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The proliferation and differentiation of pre-adipocytes are regulated by microRNAs (miRNAs) and other factors. In this study, the potential functions of bta-miR-6517 in the regulation of pre-adipocyte proliferation and differentiation were explored. The qRT-PCR, oil red O staining and CCK-8 assay were used to evaluate the role of bta-miR-6517. Further, the target gene of bta-miR-6517 was identified using bioinformatics analysis, dual-luciferase reporter system and qRT-PCR system. The results found that the overexpression of bta-miR-6517 promoted the expression of proliferation marker genes and substantially increased the adipocyte proliferation vitality in the CCK-8 assay, whereas suppressing of bta-miR-6517 had the opposite effect. Overexpression bta-miR-6517 suppressed the expression of adipogenic genes, which inhibited lipid accumulation, whereas suppressing of bta-miR-6517 had the opposite effect. Furthermore, the dual-fluorescent reporter experiment results demonstrated that bta-miR-6517 directly targeted phosphofructokinase, liver type (PFKL). When bta-miR-6517 was either overexpressed or suppressed, it negatively regulated PFKL. In conclusion, we observed that bta-miR-6517 promoted adipocyte proliferation and inhibited differentiation by targeting PFKL.

The recently emerged Omicron (B.1.1.529) variant has rapidly surpassed Delta to become the predominant circulating SARS-CoV-2 variant, given the higher transmissibility rate and immune escape ability, resulting in breakthrough infections in vaccinated individuals. A new generation of SARS-CoV-2 vaccines targeting the Omicron variant are urgently needed. Here, we developed a subunit vaccine named RBD-HR/trimer by directly linking the sequence of RBD derived from the Delta variant (containing L452R and T478K) and HR1 and HR2 in SARS-CoV-2 S2 subunit in a tandem manner, which can self-assemble into a trimer. In multiple animal models, vaccination of RBD-HR/trimer formulated with MF59-like oil-in-water adjuvant elicited sustained humoral immune response with high levels of broad-spectrum neutralizing antibodies against Omicron variants, also inducing a strong T cell immune response in vivo. In addition, our RBD-HR/trimer vaccine showed a strong boosting effect against Omicron variants after two doses of mRNA vaccines, featuring its capacity to be used in a prime-boost regimen. In mice and non-human primates, RBD-HR/trimer vaccination could confer a complete protection against live virus challenge of Omicron and Delta variants. The results qualified RBD-HR/trimer vaccine as a promising next-generation vaccine candidate for prevention of SARS-CoV-2, which deserved further evaluation in clinical trials.

The disease caused by SARS-CoV-2 infection threatens human health. In this study, we used high-pressure homogenization technology not only to efficiently drive the bacterial membrane to produce artificial vesicles but also to force the fusion protein ClyA-receptor binding domain (RBD) to pass through gaps in the bacterial membrane to increase the contact between ClyA-RBD and the membrane. Therefore, the load of ClyA-RBD on the membrane is substantially increased. Using this technology, we constructed a "ring-like" bacterial biomimetic vesicle (BBV) loaded with polymerized RBD (RBD-BBV). RBD-BBVs injected subcutaneously can accumulate in lymph nodes, promote antigen uptake and processing, and elicit SARS-CoV-2-specific humoral and cellular immune responses in mice. In conclusion, we evaluated the potential of this novel bacterial vesicle as a vaccine delivery system and provided a new idea for the development of SARS-CoV-2 vaccines.

Neurological manifestations are frequently reported in the COVID-19 patients. Neuromechanism of SARS-CoV-2 remains to be elucidated. In this study, we explored the mechanisms of SARS-CoV-2 neurotropism via our established non-human primate model of COVID-19. In rhesus monkey, SARS-CoV-2 invades the CNS primarily via the olfactory bulb. Thereafter, viruses rapidly spread to functional areas of the central nervous system, such as hippocampus, thalamus, and medulla oblongata. The infection of SARS-CoV-2 induces the inflammation possibly by targeting neurons, microglia, and astrocytes in the CNS. Consistently, SARS-CoV-2 infects neuro-derived SK-N-SH, glial-derived U251, and brain microvascular endothelial cells in vitro. To our knowledge, this is the first experimental evidence of SARS-CoV-2 neuroinvasion in the NHP model, which provides important insights into the CNS-related pathogenesis of SARS-CoV-2.

Identification of a suitable nonhuman primate (NHP) model of COVID-19 remains challenging. Here, we characterized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in three NHP species: Old World monkeys Macaca mulatta (M. mulatta) and Macaca fascicularis (M. fascicularis) and New World monkey Callithrix jacchus (C. jacchus). Infected M. mulatta and M. fascicularis showed abnormal chest radiographs, an increased body temperature and a decreased body weight. Viral genomes were detected in swab and blood samples from all animals. Viral load was detected in the pulmonary tissues of M. mulatta and M. fascicularis but not C. jacchus. Furthermore, among the three animal species, M. mulatta showed the strongest response to SARS-CoV-2, including increased inflammatory cytokine expression and pathological changes in the pulmonary tissues. Collectively, these data revealed the different susceptibilities of Old World and New World monkeys to SARS-CoV-2 and identified M. mulatta as the most suitable for modeling COVID-19.

Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell–cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.

New SARS-COV-2 vaccine strategies are still urgently needed, especially for emerging virus mutations and variants. In this study, we focused on analyzing the antigenicity and vaccine potency of linear peptide epitopes located in receptor binding motif (RBM) of spike (S) protein. Nine 12 to 16-mer overlapping peptides (P1-P9) were synthesized chemically and coupled to carrier protein KLH for the immunization in mice. Four of identified peptides were further engineered to present on the surface of recombinant Hepatitis B core antigen (HBcAg) virus-like particles (VLPs) respectively. Antisera obtained from VLPs -immunized mice demonstrated strong reactivity and affinity to S1 protein or inactivated virus and neutralizing activity against virus infection in vitro. This study indicates that recombinant VLPs empower peptides which display underprivileged antigenicity in native protein to elicit high levels of neutralizing antibody, providing potential epitope candidates and an effective delivery strategy for the development of a multi-epitope vaccine., Graphical Abstract Nine peptides (P1-P9) which cover the whole sequence of RBM of SARS-COV-2 spike protein were synthesized chemically and coupled to KLH for the immunization in mice. Among these peptides, P2, P3, P4 and P6 showed neutralizing protection against virus infection in a cell assay. The four peptides were further engineered to present on the surface of recombinant Hepatitis B core antigen (HBcAg) virus-like particles (VLPs) respectively. HBcAg-P6 VLPs dramatically elevated specific IgG level, and the antiserum demonstrated a strong reactivity and affinity to S1 protein or inactivated virus and neutralizing activity against virus infection in vitro.Unlabelled Image

The specificity of sperm–egg recognition is crucial to species independence, and two proteins (Izumo1 and JUNO) are essential for gamete adhesion/fusion in mammals. However, hybridization, which is very common in turtles, also requires specific recognition of sperm–egg binding proteins. In this study, we discovered that natural selection plays an important role in the codon usage bias of Tu-Izumo1 and Tu-JUNO. Positively selected sites and co-evolutionary analyses between Tu-Izumo1 and Tu-JUNO have been previously reported, and we confirm these results in a larger analysis containing 25 turtle species. We also showed that Tu-JUNO is expressed on the oocyte surface and that Tu-Izumo1 and Tu-JUNO interact with each other directly in different species hybridization combinations. Co-immunization assays revealed that this interaction is evolutionarily conserved in turtles. The results of avidity-based extracellular interaction screening between Tu-Izumo1 and Tu-JUNO for sperm–oocyte binding pairs (both within and across species) likely suggest that the interaction force between Izumo1 and JUNO has a certain correlation in whether the turtles can hybridize. Our results lay a theoretical foundation for the subsequent development of techniques to detect whether different turtle species can interbreed, which would provide the molecular basis for breeding management and species protection of turtles.

Ozobranchus jantseanus is the largest metazoan known to survive in liquid nitrogen without pretreatment to date; however, the mechanism underlying this tolerance remains unclear. In this study, the first analyses of histological and morphological changes in normal, frozen, and dehydrated states were performed. Adults survived after direct placement in liquid nitrogen for 96 h, with a survival rate of approximately 86.7%. The leech could withstand rapid desiccation and its survival rate after rehydration was 100% when its water loss was below about 84.8%. After freezing, desiccation, and ethanol dehydration, the leech immediately formed a hemispherical shape. Particularly during drying, an obvious transparent glass-like substance was observed on surface. Scanning electron microscopy revealed many pores on the surface of the posterior sucker, creating a sponge-like structure, which may help to rapidly expel water, and a hemispherical shape may protect the internal organs by contraction and folding reconstruction in the anterior–posterior direction. A substantial amount of mucopolysaccharides on the surface and acid cells and collagen fibers in the body, all of which contained substantial polysaccharides, may play a key protective role during freezing. Our results indicate that the resistance of leeches to ultra-low temperatures can be explained by cryoprotective dehydration/vitrification strategies. This article has an associated First Person interview with the first author of the paper., Summary: The freeze tolerance mechanism of Ozobranchus jantseanus, the largest metazoan animal requiring no pretreatment that can survive in ultra-low temperature, was first studied from the perspective of morphology.

Background and Aims Gastrointestinal (GI) manifestations have been increasingly reported in Coronavirus Disease 2019 (COVID-19) patients. However, the roles of the GI tract in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection are not fully understood. We investigated how the GI tract is involved in SARS-CoV-2 infection to elucidate the pathogenesis of COVID-19. Methods Our previously established nonhuman primate (NHP) model of COVID-19 was modified in this study to test our hypothesis. Rhesus monkeys were infected with an intragastric or intranasal challenge with SARS-CoV-2. Clinical signs were recorded after infection. Viral genomic RNA was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Host responses to SARS-CoV-2 infection were evaluated by examining inflammatory cytokines, macrophages, histopathology and mucin barrier integrity. Results Intranasal inoculation with SARS-CoV-2 led to infections and pathological changes not only in respiratory tissues but also in digestive tissues. Expectedly, intragastric inoculation with SARS-CoV-2 resulted in the productive infection of digestive tissues and inflammation in both the lung and digestive tissues. Inflammatory cytokines were induced by both types of inoculation with SARS-CoV-2, consistent with the increased expression of CD68. Immunohistochemistry and alcian blue/periodic acid-Schiff (AB-PAS) staining showed decreased Ki67, increased cleaved caspase 3 and decreased numbers of mucin-containing goblet cells, suggesting that the inflammation induced by these two types of inoculation with SARS-CoV-2 impaired the GI barrier and caused severe infections. Conclusions Both intranasal and intragastric inoculation with SARS-CoV-2 caused pneumonia and GI dysfunction in our rhesus monkey model. Inflammatory cytokines are possible connections for the pathogenesis of SARS-CoV-2 between the respiratory and digestive systems., Graphical abstract

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and Coronavirus disease (COVID-19) were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature particularly in female animals. Low levels of virus shedding and replication in tissues occurred in all three age groups. Notably, young tree shrews (6 months to 12 months) showed virus shedding at the earlier stage of infection than adult (2 years to 4 years) and old (5 years to 7 years) animals that had longer duration of virus shedding comparatively. Histopathological examine revealed that pulmonary abnormalities were the main changes but mild although slight lesions were also observed in other tissues. In summary, tree shrew is less susceptible to SARS-CoV-2 infection compared with the reported animal models and may not be a suitable animal for COVID-19 related researches. However, tree shrew may be a potential intermediate host of SARS-CoV-2 as an asymptomatic carrier.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells1,2. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.

Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes β-glucan in the fungal cell wall. C. albicans β-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how β-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates β-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated β-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated β-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis., Author summary Candida albicans is a major cause of invasive candidiasis, which has a high mortality rate. It is a constituent of the human microbiome and found in the gastrointestinal and genitourinary tracts of most healthy individuals. C. albicans is able to switch reversibly between yeast and hyphae in response to environmental cues, and the ability to switch is essential for virulence. The innate immune response is critical for host defense against fungal infections. However, the relationships between morphogenesis, host immune recognition and immune protection are not fully understood for C. albicans. This study shows that β-glucan is masked by two highly expressed yeast proteins, the endo-1,3-β-glucanase Eng1 and Yeast Wall Protein 1 Ywp1. β-glucan is exposed during yeast-to-hypha transition. Eng1 level is also dynamically regulated in response to different carbon sources and antifungal drugs. The eng1 mutant modulates virulence in the mouse model of hematogenously disseminated candidiasis in a sex and Dectin-1-dependent manner. Our data reveal that the level of β-glucan exposure in C. albicans is highly controlled to modulate the balance between immune protection and immunopathogenesis during disseminated candidiasis.

Physically interacting sperm-egg proteins have been identified using gene-modified animals in some mammal species. Three proteins are essential for sperm-egg binding: Izumo1 on the sperm surface, and JUNO and CD9 on the egg surface. Most proteins linked to reproductive function evolve rapidly among species by positive selection, and have correlated evolutionary rates to compensate for changes on both the sperm and egg. Up to now, interactions between sperm and egg proteins have not been identified in non-mammalian vertebrates, such as turtles that have interspecific hybrids that can produce surviving F1 generations. To explore the potential physical interactions of sperm-egg proteins in turtle species, the coding region of Izumo1, JUNO, and CD9 homologous genes (named Tu-Izumo1, Tu-JUNO, and Tu-CD9) in six turtle species (Mauremys reevesii, M. mutica, M. sinensis, Cistoclemmys flavomarginata, Platysternon megacephalum and Chrysemys picta bellii) were identified, amplified, and sequenced, and tissue-specific expression was analyzed in M. reevesii. We constructed phylogenetic trees and analyzed the signatures of coevolution between sperm-egg protein pairs using MirrorTree Server and linear regression methods. The results showed that Tu-Izumo1, Tu-JUNO, and Tu-CD9 proteins have correlated evolutionary rates, and that the area where Tu-Izumo1 interacts with Tu-JUNO has only one positive selection site in some turtle species. These results suggest there is a potential interaction between Tu-Izumo1 and Tu-JUNO among turtles that can interbreed, and that a significantly lower positive selection in the interaction region may be one of the reasons why turtle hybrids are so common. Further studies are required to uncover Tu-Izumo1, Tu-JUNO and Tu-CD9 protein biological functions during gamete fusion.