In particular, chromosome segment substitution lines (CSSLs) ais a powerful tool for fine mapping of quantitative traits, new gene discovery and marker-assisted breeding. In this study, 132 CSSLs were developed from a cultivated rice (Oryza sativa) cultivar (93-11) and common wild rice (Oryza rufipogon Griff. DP30) by selfing-crossing, backcrossing and marker-assisted selection (MAS). Based on the high-throughput sequencing of the 93-11 and DP30, 285 pairs of Insertion-deletions (InDel) markers were selected with an average distance of 1.23 Mb. The length of this DP30-CSSLs library was 536.4 cM. The coverage rate of substitution lines cumulatively overlapping the whole genome of DP30 was about 91.55%. DP30-CSSLs were used to analyze the variation for 17 traits leading to the detection of 36 quantitative trait loci (QTLs) with significant phenotypic effects. A cold-tolerant line (RZ) was selected to construct a secondary mapping F2 population, which revealed that qCT2.1 is in the 1.7 Mb region of chromosome 2. These CSSLs may, therefore, provide powerful tools for genome wide large-scale gene discovery in wild rice. This research will also facilitate fine mapping and cloning of QTLs and genome-wide study of wild rice. Moreover, these CSSLs will provide a foundation for rice variety improvement.

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.

In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA1 and GA4) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA3 treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes.

Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line.

Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T0 generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T1 generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice.

Generation of High Yielding and Fragrant Rice (Oryza sativa L.) Lines by CRISPR/Cas9 Targeted Mutagenesis of Three Homoeologs of Cytochrome P450 Gene Family and OsBADH2 and Transcriptome and Proteome Profiling of Revealed Changes Triggered by Mutations.

The significant increase in grain yield and quality are often antagonistic but a constant demand for breeders and consumers. Some genes related to cytochrome P450 family are known for rice organ growth but their role in controlling grain yield is still unknown. Here, we generated new rice mutants with high yield and improved aroma by simultaneously editing three cytochrome P450 homoeologs (Os03g0603100, Os03g0568400, and GL3.2) and OsBADH2 with the CRISPR/Cas9 system, and RNA-sequencing and proteomic analysis were performed to unveil the subsequent changes. High mutation efficiency was achieved in both target sites of each gene and the mutations were predominantly only deletions, while insertions were rare, and no mutations were detected in the five most likely off-target sites against each sgRNA. Mutants exhibited increased grain size, 2-acetyl-1-pyrroline (2AP) content, and grain cell numbers while there was no change in other agronomic traits. Transgene-DNA-free mutant lines appeared with a frequency of 44.44% and homozygous mutations were stably transmitted, and bi-allelic and heterozygous mutations followed Mendelian inheritance, while the inheritance of chimeric mutations was unpredictable. Deep RNA sequencing and proteomic results revealed the regulation of genes and proteins related to cytochrome P450 family, grain size and development, and cell cycle. The KEGG and hub-gene and protein network analysis showed that the gene and proteins related to ribosomal and photosynthesis pathways were mainly enriched, respectively. Our findings provide a broad and detailed basis to understand the role of CRISPR/Cas9 in rice yield and quality improvement.

Effects of biochar content on gas diffusion coefficient of soil with different compactness and air contents.

Biochar has been found to be a potentially suitable amendment for landfill cover material and agricultural soil. The addition of biochar can improve the physical (e.g., adsorption capacity) and hydrological properties (e.g., water/gas permeability) of soil. However, no experimental study is available about the effect of biochar content (BC) on the gas diffusion coefficient (DP) of soil. The present study investigated the effect of BC on DP under different degree of compaction (DOC; 85%, 90%, and 95%) and soil air contents (SAC; 5%, 10%, and 15%). It was found that DOC and BC had negligible effects on DP when SAC was low (~ 5%). In contrast, when the SAC was relatively high (~ 15%), soil with DOC of 85% had the largest DP for BC ranging from 0 to 15% (w/w). Only when the SAC was large (~ 15%), the addition of biochar generally increased DP.

Programmed Editing of Rice (Oryza sativa L.) OsSPL16 Gene Using CRISPR/Cas9 Improves Grain Yield by Modulating the Expression of Pyruvate Enzymes and Cell Cycle Proteins.

Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T0, T1, and T2 generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations.

Generation of semi-dwarf rice (Oryza sativa L.) lines by CRISPR/Cas9-directed mutagenesis of OsGA20ox2 and proteomic analysis of unveiled changes caused by mutations.

Plant height (PH) is one of the most important agronomic traits of rice, as it directly affects the yield potential and lodging resistance. Here, semi-dwarf mutant lines were developed through CRISPR/Cas9-based editing of OsGA20ox2 in an indica rice cultivar. Total 24 independent lines were obtained in T0 generation with the mean mutation rate of 73.5% including biallelic (29.16%), homozygous (47.91%) and heterozygous (16.66%) mutations, and 16 T-DNA-free lines (50%) were obtained in T1 generation without off-target effect in four most likely sites. Mutations resulted in a changed amino acid sequence of mutant plants and reduced gibberellins (GA) level and PH (22.2%), flag leaf length (FLL) and increased yield per plant (YPP) (6.0%), while there was no effect on other agronomic traits. Mutants restored their PH to normal by exogenous GA3 treatment. The expression of the OsGA20ox2 gene was significantly suppressed in mutant plants, while the expression level was not affected for other GA biosynthesis (OsGA2ox3 and OsGA3ox2) and signaling (D1, GIDI and SLR1) genes. The mutant lines showed decreased cell length and width, abnormal cell elongation, while increased cell numbers in the second internode sections at mature stage. Total 30 protein spots were exercised, and 24 proteins were identified, and results showed that OsGA20ox2 editing altered protein expression. Five proteins including, glyceraldehyde-3-phosphate dehydrogenase, putative ATP synthase, fructose-bisphosphate aldolase 1, S-adenosyl methionine synthetase 1 and gibberellin 20 oxidase 2, were downregulated in dwarf mutant lines which may affect the plant growth. Collectively, our results provide the insights into the role of OsGA20ox2 in PH and confirmed that CRISPR-Cas9 is a powerful tool to understand the gene functions.

Effects of occupation on intracerebral hemorrhage-related deaths in Inner Mongolia.

This study assessed the relationship between occupation and Intracerebral Hemorrhage-related deaths and compared the differences in ICH-related deaths rates between the eastern and midwestern regions of Inner Mongolia. We used the case-control method. Cases included Intracerebral Hemorrhage-related deaths that occurred from 2009 to 2012 in Inner Mongolia while controls included non-circulatory system disease deaths that occurred during the same period. Odds ratios (ORs) for Intracerebral Hemorrhage-related deaths were calculated using logistic regression analysis, estimated according to occupation, and adjusted for marital status and age. The Intracerebral Hemorrhage mortality rate in the eastern regions (125.19/100000) was nearly 3 times higher than that in the midwestern regions (45.31/100000). ORs for agriculture-livestock workers, service professionals and general workers, professional workers and senior officials were in descending order. The age-adjusted OR for Intracerebral Hemorrhage-related deaths was lowest in unmarried men senior officials (OR 0.37, 95% CI 0.14-0.99). The Intracerebral Hemorrhage mortality rate in the eastern regions was much higher than that of the midwestern regions, since about 90% of Intracerebral Hemorrhage-related deaths in the eastern regions were those of agriculture-livestock workers who has the largest labor intensity of any other occupation assessed.

Curcumin suppresses Notch­1 signaling: Improvements in fatty liver and insulin resistance in rats.

Curcumin is a well­known phenolic substance and has many pharmacological effects associated with metabolism. However, the exact molecular mechanisms underlying this process have yet to be determined. The Notch pathway is a signal transduction pathway involved in energy metabolism. The present study aimed to investigate the effects of curcumin administration on glucose­lipid metabolism in rats subjected to a high fat diet, and investigate changes in Notch­1 signaling. Sprague­Dawley rats (n=40) were randomly divided into four groups (10 rats/group): Control diet group, high fat diet group, high fat diet plus curcumin low dose group and high fat diet plus curcumin high dose group. Following 8 weeks of treatment with curcumin (100 mg/kg in the low dose group and 200 mg/kg in the high dose group), serum metabolic markers and hepatic gene expression patterns were investigated. No differences in body weight following 8 weeks of curcumin administration (P>0.05) were observed; however, curcumin treatment did reduce visceral fat levels (peri­epididymal and peri­renal), and decreased cholesterol, triglyceride and low­density lipoprotein levels in serum compared with the high fat diet rats that did not receive curcumin (P<0.05, P<0.01). An oral glucose tolerance test and an intraperitoneal insulin tolerance test revealed that insulin resistance was reduced (P<0.05 or P<0.01) and tissue section analysis revealed that hepatosteatosis was attenuated following treatment with curcumin. Furthermore, the protein expression of Notch­1 and its downstream target Hes­1 were suppressed. These effects were also in parallel with an upregulation of fatty acid oxidation­associated gene expression, including peroxisome proliferator­activated receptor (PPAR)­α, carnitine palmitoyltransferase 1 and PPAR­Î³ (P<0.05). In addition, curcumin administration led to a downregulation in the expression of lipogenic genes, including sterol regulatory element­binding protein, fatty acid synthase and acetyl­CoA carboxylase (P<0.05). The expression of inflammation­associated genes, including nuclear factor­κB, tumor necrosis factor­α and prostaglandin­endoperoxide synthase 2 were also suppressed. The results of the present study suggest that the hepatic Notch­1 pathway can be suppressed via curcumin treatment, which may ameliorate fatty liver and insulin resistance in rats subjected to a high fat diet.

Ping-tang Recipe () improves insulin resistance and attenuates hepatic steatosis in high-fat diet-induced obese rats.

OBJECTIVE: To investigate the therapeutic effects of Ping-tang Recipe (, PTR) on high-fat diet (HFD)-induced insulin resistance and non-alcoholic fatty liver disease (NAFLD), and to elucidate the underlying mechanisms. METHODS: Forty male SD rats were included in the study. Ten rats were fed on normal diet as normal control, and thirty rats were fed on HFD for 8 weeks to induce obesity, followed with low dose (0.42 g/kg) or high dose (0.84 g/kg) of PTR or vehicle for 8 weeks with 10 animals for each group. Glucose metabolism and insulin sensitivity were evaluated by oral glucose tolerance test and insulin tolerance test. Hepatic steatosis was measured by immunohistochemistry. Liver lipid metabolic genes were analyzed by quantitative real-time polymerase chain reaction, while AMP-activated protein kinase (AMPK) expression was examined by Western blot. RESULTS: Rats fed on HFD developed abdominal obesity, insulin resistance and NAFLD. PTR treatment reduced visceral fat (peri-epididymal and peri-renal) accumulation, improved glucose metabolism, and attenuated hepatic steatosis. The expressions of the key lipolytic regulating genes, including peroxisome proliferators-activated receptor γ co-activator 1α (PGC-1α), peroxisome proliferator-activated receptor γ (PRAR-γ) and α (PRAR-α), were up-regulated (P<0.05 or P<0.01), while the expressions of lipogenic genes such as sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FAS) and liver fatty acid-binding protein (L-FABP) were down-regulated (P<0.05 or P<0.01). In addition, PTR activated AMPK and promoted acetyl-CoA carboxylase phosphorylation in the liver. CONCLUSIONS: PTR improves insulin resistance and reverse hepatic steatosis in the rat model of HFD-induced obesity through promotion of lipolysis and reduction of lipogenesis, which involves the AMPK signaling pathway, thus representing a new therapeutic intervention for obesity related insulin resistance and NAFLD.