[Overview of new approaches to ß-thalassemia treatment].

Hemoglobinopathies are one of the most common single-gene genetic disorders globally, with approximately 1% to 5% of the global population carrying the mutated gene for thalassemia. Thalassemia are classified into transfusion-dependent thalassemia and non-transfusion-dependent thalassemia based on the need for blood transfusion. Traditional treatment modalities include blood transfusion, splenectomy, hydroxyurea therapy, and iron chelation therapy, which are now widely used for clinical treatment and constitute the main methods recommended in the ß-thalassemia treatment guidelines. However, there are multiple barriers and limitations to the application of these approaches, and there is an urgent need to explore new therapeutic approaches. With the in-depth study of the pathophysiological process of ß-thalassemia, a deeper understanding of the pathogenesis of the disease has been gained. It has been demonstrated that the pathogenesis of thalassemia is closely related to ineffective erythropoiesis (IE), imbalance in the ratio of α/ß-globin protein chains and iron overload. New therapeutic approaches are emerging for different pathogenic mechanisms. Among them, new drugs for the treatment of IE mainly include activin receptor II trap ligands, Janus kinase 2 inhibitors, pyruvate kinase activators, and glycine transporter protein 1 inhibitors. Correcting the imbalance in the hemoglobin chain is mainly due to emerging technologies such as bone marrow transplantation and gene editing. Measures in reducing iron overload are associated with inhibiting the activity of transferrin and hepcidin. These new approaches provide new ideas and options for the treatment and management of ß-thalassemia.

Study on the correlation between retrograde ductus arteriosus flow and right ventricular function evaluated by Z-score of tricuspid annular plane systolic excursion in fetuses with Ebstein anomaly.

PURPOSE: To analyze the influence of RV dysfunction evaluated by Free-angle M-mode (FAM) TAPSE Z-score on retrograde ductus arteriosus flow (RDAF) in fetuses with Ebstein anomaly (EA). METHODS: A retrospective cohort study of 30 EA and 60 normal fetuses were enrolled. The EA group was divided into two groups: with RDAF (EA-RDAF group) and without RDAF (EA-NRDAF group). FAM was used to measure TAPSE of EA and normal fetuses, and Z-scores were calculated. The differences of FAM-TAPSE Z-score, gestational week (GW), maternal age (MA), and mitral valve-tricuspid valve distance (MTD) between three groups were compared. The correlation and binary logistic regression between FAM-TAPSE Z-score, GW, MA, MTD, and RDAF were analyzed. RESULTS: FAM-TAPSE Z-score was significantly lower in EA-RDAF group compared to other groups (p < 0.05). FAM-TAPSE Z-score, GW, and MA were negatively correlated with RDAF (p < 0.05), but no correlation was found between TR, MDT, and RDAF (p > 0.05). Multivariate logistic regression showed that FAM-TAPSE Z-score was an independent influencing factor for RDAF (OR = 0.102, p < 0.05). CONCLUSION: RV dysfunction is an independent factor leading to RDAF in EA fetus, which provides a feasible theoretical basis for further study on improvement of RV function through intrauterine treatment to delay and prevent the RDAF, to avoid death cycle and improve live-birth rate.

Comparative Analysis Based on Physiological and Transcriptomic Data between Juvenile and Adult Tree Peony (Paeonia delavayi).

A long juvenile period limits the breeding process of many woody plants including tree peony. To investigate the physiological and transcriptomic differences between juvenile and adult plants of tree peony and to explore the key SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are vital in age-dependent pathways, 1-year-old and 3-year-old Paeonia delavayi plants were used to compare the relevant physiological parameters and transcriptomic profiles of the leaves in two phases of plants. The results of the physiological parameters showed that the starch content in the leaves of adult plants remained unchanged and that the soluble sugar content significantly increased compared with those in the juvenile plants. In terms of plant hormones, the contents of cytokinin-like hormone (N6-isopentenyladenine (iP)) and jasmonic acid (JA) significantly decreased, whereas the contents of auxin (indole-3-acetic acid, IAA), abscisic acid (ABA), cytokinin-like hormone (N6-isopentenyladenenosine (iPR)), and ethylene precursor (1-aminocyclopropane-1-carboxylic acid, ACC) showed no statistic difference. Transcriptome sequencing results showed that there were 194 differentially expressed genes (DEGs) between juvenile and adult plants, including 171 up-regulated DEGs and 23 down-regulated DEGs. Circadian rhythm, plant hormone signal transduction, and sugar metabolism were closely related to the juvenile-to-adult transition in P. delavayi, involving a total of 12 DEGs. In addition, a total of 13 SPL genes were identified in the transcriptome data, but only PdSPL10 (c71307.graph_c0) was differentially expressed. It was further validated via qRT-PCR analysis, indicating that PdSPL10 might be a key gene regulating the process of juvenile-to-adult in P. delavayi. Based on the above results, a hypothetical transcriptional network regulating juvenile-to-adult transition and flowering in P. delavayi was proposed. These findings provide a reference for understanding the mechanism of juvenile-to-adult transition in tree peony.

Single-cell profiling of ineffective erythropoiesis in a mouse model of ß-thalassaemia intermedia.

Beta-thalassaemia is an inherited haemoglobin disorder characterised by ineffective erythropoiesis (IE). The detailed pathogenesis of IE remains unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to examine IE in Th3/+ ß-thalassaemic mice. The results showed that the erythroid group was remarkably expanded, and genes involved in biological processes such as iron metabolism, haeme synthesis, protein folding, and response to heat were significantly upregulated from erythroid progenitors to reticulocytes in ß-thalassaemic mice. In particular, we identified a unique cell population close to reticulocytes, named ThReticulocytes, characterised by a high level of heat shock protein 70 (Hsp70) expression and dysregulation of iron metabolism and haeme synthesis signalling. Treatment of ß-thalassaemic mice with the haeme oxygenase inhibitor tin-mesoporphyrin effectively improved the iron disorder and IE, and the ThReticulocyte population and Hsp70 expression were significantly suppressed. This study revealed in detail the progression of IE at the single-cell level and possibly provided clues to find therapeutic targets in thalassaemia.

Association study of SNP locus for color related traits in herbaceous peony (Paeonia lactiflora Pall.) using SLAF-seq.

Paeonia lactiflora Pall. (P. lactiflora) is a famous ornamental plant with showy and colorful flowers that has been domesticated in China for 4,000 years. However, the genetic basis of phenotypic variation and genealogical relationships in P. lactiflora population is poorly understood due to limited genetic information, which brings about bottlenecks in the application of effective and efficient breeding strategies. Understanding the genetic basis of color-related traits is essential for improving flower color by marker-assisted selection (MAS). In this study, a high throughput sequencing of 99 diploid P. lactiflora accessions via specific-locus amplified fragment sequencing (SLAF-seq) technology was performed. In total, 4,383,645 SLAF tags were developed from 99 P. lactiflora accessions with an average sequencing depth of 20.81 for each SLAF tag. A total of 2,954,574 single nucleotide polymorphisms (SNPs) were identified from all SLAF tags. The population structure and phylogenetic analysis showed that P. lactiflora population used in this study could be divided into six divergent groups. Through association study using Mixed linear model (MLM), we further identified 40 SNPs that were significantly positively associated with petal color. Moreover, a derived cleaved amplified polymorphism (dCAPS) marker that was designed based on the SLAF tag 270512F co-segregated with flower colors in P. lactiflora population. Taken together, our results provide valuable insights into the application of MAS in P. lactiflora breeding programs.

Annular plane systolic excursion Z-scores in evaluation of heart systolic function of fetus with heart failure.

OBJECTIVE: To compare fetal tricuspid annular plane systolic excursion(TAPSE）Z-scores and mitral annular plane systolic excursion(MAPSE) Z-scores between fetuses with heart failure (HF) and normal fetuses, and to analysis the correlation between CVPS and annular plane systolic excursion(APSE) Z-score(sum of the TAPSE and MAPSE Z-score) in order to evaluate the ventricle systolic function and severity in fetuses with HF. METHODS: A total of 1012 normal fetuses and 24 fetuses with heart failure were involved. TAPSE and MAPSE were measured by free angle M-mode(FAM) echocardiography. Normal FAM-TAPSE and FAM-MAPSE Z-score models based on GA were constructed by performing a standard regression analysis followed by weighted regression of absolute residual values . Tei indexes were calculated in all fetuses with heart failure and all of them were divided into left heart failure (LHF)group and right heart failure（RHF)group by Tei index. Subsequently, FAM-MAPSE Z-scores were compared between the normal and LHF groups, FAM-TAPSE Z-scores were compared between the normal and RHF groups．FAM-APSE Z-scores (sum of the FAM-TAPSE and FAM-MAPSE Z-score) and the cardiovascular profile scores (CVPS) in 24 fetuses were calculated, the correlation was analyzed among them. RESULTS: The models used to calculate Z-score for FAM-TAPSE and FAM-MAPSE were constructed, and GA had significant correlation with them (r = 0.949, p < 0.001for all)．Compared with normal fetuses, the mean Z-scores of FAM-TAPSE and FAM-MAPSE were statistically significantly different in fetuses with HF．In the HF groups, all FAM-TAPSE and FAM-MAPSE Z-scores(22/22) were <-2. CVPS ranged from 3 to 8 (mean 5.25 out of 10) and correlated positively with FAM-APSE Z-score (r = 0.762). CONCLUSIONS: The FAM-TAPSE and FAM-MAPSE Z-scores declined in fetuses with HF and they can provide quantitative evidence in evaluation of heart systolic function, FAM-APSE Z-score correlated positively with CVPS. FAM-TAPSE, FAM-MAPSE and FAM-APSE Z-scores would be markers for assessing heart systolic function and severity in fetuses with HF.

Preliminary study on the evaluation of mitral annulus displacement in normal fetuses by automated cardiac motion quantitation.

OBJECTIVES: The aim of this study was to assess the characteristics of mitral annular plane systolic excursion (MAPSE) in different longitudinal directions in normal fetuses using a new method, automatic cardiac motion quantification (aCMQ). METHODS: A cross-sectional study was conducted in 164 fetuses with structurally normal hearts. The time-displacement curves of the septal mitral annulus (SMA) in three directions, including point A, B and C (MAPSE-SMA-A, MAPSE-SMA-B, MAPSE-SMA-C), were recorded by aCMQ. The time to peak (TTP) in three directions, including point A, B and C (TTP-SMA-A, TTP-SMA-B, TTP-SMA-C) were recorded. In the same way, various parameters of the lateral mitral annulus (LMA) were obtained including MAPSE-LMA-A, MAPSE-LMA-B, MAPSE-LMA-C, TTP-LMA-A, TTP-LMA-B and TTP-LMA-C. Free angle M-mode echocardiography (FAM) was used to obtain MAPSE of LMA (FAM-MAPSE). Finally, all the data were analyzed statistically. RESULTS: MAPSE was positively correlated with gestational age, and the difference between the second- and third-trimester groups was statistically significant. MAPSE-LMA in point B and C were greater than those of SMA. MAPSE-LMA-C and MAPSE-SMA-A were the largest in three directions.The difference between point B and C were statistically significant (p < .05), with no significant difference at point A (p > .05). There was no significant difference found in all TTP (all p > .05). The MAPSE-LMA-C was less than the FAM-MAPSE, and the differences were found significantly (p < .05), but there was better correlation (p < .05). CONCLUSIONS: The longitudinal movement of the fetal mitral annulus is comprehensive, with multiple directions and different displacements. Perpendicular to the mitral annulus is the maximum displacement. It is positively related to the gestational age. From the second trimester, the longitudinal contraction of the left ventricle wall has good synchronization. It possesses clinical value in selecting different methods and parameters during evaluating left ventricular function.

Associated risk factors with disease severity and antiviral drug therapy in patients with COVID-19.

BACKGROUND: Due to the latent onset of novel coronavirus disease 2019 (COVID-19), it is important to identify patients with increased probabilities for disease progression early in order to implement timely medical strategies. This study aimed to identify the factors associated with increased COVID-19 severity and evaluate the current antiviral drugs, especially in severe patients. METHODS: This was a retrospective observational study performed at the No. 7 Hospital of Wuhan (Wuhan, China) with hospitalized patients confirmed with COVID-19 from January 11 to March 13, 2020. Multivariable logistic regression analysis was used to identify the associated factors of severe COVID. Treatments of antivirus drugs were collected and evaluated. RESULTS: Of the 550 patients, 292 (53.1%) were female and 277 (50.4%) were > 60 years old. The most common symptom was fever (n = 372, 67.7%), followed by dry cough (n = 257, 46.7%), and dyspnea (n = 237, 43.1%), and fatigue (n = 224, 40.7%). Among the severe patients, 20.2% required invasive ventilator support and 18.0% required non-invasive ventilator. The identified risk factors for severe cases were: age ≥ 60 years (odds ratio (OR) =3.02, 95% confidence interval (CI): 1.13-8.08, P = 0.028), D-dimer > 0.243 µg/ml (OR = 2.734, 95%CI: 1.012-7.387, P = 0.047), and low oxygenation index (OR = 0.984, 95%CI: 0.980-0.989, P < 0.001). In severe cases, the benefits (relief of clinical symptoms, clinical outcome, and discharge rate) of arbidol alone was 73.3%, which was better than ribavirin (7/17, 41.2%, P = 0.029). CONCLUSIONS: Age > 60 years, D-dimer > 0.243 µg/ml, and lower oxygenation index were associated with severe COVID-19. Arbidol might provide more clinical benefits in treating patients with severe COVID-19 compared with ribavirin.

Herbaceous peony (Paeonia lactiflora Pall.) PlDELLA gene negatively regulates dormancy release and plant growth.

DELLA protein plays a significant role in plant growth and development. In this study, PlDELLA with the open reading frame of 1866 bp in length was isolated from Paeonia lactiflora. Overexpression of PlDELLA in Arabidopsis thaliana showed that seed germination was significantly repressed as it took 144∼192 h for the OEs to reach 100 % germination and it required only 60 h for the WT. The OEs were also inhibited in bolting time and in plant vegetative growth. When PlDELLA was silenced in peony by virus-induced gene silencing method, peony budbreak occurred earlier by 8∼10 d and the vegetative growth was significantly accelerated compared with the control group. These results collectively indicated that PlDELLA negatively regulated dormancy release and plant growth. During chilling process to release peony endodormancy, PlDELLA expression down-regulated, and the content of both endogenous active GAs and ABA decreased, indicating decreasing of PlDELLA expression under chilling was not caused by the known gibberellin signal transduction pathway. Besides, PlDELLA had no interaction with the four screened PlWRKYs, PlWRKY13, PlWRKY18, PlWRKY40 or PlWRKY50. These findings not only enrich the knowledge of DELLA protein family, but also provide insights into understanding the function of PlDELLA protein in endodormancy release in peony.

The WRKY transcription factor PlWRKY65 enhances the resistance of Paeonia lactiflora (herbaceous peony) to Alternaria tenuissima.

In this study, the disease resistance gene PlWRKY65 was isolated from the leaves of Paeonia lactiflora and analyzed by bioinformatics methods, and the localization of the encoded protein was explored. Quantitative real-time PCR (qRT-PCR) was also used to explore the response of this gene to Alternaria tenuissima. The results showed that the gene sequence contained multiple cis-acting elements involved in the response to hormone signaling molecules belonging to the IIe subgroup of the WRKY family, and the encoded proteins were located in the nucleus. The PlWRKY65 gene has a positive regulatory effect on A. tenuissima infection. After silencing the PlWRKY65 gene via virus-induced gene silencing (VIGS), it was found that the gene-silenced plants were more sensitive to A. tenuissima infection than the wild plants, exhibiting more severe infection symptoms and different degrees of changes in the expression of the pathogenesis-related (PR) genes. In addition, we showed that the endogenous jasmonic acid (JA) content of P. lactiflora was increased in response to A. tenuissima infection, whereas the salicylic acid (SA) content decreased. After PlWRKY65 gene silencing, the levels of the two hormones changed accordingly, indicating that PlWRKY65, acting as a disease resistance-related transcriptional activator, exerts a regulatory effect on JA and SA signals. This study lays the foundation for functional research on WRKY genes in P. lactiflora and for the discovery of candidate disease resistance genes.

An Akkermansia muciniphila subtype alleviates high-fat diet-induced metabolic disorders and inhibits the neurodegenerative process in mice.

The prevalence of obesity and diabetes, and their complicating mental disorders, severely affect public health. This study aimed to investigate the long-term effects of an Akkermansia muciniphila subtype (A. muciniphilasub) on high-fat diet-induced obesity and diabetes, and to evaluate whether this subtype can alleviate their complicated mental disorders. Whole genome sequencing and short chain fatty acid production analysis in supernatant of pure culture were performed. Female adult C57BL/6 mice were fed a high-fat diet or a normal chow diet and were gavaged with A. muciniphilasub or phosphate-buffered saline daily for 10 months. Body weight, food consumption and blood glucose were measured. At the end of the treatment period, all mice were subjected to the Y-maze test, sucrose preference test, analyses of serum, fecal microbiota analysis and histological examination. This A. muciniphilasub had 278 unique genes compared to the type strain (A. muciniphila ATCC BAA-835) and produced short chain fatty acids both. A. muciniphilasub administration significantly reduced body weight gain and improved the spatial memory of high-fat diet-fed mice. A. muciniphilasub increased Nissl bodies in neurons of the hippocampus, and restored the high-fat diet-inhibited tryptophan metabolism. The high-fat diet led to decreased serum 5-hydroxytryptamine and induced depression, which were not alleviated by A. muciniphilasub. A. muciniphilasub increased the relative fecal abundance of Bifidobacterium, and was negatively correlated with the fecal abundance of Bacteroides. The present study demonstrated the beneficial effects of this A. muciniphilasub on body weight, blood glucose control and the alleviation of the memory decay caused by a high-fat diet in mice.

PlWRKY13: A Transcription Factor Involved in Abiotic and Biotic Stress Responses in Paeonia lactiflora.

Many members of the WRKY family regulate plant growth and development. Recent studies have shown that members of the WRKY family, specifically WRKY13, play various roles in the regulation of plant stress resistance. To study the function of WRKY family members in peony, the PlWRKY13 gene (KY271095) was cloned from peony leaves. Sequence analysis and subcellular localization results revealed that PlWRKY13 has no introns, belongs to the type IIc subgroup of the WRKY family, and functions in the nucleus. The expression pattern of PlWRKY13 was analysed via real-time quantitative RT-PCR (qRT-PCR), which showed that the expression of PlWRKY13 was induced by four types of abiotic stress, low-temperature, high-temperature, waterlogging and salt stress, and was positively upregulated in response to these stresses. In addition, the expression of PlWRKY13 tended to first decrease and then increase after infection with Alternaria tenuissima. Virus-induced gene silencing (VIGS) technology was used to explore the function of PlWRKY13 in the resistance of Paeonia lactiflora to fungal infection further, and the results showed that PlWRKY13-silenced plants displayed increased sensitivity to A. tenuissima. The infection was more severe and the disease index (DI) significantly greater in the PlWRKY13-silenced plants than in the control plants, and the expression of pathogenesis-related (PR) genes was also significantly altered in the PlWRKY13-silenced plants compared with the control plants. The contents of the endogenous hormones jasmonic acid (JA) and salicylic acid (SA) were measured, and the results showed that the JA content increased gradually after infection with A. tenuissima and that JA may play an active role in the resistance of P. lactiflora to pathogen infection, while the SA content decreased after PlWRKY13 silencing. The contents of the two hormones decreased overall, suggesting that they are related to the transcription of PlWRKY13 and that PlWRKY13 may be involved in the disease-resistance pathway mediated by JA and SA. In summary, the results of our study showed that PlWRKY13 expression was induced by stress and had a positive effect on the resistance of P. lactiflora to fungal infection.

Genome sequencing of 39 Akkermansia muciniphila isolates reveals its population structure, genomic and functional diverisity, and global distribution in mammalian gut microbiotas.

BACKGROUND: Akkermansia muciniphila is one of the most dominant bacteria that resides on the mucus layer of intestinal tract and plays key role in human health, however, little is known about its genomic content. RESULTS: Herein, we for the first time characterized the genomic architecture of A. muciniphila based on whole-genome sequencing, assembling, and annotating of 39 isolates derived from human and mouse feces. We revealed a flexible open pangenome of A. muciniphila currently consisting of 5644 unique proteins. Phylogenetic analysis identified three species-level A. muciniphila phylogroups exhibiting distinct metabolic and functional features. Based on the comprehensive genome catalogue, we reconstructed 106 newly A. muciniphila metagenome assembled genomes (MAGs) from available metagenomic datasets of human, mouse and pig gut microbiomes, revealing a transcontinental distribution of A. muciniphila phylogroups across mammalian gut microbiotas. Accurate quantitative analysis of A. muciniphila phylogroups in human subjects further demonstrated its strong correlation with body mass index and anti-diabetic drug usage. Furthermore, we found that, during their mammalian gut evolution history, A. muciniphila acquired extra genes, especially antibiotic resistance genes, from symbiotic microbes via recent lateral gene transfer. CONCLUSIONS: The genome repertoire of A. muciniphila provided insights into population structure, evolutionary and functional specificity of this significant bacterium.

Phascolarctobacterium faecium abundant colonization in human gastrointestinal tract.

Phascolarctobacterium can produce short-chain fatty acids, including acetate and propionate, and can be associated with the metabolic state and mood of the host. The present study investigated the colonization characteristics of Phascolarctobacterium faecium in healthy individuals <1-80 years old in Southern China. A total of 150 fresh fecal samples were collected, and bacterial DNA was isolated from these samples for quantitative polymerase chain reaction analysis. Phascolarctobacterium faecium demonstrated a high colonization rate and abundant colonization in the human gastrointestinal tract. The colonization rate varied between 43.33-93.33%, and the abundance of Phascolarctobacterium faecium ranged between 3.22-5.76 log cells g-1 (<1 years old) and 3.06-9.33 log cells g-1 (>1 year old). The permillage of Phascolarctobacterium faecium in total bacteria ranged between 0.004-1.479. There was presence of Phascolarctobacterium faecium-like bacteria in younger individuals with a gradual increase in the number of bacteria maintained at a high level with increasing ages (between 1 and 60 years old), but with a decrease in elderly individuals (>60 years old). The results of the present study demonstrated that Phascolarctobacterium faecium is abundantly colonized in the human gastrointestinal tract.

Maximum Plant Uptakes for Water, Nutrients, and Oxygen Are Not Always Met by Irrigation Rate and Distribution in Water-based Cultivation Systems.

Growing on rooting media other than soils in situ -i.e., substrate-based growing- allows for higher yields than soil-based growing as transport rates of water, nutrients, and oxygen in substrate surpass those in soil. Possibly water-based growing allows for even higher yields as transport rates of water and nutrients in water surpass those in substrate, even though the transport of oxygen may be more complex. Transport rates can only limit growth when they are below a rate corresponding to maximum plant uptake. Our first objective was to compare Chrysanthemum growth performance for three water-based growing systems with different irrigation. We compared; multi-point irrigation into a pond (DeepFlow); one-point irrigation resulting in a thin film of running water (NutrientFlow) and multi-point irrigation as droplets through air (Aeroponic). Second objective was to compare press pots as propagation medium with nutrient solution as propagation medium. The comparison included DeepFlow water-rooted cuttings with either the stem 1 cm into the nutrient solution or with the stem 1 cm above the nutrient solution. Measurements included fresh weight, dry weight, length, water supply, nutrient supply, and oxygen levels. To account for differences in radiation sum received, crop performance was evaluated with Radiation Use Efficiency (RUE) expressed as dry weight over sum of Photosynthetically Active Radiation. The reference, DeepFlow with substrate-based propagation, showed the highest RUE, even while the oxygen supply provided by irrigation was potentially growth limiting. DeepFlow with water-based propagation showed 15-17% lower RUEs than the reference. NutrientFlow showed 8% lower RUE than the reference, in combination with potentially limiting irrigation supply of nutrients and oxygen. Aeroponic showed RUE levels similar to the reference and Aeroponic had non-limiting irrigation supply of water, nutrients, and oxygen. Water-based propagation affected the subsequent cultivation in the DeepFlow negatively compared to substrate-based propagation. Water-based propagation resulted in frequent transient discolorations after transplanting in all cultivation systems, indicating a factor, other than irrigation supply of water, nutrients, and oxygen, influencing plant uptake. Plant uptake rates for water, nutrients, and oxygen are offered as a more fundamental way to compare and improve growing systems.

[Rapid selection of recombinant orf virus expression vectors using green fluorescent protein].

OBJECTIVE: To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein (GFP) as the reporter gene. METHODS: The flanking regions of the ORFV132 of orf virus DNA were amplified by PCR to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The shuttle plasmid was transfected into OFTu cells and GFP was incorporated into orf virus IA82Delta 121 by homologous recombination. The recombinant IA82Delta121-V was selected by green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro. RESULTS: The recombinant orf virus IA82Delta121-V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the replication of the virus in vitro but reduced its virulence. CONCLUSION: Green fluorescent protein is a selectable marker for rapid, convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82Delta121-V can serve as a new expression vector for exogenous genes.

Molecular cloning and heterologous expression analysis of JrVTE1 gene from walnut (Juglans regia).

Tocopherol cyclase (VTE1) plays a key role in promoting the production of γ-tocopherol and improving total tocopherol content in photosynthetic organisms. Walnut is an important source of tocopherols in the human diet, and γ-tocopherol is the major tocopherol compound in walnut kernels. In this study, a full-length cDNA of the VTE1 gene was isolated from walnut using RT-PCR and RACE, and designated as JrVTE1. The full-length cDNA of the JrVTE1 gene contained a 1353-bp open-reading frame encoding a 451-amino-acid protein with a calculated molecular weight of 49.5 kDa. The deduced JrVTE1 protein had a considerable homology with other plant VTE1s and belonged to the tocopherol cyclase family. Functional characterization of JrVTE1 by heterologous expression was carried out in E. coli BL21 (DE3) and microshoot lines of the fruit trees jujube (Zizyphus jujuba var. spinosa) and pear (Pyrus communis) cultivar 'Old Home'. JrVTE1 in E. coli expressed as a 50 kDa protein, as expected. One or two copies of the transferred JrVTE1 gene were detected in the genomes of representative transgenic lines (from the initial transgenic plants) of jujube and pear by gel blots analysis. Over-expression of JrVTE1 in jujube and pear resulted in an accumulation of tocopherol and a shift in tocopherol composition in leaf, root and stem tissues. In the transgenic jujube, the total tocopherol content increased by 29.8 µg/g in the stems of line J3, 43.7 and 22.5 µg/g in the roots and leaves of line J1, respectively, whereas in the transgenic pear it increased by 47.3 µg/g in the leaf of line P3, and 16.7 and 10.4 µg/g in roots and stems of line P9, respectively. In the examined tissues of transgenic plants, the highest accumulation rate was the γ-tocopherol. These results indicate that JrVTE1 is one of the rate-limiting enzymes for tocopherol production and could be used to improve the tocopherol content of tree crops through genetic engineering.

Betatrophin Acts as a Diagnostic Biomarker in Type 2 Diabetes Mellitus and Is Negatively Associated with HDL-Cholesterol.

Objective. By assessing its circulating concentrations in type 2 diabetes mellitus (T2DM) patients, we aimed to explore the associations of betatrophin with various metabolic parameters and evaluate its diagnostic value in T2DM. Methods. A total of 58 non-diabetes-mellitus (NDM) subjects and 73 age- and sex-matched newly diagnosed T2DM patients were enrolled. Correlation analyses between circulating betatrophin levels and multiple metabolic parameters were performed. Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic value of betatrophin concentration in T2DM. Results. Circulating betatrophin levels were approximately 1.8 times higher in T2DM patients than in NDM individuals (median 747.12 versus 407.41 pg/mL, P < 0.001). Correlation analysis showed that betatrophin was negatively associated with high-density lipoprotein cholesterol (HDL-C) levels in all subjects. ROC curve analysis identified betatrophin as a potent diagnostic biomarker for T2DM. The optimal cut-off point of betatrophin concentration for predicting T2DM was 501.23 pg/mL. Conclusions. Serum betatrophin levels were markedly increased in newly diagnosed T2DM patients and further elevated in obese T2DM subjects. Betatrophin was negatively correlated with HDL-C levels. Our findings indicate that betatrophin could be a potent diagnostic biomarker for T2DM.

Donor foot morbidity following modified wraparound flap for thumb reconstruction: a follow-up of 69 cases.

PURPOSE: To evaluate donor foot morbidity after thumb reconstruction using a modified wraparound flap. METHODS: We observed patients who accepted thumb reconstruction with the modified wraparound flap between January 2001 and December 2009. We examined and evaluated the reconstructed thumb and donor foot. Donor morbidity was assessed on both a subjective and an objective basis using the Foot Function Index-verbal rating scales (FFI-5pt), the American Orthopaedic Foot and Ankle Society for Hallux Metatarsophalangeal-Interphalangeal (MTP-IP) scale, and gait analysis and dynamic pedodynographic measurements. RESULTS: We reviewed 69 patients; the follow-up period ranged from 6 months to 5 years, with an average of 26 months. The reconstructed thumbs had good aesthetic appearance, and static 2-point discrimination averaged 9.4 ± 2.7 mm. Full length or most of the length of the donor toes was preserved in 67 patients. The retained plantar strip was significantly enlarged from an average of 14.5 ± 1.4 mm measured at surgery to 27.8 ± 4.7 mm measured at last follow-up (p < .05), and its 2-point discrimination was 9.1 ± 2.3 mm. A total of 34 patients were available for FFI-5pt and Hallux MTP-IP scale evaluation. The FFI-5pt total score was 3.1 ± 2.7 and the total Hallux MTP-IP score was 87.9 ± 7.1. Gait analysis and dynamic pedodynographic measurements were available in 20 patients. All 5 biomechanical parameters (timing, trajectory, symmetry, average peak force, and peak pressure between donor foot and the contralateral foot) had no significant difference. CONCLUSIONS: The function of the donor foot after a modified wraparound flap for thumb reconstruction was well preserved, the degree of pain and disability in the donor foot was mild, and foot function in gait was not disturbed. Although a certain degree of restriction in interphalangeal joint motion occurred, this was nearly negligible and did not deter return to normal daily living activity, work, and recreation.