A candidate multi-epitope vaccine against porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae induces robust humoral and cellular response in mice

Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) are two of the most common pathogens involved in the porcine respiratory disease complex (PRDC) resulting in significant economic losses worldwide. Vaccination is the most effective approach to disease prevention. Since PRRSV and Mhp co-infections are very common, an efficient dual vaccine against these pathogens is required for the global swine industry. Compared with traditional vaccines, multi-epitope vaccines have several advantages, they are comparatively easy to produce and construct, are chemically stable, and do not have an infectious potential. In this study, to develop a safe and effective vaccine, B cell and T cell epitopes of PRRSV-GP5, PRRSV-M, Mhp-P46, and Mhp-P65 protein had been screened to construct a recombinant epitope protein rEP-PM that has good hydrophilicity, strong antigenicity, and high surface accessibility, and each epitope is independent and complete. After immunization in mice, rEP-PM could induce the production of high levels of antibodies, and it had good immunoreactivity with anti-rEP-PM, anti-PRRSV, and anti-Mhp antibodies. The anti-rEP-PM antibody specifically recognizes proteins from PRRSV and Mhp. Moreover, rEP-PM induced a Th1-dominant cellular immune response in mice. Our results showed that the rEP-PM protein could be a potential candidate for the development of a safe and effective multi-epitope peptide combined vaccine to control PRRSV and Mhp infections.     

N-acetyl-l-cysteine protects porcine oocytes undergoing meiotic resumption from heat stress

Heat stress (HS) is a notable risk factor for female reproductive performance. In particular, impaired oocyte maturation was thought to contribute largely to the HS-induced reproductive dysfunctions. In this study, we confirmed that oocytes undergoing GVBD were much susceptible to HS, and thus compromising subsequent embryonic development. Using N-acetyl-l-cysteine (NAC), we found supplementation of a relatively high dose NAC during in vitro maturation, can protect oocytes from HS-induced complications, and thus rescuing impaired embryonic development. Further analysis indicated that mechanisms responsible for protecting GVBD oocytes from HS by NAC may include: (1) reversing disorganized spindle assembly and inhibited extracellular signal–regulated kinase (ERK) signaling; (2) correcting erroneous H3K27me3 modification and dysregulated expression of imprinted genes; (3) alleviating increased intraoocyte reactive oxygen species accumulation and apoptosis initiation. Our study, focusing on the oocyte meiotic maturation, may provide a safe and promising strategy for protecting reproductive sows under environmental hyperthermal conditions.     

Effects of Bisphenol A on expression of genes related to amino acid transporters,insulin- like growth factor,aquaporin and amino acid release by porcine trophectoderm cells

The peri-implantation period of pregnancy is critical for conceptus development, implantation, and signaling for establishment of pregnancy. This study evaluated the effects of bisphenol A (BPA) on proliferation, adhesion, and migration of porcine trophectoderm (pTr2) cells, expression of transporters of arginine and synthesis of amino acids. All concentrations of BPA decreased proliferation and adhesion of pTr2 cells after 96 h compared to the control group. Lower concentrations of BPA (1 × 10⁻⁹, 1 × 10^-8, 10^-7M) increased (P < 0.05), but higher concentrations of BPA (1 × 10^-5, 1 × 10^-4 M) decreased migration of pTr2 cells. BPA increased expression of SLC7A1 mRNA at lower concentrations (1 × 10⁻⁹ to 1 × 10^-6M) and SL7A6, another cationic acid transporter, at higher concentrations (1 × 10^-5, 1 × 10^-4 M). BPA also down-regulated the expression of IGF1 and IGF1 receptor at concentrations of 1 × 10^-7 to 1 × 10^-4 M compared to the control group. The expression of mRNAs for aquaporins (AQP) 3 and 4 were reduced at all concentrations of BPA, but at lower concentrations of BPA, (1 × 10⁻⁹ to 1 × 10^-8M) expression of AQP9 mRNA increased and the expression of AQP11 was not affected by BPA (P > 0.05). There was an inhibitory effect of BPA on the release of synthesis of asparagine, threonine, taurine, tryptophan, and ornithine into the culture medium by pTr2 cells. Collectively, BPA adversely affected the expression of transporters for cationic amino acids like arginine, as well as AQPs, IGF1, and IGF1R associated with proliferation, migration, and adhesion of pTr2 cells. Those adverse effects would likely increase pregnancy losses during the peri-implantation period of pregnancy.     

Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva

PurposeThe long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC).MethodsPDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells.ResultsFrom day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva.ConclusionPlacing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.     

Carbohydrate moieties of procine 32 kDa enamelin

The structures of asparagine-linked oligosaccharides of porcine 32 kDa enamelin are reported. The oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The pyridylamino oligosaccharides were separated into eight kinds of oligosaccharides. The structures of these oligosaccharides were determined by a combination of a sequential exoglycosidase digestion and a two-dimensional suger mapping technique. The oligosaccharides consisted of fucose, galactose, mannose, N-acetylglucosamine, and N-acetylneuraminic acid, and were classified into two groups according to their core-sugar chain structures; one was a biantennary-type and the other was a triantennary-type oligosaccharide. The variation of the oligosaccharides in each of these groups was caused by the differences in the number, the site, and the mode of linkage of N-acetylneuraminic acid to the core-sugar chains.     

Evaluation of specialized laparoscopic suturing and tying devices.

BACKGROUND: Laparoscopic suturing and tying constitute advanced minimally invasive surgery skills. Developing proficiency in the standard methods with needle drivers is often an arduous process. Recent advances in laparoscopic instrumentations has allowed for easier methods of suturing and tying. This study investigated the hypothesis that the use of a specialized suturing device and a specialized tying device allows inexperienced medical students to suture and tie laparoscopically. METHODS: Preclinical medical students who had not received any training in open or laparoscopic surgery were included in this investigation. Each student was given a 5-minute demonstration of a specialized suturing device and a specialized tying device. The medical students were not allowed to deploy either device before actual use. After the demonstration, each student was given the device to use in a porcine model. Times were recorded and a subjective grade was given for each student. RESULTS: Twenty medical students were involved in this study. All medical students were able to complete the task of suturing and tying. The average time to suture was 104.6 seconds and the average time to tying was 31.2 seconds. The average subjective performance grade was 90 (out of 100). CONCLUSION: Specialized devices are easy to learn and use for laparoscopic suturing and tying with minimal instruction even for inexperienced medical students. Even surgeons who are not well versed in laparoscopic surgery should be able to suture and tie with certain laparoscopic instruments.     

再次液氮冷冻保存对猪主动脉瓣膜细胞活性及组织结构的影响

目的了解再次冷冻保存对主动脉瓣膜细胞活性及组织结构的影响,探讨液氮冷冻保存的主动脉瓣解冻后再次冷冻保存使用的可行性.方法将猪主动脉瓣叶在抗菌处理后按随机数字表法分成三组,每组6个瓣叶,组Ⅰ作对照,组Ⅱ、组Ⅲ控制降温速率降至-80℃后在液氮中保存,1个月后融化解冻.组Ⅲ解冻并在室温下放置15分钟后更换保存液,再次降温至-80℃后放入液氮中保存,2个月后再融化解冻.采用XTT比色法测定各组瓣膜细胞活性,用免疫荧光组织化学染色、光学显微镜、透射电子显微镜行组织学检测.结果组Ⅱ冷冻保存后瓣膜细胞活性下降到组Ⅰ的63.97%,组织结构一定程度受损;组Ⅲ瓣膜细胞活性下降至组Ⅰ的38.60%,组织结构损害也进一步加重.结论液氮冷冻保存的猪主动脉瓣一经解冻融化,不宜再次冷冻保存使用.     

Identification of cytotoxic principles from Fusarium avenaceum using bioassay-guided fractionation

The cytotoxicity of extracts from rice cultures of five Fusarium avenaceum strains against the porcine epithelial kidney cell-line PK-15 was investigated using the Alamar Blue™ assay. After the identification of known fungal metabolites, cytotoxic extracts were fractionated using semi-preparative reversed-phase HPLC and normal phase LC, and the fractions were tested for cytotoxicity. In this way, two different groups of metabolites were identified as the major cytotoxic principles of the extracts. High concentrations of enniatins, especially enniatins B and B1, inhibited the metabolic activity of PK-15 cells. Furthermore, an unidentified metabolite, produced in high amounts by a strain that produced relatively small amounts of enniatins, was also found to be cytotoxic to PK-15 cells. This study shows that enniatins, a group of cyclic depsipeptides, which have been ignored as significant contributors to the toxicity of fungal extracts, may account for most of the observed effect for F. avenaceum.     

A One–Step, Operator–Independent Method for Isolating Islets of Langerhans from the Porcine Pancreas

Abstract: Large–scale isolation of islets of Langerhans is one of the major obstacles in islet transplantation. Until now, isolation methods relied on enzymatic digestion, the duration of which relies on a decision dictated by the operator's experience. This approach has always hindered development of an automated method. The aim of this study was to develop a one–step method based on complete digestion of the pancreas. The original aspect of the technique (derived from the Ricordi method) is use of the University of Wisconsin (UW) solution in the digestion medium and a continuous flow collagenase processing circuit with local cooling and rewarming to allow tissue digestion to proceed at 37°C while settling of the cell suspension takes place at 4°C. A stopcock system permits the alternate use of two settling chambers so that while one is in the circuit, the other can be removed for cen–trifugation, resuspension of the crude islet preparation in collagenase in free UW solution, and further purification in a density gradient system. Ten experiments were performed, and 545, 750 ± 48, 670 purified pig islets were obtained per totally digested pancreas. Histological studies showed cell integrity. Insulin secretion in response to double glucose stimulation under perfusion conditions demonstrated the functional viability of the isolated islets. In conclusion, this one–step method makes it possible to obtain a high number of viable islets of Langerhans in the absence of any decision by an operator, and it should therefore provide basis for an automated method.