Neutrophil activation biomarker pentraxin 3 for diagnosis and monitoring of macrophage activation syndrome occurrence in adult-onset Still's disease.

Macrophage activation syndrome (MAS) is a potentially fatal consequence of adult-onset Still's disease (AOSD), driven by a cytokine storm. Efficient early diagnosis of AOSD-associated MAS requires a sensitive and specific biomarker. In this study, we demonstrated that pentraxin 3 (PTX3), an acute phase protein, was associated with AOSD disease activity and served as a biomarker for AOSD-MAS. PTX3 levels were significantly increased in AOSD patients compared to other autoimmune diseases and healthy controls. Plasma PTX3 levels showed positive correlations with inflammatory markers, the systemic score and the HScore. In active AOSD with MAS, PTX3 levels were higher compared to those in non-AOSD haemophagocytic lymphohistiocytosis (HLH) patients. Moreover, the PTX3's area under the curve value for distinguishing AOSD with MAS exceeded that of soluble interleukin-2 receptor, ferritin and C-reactive protein. Furthermore, plasma levels of PTX3 were associated with circulating NET-DNA levels. To fully understand the underlying mechanism of PTX3 prompting AOSD and AOSD-MAS progression, we discovered that neutrophils exhibited enhanced NET formation and mitogen-activated protein kinases (MAPK) pathway activation upon PTX3 stimulation. More importantly, PTX3-induced NET formation was effectively dampened by MAPK pathway inhibitors. These findings collectively revealed that PTX3 has a favorable correlation with disease activity and may serve as a potential biomarker to differentiate AOSD patients with MAS. Additionally, PTX3 induces NET release via the MAPK pathway, suggesting a pathogenic role in AOSD-MAS.

Neutralizing anti-IFN-γ IgG was increased in patients with systemic lupus erythematosus and associated with susceptibility to infection.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a complicated autoimmune disease, in which infection is a leading cause of death. Some SLE patients clinically presented with recurrent and refractory infections, which manifested as adult-onset immunodeficiency syndrome due to the production of anti-interferon-γ (anti-IFN-γ) autoantibodies. This study aimed to investigate the role of anti-IFN-γ autoantibodies concerning severe infections in SLE patients. METHODS: We detected serum levels of anti-IFN-γ IgG/IgM isotypes in SLE patients with severe infections (n = 55), SLE patients without severe infections (n = 120), rheumatoid arthritis (n = 24), ankylosing spondylitis (n = 24), and healthy controls (n = 60). The relationship between anti-IFN-γ autoantibodies and clinical characteristics and laboratory parameters were analyzed. We further evaluated the neutralizing ability of anti-IFN-γ IgG. RESULTS: The level of anti-IFN-γ IgG was significantly elevated in SLE patients with severe infections compared with the other groups (all p < 0.01), and the positive rates of anti-IFN-γ IgG in SLE patients with and without severe infections were 29.1% and 10.8%, respectively. Further analysis indicated that the levels of anti-IFN-γ IgG were positively associated with the SLEDAI score (r = 0.6420, p < 0.001), and it could predict the susceptibility to severe infections in SLE patients. Moreover, the inhibition and function assay showed that purified IgG from anti-IFN-γ IgG-positive SLE patients could neutralize IFN-γ, and further impair IFN-γ-induced STAT1 phosphorylation. CONCLUSIONS: The neutralizing anti-IFN-γ IgG might increase the susceptibility to infection in SLE patients, which has important implications for the treatment. Key Points • The role of anti-IFN-γ autoantibodies concerning severe infections in SLE patients remains unknown. • The results of this study reveals that anti-IFN-γ IgG levels were significantly elevated in SLE patients with severe infections. • This study suggests that neutralizing anti-IFN-γ IgG might increase the susceptibility to infection in SLE patients.

Neutrophil extracellular trap-induced intermediate monocytes trigger macrophage activation syndrome in adult-onset Still's disease.

BACKGROUND: Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disease characterized by innate immune system activation, with a high risk for macrophage activation syndrome (MAS). MAS development is associated with monocyte/macrophage activation and cytokine storm. Monocytes consist of three different subsets, classical monocytes (CMs, CD14brightCD16 -), intermediate monocytes (IMs, CD14brightCD16 +), and non-classical monocytes (NCMs, CD14dimCD16 +), each has distinct roles in inflammatory regulation. However, the frequencies and regulatory mechanism of monocyte subsets in AOSD patients have not been identified. METHODS: We performed flow cytometry, RNA sequencing, phagocytosis analysis, and enzyme-linked immunosorbent assay to evaluate monocyte subsets, cell functions, and potential biomarkers. The effect of neutrophil extracellular traps (NETs) on monocytes was determined by evaluating mRNA levels of DNA sensors, surface CD16 expression, and inflammasome pathway activation. RESULTS: Higher proportions of intermediate monocytes (IMs) were identified in active AOSD patients. IMs displayed higher expression of CD80, CD86, HLA-DR, and CD163 than CMs and NCMs. CD163 upregulation was noted on AOSD IMs, accompanied by increased phagocytic activity and elevated cytokine/chemokine production, including IL-1ß, IL-6, CCL8, and CXCL10. The frequencies of IMs were correlated with disease activity and higher in AOSD patients with MAS (AOSD-MAS). CCL8 and CXCL10 were highly expressed in RNA sequencing of monocytes from AOSD-MAS patients and plasma CXCL10 level could serve as a potential biomarker for AOSD-MAS. Moreover, DNA-sensing pathway was activated in monocytes from AOSD-MAS patients. Stimulation with NETs derived from AOSD induced DNA sensor expression, the expansion of IMs, and inflammasome pathway activation. These effects can be abrogated by DNase I treatment. CONCLUSIONS: Our results demonstrated that the proportions of IMs were elevated in AOSD and associated with MAS. The DNA component in NETs from AOSD plays an important role in the formation of IMs, shedding new light for the therapeutic target.

The landscape of innate and adaptive immune cell subsets in patients with adult-onset Still's disease.

OBJECTIVE: Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disorder. The understanding of the changes in adaptive immune cells and the crosstalk between innate and adaptive immune systems in AOSD is limited. This study aimed to examine the peripheral immune cell composition and inflammatory protein levels in AOSD patients. METHODS: Twenty-nine active AOSD patients were enrolled. Flow cytometry was used to analyze the cell populations in peripheral blood. Antibody chips were utilized to detect the protein expression profile in serum. RESULTS: In active AOSD patients, there was an increase in the percentage of classical and non-classical monocytes among peripheral blood mononuclear cells. The proportion of natural killer (NK) cells decreased, with an increase in CD56dim NK1 cells and a decrease in CD56bright NK2 cells compared with healthy controls (HC). The percentage of naïve central memory T cells was decreased, while the percentage of effector and effector memory T cells was increased among adaptive lymphocytes. The proportion of naïve B and memory B cells was decreased, while plasma cells were increased in AOSD patients, indicating activation of the adaptive immune system. Additionally, the serum levels of 40 proteins were elevated in AOSD patients, primarily involved in cytokine-cytokine receptor interaction, inflammatory response, and regulation of MAPK cascade. CONCLUSION: Our findings showed the activation of the innate and adaptive immune system in AOSD. The protein-protein interaction analysis suggested potential communication between innate and adaptive cell subsets. These findings provide new insights into the pathogenesis of the disease and the development of targeted therapies.

Inactivated SARS-CoV-2 vaccine does not increase the risk of relapse in patients with clinically inactive adult-onset Still's disease.

OBJECTIVE: A succession of cases have reported flares of adult-onset Still's disease (AOSD) after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), raising concerns. We aimed to investigate the impact of inactivated SARS-CoV-2 vaccines on disease activity in patients with AOSD. METHODS: We prospectively enrolled clinically inactive AOSD patients visiting the outpatient clinics of our department. The patients received SARS-CoV-2 vaccines (BBIBP-CorV, Sinopharm, Beijing, China) voluntarily. The occurrence of relapse in the participants was recorded during the follow-up period, and a propensity score matching (PSM) method was used to compare the relapse rates between vaccinated and unvaccinated patients. Localized and systemic symptoms were assessed in the vaccinated patients. RESULTS: A total of 122 patients with inactive AOSD were included, of which 49.2% (n = 60) voluntarily received the inactivated SARS-CoV-2 vaccine. The relapse rate did not increase significantly in vaccinated patients in comparison with unvaccinated patients (after PSM: 6.8% vs 6.8%), and no relapse occurred within 1 month after vaccination. No obvious adverse reactions were reported in 75.0% of the participants, and none of the patients reported severe reactions. CONCLUSION: Increased disease activity or relapse following vaccination with inactivated SARS-CoV-2 was rare in patients with inactive AOSD. Local and systemic adverse reactions were found to be mild and self-limiting. These safety profiles of inactivated SARS-CoV-2 vaccines in patients with AOSD may assist in eliminating vaccine hesitancy and increase the vaccination rate against SARS-CoV-2.

COVID-19 vaccine affects neither prothrombotic antibody profile nor thrombosis in primary anti-phospholipid syndrome: a prospective study.

OBJECTIVE: To explore whether inactivated coronavirus disease 2019 vaccine influences the profile of prothrombotic autoantibodies and induces thrombotic events in primary APS patients. METHODS: We enrolled 39 primary APS patients who received two doses of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine (BBIBPCorV, Sinopharm, Beijing, China) voluntarily in this prospective cohort. Prothrombotic autoantibodies were determined before vaccination and 4 weeks after the second dose of vaccination. Thrombotic disorders were evaluated via hospital site visits and assessments. RESULTS: There was no significant difference in the presence of all 11 autoantibodies detected before and 4 weeks after vaccination: for aCL, IgG (14 vs 16, P = 0.64), IgM (13 vs 19, P = 0.34), IgA (2 vs 3, P = 0.64); anti-ß2GP1, IgG (12 vs 12, P = 1.00), IgM (5 vs 8, P = 0.36), IgA (4 vs 3, P = 0.69); anti-PS/PT IgG (13 vs 16, P = 0.48), IgM (17 vs 22, P = 0.26); LAC (22 vs 28, P = 0.16); aPF4-heparin (0 vs 0, P = 1.00) and ANA (23 vs 26, P = 0.48). Notably, the distribution of the aPL profile in the pre- and post-vaccination cohorts was not affected by SARS-CoV-2 vaccination: for patients with a low-risk aPL profile (11 vs 10, P = 0.799) and patients with a high-risk aPL profile (28 vs 29, P = 0.799), respectively. Furthermore, no case exhibited symptoms of the thrombotic disorder during a minimum follow-up period of 12 weeks. There was no adjustment to the ongoing treatment regimens following SARS-CoV-2 vaccination. CONCLUSION: Inactivated SARS-CoV-2 vaccine does not influence the profile of anti-phospholipid antibodies and anti-PF4-heparin antibodies nor induces thrombotic events in primary APS patients.

Anti-Interferon Autoantibodies in Adult-Onset Immunodeficiency Syndrome and Severe COVID-19 Infection.

Adult-onset immunodeficiency syndrome due to anti-interferon (IFN)-γ autoantibodies has attracted much attention in recent years. It usually occurs in previously healthy people and usually presents as chronic, recurrent, and hard-to-control infections that can be effectively treated with aggressive antibiotic therapy. Adult-onset immunodeficiency syndrome is also referred to as AIDS-like syndrome. Anti-type I IFN (IFN-I) autoantibodies have been reported to play a significant role in the pathogenesis of coronavirus disease 2019 (COVID-19) and preexisting anti-IFN-I autoantibodies are associated with an increased risk of severe COVID-19. This review summarizes the effects of anti-IFN autoantibodies on the susceptibility and severity of various infectious diseases, including SARS-CoV-2 infection. In addition, we discuss the role of anti-IFN autoantibodies in the pathogenesis of autoimmune diseases that are characterized by recurrent infections.

Pediatric tuina for the treatment of fever in children: A protocol for systematic review and meta-analysis.

BACKGROUND: Infantile fever is a common symptom of the pediatric diseases, which is often caused by cold, food accumulation, or other pathogenic factors. Pediatric tuina is regarded as an acceptable non-pharmaceutical therapy for children with optimal effects, which has been widely used for infantile fever around China. But there is still a lack of systematic evaluation and research on its safety and effectiveness during the treatment of infantile fever. Thus the protocol is to collect clinical evidence and demonstrate the efficacy and safety of antipyretic manipulation by pediatric tuina. METHODS: The systematic electronic search will be executed in Cochrane Library (1991-2020.6), EMBASE (1980-2020.6), PubMed (1996-2020.6), WHOICTRP (2004-2020.6), Web of Science (1900-2020.6), CNKI (1994-2020.6), CBM (1994-2020.6), WANFANG (1980-2020.6), and VIP (2000-2020.6) Database. The Review Manager (V.5.3) will be use to assess the risk of bias and data analyses. The methodological quality will be assessed by using the online GRADEpro tool. If the quality of numeric data is favorable, a meta-analysis will be carried out. RESULTS: A high-quality evidence of pediatric tuina for the treatment of infantile fever. CONCLUSION: The systematic review will provide a reliable basis for judging whether pediatric tuina is safe and effective in the treatment of pediatric fever. INPLASY REGISTRATION NUMBER: INPLASY202060032.

The efficacy of Qigong exercises for post-stroke mental disorders and sleep disorders: Protocol for a systematic review and meta-analysis.

BACKGROUND: Post-stroke mental disorders (PSMDs) and post-stroke sleep disorders (PSSDs) are quite common condition in stroke's patients. Qigong has been widely applied as a replaced and useful treatment for PSMDs and PSSDs. However, the exact effects and safety of Qigong have yet be systematically investigated. Our study focused on summary of efficacy and safety of Qigong for the treatment of advanced PSMDs and PSSDs through the systematic analysis and meta-analysis, in order to provide scientific reference for the clinical. METHODS: The protocol followed Preferred Reporting Items for Systematic Review and Meta-Analyses Protocols. Relevant randomized controlled trials were only considered. Search strategy will be performed in 4 English database including Cochrane Library, PubMed, Web of Science, Excerpt Medical Database, 4 Chinese Database including Chinese Biomedical Literature Database, China National Knowledge Infrastructure, China Scientific Journal Database, Wanfang Database, and WHO International Clinical Trials Registry Platform. Papers in English or Chinese published from their inception to 30 June 2020. Study selection and data extraction will be performed independently by 2 investigators. The clinical outcomes including overall Hamilton depression scale, Hamilton anxiety scale, the mental health part of the MOS item short from health survey, Generic Quality of Life Inventory-74, Center for Epidemiologic Studies Depression Scale, Pittsburgh sleep quality index. Based on the Cochrane Assessment tool and Physiotherapy Evidence Database scale, a modified assessment form should be used to evaluate the methodological quality. Review Manager 5.3 was used for data analysis and risk of bias. RESULTS AND CONCLUSION: We provide some more practical and targeted results examine the effect of Qigong exercises for PSMDs and PSSDs in the relative meta-analysis. We find out defects or inadequacies of Qigong in previous studies. The findings of this research will provide more evidence-based guidance in clinical practice and more rigorous study.International Platform of Registered Systematic Review and Meta-Analysis Protocols (INPLASY) registration number: INPLASY202070051.

Multiple promoters driving the expression of astaxanthin biosynthesis genes can enhance free-form astaxanthin production.

Astaxanthin possesses various biological properties and is used in the animal and fish feed, food, and beverage industries. In this study, we derived zeaxanthin biosynthesis genes (crtE, crtB, crtI, crtY, and crtZ) from Erwinia uredovora and crtW from Agrobacterium aurantiacum. We fused inducible and constitutive promoters to astaxanthin biosynthesis genes to construct a novel plasmid (dubbed PTP3-6) that can effectively enhance free-form astaxanthin (FFAX) production. The PTP3-6 plasmid contains one T7 promoter, driving IPTG inducible crtW expression, and three constitutive promoters (isolated from E. uredovora) driving expression of the other zeaxanthin biosynthesis genes. Escherichia coli BL21 (DE3) cells carrying the PTP3-6 plasmid produced 8.3 mg/g dry cell weight astaxanthin, which is 69.4-fold higher than has been previously reported. Using multiple promoter fusions of astaxanthin biosynthesis genes could be applied in other hosts to enhance astaxanthin production. FFAX was identified in recombinant E. coli cells through ultra-performance liquid chromatography-mass spectrometry.

The complete chloroplast genome of agarwood producing species, Aquilaria sinensis (Lour.) Gilg: a species on IUCN red list.

The entire chloroplast genome of Aquilaria sinensis (Lour.) Gilg was identified as a circular molecule of 174,885 bp length with a typical tetrad structure, including a pair of inverted repeats (42,103 bp each), a large single copy (87,331 bp) and a small single copy (3,348 bp) regions. The A. sinensis cp genome encoded 8 rRNAs, 39 tRNAs, and 90 proteins. A phylogenetic tree was reconstructed using the 43 protein-coding genes of eight Thymelaeaceae. Two other Malvales, Abelmoschus esculentus and Durio zibethinus, were selected as the outgroup. Our phylogenetic analysis suggests that the five examined species of Aquilaria appeared a monophyletic group with robust support.

The complete mitochondrial genome of wild mung bean (Vigna radiata var. sublobata TC1966).

The entire mitogenome of wild mung bean (Vigna radiata var. sublobata TC1966) was identified as a circular molecule of 402,981 bp length. The wild mung bean mitogenome encoded 3 rRNAs, 16 tRNAs, and 33 proteins. A phylogenetic tree was reconstructed using the 18 protein-coding genes of 14 legumes and one close species, Ricinus communis. Our phylogenetic analysis suggests that the wild mung bean clustered with the Vigna radiata var. radiata, as well as, the species of Vigna and Glycine appeared as a monophyletic group. This complete mitogenome sequence provides a genomic resource for further studies in mung bean breeding and domestication.

Trithorax Group Proteins Act Together with a Polycomb Group Protein to Maintain Chromatin Integrity for Epigenetic Silencing during Seed Germination in Arabidopsis.

Polycomb group (PcG) and trithorax group (trxG) proteins have been shown to act antagonistically to epigenetically regulate gene expression in eukaryotes. The trxG proteins counteract PcG-mediated floral repression in Arabidopsis, but their roles in other developmental processes are poorly understood. We investigated the interactions between the trxG genes, ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) and ULTRAPETALA1 (ULT1), and the PcG gene EMBRYONIC FLOWER 1 (EMF1) during early development. Unexpectedly, we found that mutations in the trxG genes failed to rescue the early-flowering phenotype of emf1 mutants. Instead, emf1 atx1 ult1 seedlings showed a novel swollen root phenotype and massive deregulation of gene expression. Greater ectopic expression of seed master regulatory genes in emf1 atx1 ult1 triple than in emf1 single mutants indicates that PcG and trxG factors together repress seed gene expression after germination. Furthermore, we found that the widespread gene derepression is associated with reduced levels of H3K27me3, an epigenetic repressive mark of gene expression, and with globally altered chromatin organization. EMF1, ATX1, and ULT1 are able to bind the chromatin of seed genes and ULT1 can physically interact with ATX1 and EMF1, suggesting that the trxG and EMF1 proteins directly associate at target gene loci for EMF1-mediated gene silencing. Thus, while ATX1, ULT1, and EMF1 interact antagonistically to regulate flowering, they work together to maintain chromatin integrity and prevent precocious seed gene expression after germination.

Genome-wide analysis of GDSL-type esterases/lipases in Arabidopsis.

KEY MESSAGE: In this present study, we introduce a fundamental framework and provide information regarding the possible roles of GDSL-type esterase/lipase gene family in Arabidopsis. GDSL-type esterases/lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity, regiospecificity, and stereoselectivity. In this study, we identified 105 GDSL-type esterase/lipase genes in Arabidopsis thaliana by conducting a comprehensive computational analysis. Expression studies indicated that GDSL-type lipase proteins showed varied expression patterns. Phylogenetic tree analysis indicated that AtGELP (Arabidopsis thaliana GDSL-type esterase/lipase protein) gene family was divided into four clades. The phylogenetic analysis, combined with protein motif architectures, and expression profiling were used to predict the roles AtGELP genes. To investigate the physical roles of the AtGELP gene family, we successfully screened 88 AtGELP T-DNA knockout lines for 54 AtGELP genes from 199 putative SALK T-DNA mutants. Transgenic plants of AtGELP genes were used to elucidate the phenotypic characteristics in various developmental stages or stress conditions. Our results suggest that the AtGELP genes have diverse physical functions such as affecting the germination rate and early growth of seedlings subjected to high concentrations of glucose, or being involved in biotic stress responses.

Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene.

Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives.

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

Identification of single nucleotide polymorphism markers associated with resistance to bruchids (Callosobruchus spp.) in wild mungbean (Vigna radiata var. sublobata) and cultivated V. radiata through genotyping by sequencing and quantitative trait locus analysis.

BACKGROUND: Bruchid beetles are an important storage pest of grain legumes. Callosobruchus sp. infect mungbean (Vigna radiata) at low levels in the field, multiply during grain storage and can destroy seed stocks in a few months. Resistance against bruchid beetles has been found in wild mungbean V. radiata var. sublobata TC1966 and in cultivated mungbean line V2802. RESULTS: Bruchid resistance data were obtained from recombinant inbred line populations TC1966 (V. radiata var. sublobata) × NM92 (F12) and V2802 (V. radiata) × NM94 (F7). More than 6,000 single nucleotide polymorphic markers were generated through genotyping by sequencing (GBS) for each of these populations and were used to map bruchid resistance genes. One highly significant quantitative trait locus (QTL) associated with bruchid resistance was mapped to chromosome 5 on genetic maps of both populations, suggesting that TC1966 and V2802 contain the same resistance locus. Co-segregation of all markers associated with resistance indicated the presence of only one major resistance QTL on chromosome 5, while QTL analysis based on physical map positions of the markers suggested the presence of multiple QTLs on different chromosomes. The diagnostic capacity of the identified molecular markers located in the QTL to correctly predict resistance was up to 100 %. CONCLUSIONS: Molecular markers tightly linked to bruchid resistance loci of two different mungbean resistance sources were developed and validated. These markers are highly useful for developing resistant lines.

Genomic and transcriptomic comparison of nucleotide variations for insights into bruchid resistance of mungbean (Vigna radiata [L.] R. Wilczek).

BACKGROUND: Mungbean (Vigna radiata [L.] R. Wilczek) is an important legume crop with high nutritional value in South and Southeast Asia. The crop plant is susceptible to a storage pest caused by bruchids (Callosobruchus spp.). Some wild and cultivated mungbean accessions show resistance to bruchids. Genomic and transcriptomic comparison of bruchid-resistant and -susceptible mungbean could reveal bruchid-resistant genes (Br) for this pest and give insights into the bruchid resistance of mungbean. RESULTS: Flow cytometry showed that the genome size varied by 61 Mb (mega base pairs) among the tested mungbean accessions. Next generation sequencing followed by de novo assembly of the genome of the bruchid-resistant recombinant inbred line 59 (RIL59) revealed more than 42,000 genes. Transcriptomic comparison of bruchid-resistant and -susceptible parental lines and their offspring identified 91 differentially expressed genes (DEGs) classified into 17 major and 74 minor bruchid-resistance-associated genes. We found 408 nucleotide variations (NVs) between bruchid-resistant and -susceptible lines in regions spanning 2 kb (kilo base pairs) of the promoters of 68 DEGs. Furthermore, 282 NVs were identified on exons of 148 sequence-changed-protein genes (SCPs). DEGs and SCPs comprised genes involved in resistant-related, transposable elements (TEs) and conserved metabolic pathways. A large number of these genes were mapped to a region on chromosome 5. Molecular markers designed for variants of putative bruchid-resistance-associated genes were highly diagnostic for the bruchid-resistant genotype. CONCLUSIONS: In addition to identifying bruchid-resistance-associated genes, we found that conserved metabolism and TEs may be modifier factors for bruchid resistance of mungbean. The genome sequence of a bruchid-resistant inbred line, candidate genes and sequence variations in promoter regions and exons putatively conditioning resistance as well as markers detecting these variants could be used for development of bruchid-resistant mungbean varieties.

A novel recombinant chlorophyllase from cyanobacterium Cyanothece sp. ATCC 51142 for the production of bacteriochlorophyllide a.

Bacteriopheophorbide a (BPheid a) is used as a precursor for bacteriochlorin a (BCA), which can be used for photodynamic therapy in both in vitro and in vivo biochemical applications. This study successfully isolated and expressed a photosynthetic bacterium (Cyanothece sp. ATCC 51142) chlorophyllase called CyanoCLH, which can be used as a biocatalyst for the production of a BCA precursor by degrading bacteriochlorophyll a (BChl a). Substrate specificity and enzyme kinetic analyses were performed and the results verified that the recombinant CyanoCLH preferred hydrolyzing BChl a to produce bacteriochlorophyllide a (BChlide a), which can be converted to BPheid a by removing magnesium ion. The recombinant CyanoCLH was cloned and expressed in Escherichia coli BL-21 (DE3), and its molecular weight was 54.7 kDa. The deduced amino acid sequence of the recombinant CyanoCLH comprised a unique lipase-motif GHSLG, which differs from the GHSRG sequence of other plants and lacks a histidine of the typical and conserved catalytic triad Ser-Asp-His. The recombinant CyanoCLH was subjected to biochemical analyses, and the results indicated that its optimal pH and temperature were 7.0 and 60 °C, respectively.