Mechanism, structural and functional insights into nidovirus-induced double-membrane vesicles.

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future.

Ni, Co-Embedded MOF-Derived N-Doped Bimetallic Porous Carbon for Adsorption-Photocatalytic Degradation of Organic Dyes and Antibiotics.

An efficient protocol for photocatalytic degradation of organic dyes and antibiotics has been successfully established via MOF-derived (MOF = metal-organic framework) Ni, Co-embedded N-doped bimetallic porous carbon nanocomposites (NiCo/NC). Such a NiCo/NC nanocomposite features well-distributed structures, suitable specific surface areas, and more active sites determined by various characterization analyses. The catalyst exhibits higher photocatalytic performance and stability toward the liquid-phase degradation of methylene blue (MB) under visible light irradiation for 60 min, after the adsorption-desorption equilibrium and the thorough degradation into H2O and CO2. Radical quenching experiments further confirmed the dominant effect of electron holes h+ and superoxide radical anions ·O2- for the MB photodegradation process. NiCo/NC was also appropriate for the degradation of Rhodamine B, methyl orange, tetracycline hydrochloride, and norfloxacin. Moreover, NiCo/NC is robust, and its photocatalytic activity is basically maintained after 8 cycles. This work is expected to provide additional information for the design of MOF-derived carbon material with more excellent properties and lay the foundation for further industrial applications.

Transmissible Gastroenteritis Virus: An Update Review and Perspective.

Transmissible gastroenteritis virus (TGEV) is a member of the alphacoronavirus genus, which has caused huge threats and losses to pig husbandry with a 100% mortality in infected piglets. TGEV is observed to be recombining and evolving unstoppably in recent years, with some of these recombinant strains spreading across species, which makes the detection and prevention of TGEV more complex. This paper reviews and discusses the basic biological properties of TGEV, factors affecting virulence, viral receptors, and the latest research advances in TGEV infection-induced apoptosis and autophagy to improve understanding of the current status of TGEV and related research processes. We also highlight a possible risk of TGEV being zoonotic, which could be evidenced by the detection of CCoV-HuPn-2018 in humans.

Large-Scale CRISPR Screen of LDLR Pathogenic Variants.

Familial hypercholesterolemia (FH) is a frequently occurring genetic disorder that is linked to early-onset cardiovascular disease. If left untreated, patients with this condition can develop severe cardiovascular complications. Unfortunately, many patients remain undiagnosed, and even when diagnosed, the treatment is often not optimal. Although mutations in the LDLR gene are the primary cause of FH, predicting whether novel variants are pathogenic is not a straightforward task. Understanding the functionality of LDLR variants is crucial in uncovering the genetic basis of FH. Our study utilized CRISPR/Cas9 cytosine base editors in pooled screens to establish a novel approach for functionally assessing tens of thousands of LDLR variants on a large scale. A total of more than 100 single guide RNAs (sgRNAs) targeting LDLR pathogenic mutations were successfully screened with relatively high accuracy. Out of these, 5 sgRNAs were further subjected to functional verification studies, including 1 in the promoter, 1 in the antisense RNA, 1 in the exon, and 2 in the intron. Except for the variant caused by the sgRNA located at intron 16, the functionalities of the other LDLR variants were all downregulated. The high similarity of LDLR intron sequences may lead to some false positives. Overall, these results confirm the reliability of the large-scale screening strategy for functional analysis of LDLR variants, and the screened candidate pathogenic mutations could be used as an auxiliary means of clinical gene detection to prevent FH-induced heart disease.

An integrative analysis of genotype-phenotype correlation in Charcot Marie Tooth type 2A disease with MFN2 variants: A case and systematic review.

Dominant genetic variants in the mitofusin 2 (MFN2) gene lead to Charcot-Marie-Tooth type 2A (CMT2A), a neurodegenerative disease caused by genetic defects that directly damage axons. In this study, we reported a proband with a pathogenic variant in the GTPase domain of MFN2, c.494A > G (p.His165Arg). To date, at least 184 distinct MFN2 variants identified in 944 independent probands have been reported in 131 references. However, the field of medical genetics has long been challenged by how genetic variation in the MFN2 gene is associated with disease phenotypes. Here, by collating the MFN2 variant data and patient clinical information from Leiden Open Variant Database 3.0, NCBI clinvar database, and available related references in PubMed, we determined the mutation frequency, age of onset, sex ratio, and geographical distribution. Furthermore, the results of an analysis examining the relationship between variants and phenotypes from multiple genetic perspectives indicated that insertion and deletions (indels), copy number variants (CNVs), duplication variants, and nonsense mutations in single nucleotide variants (SNVs) tend to be pathogenic, and the results emphasized the importance of the GTPase domain to the structure and function of MFN2. Overall, three reliable classification methods of MFN2 genotype-phenotype associations provide insights into the prediction of CMT2A disease severity. Of course, there are still many MFN2 variants that have not been given clear clinical significance, which requires clinicians to make more accurate clinical diagnoses.

Erratum: HMEJ-mediated site-specific integration of a myostatin inhibitor increases skeletal muscle mass in porcine.

[This corrects the article DOI: 10.1016/j.omtn.2021.06.011.].

Enhanced Adsorption-Photocatalytic Degradation of Organic Pollutants via a ZIF-67-Derived Co-N Codoped Carbon Matrix Catalyst.

Stable and efficient photocatalytic degradation of organic pollutants has been achieved via a ZIF-67-derived Co-embedded N-doped nanoporous carbon material catalyst (Co-N/C). The catalyst features a well-distributed structure, suitable specific surface area, and more active sites according to the various characterization analyses. The photocatalytic activity of Co-N/C was evaluated by the degradation of the target pollutant Rhodamine B (RhB). As a result, RhB could establish an adsorption-desorption equilibrium in the dark within 30 min and was thoroughly degraded into H2O and CO2 by Co-N/C under 500 W visible light irradiation in 40 min. Moreover, radical-quenching experiments and reactive oxygen species monitoring were performed to further probe the plausible photodegradation mechanism of RhB. Co-N/C is also appropriate for other alternative dyes and antibiotics affording ideal removal efficiencies. After the reaction, Co-N/C could be facilely separated by an external magnetic field and reused for eight reaction cycles without obvious deactivation of its photocatalytic properties. This study is expected to provide an instructive guideline for the design of efficient and recyclable composite photocatalysts derived from metal-organic frameworks for a broad range of environmental remediation processes.

TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis.

Human telomerase is a specialized DNA polymerase whose catalytic core includes both TERT and human telomerase RNA (hTR). Telomerase in humans, which is silent in most somatic cells, is activated to maintain the telomere length (TEL) in various types of cancer cells, including melanoma. In the vast majority of tumor cells, the TERT promoter is mutated to promote proliferation and inhibit apoptosis. Here, we exploited NG-ABEmax to revert TERT -146 T to -146 C in melanoma, and successfully obtained TERT promoter revertant mutant cells. These TERT revertant mutant cells exhibited significant growth inhibition both in vitro and in vivo. Moreover, A375-146C/C cells exhibited telomere shortening and the downregulation of TERT at both the transcription and protein levels, and migration and invasion were inhibited. In addition, TERT promoter revertant mutation abrogated the inhibitory effect of mutant TERT on apoptosis via B-cell lymphoma 2 (Bcl-2), ultimately leading to cell death. Collectively, the results of our work demonstrate that reverting mutations in the TERT promoter is a potential therapeutic option for melanoma.

Porcine Epidemic Diarrhea Virus: An Updated Overview of Virus Epidemiology, Virulence Variation Patterns and Virus-Host Interactions.

The porcine epidemic diarrhea virus (PEDV) is a member of the coronavirus family, causing deadly watery diarrhea in newborn piglets. The global pandemic of PEDV, with significant morbidity and mortality, poses a huge threat to the swine industry. The currently developed vaccines and drugs are only effective against the classic GI strains that were prevalent before 2010, while there is no effective control against the GII variant strains that are currently a global pandemic. In this review, we summarize the latest progress in the biology of PEDV, including its transmission and origin, structure and function, evolution, and virus-host interaction, in an attempt to find the potential virulence factors influencing PEDV pathogenesis. We conclude with the mechanism by which PEDV components antagonize the immune responses of the virus, and the role of host factors in virus infection. Essentially, this review serves as a valuable reference for the development of attenuated virus vaccines and the potential of host factors as antiviral targets for the prevention and control of PEDV infection.

Correction: Wang et al. TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis. Biology 2022, 11, 141.

The authors would like to make the following correction to the published paper [...].

Differential Expression Analysis of tRNA-Derived Small RNAs from Subcutaneous Adipose Tissue of Obese and Lean Pigs.

Epigenetic factors, including non-coding RNA regulation, play a vital role in the development of obesity and have been well researched. Transfer RNA-derived small RNA (tsRNA) is a class of non-coding RNA proven to be involved in various aspects of mammalian biology. Here we take pigs as a model for obesity research and use tsRNA-seq to investigate the difference in tsRNA expression in the subcutaneous adipose tissue of obese and lean pigs to elucidate the role of tsRNA in obesity development. A total of 482 tsRNAs were identified in pig adipose tissue, of which 123 were significantly differentially accumulated tsRNAs compared with the control group. The tRF-5c was the main type of these tsRNAs. The largest number of tsRNAs produced was the Gly-carrying tRNA, which produced 81 tsRNAs. Functional enrichment analysis revealed that differential tsRNAs indirectly participated in MAPK, AMPK, insulin resistance, the TNF signaling pathway, adipocytokine signaling pathway, and other signaling pathways by interacting with target genes. These are involved in bioenergetic metabolic regulatory processes, suggesting that tsRNAs may influence these pathways to mediate the regulation of energy metabolism in porcine adipocytes to promote lipid deposition, thus contributing to obesity. Our findings suggest a potential function of tsRNA in regulating obesity development.

Plasma Metabolomic Profiling Reveals Preliminary Biomarkers of Pork Quality Based on pH Value.

This study aimed to identify biomarkers for pork quality evaluation. Firstly, the correlation between indicators of pork quality evaluation was investigated. The pH of pork meat at 45 min post slaughter showed a significant negative correlation with meat color indicators (r: -0.4868--0.3040). Subsequently, porcine plasma samples were further divided into low pH (pH = 6.16 ± 0.22) or high pH (pH = 6.75 ± 0.08) groups. Plasma metabolites in both sample groups were investigated using untargeted metabolomics. In total, 90 metabolites were recognized as differential metabolites using partial least squares discriminant analysis. Pathway enrichment analysis indicated these differential metabolites were enriched in amino acid metabolism and energy metabolism. Correlation analysis revealed that creatinine, L-carnitine, D-sphingosine, citraconic acid, and other metabolites may constitute novel plasma biomarkers with the pH value of pork meat. The current study provides important insights into plasma biomarkers for predicting pork quality based on pH value.

CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus.

Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.

HMEJ-mediated site-specific integration of a myostatin inhibitor increases skeletal muscle mass in porcine.

As a robust antagonist of myostatin (MSTN), follistatin (FST) is an important regulator of skeletal muscle development, and the delivery of FST to muscle tissue represents a potential therapeutic strategy for muscular dystrophies. The N terminus and FSI domain of FST are the functional domains for MSTN binding. Here, we aimed to achieve site-specific integration of FSI-I-I, including the signal peptide, N terminus, and three FSI domains, into the last codon of the porcine MSTN gene using a homology-mediated end joining (HMEJ)-based strategy mediated by CRISPR-Cas9. Based on somatic cell nuclear transfer (SCNT) technology, we successfully obtained FSI-I-I knockin pigs. H&E staining of longissimus dorsi and gastrocnemius cross-sections showed larger myofiber sizes in FSI-I-I knockin pigs than in controls. Moreover, the Smad and Erk pathways were inhibited, whereas the PI3k/Akt pathway was activated in FSI-I-I knockin pigs. In addition, the levels of MyoD, Myf5, and MyoG transcription were upregulated while that of MRF4 was downregulated in FSI-I-I knockin pigs. These results indicate that the FSI-I-I gene mediates skeletal muscle hypertrophy through an MSTN-related signaling pathway and the expression of myogenic regulatory factors. Overall, FSI-I-I knockin pigs with hypertrophic muscle tissue hold great promise as a therapeutic model for human muscular dystrophies.

Monitoring of sausage products sold in Sichuan Province, China: a first comprehensive report on meat species' authenticity determination.

Presently, there is growing concern worldwide regarding the adulteration of meat products. However, no reports on determining meat authenticity have been reported in China. To verify labelling compliance and evaluate the existence of fraudulent practices, 250 sausage samples were purchased from local markets in Sichuan Province and analysed for the presence of chicken, pork, beef, duck and genetically modified soybean DNA using real-time and end-point PCR methods, providing a Chinese case study on the problem of world food safety. In total, 74.4% (186) of the samples were properly labelled, while the other 25.6% (64) were potentially adulterated samples, which involved three illicit practices: product removal, addition and substitution. The most common mislabelling was the illegal addition of, or contamination with, duck. Therefore, meat authenticity monitoring should be routinely conducted. Additionally, the strict implementation of the nation's food safety laws, along with regular surveillance, should be compulsory to alleviate and deter meat adulteration.

Molecular marker diversity of SCN-resistant sources in soybean.

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe; HG) is one of the most destructive pests of soybean (Glycine max (L.) Merr.) in the United States. Over 100 SCN-resistant accessions within the USDA Soybean Germplasm Collection have been identified, but little is known about the genetic diversity of this SCN-resistant germplasm. The objective of this research was to evaluate the genetic variation and determine the genetic relationships among SCN-resistant accessions. One hundred twenty-two genotypes were evaluated by 85 simple sequence repeat (SSR) markers from 20 linkage groups. Non-hierarchical (VARCLUS) and hierarchical (Ward's) clustering were combined with multidimensional scaling (MDS) to determine relationships among tested lines. The 85 SSR markers produced 566 allelic fragments with a mean polymorphic information content (PIC) value of 0.35. The 122 lines were grouped into 7 clusters by 2 different clustering methods and the MDS results consistently corresponded to the assigned clusters. Assigned clusters were dominated by genotypes that possess one or more unique SCN resistance genes and were associated with geographical origins. The results of analysis of molecular variance (AMOVA) showed that the variation differences among clusters and individual lines were significant, but the differences among individuals within clusters were not significant.