GhHAM regulates GoPGF-dependent gland development and contributes to broad-spectrum pest resistance in cotton.

Cotton is a globally cultivated crop, producing 87% of the natural fiber used in the global textile industry. The pigment glands, unique to cotton and its relatives, serve as a defense structure against pests and pathogens. However, the molecular mechanism underlying gland formation and the specific role of pigment glands in cotton's pest defense are still not well understood. In this study, we cloned a gland-related transcription factor GhHAM and generated the GhHAM knockout mutant using CRISPR/Cas9. Phenotypic observations, transcriptome analysis, and promoter-binding experiments revealed that GhHAM binds to the promoter of GoPGF, regulating pigment gland formation in cotton's multiple organs via the GoPGF-GhJUB1 module. The knockout of GhHAM significantly reduced gossypol production and increased cotton's susceptibility to pests in the field. Feeding assays demonstrated that more than 80% of the cotton bollworm larvae preferred ghham over the wild type. Furthermore, the ghham mutants displayed shorter cell length and decreased gibberellins (GA) production in the stem. Exogenous application of GA3 restored stem cell elongation but not gland formation, thereby indicating that GhHAM controls gland morphogenesis independently of GA. Our study sheds light on the functional differentiation of HAM proteins among plant species, highlights the significant role of pigment glands in influencing pest feeding preference, and provides a theoretical basis for breeding pest-resistant cotton varieties to address the challenges posed by frequent outbreaks of pests.

The dual role of GoPGF reveals that the pigment glands are synthetic sites of gossypol in aerial parts of cotton.

Gossypol and the related terpenoids are stored in the pigment gland to protect cotton plants from biotic stresses, but little is known about the synthetic sites of these metabolites. Here, we showed that GoPGF, a key gene regulating gland formation, was expressed in gland cells and roots. The chromatin immunoprecipitation sequencing (ChIP-seq) analysis demonstrated that GoPGF targets GhJUB1 to regulate gland morphogenesis. RNA-sequencing (RNA-seq) showed high accumulation of gossypol biosynthetic genes in gland cells. Moreover, integrated analysis of the ChIP-seq and RNA-seq data revealed that GoPGF binds to the promoter of several gossypol biosynthetic genes. The cotton callus overexpressing GoPGF had dramatically increased the gossypol levels, indicating that GoPGF can directly activate the biosynthesis of gossypol. In addition, the gopgf mutant analysis revealed the existence of both GoPGF-dependent and -independent regulation of gossypol production in cotton roots. Our study revealed that the pigment glands are synthetic sites of gossypol in aerial parts of cotton and that GoPGF plays a dual role in regulating gland morphogenesis and gossypol biosynthesis. The study provides new insights for exploring the complex relationship between glands and the metabolites they store in cotton and other plant species.

Triterpenoids from Kochiae Fructus: Glucose Uptake in 3T3-L1 Adipocytes and α-Glucosidase Inhibition, In Silico Molecular Docking.

In this study, three new triterpenes (1-3) and fourteen known triterpenoids (4-17) were isolated from the ethanol extract of Kochiae Fructus, and their structures were elucidated by analyzing UV, IR, HR-ESI-MS, 1D, and 2D NMR spectroscopic data. Among them, compounds 6, 8, and 11-17 were isolated for the first time from this plant. The screening results of the glucose uptake experiment indicated that compound 13 had a potent effect on glucose uptake in 3T3-L1 adipocytes at 20 µM. Meanwhile, compounds 3, 9 and 13 exhibited significant inhibitory activities against α-glucosidase, with IC50 values of 23.50 ± 3.37, 4.29 ± 0.52, and 16.99 ± 2.70 µM, respectively, and their α-glucosidase inhibitory activities were reported for the first time. According to the enzyme kinetics using Lineweaver-Burk and Dixon plots, we found that compounds 3, 9 and 13 were α-glucosidase mixed-type inhibitors with Ki values of 56.86 ± 1.23, 48.88 ± 0.07 and 13.63 ± 0.42 µM, respectively. In silico molecular docking analysis showed that compounds 3 and 13 possessed superior binding capacities with α-glucosidase (3A4A AutoDock score: -4.99 and -4.63 kcal/mol). Whereas compound 9 showed +2.74 kcal/mol, which indicated compound 9 exerted the effect of inhibiting α-glucosidase activity by preferentially binding to the enzyme-substrate complex. As a result, compounds 3, 9 and 13 could have therapeutic potentials for type 2 diabetes mellitus, due to their potent hypoglycemic activities.

[Clinical study on new risk assessment and prediction system for early osteonecrosis of the femoral head].

OBJECTIVE: To establish a risk assessment and prediction system for early osteonecrosis of the femoral head (ONFH) in order to predict the collapse risk. METHODS: The risk assessment system for early necrosis and collapse of femoral head was established based on the combination of Steinberg stage, ABC typing and the proportion of the proximal sclerotic rim. Firstly, Steinberg stage system was applied. ABC typing was applied to predict risk in stage I, type C was risk free, type B was low risk, type A and type BC were medium risk, type A-C and type AB were high risk. The classification of proximal sclerotic rim was first applied when the Steinberg stage was Ⅱ-Ⅲ, and type 2 was expected to be low risk. If the classification of proximal sclerotic rimwas type 1, then the ABC typing was applied, type C was risk-free, type B was low risk, type A and type BC were medium risk, type A-C and type AB were high risk. According to this prediction system, the collapse risk of femoral head in 188 cases(301 hips) were predicted by retrospective analysis. All the hips were enrolled at the out-patient department of orthopedic in Guang'anmen Hospital attached to China Academy of Chinese Medical Science. The consistency of the prediction results of three doctors and one doctor at different times were evaluated. RESULTS: Among them, 136 cases were male, 52 were female. 75 cases were single hip, 113 were double hip. The age of the patients wa 19 to 64(42.61±12.07) years. The natural course of disease was 0.33 to 5.00(3.62±1.93) years. 206 hips in 301 hips had collapsed, with a collapse rate of 68.44%. In the risk-free group, none hip had collapsed, with a collapse rate of 0%. In the low-risk group, 9 hip in 91 hips had collapsed, with a collapse rate of 9.89%. In the medium-risk group, 12 hip in 19 hips had collapsed, with a collapse rate of 63.16%. And in the high risk group, 185 hips in 190 hips had collapsed, with a collapse rate of 97.37%. They were significantly differences in their collapse rate (P=0.00) in the following order:high-risk group> medium-risk > low-risk group > risk-free group. The prediction value of the system was high (AUC=0.95, P=0.00). The results predicted by different doctors were consistent (ICC=0.94, P=0.00), and the results predicted by the same doctor at two different times were consistent (Kappa coefficient =0.90, P=0.00). CONCLUSION: The risk assessment and prediction system for early ONFH selects different methods to predict the risk of collapse according to the imaging characteristics of different stages, which is combines with the comprehensive assessment of multiple risk factors. The system is applicable to a wide range, simple operation and convenient for clinical application.

Analysis of the Role of the Drought-Induced Gene DRI15 and Salinity-Induced Gene SI1 in Alternanthera philoxeroides Plasticity Using a Virus-Based Gene Silencing Tool.

Alternanthera philoxeroides is a notoriously invasive weed that can readily adapt to different environmental conditions. Control of this weed is difficult, and it spreads easily and causes damage to native habitats and agriculture. In this study, our goal was to investigate the molecular mechanisms that lead to the ability of A. philoxeroides to invade new habitats, to adapt to environmental stresses, and to cause damage. We developed a simple and highly effective potato virus X-based virus-induced gene silencing (VIGS) approach. The VIGS approach was first used to silence the phytoene desaturase gene, which resulted in the expected photo-bleaching phenotype. Next, the VIGS approach was used to silence two additional genes, drought-induced protein gene 15 (ApDRI15) and salinity-induced protein gene 1 (ApSI1). When ApDRI15 was knocked down, the plants were more sensitive to drought stress than the control plants, with smaller leaves, shorter internodes, and lower biomass. The ApDRI15-silenced plants had lower relative water content, lower free proline levels, and higher water loss rates than the control. Silencing of ApSI1 significantly decreased tolerance to salinity, and the ApSI1-silenced plants were withered and smaller. These results indicate that the pgR107 VIGS approach is a simple and highly effective tool for dissecting gene function in A. philoxeroides. Further experiments with the VIGS approach will enhance our understanding of the molecular mechanisms of the adaptability and plasticity of A. philoxeroides and improve our ability to combat the damage caused by this weed.

Functional characterization of a novel jasmonate ZIM-domain interactor (NINJA) from upland cotton (Gossypium hirsutum).

The jasmonic acid (JA) signalling pathway plays roles in plant development and defence against biotic and abiotic stresses. We isolated a cotton NINJA (novel interactor of JA ZIM-domain) gene, designated GhNINJA, which contains a 1305 bp open read frame. The GhNINJA gene encodes a 434 amino acid peptide. According to quantitative real-time PCR analysis, GhNINJA is preferentially expressed in roots, and its expression level is greatly induced by Verticillium dahliae infection. Through a virus-induced gene silencing technique, we developed GhNINJA-silenced cotton plants, which had significantly decreased expression of the target gene with an average expression of 6% of the control. The regenerating lateral root growth of silenced plants was largely inhibited compared to the control. Analysis by microscopy demonstrated that the cell length of the root differentiation zone in GhNINJA-silenced plants is significantly shorter than those of the control. Moreover, the silenced plants exhibited higher tolerance to V. dahliae infection compared to the control, which was linked to the increased expression of the defence marker genes PDF1.2 and PR4. Together, these data indicated that knockdown of GhNINJA represses the root growth and enhances the tolerance to V. dahliae. Therefore, GhNINJA gene can be used as a candidate gene to breed the new cultivars for improving cotton yield and disease resistance.

[Study on the Distribution of Constitutions of Chinese Medicine in Patients with Osteonecrosis of Femoral Head].

OBJECTIVE: To observe the distribution of constitution types of Chinese medicine (CM) in patients with osteonecrosis of femoral head (ONFH). METHODS: Totally 130 ONFH patients were recruited. Constitution types of CM were identified in all patients. Distribution features of constitution types of CM in ONFH patients were observed. The differences of distribution in gender, age, single or bilateral hips, course of disease, staging, cause, and region were also analyzed. RESULTS: Seventy patients were of complicated constitutions, while 60 patients were of single constitution. Among the 60 single constitution cases, yang-deficiency constitution [18 (13.9%)], damp-heat constitution [10 (7.7%)], blood-stasis constitution [7 (5.4%)], and qi-deficiency constitution [7 (5.4%)] were mainly distributed. Of the complicated constitutions, yang-deficiency dominated constitution occupied the top ratio [30 (23.1%)], followed by blood-stasis dominated constitution [15 (11.5%)], damp-heat dominated constitution [9 (6.9%)]. By putting them together, yang-deficiency constitution occupied the top constitution of CM [48 (36.9%)], followed by blood-stasis constitution [ 22 (16.9%)] and damp-heat constitution [19 (14.6%)]. The aforesaid three constitutions accounted for 68.5% of the total. There were no statistical distribution differences in gender, age, single or bilateral hips, course of disease, staging, or cause. CONCLUSION: Yang-deficiency constitution, damp-heat constitution, and blood-stasis constitution were liable constitutions of CM in ONFH patients.

[Effects of Human Gut Microbiota on Bioaccessibility of Soil Cd, Cr and Ni Using SHIME Model].

The influence of human gut micobiota on bioaccessibilities of soil Cd, Cr, and Ni were investigated in this study. Five soil samples were collected from some sites of China, and the bioaccessibilities of soil Cd, Cr, and Ni in the gastric, small intestinal, and colon phases were determined using the PBET method (physiologically based extraction test) combined with SHIME model (simulator of human intestinal microbial ecosystem). The results showed that the bioaccessibilities of Cd, Cr, and Ni in the gastric phase were 4.3%-94.0%, 6.4%-21.6%, and 11.3%-47.3%, respectively. In the small intestinal phase, the bioaccessibilities of Cr and Ni were either congruent or slightly increased, while for Cd, the values were reduced by 1.4-1.6 folds except for soil 2. In the gastric and small intestinal phases, the mean bioaccessibility of Cd was higher but that of Cr was lower. In the colon phase, the bioaccessibilities of Cr and Ni were 1.3-2.4 and 1.0-2.1 times higher than those in the small intestinal phase. Furthermore, the bioaccessibility of Cd also increased except for soil 3 and 4. Human gut micobiota could induce Cd, Cr, and Ni release from soils and increase their bioaccessibilities, which may result in high risk to human health.

Global Transcriptome Analysis Reveals Acclimation-Primed Processes Involved in the Acquisition of Desiccation Tolerance in Boea hygrometrica.

Boea hygrometrica resurrection plants require a period of acclimation by slow soil-drying in order to survive a subsequent period of rapid desiccation. The molecular basis of this observation was investigated by comparing gene expression profiles under different degrees of water deprivation. Transcripts were clustered according to the expression profiles in plants that were air-dried (rapid desiccation), soil-dried (gradual desiccation), rehydrated (acclimated) and air-dried after acclimation. Although phenotypically indistinguishable, it was shown by principal component analysis that the gene expression profiles in rehydrated, acclimated plants resemble those of desiccated plants more closely than those of hydrated acclimated plants. Enrichment analysis based on gene ontology was performed to deconvolute the processes that accompanied desiccation tolerance. Transcripts associated with autophagy and α-tocopherol accumulation were found to be activated in both air-dried, acclimated plants and soil-dried non-acclimated plants. Furthermore, transcripts associated with biosynthesis of ascorbic acid, cell wall catabolism, chaperone-assisted protein folding, respiration and macromolecule catabolism were activated and maintained during soil-drying and rehydration. Based on these findings, we hypothesize that activation of these processes leads to the establishment of an optimal physiological and cellular state that enables tolerance during rapid air-drying. Our study provides a novel insight into the transcriptional regulation of critical priming responses to enable survival following rapid dehydration in B. hygrometrica.

[Clinical observation of jianpi bushen formula to prevent collapse of osteonecrosis of femoral head].

To observe the clinical effect of Jianpi Bushen formula to prevent the collapse of osteonecrosis of femoral head (ONFH) of type phlegm and blood stasis obstructing the collaterals. 50 cases (including 73 hips) of non-collapse ONFH (ARCO I, II, III a) were selected from the out-patient department of orthopedic in Guang'anmen Hospital attached to China Academy of Chinese Medical Science. All the cases fit for diagnostic criteria were given Jianpi Bushen formula and followed up. Staging criteria was ARCO classification. Harris score evaluated the hip function. The mean follow-up time was 4.2 years (3-5.4 years). After a mean of 4.2 years (3-5.4 years) followed-up, 12 hips collapse occurred while 61 hips not (the non-collapse rate was 83.56%). Collapse did not occurred among the all 7 hips at stage ARCO I (the non-collapse rate was 100%). Among the 49 hips at stage ARCO II, 9 hips collapse occurred while 40 hips not (the non-collapse rate was 81.63%). Among the 17 hips at stage ARCO III a, 3 hips collapse occurred while 14 hips not (the non-collapse rate was 82.35%). Kaplan-Meier analysis showed the average survival time of non-collapse was 5 years (4. 8-5.2 years). The 3 year survival rate of non-collapse was 92.5%. The 4 year survival rate was 74% and the condition tended to be stability trend. The Harris score was 71.93 +/- 11.25 before treatment and 81.63 +/- 12.16 after treatment, significantly different. These results suggest that: Jianpi Bushen formula is an effective method for treating ONFH of type phlegm and blood stasis obstructing the collaterals. It can delay or prevent the collapse of femoral head and significantly improve the hip function.