Development and evaluation of immunological effects of a DNA vaccine encoding phosphoketolase family protein against Nocardia seriolae in hybrid snakehead.

Fish nocardiosis is a chronic disease mainly caused by Nocardia seriolae, which occurs in a variety of economically cultured freshwater and marine fish. Studies have shown that DNA vaccine is an effective treatment to protect fish from bacterial infection. In our previous experiment, an in vivo-induced gene of N. seriolae, encoding phosphoketolase (PK) family protein, was identified by in vivo-induced antigen technology. In the present study, the antigenic gene encoding PK family protein was analyzed by bioinformatics and further inserted into the eukaryotic expression vector pcDNA3.1-myc-his-A for DNA vaccine development. The immunological effects of pcDNA-PK DNA vaccine were assessed in hybrid snakehead (Channa maculata ♀ × Channa argus ♂), showing induction in several serum enzyme activity parameters (including LZM, SOD, ACP and AKP), increasing in specific-antibody IgM levels, as well as up-regulation in six immune-related genes (CD4, CD8α, TNFα, IL-1ß, MHCIα and MHCIIα). Moreover, an immune-protection with a relative survival rate was provided at 53.82 % following artificial challenge with N. seriolae in vaccinated fish in comparison to the control group. In summary, these results indicate that pcDNA-PK DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, which may be applied in aquaculture to control fish nocardiosis.

Magnetic Resonance Imaging Radiomics Predicts Histological Response to Neoadjuvant Chemotherapy in Localized High-grade Osteosarcoma of the Extremities.

RATIONALE AND OBJECTIVES: Research involving radiomics models based on magnetic resonance imaging (MRI) has mainly used radiomics features derived from a single MRI sequence at a single time point to develop predictive models. This study aimed to construct radiomics models based on before and after neoadjuvant chemotherapy (NAC) MRI for predicting the histological response to NAC in patients with high-grade osteosarcoma. MATERIALS AND METHODS: We included 109 patients with localized high-grade osteosarcomas of the extremities, who underwent pre- and post-NAC MRI examinations, from which radiomics features were extracted. According to the tumor necrosis rate, all patients were classified as good responders (GRs) or poor responders (PRs) and were randomly allocated into training and test sets at a 7:3 ratio. Radiomics features were extracted from T2-weighted (T2WI) and contrast-enhanced T1-weighted imaging (T1CE) of the two MRI scans to construct three models: pre-NAC, post-NAC, and combined pre-NAC and post-NAC (combined model). RESULTS: In total, 1175 radiomics features were extracted from each sequence. Following feature selection, nine radiomics features were selected for each model to construct radiomics signatures. The radiomics signatures of the pre-NAC, post-NAC, and combined models demonstrated good predictive performance for chemotherapy response in osteosarcoma. The combined model achieved the highest areas under the receiver operating curve (AUC) values of 0.999 and 0.915 in the training and test sets, respectively. The AUCs of the post-NAC model were higher than those of the pre-NAC model. CONCLUSION: MRI-based radiomics models demonstrate excellent performance in predicting the histological response to NAC in patients with high-grade osteosarcoma.

An improved sampled-data control for a nonlinear dynamic positioning ship with Takagi-Sugeno fuzzy model.

This article considered the sampled-data control issue for a dynamic positioning ship (DPS) with the Takagi-Sugeno (T-S) fuzzy model. By introducing new useful terms such as second-order term of time, an improved Lyapunov-Krasovskii function (LKF) was constructed. Additionally, the reciprocally convex method is introduced to bound the derivative of LKF. According to the constructed LKF, the sampling information during the whole sampling period was fully utilized, and less conservatism was obtained. Then, the stability condition, robust performance, mode uncertainty and sampled-data controller design were analyzed by means of the linear matrix inequality (LMI). Finally, an example was given to demonstrate the effectiveness of the proposed method.

Comparative Bleeding Risk of Brand Vs Generic Rivaroxaban in Elderly Inpatients with Atrial Fibrillation.

Objective: Atrial fibrillation (AF) is the most common abnormal heart rhythm in elderly patients. Rivaroxaban has been widely used for stroke prevention. The anticoagulant response to rivaroxaban increases with age, which may make elderly patients susceptible to adverse outcomes resulting from small differences in bioavailability between generic and brand products. Methods: We designed a cohort study of ≥65-year-old inpatients with AF. Sociodemographic and laboratory measures of qualified patients who received brand or generic rivaroxaban for at least 72 hours at the study hospital from January 2021 to June 2023 were collected retrospectively. The primary outcome was the incidence of bleeding. Results: A total of 1008 qualifying patients were included for analysis, with 626 (62.1%) receiving brand rivaroxaban and 382 (37.9%) receiving generic rivaroxaban. After propensity score matching and weighting to account for confounders, the odds ratios comparing brand vs generic rivaroxaban (95% confidence intervals) for the bleeding was 1.15 (0.72-1.82). Results from subgroup analyses of patients with age ≥85, HAS-BLED score ≥ 3, containment of antiplatelet drugs, and female patients were consistent with the primary analysis. Conclusion: It provides evidence regarding the clinical safety outcome of generic rivaroxaban in the elderly AF population that may be particularly susceptible to adverse outcomes resulting from small allowable differences in pharmacokinetics.

Development and application of whole-chromosome painting of chromosomes 7A and 8A of Arachis duranensis based on chromosome-specific single-copy oligonucleotides.

For peanut, the lack of stable cytological markers is a barrier to tracking specific chromosomes, elucidating the genetic relationships between genomes and identifying chromosomal variations. Chromosome mapping using single-copy oligonucleotide (oligo) probe libraries has unique advantages for identifying homologous chromosomes and chromosomal rearrangements. In this study, we developed two whole-chromosome single-copy oligo probe libraries, LS-7A and LS-8A, based on the reference genome sequences of chromosomes 7A and 8A of Arachis duranensis. Fluorescence in situ hybridization (FISH) analysis confirmed that the libraries could specifically paint chromosomes 7 and 8. In addition, sequential FISH and electronic localization of LS-7A and LS-8A in A. duranensis (AA) and A. ipaensis (BB) showed that chromosomes 7A and 8A contained translocations and inversions relative to chromosomes 7B and 8B. Analysis of the chromosomes of wild Arachis species using LS-8A confirmed that this library could accurately and effectively identify A genome species. Finally, LS-7A and LS-8A were used to paint the chromosomes of interspecific hybrids and their progenies, which verified the authenticity of the interspecific hybrids and identified a disomic addition line. This study provides a model for developing specific oligo probes to identify the structural variations of other chromosomes in Arachis and demonstrates the practical utility of LS-7A and LS-8A.

Identifying the in vivo-induced antigenic genes is a strategy to develop DNA vaccine against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂).

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4、CD8α、MHCIIα、TNFα、IL-1ß and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis.

Dosimetric Comparison of Commonly Used Volumetric Modulated Arc Therapy Field Arrangements Based on Flattening Filter-Free Beams for Synchronous Bilateral Breast Carcinoma Radiation Therapy.

PURPOSE: Flattening filter-free (FFF)-based volumetric modulated arc therapy (VMAT) has been shown to be feasible and significantly improves treatment efficiency and lung protection for synchronous bilateral breast irradiation (SBBI). This research compared the commonly used VMAT field arrangements using FFF beams. METHODS: Twenty-eight patients underwent SBBI were retrospectively enrolled to design irradiation plans using tangential arc VMAT (taVMAT), half arc VMAT (haVMAT), and large arc VMAT (laVMAT). Dosimetric and delivery parameters of all designed plans were recorded and compared. RESULTS: Comparable target volume coverage was observed for all field arrangements. taVMAT significantly reduced the dose to spinal cord and the volume covered by 5 Gy (V5Gy) and V7Gy of the lungs while decreasing the conformity index of the target volume. It also increased the volume covered by 105% of the prescription dose (V105%) and V107% of the target volume. haVMAT considerably decreased V20 Gy and V30 Gy of the lungs, mean dose (Dmean) and V30 Gy of the heart and the liver. It also notably reduced Dmean and V40 Gy of the left anterior descending coronary artery while increasing the beam-on time. laVMAT significantly reduced the mean treatment time (range, 113-117 seconds) compared with the other field arrangements. CONCLUSIONS: There were distinct differences in various dosimetric and delivery parameters for different field arrangements, highlighting the importance of selecting the appropriate field arrangement based on specific treatment goals and considerations. This study contributes valuable insights into the use of FFF-based VMAT techniques in SBBI.

Anticoagulation strategies in patients with extracorporeal membrane oxygenation: A network meta-analysis and systematic review.

OBJECTIVES: Extracorporeal membrane oxygenation (ECMO) plays an important role in providing temporary life support for patients with severe cardiac or pulmonary failure, but requires strict anticoagulation and monitoring. This network meta-analysis systematically explored the most effective anticoagulation and monitoring strategies for patients receiving ECMO. METHODS: MEDLINE, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials were searched up to January 31, 2023, for studies comparing unfractionated heparin (UFH), argatroban (Arg), bivalirudin (Biv), and/or nafamostat mesylate (NM) in patients receiving ECMO. The primary outcomes included device-related thrombosis, patient-related thrombosis, and major bleeding events. The secondary outcomes included ECMO survival, ECMO duration, and in-hospital mortality. RESULTS: A total of 2522 patients from 23 trials were included in the study. Biv was associated with a decreased risk of device-related thrombosis (odd ratio [OR] 0.51, 95% confidence interval [CI]: 0.33-0.84) compared with UFH, whereas NM (OR 2.2, 95% CI: 0.24-65.0) and Arg (OR 0.92, 95% CI: 0.43-2.0) did not reduce the risk of device-related thrombosis compared with UFH. Biv was superior to Arg in decreasing the risk of device-related thrombosis (OR 0.14, 95% CI: 0.03-0.51). Biv reduced the risk of patient-related thrombosis compared with UFH (OR 0.44, 95% CI: 0.18-0.85); NM (OR 0.65, 95% CI: 0.14-3.3) and Arg (OR 3.1, 95% CI: 0.94-12.0) did not decrease risk of patient-related thrombosis compared with UFH. No significant difference was observed in the risk of major bleeding between three alternatives and UFH: Biv (OR 0.54, 95% CI: 0.23-1.3), Arg (OR 1.3, 95% CI: 0.34-5.8), and NM (OR 0.60, 95% CI: 0.13-2.6). NM showed a reduced risk of in-hospital mortality compared with UFH (OR 0.27, 95% CI: 0.091-0.77), whereas Arg (OR 0.43, 95% CI: 0.15-1.2) and Biv (OR 0.75, 95% CI: 0.52-1.1) did not decrease risk of in-hospital mortality. CONCLUSIONS: Compared with UFH and Arg, Biv reduces the risk of thrombosis and appears to be a better choice for patients requiring ECMO. NM was associated with a reduced risk of in-hospital mortality.

Characterization of chromatin accessibility and gene expression reveal the key genes involved in cotton fiber elongation.

Cotton (Gossypium hirsutum L.) is an important economic crop, and cotton fiber is one of the longest plant cells, which provides an ideal model for the study of cell elongation and secondary cell wall synthesis. Cotton fiber length is regulated by a variety of transcription factors (TF) and their target genes; however, the mechanism of fiber elongation mediated by transcriptional regulatory networks is still unclear to a large extent. Here, we used a comparative assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) assay and RNA-seq analysis to identify fiber elongation transcription factors and genes using the short-fiber mutant ligon linless-2 (Li2 ) and wild type (WT). A total of 499 differential target genes were identified and GO analysis shows that differential genes are mainly involved in plant secondary wall synthesis and microtubule-binding processes. Analysis of the genomic regions preferentially accessible (Peak) has identified a number of overrepresented TF-binding motifs, highlighting sets of TFs that are important for cotton fiber development. Using ATAC-seq and RNA-seq data, we have constructed a functional regulatory network of each TF regulatory target gene and also the network pattern of TF regulating differential target genes. Further, to obtain the genes related to fiber length, the differential target genes were combined with FLGWAS data to identify the genes highly related to fiber length. Our work provides new insights into cotton fiber elongation.

Identification of an Arylnaphthalene Lignan Derivative as an Inhibitor against Dengue Virus Serotypes 1 to 4 (DENV-1 to -4) Using a Newly Developed DENV-3 Infectious Clone and Replicon.

Dengue virus (DENV) is the most widespread arbovirus, causing symptoms ranging from dengue fever to severe dengue, including hemorrhagic fever and shock syndrome. Four serotypes of DENV (DENV-1 to -4) can infect humans; however, no anti-DENV drug is available. To facilitate the study of antivirals and viral pathogenesis, here we developed an infectious clone and a subgenomic replicon of DENV-3 strains for anti-DENV drug discovery by screening a synthetic compound library. The viral cDNA was amplified from a serum sample from a DENV-3-infected individual during the 2019 epidemic; however, fragments containing the prM-E-partial NS1 region could not be cloned until a DENV-3 consensus sequence with 19 synonymous substitutions was introduced to reduce putative Escherichia coli promoter activity. Transfection of the resulting cDNA clone, plasmid DV3syn, released an infectious virus titer of 2.2 × 102 focus-forming units (FFU)/mL. Through serial passages, four adaptive mutations (4M) were identified, and addition of 4M generated recombinant DV3syn_4M, which produced viral titers ranging from 1.5 × 104 to 6.7 × 104 FFU/mL and remained genetically stable in transformant bacteria. Additionally, we constructed a DENV-3 subgenomic replicon and screened an arylnaphthalene lignan library, from which C169-P1 was identified as exhibiting inhibitory effects on viral replicon. A time-of-drug addition assay revealed that C169-P1 also impeded the internalization process of cell entry. Furthermore, we demonstrated that C169-P1 inhibited the infectivity of DV3syn_4M, as well as DENV-1, DENV-2, and DENV-4, in a dose-dependent manner. This study provides an infectious clone and a replicon for the study of DENV-3 and a candidate compound for future development against DENV-1 to -4 infections. IMPORTANCE Dengue virus (DENV) is the most prevalent mosquito-transmitted virus, and there is no an anti-dengue drug. Reverse genetic systems representative of different serotype viruses are invaluable tools for the study of viral pathogenesis and antiviral drugs. Here, we developed an efficient infectious clone of a clinical DENV-3 genotype III isolate. We successfully overcame the instability of flavivirus genome-length cDNA in transformant bacteria, an unsolved issue for construction of cDNA clones of flaviviruses, and adapted this clone to efficiently produce infectious viruses following plasmid transfection of cell culture. Moreover, we constructed a DENV-3 subgenomic replicon and screened a compound library. An arylnaphthalene lignan, C169-P1, was identified as an inhibitor of virus replication and cell entry. Finally, we demonstrated that C169-P1 exhibited a broad-spectrum antiviral effect against the infections with DENV-1 to -4. The reverse genetic systems and the compound candidate described here facilitate the study of DENV and related RNA viruses.

Anticoagulant in atrial fibrillation patients with prior intracranial haemorrhage: a meta-analysis.

BACKGROUND: The benefit of resuming anticoagulation in atrial fibrillation (AF) patients with prior intracranial haemorrhage (ICH) and which anticoagulant to choose are controversial. SUMMARY OF REVIEW: PubMed, Embase, Web of Science and the Cochrane Library were searched from their inception until 13 February 2022. Thirteen eligible articles (17 600 participants) were collected, including 11 real-world studies (n=17 296) and 2 randomised controlled trials (RCTs) (n=304). Compared with no anticoagulants, oral anticoagulation (OAC) was not associated with an increased risk of ICH recurrence (HR 0.85 (95% CI 0.57 to 1.25), p=0.41), but with a significantly increased risk of major bleeding (HR 1.66 (95% CI 1.20 to 2.30), p<0.01). Meanwhile, OAC was associated with a reduced risk of ischaemic stroke/systemic thromboembolism (IS/SE) (HR 0.54 (95% CI 0.42 to 0.70), p<0.01) and all-cause death (HR 0.38 (95% CI 0.28 to 0.52), p<0.01) compared with no anticoagulants. Furthermore, compared with warfarin, non-vitamin K antagonist oral anticoagulants (NOACs) were associated with a significant reduction of ICH recurrence (HR 0.64 (95% CI 0.49 to 0.85), p<0.01), while the risk of IS/SE and all-cause mortality were comparable between warfarin and NOACs. CONCLUSIONS: For patients with AF with prior ICH, OAC is associated with a significant reduction in IS/SE and all-cause mortality without increasing ICH recurrence, but may increase major bleeding risk. Compared with warfarin, NOACs had a better safety profile and comparable efficacy. Further larger RCTs are warranted to validate these findings.

A brassinosteroid transcriptional regulatory network participates in regulating fiber elongation in cotton.

Brassinosteroids (BRs) participate in the regulation of plant growth and development through BRI1-EMS-SUPPRESSOR1 (BES1)/BRASSINAZOLE-RESISTANT1 (BZR1) family transcription factors. Cotton (Gossypium hirsutum) fibers are highly elongated single cells, and BRs play a vital role in the regulation of fiber elongation. However, the mode of action on how BR is involved in the regulation of cotton fiber elongation remains unexplored. Here, we generated GhBES1.4 over expression lines and found that overexpression of GhBES1.4 promoted fiber elongation, whereas silencing of GhBES1.4 reduced fiber length. DNA affinity purification and sequencing (DAP-seq) identified 1,531 target genes of GhBES1.4, and five recognition motifs of GhBES1.4 were identified by enrichment analysis. Combined analysis of DAP-seq and RNA-seq data of GhBES1.4-OE/RNAi provided mechanistic insights into GhBES1.4-mediated regulation of cotton fiber development. Further, with the integrated approach of GWAS, RNA-seq, and DAP-seq, we identified seven genes related to fiber elongation that were directly regulated by GhBES1.4. Of them, we showed Cytochrome P450 84A1 (GhCYP84A1) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (GhHMG1) promote cotton fiber elongation. Overall, the present study established the role of GhBES1.4-mediated gene regulation and laid the foundation for further understanding the mechanism of BR participation in regulating fiber development.

GhBES1 mediates brassinosteroid regulation of leaf size by activating expression of GhEXO2 in cotton (Gossypium hirsutum).

KEY MESSAGE: We proposed a working model of BR to promote leaf size through cell expansion. In the BR signaling pathway, GhBES1 affects cotton leaf size by binding to and activating the expression of the E-box element in the GhEXO2 promoter region. Brassinosteroid (BR) is an essential phytohormone that controls plant growth. However, the mechanisms of BR regulation of leaf size remain to be determined. Here, we found that the BR deficient cotton mutant pagoda1 (pag1) had a smaller leaf size than wild-type CRI24. The expression of EXORDIUM (GhEXO2) gene, was significantly downregulated in pag1. Silencing of BRI1-EMS-SUPPRESSOR 1 (GhBES1), inhibited leaf cell expansion and reduced leaf size. Overexpression of GhBES1.4 promoted leaf cell expansion and enlarged leaf size. Expression analysis showed GhEXO2 expression positively correlated with GhBES1 expression. In plants, altered expression of GhEXO2 promoted leaf cell expansion affecting leaf size. Furthermore, GhBES1.4 specifically binds to the E-box elements in the GhEXO2 promoter, inducing its expression. RNA-seq data revealed many down-regulated genes related to cell expansion in GhEXO2 silenced plants. In summary, we discovered a novel mechanism of BR regulation of leaf size through GhBES1 directly activating the expression of GhEXO2.

Potential Roles of 1-Aminocyclopropane-1-carboxylic Acid Synthase Genes in the Response of Gossypium Species to Abiotic Stress by Genome-Wide Identification and Expression Analysis.

Ethylene plays a pivotal role in plant stress resistance and 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is the rate-limiting enzyme in ethylene biosynthesis. Upland cotton (Gossypium hirsutum L.) is the most important natural fiber crop, but the function of ACS in response to abiotic stress has rarely been reported in this plant. We identified 18 GaACS, 18 GrACS, and 35 GhACS genes in Gossypiumarboreum, Gossypium raimondii and Gossypiumhirsutum, respectively, that were classified as types I, II, III, or IV. Collinearity analysis showed that the GhACS genes were expanded from diploid cotton by the whole-genome-duplication. Multiple alignments showed that the C-terminals of the GhACS proteins were conserved, whereas the N-terminals of GhACS10 and GhACS12 were different from the N-terminals of AtACS10 and AtACS12, probably diverging during evolution. Most type II ACS genes were hardly expressed, whereas GhACS10/GhACS12 were expressed in many tissues and in response to abiotic stress; for example, they were highly and hardly expressed at the early stages of cold and heat exposure, respectively. The GhACS genes showed different expression profiles in response to cold, heat, drought, and salt stress by quantitative PCR analysis, which indicate the potential roles of them when encountering the various adverse conditions, and provide insights into GhACS functions in cotton's adaptation to abiotic stress.

Ultra-Broadband Tunable Terahertz Metamaterial Absorber Based on Double-Layer Vanadium Dioxide Square Ring Arrays.

Based on the phase transition of vanadium dioxide(VO2), an ultra-broadband tunable terahertz metamaterial absorber is proposed. The absorber consists of bilayer VO2 square ring arrays with different sizes, which are completely wrapped in Topas and placed on gold substrate. The simulation results show that the absorption greater than 90% has frequencies ranging from 1.63 THz to 12.39 THz, which provides an absorption frequency bandwidth of 10.76 THz, and a relative bandwidth of 153.5%. By changing the electrical conductivity of VO2, the absorption intensity can be dynamically adjusted between 4.4% and 99.9%. The physical mechanism of complete absorption is elucidated by the impedance matching theory and field distribution. The proposed absorber has demonstrated its properties of polarization insensitivity and wide-angle absorption, and therefore has a variety of application prospects in the terahertz range, such as stealth, modulation, and sensing.

High-mobility group box chromosomal protein-1 deletion alleviates osteoporosis in OVX rat model via suppressing the osteoclastogenesis and inflammation.

BACKGROUND: Osteoporosis is a skeletal metabolic disease that constitutes a great threaten to human health. However, there is currently no gold standard for its treatment. High-mobility group box chromosomal protein-1 (HMGB-1) has been reported to play an important role in various orthopedic diseases. Till now, its role in osteoporosis remains elusive. METHODS: Rats underwent ovariectomy (OVX) were used to construct a postmenopausal model of osteoporosis. Then, rats were divided into sham groups without OVX surgery, OVX model group, HMGB-1 knockdown (HMGB-1 KD) OVX model groups. The expression of HMGB1 was evaluated by qRT-PCR and western blotting. Subsequently, the changes of trabeculae were evaluated by micro-computed tomography (CT) assay. Skeletal necrosis and metabolism were further analyzed by hematoxylin-eosin (HE) staining, Alcian blue staining and Masson's trichrome staining. The contents of serum alkaline phosphatase (ALP) and osteocalcin were detected by ELISA assay. Expression of osteoclast-associated receptor (OSCAR) and tartrate-resistant acid phosphatase (TRAP) were determined to investigate the effects of HMGB-1 loss on osteoclastogenesis. RESULTS: Single HMGB-1 deletion exerted no significant effect on rat trabeculae, serum ALP and osteocalcin. Noticeably, HMGB1 knockdown dramatically ameliorated OVX-induced changes in above indexes. Trabeculae structures of OVX rats were sparse with disorder arrangement, which were greatly recovered after HMGB-1 deletion. Enhanced osteoclastogenesis was observed in OVX rats by increasing number of TRAP + cells and expression of TRAP and OSCAR, and loss of HMGB1 ameliorated osteoclastogenesis in OVA rats. Moreover, HMGB-1 deletion antagonized OVX-evoked downregulation of osteoblast activity markers osterix (OSX), collagen type I alpha 1(COL1A1) and distal-less homeobox 2 (DLX2) protein. Furthermore, loss of HMGB-1 attenuated fluctuation of inflammatory factors in OVX rats. Additionally, HMGB-1 deficiency inhibited OVX-evoked activation of the Toll-like receptor (TLR) 4/NF-κB signaling pathway. Moreover, reactivating the TLR4 signaling further aggravated OVX-induced osteoporosis, which was reversed by HMGB1 knockdown. CONCLUSION: HMGB-1 deletion alleviated OVX-triggered osteoporosis by suppressing osteoclastogenesis and inflammatory disorder via the inhibition of the TLR4 signaling. Therefore, HMGB-1 may be a promising therapeutic target for osteoporosis.

Systematic Characterization of TCP Gene Family in Four Cotton Species Revealed That GhTCP62 Regulates Branching in Arabidopsis.

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play an essential role in regulating various physiological and biochemical functions during plant growth. However, the function of TCP transcription factors in G. hirsutum has not yet been studied. In this study, we performed genome-wide identification and correlation analysis of the TCP transcription factor family in G. hirsutum. We identified 72 non-redundant GhTCP genes and divided them into seven subfamilies, based on phylogenetic analysis. Most GhTCP genes in the same subfamily displayed similar exon and intron structures and featured highly conserved motif structures in their subfamily. Additionally, the pattern of chromosomal distribution demonstrated that GhTCP genes were unevenly distributed on 24 out of 26 chromosomes, and that fragment replication was the main replication event of GhTCP genes. In TB1 sub-family genes, GhTCP62 was highly expressed in the axillary buds, suggesting that GhTCP62 significantly affected cotton branching. Additionally, subcellular localization results indicated that GhTCP62 is located in the nucleus and possesses typical transcription factor characteristics. The overexpression of GhTCP62 in Arabidopsis resulted in fewer rosette-leaf branches and cauline-leaf branches. Furthermore, the increased expression of HB21 and HB40 genes in Arabidopsis plants overexpressing GhTCP62 suggests that GhTCP62 may regulate branching by positively regulating HB21 and HB40.

Genome-wide analysis of ZAT gene family revealed GhZAT6 regulates salt stress tolerance in G. hirsutum.

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C2H2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C2H2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression.

Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.).

Brassinosteroids (BRs), an efficient plant endogenous hormone, significantly promotes plant nutrient growth adapting to biological and abiotic adversities. BRs mainly promote plant cell elongation by regulating gene expression patterns. EXORDIUM (EXO) genes have been characterized as the indicators of BR response genes. Cotton, an ancient crop, is of great economic value and its fibers can be made into all kinds of fabrics. However, EXO gene family genes have not been full identified in cotton. 175 EXO genes were identified in nine plant species, of which 39 GhEXO genes in Gossypium hirsutum in our study. A phylogenetic analysis grouped all of the proteins encoded by the EXO genes into five major clades. Sequence identification of conserved amino acid residues among monocotyledonous and dicotyledonous species showed a high level of conservation across the N and C terminal regions. Only 25% the GhEXO genes contain introns besides conserved gene structure and protein motifs distribution. The 39 GhEXO genes were unevenly distributed on the 18 At and Dt sub-genome chromosomes. Most of the GhEXO genes were derived from gene duplication events, while only three genes showed evidence of tandem duplication. Homologous locus relationships showed that 15 GhEXO genes are located on collinear blocks and that all orthologous/paralogous gene pairs had Ka > Ks values, indicating purifying selection pressure. The GhEXO genes showed ubiquitous expression in all eight tested cotton tissues and following exposure to three phytohormones, IAA, GA, and BL. Furthermore, GhEXO7_At was mainly expressed in response to BL treatment, and was predominantly expressed in the fibers. GhEXO7_At was found to be a plasma membrane protein, and its ectopic expression in Arabidopsis mediated BR-regulated plant growth and development with altered expression of DWF4, CPD, KCS1, and EXP5. Additionally, the functions of GhEXO7_At were confirmed by virus-induced gene silencing (VIGS) in cotton. This study will provide important genetic resources for future cotton breeding programs.

Identification of BR biosynthesis genes in cotton reveals that GhCPD-3 restores BR biosynthesis and mediates plant growth and development.

MAIN CONCLUSION: Brassinosteroid (BR) synthesis genes in different cotton species was comprehensively identified, and the participation of GhCPD-3 in the BR synthesis signaling pathway for regulating plant development was verified. Brassinosteroid is a natural steroidal phytohormone that plays fundamental roles in plant growth and development. In cotton, detailed characterization and functional validation of BR biosynthesis genes remain rare. Here, 16, 8 and 9 BR biosynthesis genes were identified in Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively, and their phylogenetic relationships, gene structures, conserved motifs of the encoded proteins, chromosomal locations were determined and a synteny analysis was performed. Gossypium hirsutum and Arabidopsis BR biosynthesis genes closely clustered in the phylogenetic tree and fragment duplication was likely the primary cause promoting gene family expansion in G. hirsutum. Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed their relevance as BR biosynthesis genes. GhCPD-3 was highly expressed in roots and stems and the loci of single nucleotide polymorphisms (SNPs) were significantly associated with these traits.Ectopic overexpression of GhCPD-3 in the cpd91 Arabidopsis mutant rescued the mutant phenotype by increasing plant height and leaf size in comparison to those of cpd91 and WT plants. Moreover, overexpressed GhCPD-3 in cpd91 mutants showed greater hypocotyl and root lengths than those of cpd91 and WT plants under light and dark conditions, respectively, indicating that BR actively promotes hypocotyl and root growth. Similar to CPD (CONSTITUTIVE PHOTOMORPHOGENIC DWARF), GhCPD-3 restores BR biosynthesis thereby mediating plant growth and development.