Predictive factors of poor outcome and mortality among anterior ischaemic stroke patients despite successful recanalisation in China: a secondary analysis of the CAPTURE trial.

OBJECTIVES: This work aimed to analyse the risk factors for poor outcomes and mortality among patients with anterior large vessel occlusion (LVO) ischaemic stroke, despite successful recanalisation. SETTING AND PARTICIPANTS: This study conducted a secondary analysis among patients who underwent successful recanalisation in the CAPTURE trial. The trial took place between March 2018 and September 2020 at 21 sites in China. The CAPTURE trial enrolled patients who had an acute ischaemic stroke aged 18-80 years with LVO in anterior circulation. INTERVENTIONS: Thrombectomy was immediately performed using Neurohawk or the Solitaire FR after randomisation in CAPTURE trial. Rescue treatment was available for patients with severe residual stenosis caused by atherosclerosis. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary goal was to predict poor 90-day survival or mortality within 90 days post-thrombectomy. Univariate analysis, using the χ2 test or Fisher's exact test, was conducted for each selected factor. Subsequently, a multivariable analysis was performed on significant factors (p≤0.10) identified through univariate analysis using the backward selection logistic regression approach. RESULTS: Among the 207 recruited patients, 79 (38.2%) exhibited poor clinical outcomes, and 26 (12.6%) died within 90 days post-thrombectomy. Multivariate analysis revealed that the following factors were significantly associated with poor 90-day survival: age ≥67 years, internal carotid artery (ICA) occlusion (compared with middle cerebral artery (MCA) occlusion), initial National Institutes of Health Stroke Scale (NIHSS) score ≥17 and final modified Thrombolysis in Cerebral Infarction (mTICI) score 2b (compared with mTICI 3). Additionally, the following factors were significantly associated with mortality 90 days post-thrombectomy: initial NIHSS score ≥17, ICA occlusion (compared with MCA occlusion) and recanalisation with more than one pass. CONCLUSIONS: Age, NIHSS score, occlusion site, mTICI score and the number of passes can be independently used to predict poor 90-day survival or mortality within 90 days post-thrombectomy. TRIAL REGISTRATION NUMBER: NCT04995757.

TIPE2 regulates the response of BV2 cells to lipopolysaccharide by the crosstalk between PI3K/AKT signaling and microglia M1/M2 polarization.

Tumor necrosis factor (TNF)-α-induced protein 8-like 2 (TIPE2) is a crucial negative regulator of both adaptive and innate immunity, which helps maintain the dynamic balance of the immune system by negatively regulating the signaling of T-cell receptors (TCR) and Toll-like receptors (TLR). In this study, we aimed to investigate the role and molecular mechanism of TIPE2 using a lipopolysaccharide (LPS)-induced inflammatory injury model in BV2 cells. Specifically, we constructed a BV2 cell line of TIPE2-overexpression or TIPE2-knockdown via lentiviral transfection. Our results demonstrated that overexpression of TIPE2 downregulated the expression of pro-inflammatory cytokines IL-1ß and IL-6, which was reversed by knockdown of TIPE2 in the inflammation model of BV2 cells. In addition, overexpression of TIPE2 resulted in the conversion of BV2 cells to the M2 phenotype, while the knockdown of TIPE2 promoted the transformation of BV2 cells to the M1 phenotype. Notably, our co-culture experiments with neuronal cells SH-SY5Y showed that the overexpression of TIPE2 in inflammation-injured BV2 cells exhibited a protective effect on the neuronal cells. Finally, western blot analysis demonstrated that TIPE2 significantly reduced the expression of p-PI3K, p-AKT, p-p65, and p-IκBα in LPS treated BV2 cells, and inhibited the activation of NF-κB through the dephosphorylation of PI3K/AKT. These results suggest that TIPE2 plays an important role in mediating neuroinflammatory responses and may be involved in neuroprotection by modulating the phenotypic changes of BV2 cells and regulating the pro-inflammatory responses through the PI3K/AKT and NF-κB signaling pathways. In conclusion, our study provides new insights into the crucial role of TIPE2 in regulating neuroinflammatory responses and highlights its potential as a therapeutic target for neuroprotection.

A non-linear relationship between lesion length and risk of recurrent cerebral ischemia after stenting for symptomatic intracranial stenosis with hemodynamic impairment.

Background: Stent placement can be an effective treatment for patients with symptomatic intracranial stenosis (sICAS) and hemodynamic impairment (HI). However, the association between lesion length and the risk of recurrent cerebral ischemia (RCI) after stenting remains controversial. Exploring this association can help predict patients at higher risk for RCI and develop individualized follow-up schedules. Method: In this study, we provided a post-hoc analysis of a prospective, multicenter registry study on stenting for sICAS with HI in China. Demographics, vascular risk factors, clinical variables, lesions, and procedure-specific variables were recorded. RCI includes ischemic stroke and transient ischemic attack (TIA), from month 1 after stenting to the end of the follow-up period. Smoothing curve fitting and segmented Cox regression analysis were used to analyze the threshold effect between lesion length and RCI in the overall group and subgroups of the stent type. Results: The non-linear relationship between lesion length and RCI was observed in the overall population and subgroups; however, the non-linear relationship differed by subgroup of stent type. In the balloon-expandable stent (BES) subgroup, the risk of RCI increased 2.17-fold and 3.17-fold for each 1-mm increase in the lesion length when the lesion length was <7.70 mm and >9.00 mm, respectively. In the self-expanding stent (SES) subgroup, the risk of RCI increased 1.83-fold for each 1-mm increase in the lesion length when the length was <9.00 mm. Nevertheless, the risk of RCI did not increase with the length when the lesion length was >9.00mm. Conclusion: A non-linear relationship exists between lesion length and RCI after stenting for sICAS with HI. The lesion length increases the overall risk of RCI for BES and for SES when the length was <9.00 mm, while no significant relationship was found when the length was >9.00 mm for SES.

Betulinic acid mitigates zearalenone-induced liver injury by ERS/MAPK/Nrf2 signaling pathways in mice.

Zearalenone (ZEA) is a mycotoxin commonly found in cereals and feedstuffs, which can induce oxidative stress and inflammation to cause liver damage in humans and animals. Betulinic acid (BA) is extracted from pentacyclic triterpenoids of many natural plants and has anti-inflammatory, and anti-oxidation biological activities in many studies. However, the protective effect of BA on liver injury induced by ZEA has not been reported. Therefore, this study aims to explore the protective effect of BA on ZEA-induced liver injury and its possible mechanism. In the mice experiment, ZEA exposure increased the liver index and caused histopathological impairment, oxidative damage, hepatic inflammatory responses, and increased hepatocyte apoptosis. However, when combined with BA, it could inhibit the production of ROS, up-regulate the proteins expression of Nrf2 and HO-1 and down-regulate the expression of Keap1, and alleviate oxidative damage and inflammation in the liver of mice. In addition, BA could alleviate ZEA-induced apoptosis and liver injury in mice by inhibiting the endoplasmic reticulum stress (ERS) and MAPK signaling pathways. In conclusion, this study revealed the protective effect of BA on the hepatotoxicity of ZEA for the first time, providing a new perspective for the development of ZEA antidote and the application of BA.

Citrinin Exposure Induced Testicular Damage and Spermatogenesis Disorder by Triggering Endoplasmic Reticulum Stress.

Damage to the reproductive system is the key factor leading to male infertility. Citrinin (CTN) is produced by Penicillium and Aspergillus in nature, and is definitely found in food and animal feed. Studies have revealed that CTN can cause damage to male reproductive organs and reduce fertility, but the mechanism of toxicity has not been revealed. In the present study, male Kunming mice were given different doses of CTN (0, 1.25, 5 or 20 mg/kg BW) by intragastric administration. The results demonstrated that CTN exposure caused disorder of androgen, a decline in sperm quality, and histopathological damage of testis. The inhibition of the expression of ZO-1, claudin-1 and occludin suggests that the blood-testis barrier (BTB) was damaged. Simultaneously, CTN inhibited the activity of antioxidant enzymes such as CAT and SOD, and promoted the production of MDA and ROS, resulting in oxidative damage of testis. Additionally, apoptotic cells were detected and the ratio of Bax/Bcl-2 was increased. Not only that, CTN activated the expression of endoplasmic reticulum stress (ERS)-related proteins IRE1, ATF6, CHOP, and GRP78. Interestingly, 4-Phenylbutyric Acid (4-PBA, an ERS inhibitor) treatment blocked the adverse effects of CTN exposure on male reproduction. In short, the findings suggested that CTN exposure can cause damage to mouse testis tissue, in which ERS exhibited an important regulatory role.

Establishment and application of multiplex real-time PCR for simultaneous detection of four viruses associated with porcine reproductive failure.

Many pathogens cause reproductive failure in sows suffering a broad spectrum of sequelae, including abortions, stillbirth, mummification, embryonic death, and infertility. Although various detection methods, such as polymerase chain reaction (PCR) and real-time PCR, have been widely used for molecular diagnosis, mainly for a single pathogen. In this study, we developed a multiplex real-time PCR method for the simultaneous detection of porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine parvovirus (PPV) and pseudorabies virus (PRV) associated with porcine reproductive failure. The R 2 values for the standard curve of multiplex real-time PCR of PCV2, PCV3, PPV, and PRV reached to 0.996, 0.997, 0.996, and 0.998, respectively. Importantly, the limit of detection (LoD) of PCV2, PCV3, PPV, and PRV, were 1, 10, 10, 10 copies/reaction, respectively. Meanwhile, specificity test results indicated that multiplex real-time PCR for simultaneous detection is specific for these four target pathogens and does not react with other pathogens, such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine epidemic diarrhea virus. Besides, this method had good repeatability with coefficients of variation of intra- and inter-assay less than 2%. Finally, this approach was further evaluated by 315 clinical samples for its practicality in the field. The positive rates of PCV2, PCV3, PPV, and PRV were 66.67% (210/315), 8.57% (27/315), 8.89% (28/315), and 4.13% (13/315), respectively. The overall co-infection rates of two or more pathogens were 13.65% (43/315). Therefore, this multiplex real-time PCR provides an accurate and sensitive method for the identification of those four underlying DNA viruses among potential pathogenic agents, allowing it to be applied in diagnostics, surveillance, and epidemiology.

Pseudorabies in pig industry of China: Epidemiology in pigs and practitioner awareness.

Pseudorabies virus (PRV) is widely prevalent in China, which can transmit from pigs to other mammals. Moreover, a PRV variant isolated from an acute human encephalitis case was documented recently. It is imperative to investigate PRV epidemiology in pigs, the knowledge regarding pseudorabies (PR) and self-protection behaviors upon working among relevant practitioners including pig farmers, pig cutters, and pork salesman. In the present study, 18,812 pig serum samples and 1,634 tissue samples were collected from Hunan Province during the period of 2020 to 2021 for detecting the presence of PRV gE-special antibody and nucleic acids, respectively. Meanwhile, we conducted a questionnaire survey about PR among these practitioners in China. The results showed that nearly 9% (1,840/20,192) pigs from 161 collected sites (20.17%, 161/797) were seropositive for PRV-gE antibody. Though only 2.33% tissue samples were positive for PRV nucleic acids, all the representative PRV strains were variant. It was learned that most practitioners were frequently injured when working, the injured sites mainly included hand and foot. Among the three transmission routes of PRV, the aerosol transmission route was often overlooked. Moreover, the workers lacked self-protection awareness and were poor conscious about PRV and its potential threat to humans. All the results demonstrate that PRV remains widely spread in pig populations, while the potential threats of PRV in pig industry receive less attention, suggesting that targeted educational programs to these people should be performed.

LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2.

Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus-associated disease (PCVAD), which can cause considerable economic loss to the pig industry. The diagnosis of PCVAD is complicated and requires a series of clinical, pathological, and virological methods. Therefore, a rapid, highly sensitive, on-site, and visual diagnostic approach would facilitate dealing with the spread of PCV2. In this study, we intended to establish a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). Following a series of optimizations of its reaction conditions, this LAMP-CRISPR-based PCV2 detection could be conducted in constant temperature equipment, with the result reflected in a direct visual readout way. This established PCV2 detection approach presented fine sensitivity, rapidity, specificity, and reliability, as demonstrated by a low detectable limit of 1 copy/µL, completed within an hour, no cross-reaction with main porcine DNA or RNA viruses like PCV1, PCV3, and PEDV, and a 100% coincidence rate with that of the quantitative PCR (qPCR) method in the evaluation of 30 clinical blood samples, respectively. Therefore, this novel method makes rapid, on-site, visual, highly sensitive, and specific detection of PCV2 possible, facilitating the prevention of this pathogen in the field.

Factors Influencing the Outcome of Symptomatic Intracranial Artery Stenosis With Hemodynamic Impairment After Short and Long-Term Stent Placement.

Objective: Stent placement is a feasible approach worldwidely for patients with symptomatic intracranial artery stenosis (sICAS) and hemodynamic impairment (HI) who are at high risk of recurrent stroke after medical treatment. Exploration of factors associated with poor outcomes after stent placement could help develop better individualized therapeutic strategies. Methods: This study conducted a post-hoc analysis of a prospective, multicenter registry study of stent use for sICAS with HI in China. Patient and clinical demographics, and stenotic lesion images were analyzed using univariate and multivariate Cox regression to the time until any endpoints or the end of the follow-up period. The short-term endpoint included any transient ischemic attack (TIA), stroke, or death within 1 month after stent placement. The long-term endpoints included the short-term endpoints and any TIA or stroke in the region of the affected artery that occurred more than 1 month after stent placement. Results: Two hundred and ninety two patients were included, with 13 short-term and 39 long-term endpoints. Multivariate Cox regression analysis revealed that lesions at the arterial origin or bifurcation (Hazard Ratio (HR) = 7.52; 95% CI, 1.89-29.82; p = 0.004) were significantly associated with higher short-term risk. Baseline renal insufficiency reduced the risk (HR = 0.08; 95% CI: 0.01-0.68; p = 0.021). Factors significantly associated with higher long-term risk included irregular or ulcerated plaques at the lesion (HR = 2.15; 95% CI: 1.07-4.33; p = 0.031). Subgroup analyses indicated that higher risk occurred in the older age group (age>59 years, HR = 3.73, 95% CI: 1.27-10.97, p = 0.017), and not in the younger group (age≤59 years, HR = 1.12, 95% CI: 0.42-3.03, p = 0.822). Conclusion: Irregular or ulcerated plaques in older patients and lesions at the arterial opening or bifurcation were more likely to result in adverse endpoints for stent placement during long or short -term follow-up. Investigation of these factors might facilitate the development of individualized therapeutic strategies for this population. Clinical Trial Registration: http://www.clinicaltrials.gov, identifier: NCT01968122.

Current knowledge on epidemiology and evolution of novel porcine circovirus 4.

Porcine circovirus type 4 (PCV4) is a newly emerging virus, with both PCV4 genomic DNA and specific antibodies detected in swine herds in several provinces in China and South Korea. Although the virus was first identified in 2019 in Hunan, China, retrospective research suggests that serum samples collected as early as 2008 were positive for PCV4 antibody. Infections with only PCV4 or co-infections with other pathogens have been associated with several clinical manifestations, but its pathogenesis remains to be determined. The purpose of this review was the following: (1) to characterize PCV4 epidemiology by assessing evolutionary dynamics and genetic diversity of PCV4 strains circulating in swine herds; (2) to reconstruct a computerized 3D model to analyze PCV4 Cap properties; (3) and to summarize the current evidence of PCV4-associated clinical-pathological manifestations. The origin of PCV4 is apparently distinct from other PCV, based on analysis of phylogenetic trees. Of note, PCV4 shares an ancient common ancestor with mink circoviruses. Furthermore, the amino acid residue at position 27 of the PCV4 Cap is a key benchmark to distinguish PCV4a (27S) from PCV4b (27 N), based on PCV4 strains currently available, and variation of this residue may alter Cap antigenicity. In addition, the capsid surface of PCV4 has characteristics of increased polar residues, compared to PCV2, which raises the possibility that PCV4 may target negatively charged host receptors to promote virus infection. Further studies are required, including virus isolation and culture, and more detailed characterization of molecular epidemiology and genetic diversity of PCV4 in swine herds.

Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis.

Porcine circovirus 2 (PCV2), considered one of the most globally important porcine pathogens, causes postweaning multisystemic wasting syndrome (PMWS). This virus is localized in the mitochondria in pigs with PMWS. Here, we identified, for the first time, a mitochondrial localization signal (MLS) in the PCV2 capsid protein (Cap) at the N-terminus. PK-15 cells showed colocalization of the MLS-EGFP fusion protein with mitochondria. Since the PCV2 Cap also contained a nuclear localization signal (NLS) that mediated entry into the nucleus, we inferred that the subcellular localization of the PCV2 Cap is inherently complex and dependent on the viral life cycle. Furthermore, we also determined that deletion of the MLS attenuated Cap-induced apoptosis. More importantly, the MLS was essential for PCV2 replication, as absence of the MLS resulted in failure of virus rescue from cells infected with infectious clone DNA. In conclusion, the MLS of the PCV2 Cap plays critical roles in Cap-induced apoptosis, and MLS deletion of Cap is lethal for virus rescue.

Structure, Antigenic Properties, and Highly Efficient Assembly of PCV4 Capsid Protein.

Porcine circovirus type 4 (PCV4), a recently reported circovirus, was first identified in pigs with clinical signs similar to porcine dermatitis nephropathy syndrome (PDNS), in Hunan province, China, in 2019. More knowledge regarding the assembly of capsid protein (Cap) into virus-like particles (VLPs), their structure and antigenic properties, are needed to provide new knowledge for diagnosis and further characterization of PCV4. In this study, high-level expression of PCV4 Cap was achieved in Escherichia coli with purified Cap self-assembling into VLPs (~20 nm) in vitro. Furthermore, these VLPs were internalized in vitro by PK15 and 3D4/21 cell lines. Significant structural differences between PCV4 and PCV2 capsids were demonstrated among loops (loop BC, CD, DE, EF, and GH), based on comparisons of 3D structures. In addition, five potential B cell epitopes identified in silico were mostly located in surface-exposed loops of PCV4 capsid. Cross-reaction between PCV4 and PCV2 or PCV3 conferred by humoral immune responses was deemed unlikely on the basis of ELISA and Western blotting for assessment of VLPs and using PCV4 or PCV2 VLPs. In conclusion, these studies provided new knowledge regarding PCV4 capsid surface patterns. It is noteworthy that the PCV4 VLPs prepared in our study have much potential for development of serological diagnostics for PCV4 and to further characterize this virus.

Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus.

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/µL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

High Co-infection Status of Novel Porcine Parvovirus 7 With Porcine Circovirus 3 in Sows That Experienced Reproductive Failure.

Porcine parvoviruses (PPVs) and porcine circoviruses (PCVs) infect pigs worldwide, with PPV1-7 and PCV2 infections common in pigs. Although PPV7 was only identified in 2016, co-infection of PPV7 and PCV2 is already common, and PPV7 may stimulate PCV2 replication. PCV3, a novel type of circovirus, is prevalent in pig populations worldwide and considered to cause reproductive disorders and dermatitis nephrotic syndrome. In recent studies, pigs were commonly infected with both PCV3 and PPV7. Our objective was to investigate the co-infections between PPV7 and PCV3 in samples from swine on farms in Hunan, China, and assess the potential impacts of PPV7 on PCV3 viremia. A total of 209 samples, known to be positive (105) or negative (104) for PCV3, were randomly selected from serum samples that were collected from commercial swine herds in seven regions from 2016 to 2018 in our previous studies; these samples were subjected to real-time PCR to detect PPV7. Of these samples, 23% (48/209) were positive for PPV7. Furthermore, the PPV7 positive rate was significantly higher in PCV3 positive serum (31.4%, 33/105) than in PCV3 negative serum (14.4%, 15/104). Another 62 PCV3 positive sow serum samples and 20 PCV3 positive aborted fetuses were selected from 2015 to 2016 in our other previous study. These samples were designated as being from farms with or without long-standing histories of reproductive failure (RF or non-RF), respectively, and they were also subjected to real-time PCR to detect PPV7 and to determine whether PPV7 affected PCV3 viremia. Among the 62 serum samples (39 PCV3 positive RF-serum and 23 PCV3 positive non-RF-serum), 45.1% (28/62) were positive for PPV7 and PCV3, and the PPV7 positive rate was significantly higher in PCV3 positive RF-serum (51.2%, 20/39) than in PCV3 positive non-RF-serum (34.8%, 8/23). In addition, there was a higher positive rate of PPV7 (55%, 11/20) in PCV3 positive aborted fetus samples. In addition, the copy number of PCV3 in PPV7 positive samples was significantly higher than that in PPV7 negative serum samples. Based on these findings, we concluded that PPV7 may stimulate PCV3 replication.

Endovascular recanalization for symptomatic intracranial internal carotid and middle cerebral artery occlusion lasting longer than 72 h: Experience in a single center.

OBJECTIVE: The objective of this study was to assess the safety, feasibility, and outcomes of endovascular recanalization for symptomatic intracranial internal carotid and middle cerebral artery occlusion lasting longer than 72 h. METHODS: Thirty-nine consecutive patients with symptomatic occlusion of the anterior circulation and failure of medical therapy underwent endovascular recanalization and were included in this retrospective study. Patient characteristics, atherosclerotic risk factors, successful recanalization rates, and angiographic data were collected. RESULTS: Recanalization was successful in 37 cases (94.9%). The average residual stenosis immediately after intervention was 11.6 ± 4.3%. The patients who underwent balloon angioplasty alone had similar residual stenosis to those who also underwent stent placement (15.6 ± 7.3% vs. 9.0 ± 6.4%, P = 0.184). Intra- and perioperative complications occurred in three cases (7.69%). One patient (2.7%) developed severe in-stent restenosis with transient ischemic attack symptoms at 1-year follow-up. CONCLUSIONS: Endovascular recanalization is feasible for symptomatic occlusion of the anterior circulation lasting longer than 72 h. Recanalization provides a higher success rate when performed within 6 months of the qualifying event.

Treatment of acute thrombosis during stent-assisted coil embolization of ruptured proximal posterior inferior cerebellar artery aneurysm.

Aneurysms located in the posterior inferior cerebellar artery (PICA) are not a majority of the intracranial aneurysms cases. Many challenges were addressed in the endovascular procedure to treat the disease. The authors have successfully diagnosed and treated a ruptured proximal PICA aneurysm. However, digital subtraction angiography showed acute thrombosis in the vertebral artery in the procedure, which probably could be an acute in-stent thrombosis. The tirofiban hydrochloride injection was subjected through a microcatheter, and then, the second Neuroform EZ stent was planted. In the 6-month follow-up, no recurrence of the aneurysm and complete patency of the right PICA at the site of aneurysm formation were found. We believe that the treatment of PICA aneurysms with Neuroform EZ stents could get a favorable result. Combination of tirofiban hydrochloride and Neuroform EZ stent could be an effective approach in treating acute thrombotic complications.

Immunoinformatic Analysis of T- and B-Cell Epitopes for SARS-CoV-2 Vaccine Design.

Currently, there is limited knowledge about the immunological profiles of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). We used computer-based immunoinformatic analysis and the newly resolved 3-dimensional (3D) structures of the SARS-CoV-2 S trimeric protein, together with analyses of the immunogenic profiles of SARS-CoV, to anticipate potential B-cell and T-cell epitopes of the SARS-CoV-2 S protein for vaccine design, particularly for peptide-driven vaccine design and serological diagnosis. Nine conserved linear B-cell epitopes and multiple discontinuous B-cell epitopes composed of 69 residues on the surface of the SARS-CoV-2 trimeric S protein were predicted to be highly antigenic. We found that the SARS-CoV-2 S protein has a different antigenic profile than that of the SARS-CoV S protein due to the variations in their primary and 3D structures. Importantly, SARS-CoV-2 may exploit an immune evasion mechanism through two point mutations in the critical and conserved linear neutralization epitope (overlap with fusion peptide) around a sparsely glycosylated area. These mutations lead to a significant decrease in the antigenicity of this epitope in the SARS-CoV-2 S protein. In addition, 62 T-cell epitopes in the SARS-CoV-2 S protein were predicted in our study. The structure-based immunoinformatic analysis for the SARS-CoV-2 S protein in this study may improve vaccine design, diagnosis, and immunotherapy against the pandemic of COVID-19.

Porcine Parvovirus 7: Evolutionary Dynamics and Identification of Epitopes toward Vaccine Design.

Porcine parvovirus 7 (PPV7) belonging to the genus Chapparvovirus in the family Parvoviridae, has been identified in the USA, Sweden, Poland, China, South Korea and Brazil. Our objective was to determine the phylogeny, estimate the time of origin and evolutionary dynamics of PPV7, and use computer-based immune-informatics to assess potential epitopes of its Cap, the main antigenic viral protein, for vaccines or serology. Regarding evolutionary dynamics, PPV7 had 2 major clades, both of which possibly had a common ancestor in 2004. Furthermore, PPV7 strains from China were the most likely ancestral strains. The nucleotide substitution rates of NS1 and Cap genes were 8.01 × 10-4 and 2.19 × 10-3 per site per year, respectively, which were higher than those reported for PPV1-4. The antigenic profiles of PPV7 Cap were revealed and there were indications that PPV7 used antigenic shift to escape from the host's immune surveillance. Linear B cell epitopes and CD8 T cell epitopes of Cap with good antigenic potential were identified in silico; these conserved B cell epitopes may be candidates for the PPV7 vaccine or for the development of serological diagnostic methods.

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells.

The generation of genetically modified mouse models derived from gene targeting (GT) in mouse embryonic stem (ES) cells (mESCs) has greatly advanced both basic and clinical research. Our previous finding that gene targeting at the Myh9 exon2 site in mESCs has a pronounced high homologous recombination (HR) efficiency (>90%) has facilitated the generation of a series of nonmuscle myosin II (NM II) related mouse models. Furthermore, the Myh9 gene locus has been well demonstrated to be a new safe harbor for site-specific insertion of other exogenous genes. In the current study, we intend to investigate the molecular biology underlying for this high HR efficiency from other aspects. Our results confirmed some previously characterized properties and revealed some unreported observations: 1) The comparison and analysis of the targeting events occurring at the Myh9 and several widely used loci for targeting transgenesis, including ColA1, HPRT, ROSA26, and the sequences utilized for generating these targeting constructs, indicated that a total length about 6 kb with approximate 50% GC-content of the 5' and 3' homologous arms, may facilitate a better performance in terms of GT efficiency. 2) Despite increasing the length of the homologous arms, shifting the targeting site from the Myh9 exon2, to intron2, or exon3 led to a gradually reduced GT frequency (91.7, 71.8 and 50.0%, respectively). This finding provides the first evidence that the HR frequency may also be associated with the targeting site even in the same locus. Meanwhile, the decreased trend of the GT efficiency at these targeting sites was consistent with the reduced percentage of simple sequence repeat (SSR) and short interspersed nuclear elements (SINEs) in the sequences for generating the targeting constructs, suggesting the potential effects of these DNA elements on GT efficiency; 3) Our series of targeting experiments and analyses with truncated 5' and 3' arms at the Myh9 exon2 site demonstrated that GT efficiency positively correlates with the total length of the homologous arms (R = 0.7256, p<0.01), confirmed that a 2:1 ratio of the length, a 50% GC-content and the higher amount of SINEs for the 5' and 3' arms may benefit for appreciable GT frequency. Though more investigations are required, the Myh9 gene locus appears to be an ideal location for identifying HR-related cis and trans factors, which in turn provide mechanistic insights and also facilitate the practical application of gene editing.