Cloning and characterization of the MYB transcription factor gene GhTT2 in Gossypium hirsutum.

As one of the important secondary metabolites, proanthocyanidins (PAs) are not only a defense mechanism for plants to cope with biotic and abiotic stresses, but also a key factor affecting the development and quality of plants. Although the biosynthetic and metabolic pathways of proanthocyanidins have been basically clarified in the model plants, the regulatory mechanism in cotton has not been fully elucidated. In this work, a transcription factor gene GhTT2 (transparent testa 2) was cloned from Gossypium hirsutum. Its gene structure, expression pattern, subcellular localization, and function were further analyzed. The results show that the GhTT2 has a typical MYB domain and is predominantly expressed in fibers. Its transcription level was negatively correlated with anthocyanin content. The GhTT2-GFP fusion protein is located in the nucleus. Moreover, yeast transformation results show that GhTT2 has obvious transcriptional activation characteristics. Furthermore, the content of proanthocyanidins in GhTT2-silenced cottons is significantly reduced, indicating that GhTT2 may be involved in regulation of the proanthocyanidins biosynthesis in Gossypium hirsutum. These results provide a reference for further elucidating the molecular mechanisms of MYB transcription factors involved in the regulation of the biosynthetic pathway of PAs.

iTRAQ-based comparative proteomic analysis provides insights into somatic embryogenesis in Gossypium hirsutum L.

KEY MESSAGE: iTRAQ based proteomic identified key proteins and provided new insights into the molecular mechanisms underlying somatic embryogenesis in cotton. Somatic embryogenesis, which involves cell dedifferentiation and redifferentiation, has been used as a model system for understanding molecular events of plant embryo development in vitro. In this study, we performed comparative proteomics analysis using samples of non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryo (SE) using the isobaric tags for relative and absolute quantitation (iTRAQ) technology. In total, 5892 proteins were identified amongst the three samples. The majority of these proteins (93.4%) were found to have catalytic activity, binding activity, transporter activity or structural molecular activity. Of these proteins, 1024 and 858 were differentially expressed in NEC versus EC and EC versus SE, respectively. Compared to NEC, EC had 452 and 572 down- and up-regulated proteins, respectively, and compared to EC, SE had 647 and 221 down- and up-regulated proteins, respectively. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis indicated that genetic information transmission, plant hormone transduction, glycolysis, fatty acid biosynthesis and metabolism, galactose metabolism were the top pathways involved in somatic embryogenesis. Our proteomics results not only confirmed our previous transcriptomic results on the role of the polyamine metabolic pathways and stress responses in cotton somatic embryogenesis, but identified key proteins important for cotton somatic embryogenesis and provided new insights into the molecular mechanisms underlying somatic embryogenesis in cotton.

De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

Plant regeneration via somatic embryogenesis (SE) is the key step for genetic improvement of cotton (Gossypium hirsutum L.) through genetic engineering mediated by Agrobacteria, but the molecular mechanisms underlying SE in cotton is still unclear. Here, RNA-Sequencing was used to analyze the genes expressed during SE and their expression dynamics using RNAs isolated from non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryos (SEs). A total of 101, 670 unigenes were de novo assembled. The genes differentially expressed (DEGs) amongst NEC, EC and SEs were identified, annotated and classified. More DEGs were found between SEs and EC than between EC and NEC. A significant number of DEGs were related to hormone homeostasis, stress and ROS responses, and metabolism of polyamines. To confirm the expression dynamics of selected DEGs involved in various pathways, experiments were set up to investigate the effects of hormones (Indole-3-butytric acid, IBA; Kinetin, KT), polyamines, H2O2 and stresses on SE. Our results showed that exogenous application of IBA and KT positively regulated the development of EC and SEs, and that polyamines and H2O2 promoted the conversion of EC into SEs. Furthermore, we found that low and moderate stress is beneficial for proliferation of EC and SEs formation. Together, our global analysis of transcriptomic dynamics reveals that hormone homeostasis, polyamines, and stress response synergistically regulating SE in cotton.

Efficacy of Continuous Positive Airway Pressure in the Treatment of Chronic Obstructive Pulmonary Disease Combined With Respiratory Failure.

Our aim is to investigate the efficacy of continuous positive airway pressure (CPAP) in chronic obstructive pulmonary disease (COPD) combined with respiratory failure. Electronic and manual searches were applied to retrieve published studies relevant to CPAP, COPD, and respiratory failure. The retrieved studies were screened based on stringent inclusion and exclusion criteria to identify high-quality clinical studies for meta-analysis. Comprehensive meta-analysis 2.0 was conducted to statistical analysis. Initially, we retrieved 958 studies based on stringent inclusion and exclusion criteria, 10 studies, containing a total of 372 patients with COPD combined with respiratory failure, were selected for meta-analysis. The result of our meta-analysis revealed that partial pressure of carbon dioxide (PaCO2) in patients with COPD combined with respiratory failure was significantly lower 24 hours after CPAP treatment, and partial pressure of oxygen (PaO2) and pH were markedly higher 24 hours after CPAP treatment in the same patient group, indicating statistically significant differences as a result of CPAP treatment. Subgroup analysis based on ethnicity demonstrated that PaCO2 in patients with COPD combined with respiratory failure is evidently lower 24 hours after CPAP treatment among whites, but no such statistical correlation was observed among Asians. The results of this meta-analysis indicate that CPAP can effectively improve the respiratory function of patients with COPD combined with respiratory failure, and CPAP can be an effective way of treating COPD.

Transarticular fixation by hook plate versus coracoclavicular stabilization by single multistrand titanium cable for acute Rockwood grade-V acromioclavicular joint dislocation: a case-control study.

BACKGROUND: Hook plate (HP) is popularly used for acute and severely displaced acromioclavicular (AC) dislocations. However, subacromial impingement and acromion osteolysis induced by transarticular fixation are notorious. The current case-control study was to compare transarticular fixation by HP to coracoclavicular (CC) stabilization by single multistrand titanium cable (MSTC). METHODS: Between January 2006 and August 2009, 24 patients with acute AC dislocations were surgically treated by open reduction and transarticular fixation with HP. These patients were matched to a series of 24 patients, who were managed by CC stabilization with MSTC in the same period. All AC dislocations were graded as Rockwood type V. Implant was removed 8-12 months after the primary operation in all patients, and 12 months at least were needed to assess the maintenance of AC joint. Functional results were evaluated before implant removal as well as in the last follow-up based on Constant-Murley criteria. RESULTS: There were no differences of demographic data including age, dominant gender and side, injury-to-surgery interval, operation time and follow-up period. In terms of functionality, Constant score was 95.8 ± 4.1 in MSTC group, while 76.7 ± 8.0 in HP group before implant removal (P < 0.001). In detail, MSTC was superior to HP in pain, ROM and activities. Constant score was significantly improved to 86.1 ± 5.7 after hardware removal for patients in HP (P < 0.001). Degenerative change of acromioclavicular joint presented in 16 patients (66.7%) in patients treated by HP, while it was found in only 3 patients (12.5%) treated by MSTC (P < 0.001). CONCLUSIONS: MSTC is superior to HP for the treatment of Rockwood type-V acromioclavicular dislocation both before and after removal of the implant. Hardware removal is of great benefits for functional improvement in patients treated by HP.

Late surgery for acetabular fractures in a Chinese level I trauma centre: surgical experience and outcomes.

PURPOSE: Delayed surgical management of acetabular fractures, often necessary due to life-threatening concomitant injuries, is a great challenge because delays may potentially increase complications and decrease outcomes. We report clinical outcomes of 61 acetabular fractures treated by delayed open reduction and internal fixation (ORIF) with an injury-to-surgery interval (ISI) of 22-399 days. METHODS: Operations were performed between April 2001 and December 2008. There were 61 cases (42 men 19 women), with an average age of 38 years. All patients were followed for an average of 82 months. Demographic data, fracture pattern, ISI, concomitant injuries, surgical approach, complications and clinical outcomes were recorded and analysed. There were 16 simple fractures (26.2%) and 45 associated fractures (73.8%). Matta criteria were used to evaluate reduction quality. The Merle d'Aubigné and Postel scoring system was employed to assess post-operative functionality. RESULTS: Anatomical reduction was achieved in 45 cases (73.8%). The clinical result was excellent in 38 cases, good in 13, fair in six and poor in four. Osteonecrosis of the femoral head was observed in three cases, and heterotopic ossification was found in 28 cases. Four patients had transient palsy of the sciatic nerve. CONCLUSIONS: ORIF for fresh acetabular fractures might yield a better prognosis; however, for delayed acetabular fractures, clinical outcomes are also predictable when sophisticated surgical techniques are employed. Our results indicate that delayed ORIF could yield satisfactory clinical outcomes in the majority of patients with acetabular fractures.

Subtrochanteric femur fracture treated by intramedullary fixation.

PURPOSE: To discuss surgical technique, operative efficacy and clinical outcome of intramedullary fixation in the treatment of subtrochanteric femur fractures. METHODS: From February 2011 to February 2013, 76 cases of subtrochanteric femur fractures were treated by intramedullary fixation in our hospital, including 53 males and 23 females, with the age range of 37 -72 years (mean 53.5 years). According to Seinsheimer classification, there were 2 cases of type I, 7 type II, 15 type III, 23 type IV and 29 type V. Firstly, all patients underwent closed reduction with the guidance of C-arm fluoroscopy in a traction table. Two cases of type I and 3 cases of type III fractures had ideal closed reduction followed by internal fixation. The others needed additional limited open reduction. Radiographic examination was used to evaluate callus formation and fracture healing in postoperative 1, 3, 6 and 12 months follow-up. Functional recovery was evaluated by Harris Hip Scoring (HHS) system. RESULTS: Patients were followed up for 6-12 months. All fractures were healed except one patient with delayed union. The average bone union time was 4.5 months. According to HHS system, 65 cases were considered as excellent in functional recovery, 8 good, 2 fair and 1 poor. The proportion of the patients with excellent and good recovery was 96.05%. CONCLUSION: Intramedullary fixation is feasible for the treatment of subtrochanteric femur fracture. The accuracy of intraoperative reduction and surgical skill are important for the clinical outcome and the patients' prognosis.

Prognostic significance of VEGF expression in osteosarcoma: a meta-analysis.

Vascular endothelial growth factor (VEGF) is considered as a prime mediator of angiogenesis and has been implicated in carcinogenesis and metastasis. Various studies examined the relationship between VEGF overexpression with the clinical outcome in patients with osteosarcoma but yielded conflicting results. Electronic databases updated to April 2013 were searched to find relevant studies. A meta-analysis was conducted with eligible studies which quantitatively evaluated the relationship between VEGF overexpression and survival of patients with osteosarcoma. Survival data were aggregated and quantitatively analyzed. We performed a meta-analysis of eight studies that evaluated the correlation between VEGF overexpression and survival in patients with osteosarcoma. Combined hazard ratios suggested that VEGF overexpression had an unfavorable impact on overall survival (hazard ratio (HR) = 1.75, 95% confidence interval (CI): 1.21-2.28) in patients with osteosarcoma for overall populations, 2.37 (1.35-3.39) in Asian studies but not in non-Asian studies (HR = 1.51, 95% CI: 0.89-2.14). No significant heterogeneity was observed among all studies. VEGF overexpression indicates a poor prognosis for patients with osteosarcoma. However, the prognostic value of VEGF on survival in osteosarcoma patients still needs further large-scale prospective trials to be clarified.

Identification of the proteins in green cotton fiber using a proteomics-based approach.

The proteome of 21 DPA (days post-anthesis) of green cotton fiber was analyzed using two-dimensional gel electrophoresis to yield the first reference proteome map. Of 220 individual spots that were excised and analyzed by MALDI-TOF/TOF MS, 156 were identified and cataloged according to their functions. Twelve classes of proteins were identified. Many of these proteins were related to carbohydrate metabolism and energy production. Other notable functional classes included oxidoreductase, cell wall-related and cytoskeleton proteins. This study gives a snapshot of the proteome in 21 DPA green cotton fiber and advances our understanding of molecular mechanisms related to pigment biosynthesis in green cotton fiber.

High codon adaptation in citrus tristeza virus to its citrus host.

BACKGROUND: Citrus tristeza virus (CTV), a member of the genus Closterovirus within the family Closteroviridae, is the causal agent of citrus tristeza disease. Previous studies revealed that the negative selection, RNA recombination and gene flow were the most important forces that drove CTV evolution. However, the CTV codon usage was not studied and thus its role in CTV evolution remains unknown. RESULTS: A detailed comparative analysis of CTV codon usage pattern was done in this study. Results of the study show that although in general CTV does not have a high degree of codon usage bias, the codon usage of CTV has a high level of resemblance to its host codon usage. In addition, our data indicate that the codon usage resemblance is only observed for the woody plant-infecting closteroviruses but not the closteroviruses infecting the herbaceous host plants, suggesting the existence of different virus-host interactions between the herbaceous plant-infecting and woody plant-infecting closteroviruses. CONCLUSION: Based on the results, we suggest that in addition to RNA recombination, negative selection and gene flow, host plant codon usage selection can also affect CTV evolution.

Minimally invasive percutaneous osteosynthesis for proximal humeral shaft fractures with the PHILOS through the deltopectoral approach.

PURPOSE: The aim of this study was to evaluate clinical outcomes and complications following minimally invasive plate osteosynthesis (MIPO) with the proximal humeral internal locking system (PHILOS) for treating proximal humeral shaft fracture through the deltopectoral approach. METHODS: Between November 2008 and March 2010, 74 patients with unilateral proximal humeral shaft fractures were treated using the MIPO technique with the PHILOS through the deltopectoral approach. Patients received an average follow-up of 16.9 (range, 12-24) months, and the final follow-up included anteroposterior and lateral imaging and recording of postoperative complications. The Constant-Murley shoulder score was used to evaluate function. RESULTS: No intraoperative complications occurred. Postoperative complications included subacromial impingement in four patients. There was no deep infection, neurovascular damage, breakage or implant loosening. All fractures united in an average time of 17.4 (15-25) weeks. In terms of function, the Constant-Murley score was 85.8 points on average (range, 67-100). The range of motion of the involved shoulder was satisfactory, and pain-free in 83.8 % of patients. CONCLUSIONS: Using the MIPO technique with the PHILOS through the deltopectoral approach is a valid and safe method of treating proximal humeral shaft fractures.

[Application of inferior vena cava filters in prevention of peri-operative pulmonary embolism in patients with bone fracture combined with deep venous thrombosis: a report of 572 cases].

OBJECTIVES: To evaluate the application of inferior vena cava filter (IVCF) in prevention of peri-operative pulmonary embolism in patients with bone fracture combined with deep venous thrombosis (DVT). METHODS: From July 2007 to April 2011, 572 cases of bone fractures consequent with DVT that confirmed by Duplex were deployed IVCF to avoid fatal pulmonary embolism in the para-operative period. There were 318 male and 254 female patients with a mean age of (51±23) years (ranging from 16 to 94 years). RESULTS: All the operations of deployment of the IVCFs were successfully. There were 174 permanent and 399 temporary IVCFs deployed. In all 399 temporary filters, 389 filters were taken out successfully. The mean station time in the IVC of the temporary filters was 22.8 days (7 to 60 days), among which, 118 were found of captured thrombus. After the follow-up of mean 16 months (3 to 40 months) of 566 patients, no pulmonary embolism occurred, but symptomatic DVT were revealed in 15 patients, and the conditions were improved after anticoagulation treatment. No serious complication of post thrombotic syndrome occurred. CONCLUSION: IVCF deployment can prevent the trauma patients with deep vein thrombosis of fatal pulmonary embolism in the peri-operative period, which may also offer a safe condition to the orthopedic operations.

Two new supramolecular Ag(I) coordination polymers: luminescent properties and treatment activity on glioblastoma.

Two new Ag(I) coordination polymers, namely [Ag(bpp)]·0.5 n(1,5-NDSA)·n(H2O) (1) and [Ag2(bpp)2]n·n(2,7-NDSA)·2 n(H2O)·n(CH3CN) (2) (Na2(1,5-NDSA) = sodium 1,5-naphthalenedisulfonate dibasic, Na2(2,7-NDSA) = sodium 1,5-naphthalenedisulfonate dibasic, bpp is 1,3-bis(4-pyridyl)propane), were generated via the solution evaporation method under room temperature. Moreover, the solids of these two compounds display strong luminescence emission at RT. And the application values of the compounds against the glioblastoma treatment were determined, and the corresponding mechanism was simultaneously tested. The analysis of CCK-8 was first implemented and the glioblastoma viability was measured. The real-time RT-PCR was next performed, and the signaling pathway activation of VEGF in glioblastoma cells was tested after treating by the above compound.

Identification and Functional Characterization of a Microtubule-Associated Protein, GhCLASP2, From Upland Cotton (Gossypium hirsutum L.).

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins (MAPs) involved in regulation of dynamics of microtubules (MTs) that play an important role in plant growth and development. In this study, we identified cotton CLASP genes and investigated the function of GhCLASP2. GhCLASP2 was mainly expressed in stem and developing fibers, especially in fibers of the secondary cell wall deposition stage. Ectopic expression of GhCLASP2 in Arabidopsis increased the branching number of leaf trichomes and rescued the defective phenotypes of clasp-1. In cotton, overexpression of GhCLASP2 increased fiber strength, probably related to enhanced expression levels of tubulin, cellulose synthase, and expansin genes. Suppression of GhCLASP2 caused shorter internodes and semi-dwarfism, abnormal flower stigma, aborted anthers without pollen grains, and sterility. These changed phenotypes were similar to those observed in the Arabidopsis clasp-1 mutant. GhCLASP2 was co-localized with MTs according to transient experiment. These results suggest that GhCLASP2 functions similarly as AtCLASP, acting as a MAP and controlling cotton growth and development by regulating MTs.

Function analysis of promoter trapping system after inserted into cotton (Gossypium hirsutum L. ) genome.

The technique of promoter trapping was developed to investigate its viability in cotton ( Gossypium hirsutum L.) functional genomics. 141 independent transformants of cotton were generated via Agrobacterium tumefaciens mediated transformation, of which 97% showed positive by PCR detection. The reporter, GUS gene, was expressed to different extent in different organs, with a frequency of 48% in roots, 9.2% in vascular bundles of stem, 5.2% in leaves, and 51% in flowers. Meanwhile, we discovered that there existed great differences in expression patterns among different transgenic lines. Their GUS expression patterns were organ- or tissue-specific or ubiquitous in all of the plants. The promoter trapping system developed here was characterized as an effective method for creating mutants with diverse reporter gene expression patterns, which laid a solid foundation for further research of functional genomics in cotton.

Polyamine and Its Metabolite H2O2 Play a Key Role in the Conversion of Embryogenic Callus into Somatic Embryos in Upland Cotton (Gossypium hirsutum L.).

The objective of this study was to increase understanding about the mechanism by which polyamines (PAs) promote the conversion of embryogenic calli (EC) into somatic embryos in cotton (Gossypium hirsutum L.). We measured the levels of endogenous PAs and H2O2, quantified the expression levels of genes involved in the PAs pathway at various stages of cotton somatic embryogenesis (SE), and investigated the effects of exogenous PAs and H2O2 on differentiation and development of EC. Putrescine (Put), spermidine (Spd), and spermine (Spm) significantly increased from the EC stage to the early phase of embryo differentiation. The levels of Put then decreased until the somatic embryo stage whereas Spd and Spm remained nearly the same. The expression profiles of GhADC genes were consistent with changes in Put during cotton SE. The H2O2 concentrations began to increase significantly at the EC stage, during which time both GhPAO1 and GhPAO4 expressions were highest and PAO activity was significantly increased. Exogenous Put, Spd, Spm, and H2O2 not only enhanced embryogenic callus growth and embryo formation, but also alleviated the effects of D-arginine and 1, 8-diamino-octane, which are inhibitors of PA synthesis and PAO activity. Overall, the results suggest that both PAs and their metabolic product H2O2 are essential for the conversion of EC into somatic embryos in cotton.

A novel fluid resuscitation protocol: provide more protection on acute kidney injury during septic shock in rats.

OBJECTIVE: To investigate the therapeutic effects of a novel fluid resuscitation protocol (early fluid resuscitation plus 2% hydrogen inhalation) on acute kidney injury during septic shock induced by lipopolysaccharide in rats. METHODS: Sixty male Wistar rats were randomly divided into four groups (n = 15 per group): control group (C), septic shock group (S), septic shock with early fluid resuscitation group (R), and septic shock with early fluid resuscitation plus 2% hydrogen inhalation group (R+R+H2). The rats were ventilated, and a 2% hydrogen mixture was used in Group R+H2. Lipopolysaccharide (10 mg/kg) was administered to establish the septic shock model in rats and fluid resuscitation was performed in Groups R and R+R+H2. RESULTS: Fluid resuscitation with 2% hydrogen inhalation decreased serum creatinine, blood urea nitrogen, and neutrophil gelatinase-associated lipocalin. It also reduced oxidative stress injury and decreased renal tumor necrosis factor-α and interleukin-6 levels compared with fluid resuscitation alone. CONCLUSION: Early fluid resuscitation plus 2% hydrogen inhalation provided more protection against AKI during septic shock.

Toll-like receptor 4 is involved in myocardial damage following paraquat poisoning in mice.

The ingestion of the herbicide paraquat (PQ) can cause multiple organ injury including cardiac lesions. However, the underlying mechanism of myocardial damage is not known. Toll-like receptor 4 (TRL4) is a pattern-recognition receptor in the innate immune response to microbial pathogens. TLR4 is involved in heart dysfunction such as septic shock or myocardial ischemia. We investigated whether TLR4 would be linked to the pathogenesis of heart disease due to PQ exposure. Wild type mice (WT) and TLR4-deficient mice were injected intraperitoneally with 75mg/kg of PQ to induce myocardial damage and tested for echocardiographic assessment, histopathology, pro-inflammatory cytokine and TLR4 expression. WT mice after PQ exposure displayed deteriorate cardiac function, pathological damages, increased TLR4 mRNA and protein levels as well as myocardial TNF-α and IL-1ß levels. Compared with WT mice, TLR4-deficient mice were significantly resistant to the PQ-induced injury. We concluded that the TLR4 was required as a mediator and played an important role in myocardial damage due to PQ.

Exendin-4, a glucagon-like peptide-1 receptor agonist, inhibits cell apoptosis induced by lipotoxicity in pancreatic ß-cell line.

Lipotoxicity plays an important role in the underlying mechanism of type 2 diabetes mellitus. Prolonged exposure of pancreatic ß-cells to elevated concentrations of fatty acid is associated with ß-cell apoptosis. Recently, glucagon-like peptide-1 (GLP-1) receptor agonists have been reported to have direct beneficial effects on ß-cells, such as anti-apoptotic effects, increased ß-cell mass, and improvement of ß-cell function. The mechanism of GLP-1 receptor agonists' protection of pancreatic ß-cells against lipotoxicity is not completely understood. We investigated whether the GLP-1 receptor agonist exendin-4 promoted cell survival and attenuated palmitate-induced apoptosis in murine pancreatic ß-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4mM) for 24h caused a significant increase in cell apoptosis, which was inhibited by exendin-4. Exposure of MIN6 cells to exendin-4 caused rapid activation of protein kinase B (PKB) under lipotoxic conditions. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of exendin-4 on MIN6 cells. Exendin-4 also inhibited the mitochondrial pathway of apoptosis and down-regulated Bax in MIN6 cells. Exendin-4 enhanced glucose-stimulated insulin secretion in the presence of palmitate. Our findings suggest that exendin-4 may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB and inhibition of the mitochondrial pathway.