Intestinal segment and vitamin D3 concentration affect gene expression levels of calcium and phosphorus transporters in broiler chickens.

Two experiments were conducted in this research. Experiment 1 investigated the spatial expression characteristics of calcium (Ca) and phosphorus (P) transporters in the duodenum, jejunum, and ileum of 21-day-old broilers provided with adequate nutrient feed. Experiment 2 evaluated the effects of dietary vitamin D3 (VD3) concentration (0, 125, 250, 500, 1,000, and 2,000 IU/kg) on growth performance, bone development, and gene expression levels of intestinal Ca and P transporters in 1-21-day-old broilers provided with the negative control diet without supplemental VD3. Results in experiment 1 showed that the mRNA levels of calcium-binding protein 28-kDa (CaBP-D28k), sodium-calcium exchanger 1 (NCX1), plasma membrane calcium ATPase 1b (PMCA1b), and IIb sodium-phosphate cotransporter (NaPi-IIb) were the highest in the broiler duodenum. By contrast, the mRNA levels of inorganic phosphate transporter 1 (PiT-1) and 2 (PiT-2) were the highest in the ileum. Results in experiment 2 showed that adding 125 IU/kg VD3 increased body weight gain (BWG), feed intake (FI), bone weight, and percentage and weight of Ca and P in the tibia and femur of 1-21-day-old broilers compared with the negative control diet (p < 0.05). The rise in dietary VD3 levels from 125 to 1,000 IU/kg further increased the BWG, FI, and weights of the bone, ash, Ca, and P (p < 0.05). No difference in growth rate and leg bone quality was noted in the broilers provided with 1,000 and 2,000 IU/kg VD3 (p > 0.05). Supplementation with 125-2,000 IU/kg VD3 increased the mRNA abundances of intestinal Ca and P transporters to varying degrees. The mRNA level of CaBP-D28k increased by 536, 1,161, and 28 folds in the duodenum, jejunum, and ileum, respectively, after adding 1,000 IU/kg VD3. The mRNA levels of other Ca and P transporters (PMCA1b, NCX1, NaPi-IIb, PiT-1, and PiT-2) increased by 0.57-1.74 folds by adding 1,000-2,000 IU/kg VD3. These data suggest that intestinal Ca and P transporters are mainly expressed in the duodenum of broilers. Moreover, the addition of VD3 stimulates the two mineral transporter transcription in broiler intestines.

Age quadratically affects intestinal calcium and phosphorus transporter gene expression in broiler chickens.

OBJECTIVE: This research aimed to evaluate the effects of age on growth, tibia development, and intestinal calcium (Ca) and phosphorus (P) transporter gene expressions in broiler chickens. METHODS: A total of 224 male Arbor Acres broilers were fed with nutrient-adequate diets and reared in eight cages (28 broilers per cage). Eight broilers (one broiler per cage) were selected and killed at 5, 10, 15, 20, 25, 30, 35, and 40 days of age, respectively. RESULTS: Body weight continuously increased with age of broiler chickens from 5 to 40 days. The bone weight, ash weight, diameter, and length of the tibia also increased with broiler age. By contrast, the tibia ash, Ca, and P percentages quadratically changed with age (p<0.001), and the highest values of mineral contents were observed at 20, 25, and 25 days of age, respectively. The mRNA abundances of calcium-binding protein 28-kDa (CaBP-D28k), sodium-calcium exchanger 1 (NCX1), and plasma membrane ATPase 1b (PMCA1b) increased from 5 to 25 days and then decreased up to 40 days. Similar results were noted in the mRNA abundances of IIb sodium-phosphate cotransporter (NaPi-IIb), inorganic phosphate transporter 1 (PiT-1), inorganic phosphate transporter 2 (PiT-2), nuclear vitamin D receptor (nVDR), and membrane vitamin D receptor (mVDR). The mRNA abundances of Ca and P transporters and VDRs were the highest at 25 days of age. CONCLUSION: These data indicate that age quadratically affects intestinal Ca and P transporter gene expression and mineral absorption capacity in broiler chickens.

One-pot synthesis of Au-M@SiO2 (M = Rh, Pd, Ir, Pt) core-shell nanoparticles as highly efficient catalysts for the reduction of 4-nitrophenol.

The conversion of p-nitrophenol (4-NP) to p-aminophenol (4-AP) is of great significance for pharmaceutical and material manufacturing. In this work, Au-M@SiO2 (M = Rh, Pd, Ir, Pt) nanoparticles (NPs) with core-shell structures, which are expected to be excellent catalysts for the transformation of 4-NP to 4-AP, were synthesized by a facile one-pot one-step method. The structure and composition of the NPs were characterized through transmission electron microscopy, X-ray powder diffraction and X-ray photoelectron spectroscopy. Au-M@SiO2 (M = Rh, Pd, Ir, Pt) core-shell NPs showed excellent catalytic activity in the reduction of 4-NP, which is superior to most catalysts reported in the previous literature. The enhanced catalytic activity of Au-M@SiO2 core-shell NPs is presumably related to the bimetallic synergistic effect. This study provides a simple strategy to synthesize core-shell bimetallic NPs for catalytic applications.

Immunization with a combination of recombinant Brucella abortus proteins induces T helper immune response and confers protection against wild-type challenge in BALB/c mice.

Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. Four proteins were cloned, expressed and purified, and their immunocompetence was analysed. BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV. Cellular and humoral immune responses were determined by ELISA. Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum. A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV. In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo. Further investigation of the CSV showed a superior protective effect in mice against brucellosis. The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51. Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis.

Optimal coordinated control of hybrid AC/VSC-HVDC system integrated with DFIG via cooperative beetle antennae search algorithm.

Nowadays, with the significant integration of various renewable energy, hybrid alternating current/ voltage source converter based high voltage direct current (AC/VSC-HVDC) system integrated with doubly-fed induction generator (DFIG) has achieved rapidly development in smart grid. A proper control system design for hybrid AC/VSC-HVDC system plays a very crucial role for a reliable and effective power transmission. Hence, this paper designs a novel cooperative beetle antenna search (CBAS) algorithm for optimal coordinated control of hybrid AC/VSC-HVDC system integrated with DFIG. Compared with original beetle antennae search (BAS) algorithm, CBAS algorithm can significantly improve searching efficiency via an efficient cooperation with a group of multiple beetles instead of a single beetle. Particularly, CBAS algorithm can effectively escape from local optimums thanks to its dynamic balance mechanism, which can maintain appropriate trade-off between global exploration and local exploitation. Moreover, three case studies are undertaken to validate the effectiveness and superiorities and effectiveness of CBAS algorithm compared against that of other traditional meta-heuristic algorithms. Especially, the average results of fitness function acquired by CBAS algorithm is merely 46.05%, 41.18%, and 47.82% of that of PSO, GA, and BAS algorithm, respectively.

Integrated System for Purification and Assembly of PCV Cap Nano Vaccine Based on Targeting Peptide Ligand.

PURPOSE: The vaccine design has shifted from attenuated or inactivated whole pathogen vaccines to more pure and defined subunit vaccines. The purification of antigen proteins, especially the precise display of antigen regions, has become a key step affecting the effectiveness of subunit vaccines. MATERIALS AND METHODS: This work presents the application of molecular docking for a peptide ligand designed for PCV2 Cap purification and assembly in one step. Based on the PCV2 Cap protein affinity peptide (L11-DYWWQSWE), the amino terminal of PCV2 Cap was covalently coupled with the polylactic acid-glycolic acid copolymer (PLGA) carboxyl terminal through the EDC/NHS method. RESULTS: The PLGA had an average diameter of 106 nm. The average diameter increased to 122 nm after the PCV2 Cap protein conjugation, and the Zeta potential shifted from -13.7 mV to -9.6 mV, indicating that the PCV2 Cap protein stably binds to the PLGA. Compared with the free PCV2 Cap protein group, the neutralizing antibody titer was significantly increased on the 14th day after the PLGA-Cap immunization (P < 0.05). The neutralizing antibody level was extremely significant on the 28th day (P < 0.001). The CCK-8 analysis showed that PLGA-Cap had an obvious cytotoxic effect on RAW264.7 cells at the PLGA nanoparticle concentration up to 200 µg/mL but had no obvious cytotoxic effect on DC2.4 cells. Compared with the Cap protein group, the antigen-presenting cells had a stronger antigen uptake capacity and a higher fluorescence in the PLGA-Cap group. The immune effect showed that the level of the neutralizing antibody produced by this structure is much better than that of purified protein and helps improve the immune system response. CONCLUSION: This technology provides a potential new perspective for the rapid enrichment of the antigen protein with the affinity peptide ligand.

Ethylene-responsive factor 4 is associated with the desirable rind hardness trait conferring cracking resistance in fresh fruits of watermelon.

Fruit rind plays a pivotal role in alleviating water loss and disease and particularly in cracking resistance as well as the transportability, storability and shelf-life quality of the fruit. High susceptibility to cracking due to low rind hardness is largely responsible for severe annual yield losses of fresh fruits such as watermelon in the field and during the postharvest process. However, the candidate gene controlling the rind hardness phenotype remains unclear to date. Herein, we report, for the first time, an ethylene-responsive transcription factor 4 (ClERF4) associated with variation in rind hardness via a combinatory genetic map with bulk segregant analysis (BSA). Strikingly, our fine-mapping approach revealed an InDel of 11 bp and a neighbouring SNP in the ClERF4 gene on chromosome 10, conferring cracking resistance in F2 populations with variable rind hardness. Furthermore, the concomitant kompetitive/competitive allele-specific PCR (KASP) genotyping data sets of 104 germplasm accessions strongly supported candidate ClERF4 as a causative gene associated with fruit rind hardness variability. In conclusion, our results provide new insight into the underlying mechanism controlling rind hardness, a desirable trait in fresh fruit. Moreover, the findings will further enable the molecular improvement of fruit cracking resistance in watermelon via precisely targeting the causative gene relevant to rind hardness, ClERF4.

Neoadjuvant dose-modified docetaxel in squamous cell carcinoma of the head and neck: A phase 3 study.

OBJECTIVE: To evaluate the efficacy and safety of dose-modified docetaxel plus cisplatin and 5-fluorouracil (TPF) in Chinese patients with squamous cell carcinoma of the head and neck (SCCHN). MATERIALS AND METHODS: This Phase III, open-label, multi-center study included Chinese adults with previously untreated TNM Stage III or IV SCCHN (NCT00995293). Patients were randomized (1:1) to induction chemotherapy with TPF (docetaxel 60 mg/m2 and cisplatin 60 mg/m2 on day 1 and 5-FU 750 mg/m2  per day continuous IV infusion on days 1-5) or PF (cisplatin 75 mg/m2 on day 1 and 5-FU 750 mg/m2  per day on days 1-5) every 3 weeks for 3-4 cycles. The primary endpoint was progression-free survival (PFS). RESULTS: Median PFS in the TPF (n = 108) and PF (n = 111) groups was 400 days and 342 days (HR = 0.75; 95% CI, 0.53─1.06; p = .227), respectively. Overall response rate was higher for TPF versus PF (76.3% vs. 52.9%; p = .001), although this equalized following radiotherapy (75.0% vs. 73.9%). In the TPF and PF groups, ≥1 treatment-emergent adverse event was experienced by 104 (94.5%) and 110 (93.2%) patients, respectively. CONCLUSION: Adding dose-modified docetaxel to PF did not significantly improve PFS but may increase anti-tumor activity in Chinese patients with locally advanced SCCHN.

Design and preliminary application of affinity peptide based on the structure of the porcine circovirus type II Capsid (PCV2 Cap).

BACKGROUND: Affinity peptides, as a core part of affinity chromatography, play an important role in the purification of target molecules. METHODS: Here we describe the use of molecular docking technology for virtual screening of affinity peptides that specifically recognize the PCV2 Cap protein for the first time. Thirteen candidate peptides with high scores were obtained and then further characterized. Experimentally, the affinity and sensitivity of the peptides studied were identified by ELISA and LSPR, respectively. In order to investigate the purification effect of a selected peptide (L11) for the recombinant PCV2 Cap protein, it was coupled to NHS agarose magnetic beads as an affinity adsorbent (NaMB-L11); and the ligand density of the affinity adsorbent and pH value in the purification of the recombinant PCV2 Cap protein were optimized. RESULTS: Our data showed that the peptide L11- DYWWQSWE has the smallest KD = 103 nM with higher specificity for PCV2 Cap protein recognition. The NaMB-L11 affinity adsorbent yielded a purified Cap sample with 98% purity at 90% recovery in a single step. CONCLUSION: Based on the structure, we obtained a high affinity peptide L11 binding to the PCV2 Cap protein by molecular docking technology. It not only provides a theoretical basis for the design of PCV2 Cap affinity peptide, but a new method for the purification of the PCV2 Cap protein.

Rapid detection of giant salamander iridovirus by cross-priming amplification.

Giant salamander iridovirus (GSIV) belongs to the epizootic genus Ranavirus, and is the cause of epidemic diseases associated with high mortality and great losses to artificial breeding and farming. Here, we established a simple, accurate, and reliable cross-priming amplification (CPA) method to detect GSIV. The CPA assay targets the major caspid protein gene of the GSIV genome to design crossing primer pairs, and the reaction conditions were optimized, including optimal concentrations of the primers, betaine, dNTPs, Mg2+, and Bst DNA polymerase, and reaction conditions. The sensitivity was shown to be 10 times higher than that of conventional polymerase chain reaction (PCR), and the specificity was 100%. The results were identified on nucleic acid strips within 3-5 min. Application of the CPA and PCR to 54 samples of giant salamander showed a positive rate of 72.22% and 74.07%, respectively, demonstrating high coincidence (94.44%, kappa = 8.7, P < 0.0001). The sensitivity of the CPA assay was 97.50% and the specificity was 92.86%. Thus, the CPA assay is as effective as conventional PCR, but with added practical advantages of simplicity and an almost instrument-free platform, which will be useful for both laboratories and giant salamander farms.

Comparison of two docking methods for peptide-protein interactions.

BACKGROUND: The importance of peptides in regulatory interactions has caused peptide-protein docking to attract the attention of many researchers. A variety of methods for molecular modeling of peptide-protein docking, such as local search and global search, are currently used. RESULTS: The interactions of 11 peptides and CSFV E2 protein were evaluated by the GalaxyPepDock and FlexX/ SYBYL programs, respectively. The assessment scores of all the peptides were correlated with their KD values. The final results showed that a moderate correlation coefficient was represented between KD values and CScores of predicted models by FlexX/ SYBYL. CONCLUSION: Our results demonstrate that considering the flexibility of the peptide is better than searching for more potential binding sites on the target protein surface while performing peptide-protein molecular docking. These data provide reasonable evidence for the molecular design of peptides and guidance for the functional assignment of target proteins. © 2018 Society of Chemical Industry.

Factors influencing the incidence of sinusitis in nasopharyngeal carcinoma patients after intensity-modulated radiation therapy.

The aim of the study was to investigate the incidence of sinusitis in nasopharyngeal carcinoma (NPC) patients before and after intensity-modulated radiation therapy (IMRT) and to analyze factors associated with the incidence of sinusitis following IMRT. We retrospectively analyzed 283 NPC patients who received IMRT in our hospital from March 2009 to May 2011. The diagnostic criteria for sinusitis are based on computed tomography (CT) or magnetic resonance imaging (MRI) findings. CT or MRI scans were performed before and after IMRT to evaluate the incidence of sinusitis. Factors influencing the incidence of sinusitis were analyzed by log-rank univariate and logistic multivariate analyses. Among the 283 NPC patients, 128 (45.2 %) suffered from sinusitis before radiotherapy. The incidence rates of sinusitis in patients with T1, T2, T3, and T4 NPC before radiotherapy were 22.6, 37.5, 46.8, and 61.3 %, respectively (χ 2 = 14.548, p = 0.002). Among the 155 NPC patients without sinusitis before radiotherapy, the incidence rates of sinusitis at the end of radiotherapy and at 1, 3, 6, 9, 12, and 18 months after radiotherapy were 32.9, 43.2, 61.3, 68.4, 73.5, 69.7, and 61.3 %, respectively (χ 2 = 86.461, p < 0.001). Univariate analysis showed that T stage, invasion of the nasal cavity, nasal irrigation, and radiation dose to the nasopharynx were associated with the incidence of sinusitis in NPC patients after IMRT (p = 0.003, 0.006, 0.002, and 0.020). Multivariate analysis showed that T stage, invasion of the nasal cavity, and nasal irrigation were influential factors for the incidence of sinusitis in NPC patients after IMRT (p = 0.002, 0.002, and 0.000). There was a higher incidence of sinusitis with higher T stage among NPC patients before radiotherapy, and the incidence of sinusitis in NPC patients after IMRT was high (45.2 %). The incidence of sinusitis increased rapidly within the first 3 months after IMRT, and the number of sinusitis cases peaked at 6-9 months after IMRT and showed a trend toward stabilization after 1 year. Advanced T stage, invasion of the nasal cavity, and nasal irrigation were positively associated with the incidence of sinusitis in NPC patients after IMRT.

Hepatitis C and alcohol exacerbate liver injury by suppression of FOXO3.

Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3(-/-) mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2(+/-)) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol-induced liver injury.

The ClC-3 chloride transport protein traffics through the plasma membrane via interaction of an N-terminal dileucine cluster with clathrin.

ClC-3 is a ubiquitously expressed chloride transport protein that is present in synaptic vesicles and endosome/lysosome compartments. It is largely intracellular but has been observed at the plasma membrane as well. The aim of this study was to identify the pathways and regulation of ClC-3 trafficking to intracellular sites. At the steady state, approximately 94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [(35)S]methionine and biotinylation demonstrated that about 25% of newly synthesized ClC-3 traffics through the plasma membrane. We used both immunofluorescence microscopy and biotinylation assays to assess the trafficking of ClC-3. Plasma membrane ClC-3 was rapidly endocytosed (t((1/2)) approximately 9 min); a portion entered a recycling pool that returned to the cell surface after internalization, and the remainder trafficked to more distal intracellular compartments. ClC-3 associated with clathrin at the plasma membrane. Coimmunoprecipitation and glutathione S-transferase pulldown assays demonstrated that the N terminus of ClC-3 binds to clathrin. Alanine replacement of a dileucine acidic cluster within the cytosolic N terminus (amino acids 13-19) resulted in a molecule that had decreased endocytosis and increased surface expression. This replacement also abolished interaction with clathrin as assessed both by coimmunoprecipitation and glutathione S-transferase pulldown assays. We conclude that ClC-3 is primarily an intracellular transport protein that is transiently inserted into the plasma membrane where it is rapidly endocytosed. Internalization of ClC-3 depends on the interaction between an N-terminal dileucine cluster and clathrin.

A mutation inactivating the methyltransferase gene in avirulent Madrid E strain of Rickettsia prowazekii reverted to wild type in the virulent revertant strain Evir.

Rickettsia prowazekii Madrid E (E) strain is an effective vaccine, but can revert to virulent status when passaged in animals. The aim of this study is to identify the reverse mutation that may determine the virulence of R. prowazekii by comparing the genetic structures of E strain and its virulent revertant Evir strain. We determined that the gene (Rp028/Rp027) encoding the methyltransferase was mutated by frameshift in avirulent E strain but not in virulent revertant Evir strain and wild type virulent Breinl strain. We conclude that the mutation in the E strain gene reverts to wild type in the virulent revertant Evir strain. Whether the mutation plays an essential role in the attenuation of E strain needs to be further investigated.

[Effects of c-jun on hCG-induced testosterone secretion of rat Leydig cells in vitro].

OBJECTIVE: To investigate the effects of c-jun on hCG-induced testosterone secretion in isolated rat Leydig cells by antisense oligodeoxynucleotides(ASODNs). METHODS: c-jun ASODNs were used to antagonise the effects of c-jun, hCG was used to induce the testosterone secretion of LC cultured in vitro and testosterone was measured by radioimmunoassay. RESULTS: The testosterone secretion of LC in vitro could be induced by hCG, which was a good model for the functional study of LC. c-jun ASODNs decreased the hCG-induced testosterone secretion of LC in a dose-dependent manner(P < 0.05). CONCLUSION: It is suggested that c-jun proto-oncogene enhances the testosterone secretion of LC.