Isolation of novel simian adenoviruses from macaques for development of a vector for human gene therapy and vaccines.

IMPORTANCE: Adenoviruses are widely used in gene therapy and vaccine delivery. Due to the high prevalence of human adenoviruses (HAdVs), the pre-existing immunity against HAdVs in humans is common, which limits the wide and repetitive use of HAdV vectors. In contrast, the pre-existing immunity against simian adenoviruses (SAdVs) is low in humans. Therefore, we performed epidemiological investigations of SAdVs in simians and found that the SAdV prevalence was as high as 33.9%. The whole-genome sequencing and sequence analysis showed SAdV diversity and possible cross species transmission. One isolate with low level of pre-existing neutralizing antibodies in humans was used to construct replication-deficient SAdV vectors with E4orf6 substitution and E1/E3 deletion. Interestingly, we found that the E3 region plays a critical role in its replication in human cells, but the absence of this region could be compensated for by the E4orf6 from HAdV-5 and the E1 expression intrinsic to HEK293 cells.

Knockdown of VEGFB/VEGFR1 Signaling Promotes White Adipose Tissue Browning and Skeletal Muscle Development.

It has been demonstrated that vascular endothelial growth factor B (VEGFB) and vascular endothelial growth factor receptor 1 (VEGFR1) play a vital role in regulating vascular biological function. However, the role of VEGFB and VEGFR1 in regulating fat deposition and skeletal muscle growth remains unclear. Therefore, this study was conducted to investigate the effects of VEGFB and VEGFR1 on fat deposition and skeletal muscle growth in mice. Our results showed that knockdown of VEGFB decreased body weight and iWAT index, stimulated the browning of mice iWAT with increased expression of UCP1, decreased the diameters of adipocytes, and elevated energy expenditure. In contrast, knockdown of VEGFB increased gastrocnemius (GAS) muscle index with increased proliferation of GAS muscle by expression of PCNA and Cyclin D1. Meanwhile, knockdown of endothelial VEGFR1 induced the browning of iWAT with increased expression of UCP1 and decreased diameters of adipocytes. By contrast, knockdown of endothelial VEGFR1 inhibited GAS muscle differentiation with decreased expression of MyoD. In conclusion, these results suggested that the loss of VEGFB/VEGFR1 signaling is associated with enhanced browning of inguinal white adipose tissue and skeletal muscle development. These results provided new insights into the regulation of skeletal muscle growth and regeneration, as well as fat deposition, suggesting the potential application of VEGFB/VEGFR1 as an intervention for the restriction of muscle diseases and obesity and related metabolic disorders.

VEGFB Promotes Myoblasts Proliferation and Differentiation through VEGFR1-PI3K/Akt Signaling Pathway.

It has been demonstrated that vascular endothelial growth factor B (VEGFB) plays a vital role in regulating vascular biological function. However, the role of VEGFB in regulating skeletal muscle cell proliferation and differentiation remains unclear. Thus, this study aimed to investigate the effects of VEGFB on C2C12 myoblast proliferation and differentiation and to explore the underlying mechanism. For proliferation, VEGFB significantly promoted the proliferation of C2C12 myoblasts with the upregulating expression of cyclin D1 and PCNA. Meanwhile, VEGFB enhanced vascular endothelial growth factor receptor 1 (VEGFR1) expression and activated the PI3K/Akt signaling pathway in a VEGFR1-dependent manner. In addition, the knockdown of VEGFR1 and inhibition of PI3K/Akt totally abolished the promotion of C2C12 proliferation induced by VEGFB, suggesting that VEGFB promoted C2C12 myoblast proliferation through the VEGFR1-PI3K/Akt signaling pathway. Regarding differentiation, VEGFB significantly stimulated the differentiation of C2C12 myoblasts via VEGFR, with elevated expressions of MyoG and MyHC. Furthermore, the knockdown of VEGFR1 rather than NRP1 eliminated the VEGFB-stimulated C2C12 differentiation. Moreover, VEGFB activated the PI3K/Akt/mTOR signaling pathway in a VEGFR1-dependent manner. However, the inhibition of PI3K/Akt/mTOR blocked the promotion of C2C12 myoblasts differentiation induced by VEGFB, indicating the involvement of the PI3K/Akt pathway. To conclude, these findings showed that VEGFB promoted C2C12 myoblast proliferation and differentiation via the VEGFR1-PI3K/Akt signaling pathway, providing new insights into the regulation of skeletal muscle development.

Molecular Typing and Rapid Identification of Human Adenoviruses Associated With Respiratory Diseases Using Universal PCR and Sequencing Primers for the Three Major Capsid Genes: Penton Base, Hexon, and Fiber.

Human adenoviruses (HAdVs) within species B, C, and E are responsible for highly contagious and potentially severe respiratory disease infections. The traditional method to type these pathogens was based on virus neutralization and hemagglutination assays, which are both time-consuming and difficult, particularly due to the nonavailability of reagents. Subsequent molecular typing based on the partial characterization of the hexon gene and/or the restriction enzyme analysis (REA) of the genomes is inadequate, particularly in identifying recombinants. Here, a rapid, simple, and cost-effective method for molecular typing HAdV respiratory pathogens is presented. This incorporates three pairs of universal PCR primers that target the variable regions of the three major capsid genes, i.e., hexon, penton base, and fiber genes, that span the genome. The protocol enables typing and characterization of genotypes within species B, C, and E, as well as of some genotypes within species D and F. To validate this method, we surveyed 100 children with HAdV-associated acute respiratory infections identified by direct immunofluorescence (Hong Kong; July through October, 2014). Throat swab specimens were collected and analyzed by PCR amplification and sequencing; these sequences were characterized by BLAST. HAdVs were detected in 98 out of 100 (98%) samples, distributing as follows: 74 HAdV-B3 (74%); 10 HAdV-E4 (10%); 7 HAdV-C2 (7%); 2 HAdV-C6 (2%); 1 HAdV-B7 (1%); 1 HAdV-C1 (1%); 2 co-infection (2%); and 1 novel recombinant (1%). This study is the first detailed molecular epidemiological survey of HAdVs in Hong Kong. The developed method allows for the rapid identification of HAdV respiratory pathogens, including recombinants, and bypasses the need for whole genome sequencing for real-time surveillance of circulating adenovirus strains in outbreaks and populations by clinical virologists, public health officials, and epidemiologists.

Tracking SARS-CoV-2 Omicron diverse spike gene mutations identifies multiple inter-variant recombination events.

The current pandemic of COVID-19 is fueled by more infectious emergent Omicron variants. Ongoing concerns of emergent variants include possible recombinants, as genome recombination is an important evolutionary mechanism for the emergence and re-emergence of human viral pathogens. In this study, we identified diverse recombination events between two Omicron major subvariants (BA.1 and BA.2) and other variants of concern (VOCs) and variants of interest (VOIs), suggesting that co-infection and subsequent genome recombination play important roles in the ongoing evolution of SARS-CoV-2. Through scanning high-quality completed Omicron spike gene sequences, 18 core mutations of BA.1 (frequency >99%) and 27 core mutations of BA.2 (nine more than BA.1) were identified, of which 15 are specific to Omicron. BA.1 subvariants share nine common amino acid mutations (three more than BA.2) in the spike protein with most VOCs, suggesting a possible recombination origin of Omicron from these VOCs. There are three more Alpha-related mutations in BA.1 than BA.2, and BA.1 is phylogenetically closer to Alpha than other variants. Revertant mutations are found in some dominant mutations (frequency >95%) in the BA.1. Most notably, multiple characteristic amino acid mutations in the Delta spike protein have been also identified in the "Deltacron"-like Omicron Variants isolated since November 11, 2021 in South Africa, which implies the recombination events occurring between the Omicron and Delta variants. Monitoring the evolving SARS-CoV-2 genomes especially for recombination is critically important for recognition of abrupt changes to viral attributes including its epitopes which may call for vaccine modifications.

Cytochrome P450 epoxygenase-derived EPA and DHA oxylipins 17,18-epoxyeicosatetraenoic acid and 19,20-epoxydocosapentaenoic acid promote BAT thermogenesis and WAT browning through the GPR120-AMPKα signaling pathway.

The mechanisms whereby fish oil rich in EPA and DHA promotes BAT thermogenesis and WAT browning are not fully understood. Thus, this study aimed to investigate the effects of cytochrome P450 (CYP) epoxygenase-derived EPA and DHA oxylipins 17,18-EpETE and 19,20-EpDPE on BAT thermogenesis and WAT browning and explore the underlying mechanism. Stromal vascular cells (SVCs) were subjected to 17,18-EpETE or 19,20-EpDPE treatment and mice were treated with the CYP epoxygenase inhibitor, the thermogenic marker genes were detected and the involvement of GPR120 and AMPKα were assessed. The in vitro results indicated that 17,18-EpETE and 19,20-EpDPE induced brown and beige adipocyte thermogenesis, with increased expression of thermogenic marker gene UCP1 in differentiated SVCs. Meanwhile, the expression of GPR120 and phosphorylation of AMPKα were increased in response to these two oxylipins. However, the inhibition of GPR120 and AMPKα inhibited the promotion of adipocyte thermogenesis. In addition, in the presence of CYP epoxygenase inhibitor MS-PPOH, EPA and DHA had no effect on increasing UCP1 expression in differentiated SVCs. Consistent with the in vitro results, the in vivo findings demonstrated that fish oil had no body fat-lowering effects and no effects on enhancing energy metabolism, iBAT thermogenesis and iWAT browning in mice fed HFD after intraperitoneal injection of CYP epoxygenase inhibitor SKF-525A. Moreover, fish oil had no effect on the elevation of GPR120 expression and activation of AMPKα in iBAT and iWAT in mice fed HFD after intraperitoneal injection of SKF-525A. In summary, our results showed that CYP epoxygenase-derived EPA and DHA oxylipins 17,18-EpETE and 19,20-EpDPE promoted BAT thermogenesis and WAT browning through the GPR120-AMPKα signaling pathway, which might contribute to the thermogenic and anti-obesity effects of fish oil.

Discrete unified gas kinetic scheme for electrostatic plasma and its comparison with the particle-in-cell method.

In this paper, we present a finite-volume direct kinetic method, the so-called discrete unified gas kinetic scheme (DUGKS), for electrostatic plasma. One key feature of this method is the semi-implicit unsplitting treatment of particle transport and collision, and thus the time step in current DUGKS is not limited by the particle collision time. In addition, a fourth-order compact MUSCL scheme with a positivity preserving limiter is implemented in the interface reconstruction, which enables present DUGKS to preserve the favorable conservative property and positivity of distribution function. Combined with this MUSCL method, the semi-Lagrangian scheme is used for the particle transport in velocity space to remove Courant-Friedricks-Lewy restriction induced by the large electric force. As a result, the proposed DUGKS becomes an efficient and stable multiscale scheme. Several numerical experiments, including plasma sheath, linear Landau damping, collisional nonlinear Landau damping, and plasma ion acceleration, are performed to validate current DUGKS. A comparative study of current DUGKS with a general particle in cell (PIC) method which could handle particle collision in a conservative way is also presented. Theory and numerical experiments demonstrate that DUGKS is preferred for plasma flows involving small electrostatic perturbation and high collision regimes, while the PIC method is desired for the field- dominated plasma flows involving a wide range of velocities.