Genome-wide characterization and expression analysis of α-amylase and ß-amylase genes underlying drought tolerance in cassava.

BACKGROUND: Starch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to both biological and abiotic stresses. The α-amylase (AMY) and the ß-amylase (BAM) are important enzymes that catalyze the hydrolysis of plant starch. Cassava (Manihot esculenta Crantz) is treated as one of the most drought-tolerant crops. However, the mechanisms of how AMY and BAM respond to drought in cassava are still unknown. RESULTS: Six MeAMY genes and ten MeBAM genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. Tandem and fragment replications play important roles in the amplification of MeAMY and MeBAM genes. Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at the N-terminus, which may have transcription factor functions. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are proven as critical genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity were significantly altered in cassava under different levels of drought stress. CONCLUSIONS: These results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

The role of iTr35 cells in the inflammatory response and fibrosis progression of systemic sclerosis.

OBJECTIVE: To evaluate the role of induced immunosuppressive T regulatory (iTr) 35 cells in SSc-related inflammation and fibrosis. METHODS: Sixty-eight SSc patients were enrolled in this study. Subsets of iTr35 and Tr1 were measured by flow cytometry. IL-35 and IL-10 levels were measured using ELISA. Expressions of iTr35, Tr1, fibrosis-related genes and proteins associated with signalling pathways were determined using immunofluorescence, western blot and immunohistochemistry assays. RESULTS: In peripheral blood, the proportions of the iTr35 cells were higher and Tr1 cells were lower than the control group. Similarly, IL-35 expression was increased, while IL-10 levels were decreased. In fibroblasts from skin tissue, the expression levels of EBI3, IL-12Ap35, Foxp3 and IL-10 were decreased, but collagen I, TGF-ß, alpha smooth muscle actin (α-SMA) and fibronectin levels were increased. Phosphorylated STAT3/6 were increased, but iTr35 and Tr1 cell levels were significantly decreased. When CD4+ cells were incubated with both recombinant human (rh)IL-35 and rhIL-10, the cell numbers of iTr35 and Tr1 were greater than the same type of cells treated with rhIL-35 or rhIL-10 alone. However, the viability of conventional CD4+ T cells was decreased by gradually increasing iTr35 cells. Moreover, iTr35 cells affected α-SMA expression through the STAT3/6 signalling pathway. CONCLUSION: Both iTr35 and Tr1 cells are involved in SSc-related inflammation and fibrosis. IL-35 can induce iTr35 cells, showing a synergistic effect with IL-10. We also found that iTr35 cells can inhibit T cell proliferation and differentiation via the STAT3/6 signalling pathway, thereby causing fibrosis.

Altered CD4+ T cell and cytokine levels in peripheral blood and skin samples from systemic sclerosis patients and IL-35 in CD4+ T cell growth.

OBJECTIVE: This study explored the role of IL-35 in CD4+ T lymphocyte and human skin fibroblast (HSF) activity and cytokine levels in systemic sclerosis. METHODS: Blood and skin biopsies were collected from 41 patients and 39 healthy controls to assess CD4+ T lymphocytes and IL-35-related factors. CD4+ T lymphocytes were co-cultured with HSFs, recombinant human IL-35 and IL-35 mAb to evaluate the cell viability, activation of CD4+ T lymphocytes and HSF cells. RESULTS: The proportion of blood Th1/Th2 was lower and Th17/Treg was higher in patients than in controls (P < 0.05). IL-35 and IL-17A levels were higher and IFN-γ, IL-10 and TGF-ß levels were lower in patients than in controls. IL-17A, forkhead box P3, TGF-ß1 and collagen type I (COL-1) mRNA and phospho (p)-signal transducer and activator of transcription (STAT) 1 and p-STAT4 were higher in skin tissues from patients than in those from controls (P < 0.05). IL-6 levels were higher, whereas IL-10 levels were lower in cell culture supernatants. α-Smooth muscle actin (α-SMA) and COL-1 proteins and Ki67 positivity were higher in CD4+ T + HSF cells from patients than in those from controls. Recombinant human IL-35 treatment inhibited proliferation (P < 0.001), but increased IL-10 and decreased IL-17A, α-SMA and COL-1 secretion into the conditioned medium of CD4+ T lymphocytes + HSFs from patients compared with those from controls. IL-35 mAb blocked the effects of IL-35 in CD4+ T + HSF cells (P < 0.05). CONCLUSIONS: IL-35 plays an inhibitory role in CD4+ T lymphocyte proliferation but induces Treg cell differentiation by STAT1 signalling activation, HSF proliferation and collagen expression in systemic sclerosis.

Manure application maintained the CO2 fixation activity of soil autotrophic bacteria but changed its ecological characteristics in an entisol of China.

The response of soil autotrophs to anthropogenic activities has attracted increasing attention against the background of global change. Here, three entisol plots under different fertilizing regimes, including no fertilization (CK), manure (M), and a combined application of chemical fertilizer and manure (NPKM) were selected, and then the soil RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity and cbbl (gene encoding the large subunit of RubisCO) composition were measured to indicate the activity and community of autotrophic bacteria, respectively. The results revealed that the RubisCO activity of CK showed no difference from that of M but was significantly higher than that of NPKM. The CK and M had the lowest and highest soil cbbl abundance, respectively. The α-diversity of soil cbbl-carrying bacteria showed no significant difference among these treatments, whereas they showed significantly different community structures of cbbl-carrying bacteria. Meanwhile, compared with CK, M had significantly lower abundances of bacterial species with the functions of nitrogen fixation (Azoarcus sp.KH32C) or detoxification (Methylibium petroleiphilum), indicating that manure application might have an inhibiting potential to some beneficial autotrophic bacterial species in this entisol.

Efficacy and safety of different Janus kinase inhibitors combined with methotrexate for the treatment of rheumatoid arthritis: a single-center randomized trial.

OBJECTIVE: To compare the efficacy and safety between baricitinib (BARI) and tofacitinib (TOFA) for the treatment of the rheumatoid arthritis (RA) patients receiving methotrexate (MTX) in clinical practice. METHODS: This retrospective study recruited 179 RA patients treated with BARI (2-4 mg/d) or TOFA (10 mg/d) at The First Affiliated Hospital of Guangxi Medical University from September 2019 to January 2022. The rate of low disease activity (LDA) was used as the primary end point. Secondary end points included the Disease Activity Scale-28 (DAS-28)-C-reactive protein (CRP); the rate of DAS28-CRP remission; visual analogue scale (VAS) for pain, swollen joint, and tender joint counts; and adverse events at the 6-month follow-up. Several factors affecting LDA achievement were also analyzed. RESULTS: Seventy-four patients were treated with BARI and 105 were treated with TOFA, including 83.24% females, with a median (IQR) age of 56.0 (53.0-56.0) years old and disease duration of 12.0 (6.0-12.0) months. There was no difference of the rate of LDA between the BARI and TOFA treatment groups. All disease indices in the two groups were significantly improved, including a significantly lower VAS in the BARI group (P < 0.05), reflecting the drug efficacy after 1 and 6 months of treatment. The incidence of adverse reactions was similar in these two groups. CONCLUSION: The treatment efficacy and safety of BARI and TOFA in the RA patients were similar, but BARI was more effective in pain relief than TOFA. An older baseline age was more likely to achieve LDA in the BARI group, while a low baseline erythrocyte sedimentation rate (ESR) was more likely to achieve LDA in the TOFA group.

Therapeutic effects of thalidomide on patients with systemic sclerosis-associated interstitial lung disease.

Objective: To evaluate the clinical efficacy of thalidomide in patients with systemic sclerosis-associated interstitial lung disease. Methods: Ninety-six systemic sclerosis-associated interstitial lung disease patients who received basic glucocorticoid treatment and admitted between 2016 and 2020 were included in this study, including 48 cases in the thalidomide group (combination of thalidomide and cyclophosphamide) and 48 cases in control group (cyclophosphamide monotherapy). Evaluation items included clinical symptoms, modified Rodnan skin score, pulmonary function test, chest high-resolution computed tomography scores, and adverse effects between two groups after 24 weeks of treatment. Results: Remarkable improvements in several aspects were found in the thalidomide group, including modified Rodnan skin score, expiratory dyspnea score, cough visual analog scale score, total ground-glass opacity score, and total interstitial lung disease score. Compared to the control group, improvements in the thalidomide group were found, such as significantly decreased cough visual analog scale score and expectoration; increased number of platelets; improved pulmonary fibrosis (p = 0.056), and reduced carbon monoxide diffusing capacity (p = 0.053). There were no statistically significant differences in the expiratory dyspnea score and predicted forced vital capacity between the two groups. Patients who experienced at least one adverse event in the control group and thalidomide group were 33.3% and 64.6% (p = 0.002); while those with serious adverse events were 8.3% versus 12.5% (p = 0.504). Venous thrombosis was found in one case in the thalidomide group. Conclusion: Thalidomide combined with cyclophosphamide can improve the symptoms of cough and expectoration in patients with systemic sclerosis-associated interstitial lung disease, and may slightly delay the progression of pulmonary fibrosis, but with the possibility of an increased risk of adverse events.

Long-term fertilization coupled with rhizobium inoculation promotes soybean yield and alters soil bacterial community composition.

Microbial diversity is an important indicator of soil fertility and plays an indispensable role in farmland ecosystem sustainability. The short-term effects of fertilization and rhizobium inoculation on soil microbial diversity and community structure have been explored extensively; however, few studies have evaluated their long-term effects. Here, we applied quantitative polymerase chain reaction (qPCR) and amplicon sequencing to characterize the effect of 10-year fertilizer and rhizobium inoculation on bacterial communities in soybean bulk and rhizosphere soils at the flowering-podding and maturity stages. Four treatments were examined: non-fertilization control (CK), phosphorus and potassium fertilization (PK), nitrogen and PK fertilization (PK + N), and PK fertilization and Bradyrhizobium japonicum 5821 (PK + R). Long-term co-application of rhizobium and PK promoted soybean nodule dry weight by 33.94% compared with PK + N, and increased soybean yield by average of 32.25%, 5.90%, and 5.00% compared with CK, PK, and PK + N, respectively. The pH of PK + R was significantly higher than that of PK and PK + N at the flowering-podding stage. The bacterial abundance at the flowering-podding stage was positively correlated with soybean yield, but not at the maturity stage. The significant different class Gemmatimonadetes, and the genera Gemmatimonas, and Ellin6067 in soil at the flowering-podding stage were negatively correlated with soybean yield. However, the bacterial community at class and genus levels at maturity had no significant effect on soybean yield. The key bacterial communities that determine soybean yield were concentrated in the flowering-podding stage, not at maturity stage. Rhizosphere effect, growth period, and treatment synergies resulted in significant differences in soil bacterial community composition. Soil organic matter (OM), total nitrogen (TN), pH, and available phosphorus (AP) were the main variables affecting bacterial community structure. Overall, long-term co-application of rhizobium and fertilizer not only increased soybean yield, but also altered soil bacterial community structure through niche reconstruction and microbial interaction. Rhizobium inoculation plays key role in reducing nitrogen fertilizer application and promoting sustainable agriculture practices.

GmBTB/POZ promotes the ubiquitination and degradation of LHP1 to regulate the response of soybean to Phytophthora sojae.

Phytophthora sojae is a pathogen that causes stem and root rot in soybean (Glycine max [L.] Merr.). We previously demonstrated that GmBTB/POZ, a BTB/POZ domain-containing nuclear protein, enhances resistance to P. sojae in soybean, via a process that depends on salicylic acid (SA). Here, we demonstrate that GmBTB/POZ associates directly with soybean LIKE HETEROCHROMATIN PROTEIN1 (GmLHP1) in vitro and in vivo and promotes its ubiquitination and degradation. Both overexpression and RNA interference analysis of transgenic lines demonstrate that GmLHP1 negatively regulates the response of soybean to P. sojae by reducing SA levels and repressing GmPR1 expression. The WRKY transcription factor gene, GmWRKY40, a SA-induced gene in the SA signaling pathway, is targeted by GmLHP1, which represses its expression via at least two mechanisms (directly binding to its promoter and impairing SA accumulation). Furthermore, the nuclear localization of GmLHP1 is required for the GmLHP1-mediated negative regulation of immunity, SA levels and the suppression of GmWRKY40 expression. Finally, GmBTB/POZ releases GmLHP1-regulated GmWRKY40 suppression and increases resistance to P. sojae in GmLHP1-OE hairy roots. These findings uncover a regulatory mechanism by which GmBTB/POZ-GmLHP1 modulates resistance to P. sojae in soybean, likely by regulating the expression of downstream target gene GmWRKY40.

The 26S Proteasome Regulatory Subunit GmPSMD Promotes Resistance to Phytophthora sojae in Soybean.

Phytophthora root rot, caused by Phytophthora sojae is a destructive disease of soybean (Glycine max) worldwide. We previously confirmed that the bHLH transcription factor GmPIB1 (P. sojae-inducible bHLH transcription factor) reduces accumulation of reactive oxygen species (ROS) in cells by inhibiting expression of the peroxidase-related gene GmSPOD thus improving the resistance of hairy roots to P. sojae. To identify proteins interacting with GmPIB1 and assess their participation in the defense response to P. sojae, we obtained transgenic soybean hairy roots overexpressing GmPIB1 by Agrobacterium rhizogenes mediated transformation and examined GmPIB1 protein-protein interactions using immunoprecipitation combined with mass spectrometry. We identified 392 proteins likely interacting with GmPIB1 and selected 20 candidate genes, and only 26S proteasome regulatory subunit GmPSMD (Genbank accession no. XP_014631720) interacted with GmPIB1 in luciferase complementation and pull-down experiments and yeast two-hybrid assays. Overexpression of GmPSMD (GmPSMD-OE) in soybean hairy roots remarkably improved resistance to P. sojae and RNA interference of GmPSMD (GmPSMD -RNAi) increased susceptibility. In addition, accumulation of total ROS and hydrogen peroxide (H2O2) in GmPSMD-OE transgenic soybean hairy roots were remarkably lower than those of the control after P. sojae infection. Moreover, in GmPSMD-RNAi transgenic soybean hairy roots, H2O2 and the accumulation of total ROS exceeded those of the control. There was no obvious difference in superoxide anion (O2 -) content between control and transgenic hairy roots. Antioxidant enzymes include peroxidase (POD), glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) are responsible for ROS scavenging in soybean. The activities of these antioxidant enzymes were remarkably higher in GmPSMD-OE transgenic soybean hairy roots than those in control, but were reduced in GmPSMD-RNAi transgenic soybean hairy roots. Moreover, the activity of 26S proteasome in GmPSMD-OE and GmPIB1-OE transgenic soybean hairy roots was significantly higher than that in control and was significantly lower in PSMD-RNAi soybean hairy roots after P. sojae infection. These data suggest that GmPSMD might reduce the production of ROS by improving the activity of antioxidant enzymes such as POD, SOD, GPX, CAT, and GmPSMD plays a significant role in the response of soybean to P. sojae. Our study reveals a valuable mechanism for regulation of the pathogen response by the 26S proteasome in soybean.

Analysis of leaf morphology, secondary metabolites and proteins related to the resistance to Tetranychus cinnabarinus in cassava (Manihot esculenta Crantz).

Constitutive resistance of plant can be divided into physical and chemical barriers. Cassava (Manihot esculenta Crantz) is susceptible to mites, especially Tetranychus cinnabarinus. Although significant differences in the resistance to T. cinnabarinus are observed in different cassava cultivars, limited research has been done on the mechanism accounting for the resistance. The aim of this study was to explore the mechanism of resistance to T. cinnabarinus by comparing morphology, secondary metabolites and proteins in different cassava cultivars. The anatomical structure of leaves showed that the cassava cultivar Xinxuan 048 (XX048), which showed a stronger resistance to T. cinnabarinus in both greenhouse testing and three years field evaluation tests (2016-2018), had thicker palisade tissue, spongy tissue, lower epidermis and leaf midrib tissue compared to cultivar Guire 4 (GR4). Greenhouse evaluation demonstrated that originally these cultivars were different, leading to differences in constitutive levels of metabolites. The proteomic analysis of protected leaves in XX048 and GR4 revealed that up-regulated differentially expressed proteins (DEPs) were highly enriched in secondary metabolic pathways, especially in the biosynthesis of flavonoids. This study not only provides a comprehensive data set for overall proteomic changes of leaves in resistant and susceptible cassava, but also sheds light on the morphological characteristics of cassava-mite interaction, secondary metabolite defense responses, and molecular breeding of mite-resistant cassava for effective pest control.

Efficacy and safety of different Janus kinase inhibitors combined with methotrexate for the treatment of rheumatoid arthritis: a single-center randomized trial

Abstract Objective To compare the efficacy and safety between baricitinib (BARI) and tofacitinib (TOFA) for the treatment of the rheumatoid arthritis (RA) patients receiving methotrexate (MTX) in clinical practice. Methods This retrospective study recruited 179 RA patients treated with BARI (2-4 mg/d) or TOFA (10 mg/d) at The First Affiliated Hospital of Guangxi Medical University from September 2019 to January 2022. The rate of low disease activity (LDA) was used as the primary end point. Secondary end points included the Disease Activity Scale-28 (DAS-28)-C-reactive protein (CRP); the rate of DAS28-CRP remission; visual analogue scale (VAS) for pain, swollen joint, and tender joint counts; and adverse events at the 6-month follow-up. Several factors affecting LDA achievement were also analyzed. Results Seventy-four patients were treated with BARI and 105 were treated with TOFA, including 83.24% females, with a median (IQR) age of 56.0 (53.0-56.0) years old and disease duration of 12.0 (6.0-12.0) months. There was no difference of the rate of LDA between the BARI and TOFA treatment groups. All disease indices in the two groups were significantly improved, including a significantly lower VAS in the BARI group (P < 0.05), reflecting the drug efficacy after 1 and 6 months of treatment. The incidence of adverse reactions was similar in these two groups. Conclusion The treatment efficacy and safety of BARI and TOFA in the RA patients were similar, but BARI was more effective in pain relief than TOFA. An older baseline age was more likely to achieve LDA in the BARI group, while a low baseline erythrocyte sedimentation rate (ESR) was more likely to achieve LDA in the TOFA group.