Biocompatible Preparation of Beta-Lactoglobulin/Chondroitin Sulfate Carrier Nanoparticles and Modification of Their Colloidal and Hydropathic Properties by Tween 80.

The electrostatic complexation of the protein beta-lactoglobulin (ß-LG) with the anionic polysaccharide chondroitin sulfate (CS) and the subsequent stabilization by thermal treatment were studied to achieve the well-defined nanoparticles (NPs). The formation of the well-defined NPs was obtained at pH 4 with a hydrodynamic radius from 60 to 80 nm. NP aggregation was observed at pH 1.5 because of the loss of the anionic charge of chondroitin sulfate on the surface of the NPs. After thermal treatment, the NPs exhibited stability against a pH increase to pH 7 while a stronger aggregation at pH 1.5 was observed. Core-shell structures were found at pH 7 after thermal treatment, indicating a possible mechanism of partial disintegration. The addition of Tween 80 (T80) before thermal treatment led to the formation of T80 self-assemblies inside the NPs. This caused an increase in the hydrophobicity of the inner and outer surfaces of the NPs as it was observed by fluorescence spectroscopy. The ζ-potential of the complexes and NPs was about -20 mV while the presence of T80 did not affect it. FTIR spectra verified changes of the secondary structure of ß-LG in its complexes with CS and T80. The thermally treated NPs exhibited high surface and overall hydrophobicity and stability in high salinity and biocompatible solutions. The thermally treated NPs showed colloidal and physicochemical stability for 1 month, which were enhanced by the addition of T80. Due to the nature of the precursors and their colloidal properties, the NPs are highly promising for applications as biocompatible drug delivery nanocarriers while T80 acts as an agent to modify their properties.

Genetic and Pathogenic Analysis of a Novel Porcine Epidemic Diarrhea Virus Strain Isolated in the Republic of Korea.

Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), emerges annually in several Asian countries. Its major symptoms include watery diarrhea, vomiting, anorexia, and dehydration. PED outbreaks incur significant economic losses. The efficacy of vaccines is limited by viral mutations and insufficient intestinal mucosal immunity. Therefore, new vaccines against these recent variants are urgently needed. Herein, we isolated and genetically characterized a novel Korean PEDV strain using NGS. Comparative genomic analysis demonstrated that the CKK1-1 strain belonged to genogroup 2. The isolated strain was cultured in sodium-glycochenodeoxycholic acid for 180 passages. Typically, PEDV isolation and passage require proteases, such as trypsin. However, the CKK1-1 strain adapted to this atypical culture condition, achieving a high titer of 8.83 ± 0.14 log TCID50/mL. In vitro biological analysis revealed no cell syncytium formation without trypsin; however, a cell-lysis-type cytopathic effect was noted. Notably, pathogenicity evaluation showed that CKK1-1 p0 exhibited naturally weakened virulence in five-day-old piglets, while piglets administered with CKK1-1 p180 exhibited 100% survival and reduced clinical symptoms. Collectively, our data demonstrate that this Korean PEDV strain, attenuated through atypical culture conditions with Na-glycochenodeoxycholic acid, has potential as a vaccine candidate, providing valuable insights into the genetic variation in and pathogenicity of PEDV.

Application of Methods Detecting Xenotransplantation-Relevant Viruses for Screening German Slaughterhouse Pigs.

Detection methods have been developed to prevent transmission of zoonotic or xenozoonotic porcine viruses after transplantation of pig organs or cells to the recipient (xenotransplantation). Eleven xenotransplantation-relevant viruses, including porcine cytomegalovirus, porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses -1, -2, -3 (PLHV-1, 2, 3), porcine parvovirus (PPV), porcine circovirus 2, 3, 4 (PCV2, 3, 4), hepatitis E virus genotype 3 (HEV3), porcine endogenous retrovirus-C (PERV-C), and recombinant PERV-A/C have been selected. In the past, several pig breeds, minipigs, and genetically modified pigs generated for xenotransplantation had been analyzed using these methods. Here, spleen, liver, and blood samples from 10 German slaughterhouse pigs were screened using both PCR-based and immunological assays. Five viruses: PCMV/PRV, PLHV-1, PLHV-3, and PERV-C, were found in all animals, and PCV3 in one animal. Some animals were latently infected with PCMV/PRV, as only virus-specific antibodies were detected. Others were also PCR positive in the spleen and/or liver, indicative of an ongoing infection. These results provide important information on the viruses that infect German slaughterhouse pigs, and together with the results of previous studies, they reveal that the methods and test strategies efficiently work under field conditions.

Phylogenetic and Evolutionary Analysis of Porcine Epidemic Diarrhea Virus in Guangxi Province, China, during 2020 and 2024.

The variant porcine epidemic diarrhea virus (PEDV) has caused considerable economic losses to the global pig industry since 2010. In this study, a total of 5859 diarrhea samples were collected from different pig farms in China's Guangxi province during January 2020 and March 2024 and tested for PEDV using RT-qPCR. The positivity rate of PEDV was 11.90% (697/5859). Ninety-two PEDV-positive samples were selected based on sampling time, and the sampling region for amplification, sequencing, and analysis of the S1, M, and N genes. Phylogenetic analysis of the S1 gene revealed that all strains from Guangxi province were distributed in three subgroups, i.e., 81.5% (75/92) in the G2a subgroup, 4.3% (4/92) in the G2b subgroup, and 14.1% (13/92) in the G2c subgroup. The sequence analysis revealed that the S1 gene sequences from Guangxi province had higher homology with the variant strains than with the classical strains, showing as high as 99.2% with the variant strain AJ1102 and only 94.3% with the classical strain CV777. Recombination analysis revealed that the GX-BS08-2023 strain (G2c) from Guangxi province originated from inter-lineage recombination between the GX-BS09-2023 (G2a) and CH-JN547228-2011 (G1a) strains. In addition, the S1 gene of the G2a and G2b subgroup strains shared many mutations and insertions. There were common mutations of N143D and P235L in the G2a subgroup. Evolutionary analysis revealed that all Guangxi strains belonged to the G2 genotype. These strains have spread rapidly since the PEDV variant strains that emerged in 2010, weakened until 2021, and then remained stable. In conclusion, the results revealed the latest genetic evolution of circulating PEDV strains in Guangxi province in recent years, providing important information for preventing and controlling PEDV infection. Currently, the G2a subgroup strains are the predominant strains circulating in pig herds in Guangxi province, southern China.

Real-time surgical simulation training for recurrent laryngeal nerve and facial nerve dissection.

BACKGROUND: Head and neck surgical simulation training (SST) is an important part in otolaryngology head and neck surgical education. In this study, we provide a live porcine model for SST in recurrent laryngeal nerve (RLN) and facial nerve (FN) dissection for otolaryngology head and neck residents. METHODS: A lecture with surgical manual is provided to illustrate the surgical landmarks of pig, and step-by-step procedures for thyroid and parotid surgery, as well as neck dissection. We used 4-month-old pig weighting 32 kg for the SST. The mentor demonstrated result of RLN injury with continuous nerve monitoring. Participants used monopolar stimulation probe (4 pulse/s, 100 µs, 3-8 mA; Medtronic) to identify and intermittent monitor the RLN and FN during the SST. After the dissection course, we conducted a questionnaire survey to check the effectiveness of this training model. RESULTS: Total 30 participants were recruited, including 16 female and 14 male resident doctors. There were 1, 4 and 25 learners for 3rd year, 4th and 5th years residents, respectively. Before this training course, 53 % (16/30) and 63 % (19/30) had successful experience in finding the RLN and FN, respectively. After the SST, all of our participants had successful identify the RLN and FN (p-value <0.01); all had positive response to stimulation and familiar with the procedure. CONCLUSIONS: The live porcine model is effectiveness in SST for RLN and FN dissection. Live porcine model with real-time RLN and FN monitoring should be provided for otolaryngology head and neck resident training.

Assessing the Impact of PRESERFLO MicroShunt on Intraocular Pressure in Porcine Eyes Ex Vivo Using Infusion Pump System.

To evaluate the effectiveness of the PRESERFLO MicroShunt (PFM) in reducing intraocular pressure (IOP) ex vivo in porcine eyes using an infusion pump system and to simulate various IOP conditions, In this study, porcine eyes received increasing flows between 2 and 20 µL/min. IOP measurements were taken under conditions with and without the PFM [PFM (+) and PFM (-), respectively]. In the PFM (-) group, IOP increased from 7.4 mmHg to 46.3 mmHg as the flow rate increased from 2 µL/min to 20 µL/min. The rate of IOP reduction (%ΔIOP) rose with increasing flow rates, although the absolute IOP values achieved with the PFM insertion also increased. The correlation between IOPs in the PFM (-) conditions and the %ΔIOP was modeled as %ΔIOP = 22.4 Ln [PFM(-) IOP] - 41.7. According to this equation, IOP reduction by PFM insertion is 0% at IOPs of 6.4 mmHg or lower. IOP reductions of 10%, 20%, 30%, and 40% were observed when the pre-insertion IOPs were 10.1, 15.7, 24.6, and 38.4 mmHg, respectively. Achievable post-insertion IOP levels of ≤21 mmHg, ≤18 mmHg, ≤15 mmHg, and ≤12 mmHg corresponded to the initial IOPs of 33 mmHg, 26 mmHg, 20 mmHg, and 14.8 mmHg, respectively. In conclusion, the PFM effectively reduced IOP within a specific range of IOP values in an ex vivo experimental system. In clinical situations, the PFM is unlikely to be effective at low IOP levels. At higher levels, the PFM reduces IOP, but it may be insufficient to achieve the target IOP.

Mitochondria of Porcine Oocytes Synthesize Melatonin, Which Improves Their In Vitro Maturation and Embryonic Development.

The in vitro maturation efficiency of porcine oocytes is relatively low, and this limits the production of in vitro porcine embryos. Since melatonin is involved in mammalian reproductive physiology, in this study, we have explored whether endogenously produced melatonin can help in porcine oocyte in vitro maturation. We have found, for the first time in the literature, that mitochondria are the major sites for melatonin biosynthesis in porcine oocytes. This mitochondrially originated melatonin reduces ROS production and increases the activity of the mitochondrial respiratory electron transport chain, mitochondrial biogenesis, mitochondrial membrane potential, and ATP production. Therefore, melatonin improves the quality of oocytes and their in vitro maturation. In contrast, the reduced melatonin level caused by siRNA to knockdown AANAT (siAANAT) is associated with the abnormal distribution of mitochondria, decreasing the ATP level of porcine oocytes and inhibiting their in vitro maturation. These abnormalities can be rescued by melatonin supplementation. In addition, we found that siAANAT switches the mitochondrial oxidative phosphorylation to glycolysis, a Warburg effect. This metabolic alteration can also be corrected by melatonin supplementation. All these activities of melatonin appear to be mediated by its membrane receptors since the non-selective melatonin receptor antagonist Luzindole can blunt the effects of melatonin. Taken together, the mitochondria of porcine oocytes can synthesize melatonin and improve the quality of oocyte maturation. These results provide an insight from a novel aspect to study oocyte maturation under in vitro conditions.

Intensive Surveillance of Porcine-Rhesus Kidney Xenotransplant Using Different Ultrasound Techniques.

BACKGROUND: Significant progress has been made in kidney xenotransplantation in the past few years, and this field is accelerating towards clinical translation. Therefore, surveillance of the xenograft with appropriate tools is of great importance. Ultrasonography has been widely used in kidney allotransplantation and served as an economical and non-invasive method to monitor the allograft. However, questions remain whether the ultrasonographic criteria established for human kidney allograft could also be applied in xenotransplantation. METHODS: In the current study, we established a porcine-rhesus life sustaining kidney xenotransplantation model. The xenograft underwent intensive surveillance using gray-scale, colorful Doppler ultrasound as well as 2D shear wave elastography. The kidney growth, blood perfusion, and cortical stiffness were measured twice a day. These parameters were compared with the clinical data including urine output, chemistry, and pathological findings. RESULTS: The observation continued for 16 days after transplantation. Decline of urine output and elevated serum creatinine were observed on POD9 and biopsy proven antibody-mediated rejection was seen on the same day. The xenograft underwent substantial growth, with the long axis length increased by 32% and the volume increased by threefold at the end of observation. The resistive index of the xenograft arteries elevated in response to rejection, together with impaired cortical perfusion, while the peak systolic velocity (PSV) was not compromised. The cortical stiffness also increased along with rejection. CONCLUSION: In summary, the ultrasound findings of kidney xenograft shared similarities with those in allograft but possessed some unique features. A modified criteria needs to be established for further application of ultrasound in kidney xenotransplantation.

Docosahexaenoic acid supplementation during porcine oocyte in vitro maturation improves oocyte quality and embryonic development by enhancing the homeostasis of energy metabolism.

Although supplementation with docosahexaenoic acid (DHA) during porcine oocyte IVM is well-established, the available data are limited due to the lack of consistency. Moreover, to our knowledge, the anti-oxidant effects of DHA on porcine oocytes have not been reported. Hence, this study aimed to examine the effects of DHA supplementation on the regulation of energy metabolism during porcine oocyte maturation to improve oocyte maturation and embryonic development. By supplementing the IVM medium with various DHA concentrations, 25 µM DHA was identified as the optimal concentration which improved intraoocyte glutathione content and enhanced embryonic development after parthenogenesis. Compared to embryos derived from the control group, those derived from SCNT or IVF showed significantly improved blastocyst formation upon DHA supplementation during IVM. In addition, various transcription factors associated with oocyte development and apoptosis in mature oocytes were beneficially regulated in the DHA-treated oocytes. Moreover, DHA improved the AMP-activated protein kinase (AMPK)-regulatory ability of porcine oocytes and ameliorated nuclear maturation and embryonic development, which were decreased by artificially downregulating AMPK. To our knowledge, this is the first study to examine the effects of DHA as an AMPK regulator on oocyte maturation and embryo development in pigs. Furthermore, DHA addition to the IVM medium upregulated the relative expression of genes associated with mitochondrial potential and lipid metabolism. Therefore, the membrane potential of mitochondria (evaluated based on the JC-1 aggregate/JC-1 monomer ratio) and the levels of fatty acids and lipid droplets in matured oocytes increased, resulting in increased ATP synthesis. In conclusion, the DHA treatment of porcine oocytes with 25 µM DHA during IVM enhances the homeostasis of energy metabolism by improving mitochondrial function and lipid metabolism, leading to improved quality of matured oocytes and enhanced embryonic developmental potential of in vitro produced (IVP) embryos. Thus, 25 µM DHA supplementation could serve as a tool for improving the quality of IVP embryos. The study findings provide a basis for further research on improving the production efficiency of cloned animals by securing high-quality matured oocytes and enhancing energy metabolism in mammalian oocytes, including those of pigs.

Prediction of Left Ventricle Pressure Indices Via a Machine Learning Approach Combining ECG, Pulse Oximetry, and Cardiac Sounds: a Preclinical Feasibility Study.

Heart failure (HF) is defined as the inability of the heart to meet body oxygen demand requiring an elevation in left ventricular filling pressures (LVP) to compensate. LVP increase can be assessed in the cardiac catheterization laboratory, but this procedure is invasive and time-consuming to the extent that physicians rather rely on non-invasive diagnostic tools. In this work, we assess the feasibility to develop a novel machine-learning (ML) approach to predict clinically relevant LVP indices. Synchronized invasive (pressure-volume tracings) and non-invasive signals (ECG, pulse oximetry, and cardiac sounds) were collected from anesthetized, closed-chest Göttingen minipigs. Animals were either healthy or had HF with reduced ejection fraction and circa 500 heartbeats were included in the analysis for each animal. The ML algorithm showed excellent prediction of LVP indices estimating, for instance, the end-diastolic pressure with a R2 of 0.955. This novel ML algorithm could assist clinicians in the care of HF patients.

Host cells reprogram lipid droplet synthesis through YY1 to resist PRRSV infection.

Metabolism in host cells can be modulated after viral infection, favoring viral survival or clearance. Here, we report that lipid droplet (LD) synthesis in host cells can be modulated by yin yang 1 (YY1) after porcine reproductive and respiratory syndrome virus (PRRSV) infection, resulting in active antiviral activity. As a ubiquitously distributed transcription factor, there was increased expression of YY1 upon PRRSV infection both in vitro and in vivo. YY1 silencing promoted the replication of PRRSV, whereas YY1 overexpression inhibited PRRSV replication. PRRSV infection led to a marked increase in LDs, while YY1 knockout inhibited LD synthesis, and YY1 overexpression enhanced LD accumulation, indicating that YY1 reprograms PRRSV infection-induced intracellular LD synthesis. We also showed that the viral components do not colocalize with LDs during PRRSV infection, and the effect of exogenously induced LD synthesis on PRRSV replication is nearly lethal. Moreover, we demonstrated that YY1 affects the synthesis of LDs by regulating the expression of lipid metabolism genes. YY1 negatively regulates the expression of fatty acid synthase (FASN) to weaken the fatty acid synthesis pathway and positively regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ) to promote the synthesis of LDs, thus inhibiting PRRSV replication. These novel findings indicate that YY1 plays a crucial role in regulating PRRSV replication by reprogramming LD synthesis. Therefore, our study provides a novel mechanism of host resistance to PRRSV and suggests potential new antiviral strategies against PRRSV infection.IMPORTANCEPorcine reproductive and respiratory virus (PRRSV) has caused incalculable economic damage to the global pig industry since it was first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. It is well known that viruses are parasitic pathogens, and the completion of their replication life cycle is highly dependent on host cells. A better understanding of host resistance to PRRSV infection is essential for developing safe and effective strategies to control PRRSV. Here, we report a crucial host antiviral molecule, yin yang 1 (YY1), which is induced to be expressed upon PRRSV infection and subsequently inhibits virus replication by reprogramming lipid droplet (LD) synthesis through transcriptional regulation. Our work provides a novel antiviral mechanism against PRRSV infection and suggests that targeting YY1 could be a new strategy for controlling PRRSV.

Dehydroevodiamine inhibits PEDV through regulateing ERK1/2 MAPK pathway in Vero cells.

Porcine epidemic diarrhea virus (PEDV) results in severe economic losses to the swine industry due to its widespread prevalence and high mortality. Currently, there is no effective treatment against PEDV. New antiviral therapies are urgently needed to control this highly contagious pathogen. In this research, the anti-PEDV activity and mechanism of Dehydroevodiamine (DHED) were investigated in vitro. Our results showed that DHED exerted satisfactory anti-PEDV activity by ameliorating cytopathic effects (CPEs), reducing virus titer, and inhibiting PEDV N protein expression and gene transcription dose-dependently. The antiviral mechanism of DHED is related to its inhibition of the entry, replication, and assembly stages of PEDV life cycle. In addition, DHED can regulate the MAPK signaling pathway, and suppress phosphorylated ERK1/2 activation, thus exerting antiviral effects. In conclusion, our research confirmed the anti-PEDV activity and mechanism of DHED, preliminarily providing a new strategy for anti-PEDV drug development.

Ultrasound-assisted plasma-activated water thawing of porcine longissimus dorsi: Effects on physicochemical, thermal stability, rheological, and structural properties of myofibrillar protein.

This study evaluated the effects of five thawing methods (air thawing (AT), water thawing (WT), plasma-activated water thawing (PT), ultrasound-assisted water thawing (UWT) and ultrasound-assisted plasma-activated water thawing (UPT)) on the physicochemical, thermal stability, rheological, and structural properties of porcine longissimus dorsi myofibrillar protein (MP). UPT treatment significantly improved protein solubility (73.10%) and reduced protein turbidity (0.123) compared with AT, WT, and PT treatments (P < 0.05). UPT treatment reduced the MP particle size (635.50 nm) and zeta potential (-6.38 mV) compared with AT and WT treatments (P < 0.05), which was closer to that of the fresh sample. UPT treatment also maintained the MP surface hydrophobicity and thermal stability. UPT treatment improved the MP rheological properties of the sample. In addition, UPT treatment effectively protected the MP secondary and tertiary structures. In conclusion, UPT treatment better maintained the MP physicochemical, thermal stability, rheological, and structural properties of thawed porcine longissimus dorsi. Therefore, UPT treatment can be considered as an effective thawing method.

RPA-CRISPR-Cas13a-assisted detection method of transmissible gastroenteritis virus.

Background and aim: Transmissible gastroenteritis virus (TGEV) is a highly contagious gastrointestinal virus that causes diarrhea, vomiting, anorexia, dehydration, and weight loss in piglets. In clinical practice, it often occurs in mixed infections with other pathogens, and is therefore difficult to diagnose and prevent. It mainly harms piglets of about 2 weeks old, causing huge losses on farms. The clinical confirmation of TGEV usually requires a laboratory diagnosis, but traditional PCR and immunofluorescence assays have some limitations. Moreover, most farms in China are ill-equipped to accurately diagnose the disease. Therefore, a new detection method with high sensitivity and specificity and less dependence on instrumentation is required. Methods: We used recombinase polymerase amplification (RPA), combined with the nuclease characteristics of the activated Cas13a protein to establish a visual CRISPR-Cas13a-assisted detection method for TGEV by adding a reporter RNA with fluorescent and quenching moieties to the system. Result: We selected the optimal RPA primer and best CRISPR RNA (crRNA). The reaction system was optimized and its repeatability, specificity, and sensitivity verified. The TGEV detection system did not cross-react with other common diarrhea viruses, and its detection limit was 101 copies, which is similar with the sensitivity of qPCR. We successfully established an RPA-CRISPR-Cas13a-assisted detection method, and used this detection system to analyze 123 pig blood samples. qPCR was used as the gold standard method. The sensitivity, specificity, positive coincidence rate, and negative coincidence rate of the new method were 100, 98.93, 96.66, and 100%, respectively.

PRRSV infection facilitates the shedding of soluble CD163 to induce inflammatory responses.

Porcine reproductive and respiratory syndrome (PRRS), which poses substantial threats to the global pig industry, is primarily characterized by interstitial pneumonia. Cluster of differentiation 163 (CD163) is the essential receptor for PRRSV infection. Metalloproteinase-mediated cleavage of CD163 leads to the shedding of soluble CD163 (sCD163), thereby inhibiting PRRSV proliferation. However, the exact cleavage site in CD163 and the potential role of sCD163 in inflammatory responses during PRRSV infection remain unclear. Herein, we found that PRRSV infection increased sCD163 levels, as demonstrated in primary alveolar macrophages (PAMs), immortalized PAM (IPAM) cell lines, and sera from PRRSV-infected piglets. With LC-MS/MS, Arg-1041/Ser-1042 was identified as the cleavage site in porcine CD163, and an IPAM cell line with precise mutation at the cleavage site was constructed. Using the precisely mutated IPAM cells, we found that exogenous addition of sCD163 protein promoted inflammatory responses, while mutation at the CD163 cleavage site suppressed inflammatory responses. Consistently, inhibition of sCD163 using its neutralizing antibodies reduced PRRSV infection-triggered inflammatory responses. Importantly, sCD163 promoted cell polarization from M2 to M1 phenotype, which in turn facilitated inflammatory responses. Taken together, our findings identify sCD163 as a novel proinflammatory mediator and provide valuable insights into the mechanisms underlying the induction of inflammatory responses by PRRSV infection.

Complex coacervation of low methoxy pectin with three types of gelatins for the encapsulation of fish oil.

Herein, the complex coacervation of low methoxy pectin (LMP) with three types of gelatins was explored to encapsulate fish oil. The fish oil@gelatin-LMP complex coacervates with good precipitation separation could be obtained at low gelatin concentrations (Fish gelatin, FG: 10-80 mg/mL; porcine skin gelatin, PSG: 10-40 mg/mL; bovine skin gelatin, BSG: 10-80 mg/mL), high gelatin: fish oil mass ratios (4:1-1:1), appropriate gelatin: LMP mass ratios (3:1-12:1 for FG and PSG, 6:1 for BSG), and appropriate pH (FG: 4.90-5.50; PSG: 4.80-5.40; BSG: 4.10-4.50). FG induced similar loading ability, lower encapsulation ability, and comparable peroxide values to the mammalian gelatins. FG induced higher or similar free fatty acid released percentages to mammalian gelatins in the in vitro gastrointestinal model at low gelatin concentrations (10-40 mg/mL). These results provided useful information to understand the protein-polysaccharide complex coacervation to encapsulate oil-based bioactive substances.

The feasibility and safety of biomaterials for posterior scleral reinforcement in rabbits.

PURPOSE: To explore the feasibility and safety of biomaterials for posterior scleral reinforcement (PSR) in rabbits. Methods: Decellularization and genipin crosslink were applied to the fresh bovine pericardium and porcine endocranium, and then mechanical properties, suture retention strength, and stability were tested. PSR operation was performed on 24 rabbit eyes using treated biological materials. Ophthalmic examination was performed regularly before and after PSR operation (1 week, 1 month, 3 months, 6 months). To evaluate the effectiveness, A ultrasound, diopter, and optical coherence tomography (OCT) were conducted. General condition, fundus photograph (FP), and pathological examination were recorded to evaluate the safety. Results: Compared with genipin crosslinked bovine pericardium (Gen-BP) (21.29 ± 13.29 Mpa), genipin crosslinked porcine endocranium (Gen-PE) (34.85 ± 3.67 Mpa, P < 0.01) showed a closer elastic modulus to that of genipin crosslinked human sclera. There were no complications or toxic reactions directly related to the materials. Capillary hyperplasia, inflammatory cell infiltration, and collagen fiber deposition were observed, and the content of type I collagen fibers increased after PSR. Overall, the choroidal thickness of treated eyes was significantly thickened at different time points after PSR, which were 96.84 ± 21.08µm, 96.72 ± 22.00µm, 90.90 ± 16.57µm, 97.28 ± 14.74µm, respectively. The Gen-PE group showed changes that were almost consistent with the overall data. Conclusion: Gen-BP and Gen-PE are safe biological materials for PSR. The Gen-PE group demonstrated more significant advantages over the Gen-BP group in terms of material properties. .

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses.

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/µL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Design of multi-epitope vaccine against porcine rotavirus using computational biology and molecular dynamics simulation approaches.

Porcine Rotavirus (PoRV) is a significant pathogen affecting swine-rearing regions globally, presenting a substantial threat to the economic development of the livestock sector. At present, no specific pharmaceuticals are available for this disease, and treatment options remain exceedingly limited. This study seeks to design a multi-epitope peptide vaccine for PoRV employing bioinformatics approaches to robustly activate T-cell and B-cell immune responses. Two antigenic proteins, VP7 and VP8*, were selected from PoRV, and potential immunogenic T-cell and B-cell epitopes were predicted using immunoinformatic tools. These epitopes were further screened according to non-toxicity, antigenicity, non-allergenicity, and immunogenicity criteria. The selected epitopes were linked with linkers to form a novel multi-epitope vaccine construct, with the PADRE sequence (AKFVAAWTLKAAA) and RS09 peptide attached at the N-terminus of the designed peptide chain to enhance the vaccine's antigenicity. Protein-protein docking of the vaccine constructs with toll-like receptors (TLR3 and TLR4) was conducted using computational methods, with the lowest energy docking results selected as the optimal predictive model. Subsequently, molecular dynamics (MD) simulation methods were employed to assess the stability of the protein vaccine constructs and TLR3 and TLR4 receptors. The results indicated that the vaccine-TLR3 and vaccine-TLR4 docking models remained stable throughout the simulation period. Additionally, the C-IMMSIM tool was utilized to determine the immunogenic triggering capability of the vaccine protein, demonstrating that the constructed vaccine protein could induce both cell-mediated and humoral immune responses, thereby playing a role in eliciting host immune responses. In conclusion, this study successfully constructed a multi-epitope vaccine against PoRV and validated the stability and efficacy of the vaccine through computational analysis. However, as the study is purely computational, experimental evaluation is required to validate the safety and immunogenicity of the newly constructed vaccine protein.

Exosomal ssc-miR-1343 targets FAM131C to regulate porcine epidemic diarrhea virus infection in pigs.

The porcine epidemic diarrhea virus (PEDV) causes diarrhea in piglets, thereby causing very significant economic losses for the global swine industry. In previous studies, it has been confirmed that microRNAs (miRNAs) play an important role in the infection caused by PEDV. However, the precise molecular mechanism of miRNAs in the regulation of PEDV infection is still not fully understood. In the present study, we utilized miRNA-seq analysis to identify ssc-miR-1343 with differential expression between PEDV-infected and normal piglets. The expression of ssc-miR-1343 was detected in isolated exosomes, and it was found to be significantly higher than that in the controls following PEDV infection. The ssc-miR-1343 mimic was found to decrease PEDV replication, whereas the ssc-miR-1343 inhibitor was observed to increase PEDV replication, and ssc-miR-1343 was delivered by exosomes during PEDV infection. Mechanistically, ssc-miR-1343 binds to the 3'UTR region of FAM131C, down-regulating its expression, and FAM131C has been shown to enhance PEDV replication through simultaneously suppressing pathways associated with innate immunity. The ssc-miR-1343/FAM131C axis was found to upregulate the host immune response against PEDV infection. In conclusion, our findings indicate that the transport of ssc-miR-1343 in exosomes is involved in PEDV infection. This discovery presents a new potential target for the development of drugs to treat PEDV.