All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein-protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co-transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all-in-one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single-tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high-throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS-guided GA-mediated AioFFP vector toolkit is cost-effective, easy to use and will promote functional genomics research in plants.

Risk factors of first thrombosis in obstetric antiphospholipid syndrome.

OBJECTIVE: There is limited evidence on long-term thrombosis risk in patients with obstetric antiphospholipid syndrome (OAPS). This study aimed to investigate the clinical features and risk factors associated with the first thrombosis in patients with isolated OAPS. METHODS: Data from patients with isolated OAPS were collected. All patients were followed up until the first thrombotic event during or after delivery or until the end of the study. Logistic regression analysis identified independent risk factors associated with the first thrombosis in patients with isolated OAPS. RESULTS: The study enrolled 186 patients with OAPS. During a mean 5.4-year follow-up, 11 (5.9%) patients experienced thrombotic events. Multivariate binary logistic regression analysis revealed that triple-positive antiphospholipid antibodies (aPLs, OR=11.662, 95% CI=2.117 to 64.243, p=0.005) and hypocomplementemia (OR=9.047, 95% CI=1.530 to 53.495, p=0.015) were identified as independent risk factors for the first thrombosis in OAPS, after adjustment for low-dose aspirin and hydroxychloroquine. CONCLUSIONS: Triple-positive aPLs and hypocomplementemia are risk factors for the first thrombosis in patients with OAPS.

Recent advances in the biosynthesis of fungal glucan structural diversity.

Glucans are the most abundant class of macromolecule polymers in fungi, which are commonly found in Ascomycota and Basidiomycota. Fungal glucans are not only essential for cell integrity and function but also crucial for the immense industrial interest in high value applications. They present a variety of structural characteristics at the nanoscale due to the high regulation of genes and the involvement of stochastic processes in synthesis. However, although recent findings have demonstrated the genes of glucans synthesis are relatively conserved across diverse fungi, the formation and organization of diverse glucan structures is still unclear in fungi. Here, we summarize the structural features of fungal glucans and the recent developments in the mechanisms of glucans biosynthesis. Furthermore, we propose the engineering strategies of targeted glucan synthesis and point out the remaining challenges in the synthetic process. Understanding the synthesis process of diverse glucans is necessary for tailoring high value glucan towards specific applications. This engineering strategy contributes to enable the sustainable and efficient production of glucan diversity.

Deficiency of peripheral CLA+ Tregs and clinical relevance in Behcet's syndrome.

BACKGROUND: Autoimmune responses have been suggested to involvement in patients with Behcet's syndrome (BS). There has been growing attention towards the roles of cutaneous lymphocyte antigen (CLA)+ regular T cells (Tregs) in autoimmune diseases. The role of CLA+ Tregs in BS is still uncertain. This study aims to clarify the impact of CLA+ Tregs on BS. METHODS: We collected peripheral blood from a total of 107 patients with BS and 114 healthy controls (HCs). The number of CLA+ Tregs, natural killer (NK) cells, B cells, and several subtypes of CD4+ T cells were detected using flow cytometry and compared between patients and HCs. RESULTS: The absolute number and proportion of CLA+ Tregs among CD4+ T lymphocytes and CD4+ Tregs were lower in patients with BS than in HCs. CLA+ Tregs were positively related with NK cells (r = 0.500, P < 0.001) and B cells (r = 0.470, P < 0.001) and negatively related with effector T cells (r=-0.402, P < 0.001) in patients with BS. Patients with BS and arterial aneurysms had CLA+ Treg cell deficiency. A decreased proportion of CLA+ Tregs was associated with arterial aneurysms in patients with BS. The proportion of CLA+ Tregs in patients with BS increased with corticosteroids and immunosuppressants. CONCLUSION: CLA+ Tregs decrease in association with arterial aneurysm in patients with BS. CLA+ Tregs may be a predictor of response to BS treatment.

Dynamics of α-glucan from Agrocybe cylindracea water extract at different developmental stages and its structure characteristics.

Polysaccharides are the important bioactive macromolecules in Agrocybe cylindracea, but their changes are as yet elusive during developmental process. This study investigated the dynamic changes of polysaccharides from A. cylindracea fruiting body water extract at four developmental stages and its structure characteristics. Results revealed that the polysaccharides from A. cylindracea water extract significantly increased at the pileus expansion stage and the increased fraction could be α-glucan. The further purification and identification indicated that this α-glucan was a glycogen. It had typical morphology of ß particles with a molecular weight of 1375 kDa. Its backbone comprised α-D-(1 â†’ 4)-Glcp and α-D-(1 â†’ 4,6)-Glcp residues at a ratio of 5:1, terminated by α-D-Glcp residue. Rheological behavior suggested that it was a Newtonian fluid at the concentration of 1 %. In addition, despite both the glycogen and natural starch were composed of D-glucose, they exhibited the entirely distinct Maltese cross characteristic and unique crystalline structure. This study is the first to demonstrate the presence of abundant glycogen in the pileus expansion stage of A. cylindracea, which provides new insights on the change patterns of fungal polysaccharides.

Hemorrhage complications in obstetric antiphospholipid syndrome: Risk factors and association with adverse pregnancy outcomes.

Background: Bleeding complications are recognized as relatively infrequent manifestations of antiphospholipid syndrome (APS), and the safety of antithrombotic therapy during pregnancy is of concern. This study aims to assess the risk factors and possible associations between bleeding complications and adverse pregnancy outcomes (APOs) in patients with APS. Methods: A retrospective cohort study was conducted at the Peking University People's Hospital. The clinical and immunologic features, bleeding complications, treatment, and pregnancy outcomes of patients with APS were collected. Univariate and multivariate logistic regression analyses were applied to assess the associations between APOs and bleeding complications. Results: A total of 176 participants with obstetric APS were included in the analysis. There were 66 (37.50%) patients with APS with hemorrhage complications and 86 (48.86%) patients with APS with APOs. Mucocutaneous hemorrhage was associated with APOs including fetal death after 12 weeks [odds ratio (OR) = 10.73, 95% confidence interval (CI): 1.61-71.74, p = 0.014], preterm delivery prior to 34 weeks (OR = 8.30, 95% CI: 2.31-29.84, p = 0.001), and small for gestational age (OR = 4.17, 95% CI: 1.22-14.21, p = 0.023) in univariate logistic regression analyses. It also independently associated with preterm delivery prior to 34 weeks (OR = 40.29, 95% CI: 1.45-1121.32, p = 0.030) in multivariate logistic regression analyses. Receiver operating characteristic (ROC) analysis evaluating the accuracy of these factors for preterm delivery prior to 34 weeks showed that the area under ROC curve was 0.871. Conclusion: The study shows that mucocutaneous hemorrhage may be an indication of the occurrence of APOs in obstetric patients with APS.

Thrombocytopenia in the first trimester predicts adverse pregnancy outcomes in obstetric antiphospholipid syndrome.

Background: Thrombocytopenia is a common manifestation of antiphospholipid syndrome (APS), and is a main concern for bleeding on the standard treatment of low dose aspirin (LDA) and low molecular weight heparin (LMWH) in obstetric APS (OAPS). Objective: This study assesses the possible relationship between thrombocytopenia during the first trimester and adverse pregnancy outcomes (APOs) in OAPS patients. Methods: A case-control study was conducted at Peking University People's Hospital, Beijing, China. The clinical, immunologic, and pregnancy outcomes of the OAPS patients were collected. Univariate and multivariate logistic regression analyses were applied to assess the relationship between APOs and thrombocytopenia in the first trimester. Results: A total of 115 participants were included in the analysis. There were no difference on antepartum and postpartum hemorrhage between the two groups. The gestational age in the thrombocytopenia group was less than that in the control group (34.12 ± 8.44 vs. 37.44 ± 3.81 weeks, p = 0.002). Hypocomplementemia, double aPL positive, and high titers of anti-ß2 glycoprotein I were more frequent in APS patients with thrombocytopenia (p < 0.05). Compared to the control group, thrombocytopenia in the first trimester was correlated with SGA (12.12% vs. 31.25%, p = 0.043), premature birth <37 weeks (16.16% vs 43.75%, p = 0.010) and intrauterine fetal death (2.02% vs 12.50%, p = 0.033). Thrombocytopenia in first-trimester independently increased the risk of preterm birth <37 weeks (OR = 5.40, 95% CI: 1.35-21.53, p = 0.02) after adjusting for demographic and laboratory factors. After adding medication adjustments, these factors above become insignificant (p > 0.05). Of note, the number of platelets increased after delivery in 14 thrombocytopenia patients with live fetuses (p = 0.03). Conclusion: This study demonstrates that thrombocytopenia in the first trimester increases the risks of preterm birth in women with APS. The effective OAPS treatments may improve pregnancy outcomes and not increase the risk of antepartum and postpartum hemorrhage.

Characteristics and mortality in primary Sjögren syndrome-related interstitial lung disease.

ABSTRACT: Aim of the study was to determine the characteristics and prognosis, and to identify the risk factors for mortality in patients with primary Sjögren syndrome (pSS) with interstitial lung disease (pSS-ILD).A total of 1422 patients with SS were screened and 178 patients with pSS-ILD were recruited. The medical records and outcomes were retrospectively reviewed. Overall survival and case control study were performed to explore the predictors of death.Among 178 pSS-ILD patients, 87.1% were women. Mean age was 61.59 ±â€Š11.69-year-old. Median disease duration was 72.0 (24.0, 156.0) months. Nonspecific interstitial pneumonia was the predominant high-resolution computed tomography pattern (44.9%). Impairment in diffusion capacity was the most common abnormality of pulmonary function test (75.8%) and the most severe consequence. Type 1 respiratory failure and hypoxia were observed in 15.0% and 30.0% patients, respectively. Mean survival time after confirmation of pSS-ILD diagnosis was 9.0 (6.8, 13.0) years. The 10-year survival rate for all patients with pSS-ILD was 81.7%. Forty-four (24.7%) of 178 patients died during the follow-up period. The most predominant cause of death was respiratory failure (n = 27). Twenty-seven patients died of ILD and formed study group. The 78 patients who survived formed control group. Age and smoking were risk factors for mortality in patients with pSS-ILD. In addition, severity of ILD, as reflected by high-resolution computed tomography, pulmonary function test, and arterial blood gas, was an independent risk factor. However, inflammation status (erythrocyte sedimentation rate, C-reactive protein) and anti-Sjögren syndrome-related antigen A and anti-Sjögren syndrome-related antigen B were not.ILD is a severe complication of pSS. Age, smoking, and severity of lung involvement are more critical for prognosis rather than inflammation status and autoantibodies.

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.