An inducible hACE2 transgenic mouse model recapitulates SARS-CoV-2 infection and pathogenesis in vivo.

The classical manifestation of COVID-19 is pulmonary infection. After host cell entry via human angiotensin-converting enzyme II (hACE2), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus can infect pulmonary epithelial cells, especially the AT2 (alveolar type II) cells that are crucial for maintaining normal lung function. However, previous hACE2 transgenic models have failed to specifically and efficiently target the cell types that express hACE2 in humans, especially AT2 cells. In this study, we report an inducible, transgenic hACE2 mouse line and showcase three examples for specifically expressing hACE2 in three different lung epithelial cells, including AT2 cells, club cells, and ciliated cells. Moreover, all these mice models develop severe pneumonia after SARS-CoV-2 infection. This study demonstrates that the hACE2 model can be used to precisely study any cell type of interest with regard to COVID-19-related pathologies.

High Levels of Extracellular Potassium Can Delay Myxoma Virus Replication by Preventing Release of Virions from the Endosomes.

Potassium (K+) is one of the most abundant cations in the human body. Under normal conditions, the vast majority of K+ is found within cells, and the extracellular [K+] is tightly regulated to within 3.0 to 5.0 mM. However, it has recently been shown that high levels of localized necrosis can increase the extracellular concentration of K+ to above 50 mM. This raises the possibility that elevated extracellular K+ might influence a variety of biological processes that occur within regions of necrotic tissue. For example, K+ has been shown to play a central role in the replication cycles of numerous viral families, and in cases of lytic infection, localized regions containing large numbers of necrotic cells can be formed. Here, we show that the replication of the model poxvirus myxoma virus (MYXV) is delayed by elevated levels of extracellular K+. These increased K+ concentrations alter the cellular endocytic pathway, leading to increased phagocytosis but a loss of endosomal/lysosomal segregation. This slows the release of myxoma virus particles from the endosomes, resulting in delays in genome synthesis and infectious particle formation as well as reduced viral spread. Additionally, mathematical modeling predicts that the extracellular K+ concentrations required to impact myxoma virus replication can be reached in viral lesions under a variety of conditions. Taken together, these data suggest that the extracellular [K+] plays a role in determining the outcomes of myxoma infection and that this effect could be physiologically relevant during pathogenic infection. IMPORTANCE Intracellular K+ homeostasis has been shown to play a major role in the replication of numerous viral families. However, the potential impact of altered extracellular K+ concentrations is less well understood. Our work demonstrates that increased concentrations of extracellular K+ can delay the replication cycle of the model poxvirus MYXV by inhibiting virion release from the endosomes. Additionally, mathematical modeling predicts that the levels of extracellular K+ required to impact MYXV replication can likely be reached during pathogenic infection. These results suggest that localized viral infection can alter K+ homeostasis and that these alterations might directly affect viral pathogenesis.

Lethal Phenotype-Based Database Screening Identifies Ceramide as a Negative Regulator of Primitive Streak Formation.

In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation has been challenging. We performed comprehensive screening of 2 knockout mouse databases based on the fact that failure of PrS formation is lethal. We identified 812 genes involved in various cellular functions and responses that might be linked to PrS formation, with the category of greatest abundance being "Metabolism." In this study, we focused on genes of sphingolipid metabolism and investigated their roles in PrS formation using an in vitro mouse ES cell differentiation system. We show here that elevated intracellular ceramide negatively regulates gene expression essential for PrS formation and instead induces neurogenesis. In addition, sphingosine-1-phosphate (a ceramide derivative) positively regulates neural maturation. Our results indicate that ceramide regulates both PrS formation and the induction of neural differentiation.

Identification of IFI44L as a new candidate molecular marker for systemic lupus erythematosus.

OBJECTIVES: We screened the type I interferon signal pathway factor involved in the pathogenesis of systemic lupus erythematosus (SLE) by whole-genome sequencing in SLE patients and initially analyse their potential functions. METHODS: Use high-throughput sequencing technology to sequence mRNAs on peripheral blood mononuclear cells from SLE patients and healthy controls,and screen out differentially expressed genes related to the type I interferon (IFN) pathway. Quantitative reverse transcription PCR (RT-qPCR) was utilised to verify the expression of the IFI44L gene in SLE patients and healthy controls, and the correlation between its expression level and clinical test indicators of SLE patients were analysed. The receiver operating characteristic (ROC) analyses were conducted to explore the value of IFI44L for SLE diagnosis. Cell counting kit-8 assay and flow cytometry were used to detect the effects of IFI44L on cell proliferation, apoptosis, and cell cycle. RESULTS: A total of 122 genes were significantly up-regulated and 34 genes were significantly down-regulated in the SLE group compared with the healthy control group in this research. The significantly up-regulated IFI44L in SLE patients was verified by RT-qPCR (p<0.01), furthermore, male SLE patients were significantly higher than that in female SLE patients (p<0.05). Moreover, ROC analyses proved IFI44L may have diagnostic value for SLE. Meanwhile, IFI44L expression level was significantly correlated with platelets, mean platelet volume, red blood cell distribution width to platelet ratio, complement component 3, and C-reactive protein (p<0.05). In addition, under the action of high interferon, IFI44L can resist the proapoptotic effect of IFN-α and improve the proliferation activity of cells. CONCLUSIONS: IFI44L may play an important role in SLE pathology through abnormal regulation of the type I interferon signalling pathway.

A dePEGylated Lipopeptide-Based Pan-Coronavirus Fusion Inhibitor Exhibits Potent and Broad-Spectrum Anti-HIV-1 Activity without Eliciting Anti-PEG Antibodies.

We previously identified a lipopeptide, EK1C4, by linking cholesterol to EK1, a pan-CoV fusion inhibitory peptide via a polyethylene glycol (PEG) linker, which showed potent pan-CoV fusion inhibitory activity. However, PEG can elicit antibodies to PEG in vivo, which will attenuate its antiviral activity. Therefore, we designed and synthesized a dePEGylated lipopeptide, EKL1C, by replacing the PEG linker in EK1C4 with a short peptide. Similar to EK1C4, EKL1C displayed potent inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses. In this study, we found that EKL1C also exhibited broad-spectrum fusion inhibitory activity against human immunodeficiency virus type 1 (HIV-1) infection by interacting with the N-terminal heptad repeat 1 (HR1) of viral gp41 to block six-helix bundle (6-HB) formation. These results suggest that HR1 is a common target for the development of broad-spectrum viral fusion inhibitors and EKL1C has potential clinical application as a candidate therapeutic or preventive agent against infection by coronavirus, HIV-1, and possibly other class I enveloped viruses.

Effects of L-Cysteine and γ-Aminobutyric Acid Treatment on Postharvest Quality and Antioxidant Activity of Loquat Fruit during Storage.

Sichuan is the China's leading producer of loquat, with the largest cultivation area and yield ranked first in China. Loquat is a seasonal fruit highly appreciated by consumers; however, the fruit is prone to browning and lignification after harvest, affecting its storage quality. The effects of L-Cysteine (L-Cys, 0.01, 0.05, 0.1, 0.2%) and γ-aminobutyric acid (GABA, 0.025, 0.05, 0.075, 0.1%) on the sensory quality and antioxidant activity of loquat fruit during cold storage at 4 °C for 35 days and simulated shelf life for 5 days were investigated. The results showed that after 40 days of storage, compared with the control, 0.05% L-Cys and 0.05% GABA treatment of 'Zaozhong No. 6' loquat fruit effectively reduced the weight loss rate, browning index, decay index, respiratory rate, firmness, and lignin content and slowed the decreases in total soluble solids, soluble sugar, titratable acidityand vitamin C contents. The application of 0.05% L-Cys and 0.05% GABA significantly increased the contents of total phenols, total flavonoids, flavanols, and carotenoids; delayed the increase of relative electric conductivity, MDA, POD, and PPO activities; and significantly enhanced the activities of SOD and CAT, DPPH free radical scavenging ability, and FRAP, thereby improving antioxidant capacity. In summary, 0.05% L-Cys and 0.05% GABA treatment promotes the quality of loquat fruit after 40 days of storage, and significantly enhances antioxidant capacity, thus delaying senescence after harvest.

Targeting the lysosome: Mechanisms and treatments for nonalcoholic fatty liver disease.

The multiple functions of the lysosome, including degradation, nutrient sensing, signaling, and gene regulation, enable the lysosome to regulate lipid metabolism at different levels. In this review, I summarize the recent studies on lysosomal regulation of lipid metabolism and the alterations of the lysosome functions in the livers affected by nonalcoholic fatty liver disease (NAFLD). NAFLD is a highly prevalent lipid metabolic disorder. The progression of NAFLD leads to nonalcoholic steatohepatitis (NASH) and other severe liver diseases, and thus the prevention and treatments of NAFLD progression are critically needed. Targeting the lysosome is a promising strategy. I also discuss the current manipulations of the lysosome functions in the preclinical studies of NAFLD and propose my perspectives on potential future directions.

Development of a mechanism for reconstruction of terahertz single-frequency images of biological samples.

Algorithmic mechanisms are used to improve terahertz (THz) image quality, which is critical to a biological sample analysis. A complete mechanism for the super-resolution reconstruction and evaluation of THz biological sample images was constructed in this study. With eucalyptus leaves as an example, the THz spectral region screening technique was adopted to select the characteristic frequencies for imaging, and the THz single-frequency images were reconstructed with the single-image super-resolution image reconstruction technique. The THz super-resolution reconstructed images without ideal reference were evaluated after the introduction of three no-reference image evaluation criteria considering the diversity and complexity of organisms. The results show that the THz image reconstruction mechanism proposed in this study led to an increase in resolution and a decrease in noise. At the same time, the imaging quality of biological samples was considerably improved, and the detailed information was enriched. These provide a reference for a THz imaging analysis of leaves and other biological samples.

Peptide-Based HIV Entry Inhibitors.

The development of peptide-based HIV entry inhibitors has made an important contribution to the stock of anti-HIV drugs. In particular, the peptide-based anti-HIV drugs enfuvirtide and albuvirtide were approved for clinical use by the U.S. FDA and CFDA in 2003 and 2018, respectively. Peptide-based HIV entry inhibitors exert antiviral activity by targeting the early stage of viral infection, i.e., binding of a viral surface protein to the receptor(s) on the host cell and the subsequent fusion between the viral and host cell membranes. Therefore, they are particularly useful for HIV-infected patients who have failed to respond to the highly active antiretroviral drugs (ARD) targeting the late stage of HIV replication, such as reverse transcriptase inhibitors and protease inhibitors. In this chapter, we will focus on the past, current, and future trends in research and development of peptide-based HIV entry inhibitors.

Randomized, Double-Blinded, Placebo-Controlled Phase 2 Trial of an Inactivated Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine in Healthy Adults.

BACKGROUND: We evaluated an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine for immunogenicity and safety in adults aged 18-59 years. METHODS: In this randomized, double-blinded, controlled trial, healthy adults received a medium dose (MD) or a high dose (HD) of the vaccine at an interval of either 14 days or 28 days. Neutralizing antibody (NAb) and anti-S and anti-N antibodies were detected at different times, and adverse reactions were monitored for 28 days after full immunization. RESULTS: A total of 742 adults were enrolled in the immunogenicity and safety analysis. Among subjects in the 0, 14 procedure, the seroconversion rates of NAb in MD and HD groups were 89% and 96% with geometric mean titers (GMTs) of 23 and 30, respectively, at day 14 and 92% and 96% with GMTs of 19 and 21, respectively, at day 28 after immunization. Anti-S antibodies had GMTs of 1883 and 2370 in the MD group and 2295 and 2432 in the HD group. Anti-N antibodies had GMTs of 387 and 434 in the MD group and 342 and 380 in the HD group. Among subjects in the 0, 28 procedure, seroconversion rates for NAb at both doses were both 95% with GMTs of 19 at day 28 after immunization. Anti-S antibodies had GMTs of 937 and 929 for the MD and HD groups, and anti-N antibodies had GMTs of 570 and 494 for the MD and HD groups, respectively. No serious adverse events were observed during the study period. CONCLUSIONS: Adults vaccinated with inactivated SARS-CoV-2 vaccine had NAb as well as anti-S/N antibody and had a low rate of adverse reactions. CLINICAL TRIALS REGISTRATION: NCT04412538.

Effect of oleic acid on the viability of different freeze-dried Lactiplantibacillus plantarum strains.

Freeze drying is one of the most convenient ways to preserve microorganisms, but in the freeze-drying process, strains will inevitably suffer varying degrees of damage under different conditions. The deterioration of cell membrane integrity is one of the main forms of damage. The type and ratio of fatty acids in the cell membrane affect its characteristics. Therefore, it is worth investigating whether certain fatty acids can increase freeze-drying resistance. In this study, we found that adding a low concentration of oleic acid to a cryoprotectant could increase survival rate of strains of Lactiplantibacillus plantarum following freeze drying, and the optimal concentration of oleic acid was determined to be 0.001%. When 0.001% oleic acid was added to phosphate-buffered saline, the freeze-drying survival rate of L. plantarum increased by up to 6.63 times. Adding 0.001% oleic acid to sorbitol, the survival rate of L. plantarum increased by as much as 3.65 times. The 0.001% oleic acid-sucrose cryoprotectant resulted in a freeze-drying survival rate of L. plantarum of about 90%, a 2.26-fold improvement compared with sucrose alone. Although the effect of oleic acid depends on the cryoprotectants used and the strain treated, addition of oleic acid showed significant improvement overall. Further experiments showed that adding a low concentration of oleic acid to the cryoprotectants improved the freeze-drying survival rate of L. plantarum by maintaining cell membrane integrity and lactate dehydrogenase activity. Our findings provide a new strategy for safeguarding bacterial viability in commonly used cryoprotectants by the addition of a common food ingredient, which may be extensively applied in the food industry.

Influence of freezing temperature before freeze-drying on the viability of various Lactobacillus plantarum strains.

Although freeze-drying is an excellent method for preserving microorganisms, it inevitably reduces cell activity and function. Moreover, probiotic strains differ in terms of their sensitivity to the freeze-drying process. Therefore, it is necessary to optimize the variables relevant to this process. The pre-freezing temperature is a critical parameter of the freeze-drying process, but it remains unclear whether the optimal pre-freezing temperature differs among strains and protectants. This study explored the effects of 4 different pre-freezing temperatures on the survival rates of different Lactobacillus plantarum strains after freeze-drying in the presence of different protectants. Using phosphate-buffered saline solution and sorbitol as protectants, pre-freezing at -196°C, -40°C, and -20°C ensured the highest survival rates after freeze-drying for AR113, AR307, and WCFS1, respectively. Using trehalose, pre-freezing at -20°C ensured the best survival rate for AR113, and -60°C was the best pre-freezing temperature for AR307 and WCFS1. These results indicate that the pre-freezing temperature can be changed to improve the survival rate of L. plantarum, and that this effect is strain-specific. Further studies have demonstrated that pre-freezing temperature affected viability via changes in cell membrane integrity, membrane permeability, and lactate dehydrogenase activity. In summary, pre-freezing temperature is a crucial factor in L. plantarum survival after freeze-drying, and the choice of pre-freezing temperature depends on the strain and the protectant.

Identification of nacre matrix protein genes hic14 and hic19 and their roles in crystal growth and pearl formation in the mussel Hyriopsis cumingii.

Biological mineralization is a highly programmed process in which inorganic minerals reassociate under the strict control of the extracellular matrix to form minerals with special functions and patterns. Shells are biominerals, and their synthesis is finely regulated by organic matrix including matrix proteins, polysaccharides, lipids, pigments, free amino acids, and small peptides. In this study, two matrix protein genes, hic14 and hic19, were isolated from the mantle of the mussel Hyriopsis cumingii. Tissue expression analysis showed that both proteins were expressed mainly in the mantle, and in situ hybridization of mantle tissues showed that they were specifically expressed in the dorsal epithelial cells of mantle pallial. Therefore, hic14 and hic19 were both nacreous layer matrix proteins. In the pearl insertion experiment, hic14 and hic19 kept low expression during pearl sac formation and disordered calcium carbonate deposition, and increased significantly during pearl nacre accumulation, which showed that both proteins participated in the mineralization of pearl nacre. In the RNA interference experiment, shell nacre tablet growth was inhibited after crystal nucleation due to the decreased expression of hic14, and crystal morphology and arrangement of nacre were highly modified after expression of hic19 was inhibited. These results provided further evidence that both hic14 and hic19 participated in nacreous layer biomineralization.

RNA gene profile variation in peripheral blood mononuclear cells from rhesus macaques immunized with Hib conjugate vaccine, Hib capsular polysaccharide and TT carrier protein.

BACKGROUND: The Haemophilus influenzae type b (Hib) conjugate vaccine has been widely used in children to prevent invasive Hib disease because of its strong immunogenicity and antibody response induction relative to the capsular polysaccharide (CPS) antigen. The data from vaccine studies suggest that the conjugate vaccine contains carrier proteins that enhance and/or regulate the antigen's immunogenicity, but the mechanism of this enhancement remains unclear. METHODS: To explore the immunological role of the conjugate vaccine, we compared the immune responses and gene profiles of rhesus macaques after immunization with CPS, carrier protein tetanus toxoid (TT) or conjugate vaccine. RESULTS: A distinct immune response was induced by the Hib conjugate vaccine but not by CPS or carrier protein TT. The genes that were dynamically regulated in conjunction with the macaque immune responses to the conjugate vaccine were investigated. CONCLUSIONS: We propose that these genes are involved in the induction of specific immunity that is characterized by the appearance and maintenance of antibodies against Hib.

Mechanisms and functions of lysosome positioning.

Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation. In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration. The involvement of lysosomes in most of these processes is now known to depend on the ability of lysosomes to move throughout the cytoplasm. Here, we review recent findings on the mechanisms that mediate the motility and positioning of lysosomes, and the importance of lysosome dynamics for cell physiology and pathology.

An inactivated enterovirus 71 vaccine in healthy children.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease in children and may be fatal. A vaccine against EV71 is needed. METHODS: We conducted a randomized, double-blind, placebo-controlled phase 3 trial involving healthy children 6 to 71 months of age in Guangxi Zhuang Autonomous Region, China. Two doses of an inactivated EV71 vaccine or placebo were administered intramuscularly, with a 4-week interval between doses, and children were monitored for up to 11 months. The primary end point was protection against hand, foot, and mouth disease caused by EV71. RESULTS: A total of 12,000 children were randomly assigned to receive vaccine or placebo. Serum neutralizing antibodies were assessed in 549 children who received the vaccine. The seroconversion rate was 100% 4 weeks after the two vaccinations, with a geometric mean titer of 170.6. Over the course of two epidemic seasons, the vaccine efficacy was 97.4% (95% confidence interval [CI], 92.9 to 99.0) according to the intention-to-treat analysis and 97.3% (95% CI, 92.6 to 99.0) according to the per-protocol analysis. Adverse events, such as fever (which occurred in 41.6% of the participants who received vaccine vs. 35.2% of those who received placebo), were significantly more common in the week after vaccination among children who received the vaccine than among those who received placebo. CONCLUSIONS: The inactivated EV71 vaccine elicited EV71-specific immune responses and protection against EV71-associated hand, foot, and mouth disease. (Funded by the National Basic Research Program and others; ClinicalTrials.gov number, NCT01569581.).

Comparison of III-V/Si on-chip lasers with etched facet reflectors.

Electrically pumped heterogeneously integrated III-V/SiO2 semiconductor on-chip lasers with different types of etched facet reflectors are designed and fabricated and their lasing performances are characterized and compared. The III-V quantum-well-based epitaxial layers are bonded on silica-on-silicon substrates and fabricated to form Fabry-Perot lasers with dry-etched rear facets. Three types of reflectors are demonstrated, which are etched facets terminated by air, benzocyclobutene, and metal with a thin layer of SiO2 insulator in-between. The laser devices are characterized and compared, including lasing threshold, external quantum efficiency, and output power, and show the impact of different types of etched facet reflectors on lasing performance.

Graded-index thin-film stack for cladding and coupling.

A graded-index multilayer thin-film stack is optimized to act as a cladding layer on top of a silicon (Si) nanowaveguide and also a collimator for chip coupling where the waveguide ends. The numerical example shows an optimized graded-index profile from 2.35 to 1.45 provides an optical coupling to the standard single-mode fiber with efficiency close to 90% while retaining tight light confinement for the Si nanowaveguide. The corresponding material realization of a graded-index profile with a Si-rich nitride SiNx/SiON/SiO2 system is explored using inductively coupled plasma chemical vapor deposition, and a SiNx cladded Si waveguide is demonstrated.

Immunity and clinical efficacy of an inactivated enterovirus 71 vaccine in healthy Chinese children: a report of further observations.

BACKGROUND: To investigate the long-term effects on immunity of an inactivated enterovirus 71 (EV71) vaccine and its protective efficacy. METHODS: A sub-cohort of 1,100 volunteers from Guangxi Province in China was eligible for enrolment and randomly administered either the EV71 vaccine or a placebo on days 0 and 28 in a phase III clinical trial and then observed for the following 2 years with approval by an independent ethics committee of Guangxi Zhuang Autonomous Region, China. Serum samples from the 350 participants who provided a full series of blood samples (at all the sampling points) within the 2-year period were collected. Vaccine-induced immune effects, including the neutralizing antibody titres and cross-protection against different genotypes of EV71, were examined. This study also evaluated the protective efficacy of this vaccine based upon clinical diagnosis. RESULTS: This sub-cohort showed a >60% drop-out rate over 2 years. The seroconversion rates among the 161 immunized subjects remained >95% at the end of study. The geometric mean titres of neutralizing antibodies (anti-genotype C4) 360 days after vaccination in 350 subjects were 81.0 (subjects aged 6-11 months), 98.4 (12-23 months), 95.0 (24-35 months), and 81.8 (36-71 months). These titres subsequently increased to 423.1, 659.0, 545.0, and 321.9, respectively, at 540 days post-immunization (d.p.i.), and similar levels were maintained at 720 d.p.i. Higher IFN-γ/IL-4-specific responses to the C4 genotype of EV71 and cross-neutralization reactivity against major EV71 genotype strains were observed in the vaccine group compared to those in the placebo group. Five EV71-infected subjects were observed in the placebo-treated control group and none in the vaccine-immunized group in per-protocol analysis. CONCLUSION: These results are consistent with the induction of dynamic immune responses and protective efficacy of the vaccine against most circulating EV71 strains. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov, NCT01569581, Trial registration date: March 2012.

Heterogeneously integrated III-V laser on thin SOI with compact optical vertical interconnect access.

A new heterogeneously integrated III-V/Si laser structure is reported in this report that consists of a III-V ridge waveguide gain section on silicon, III-V/Si optical vertical interconnect accesses (VIAs), and silicon-on-insulator (SOI) nanophotonic waveguide sections. The III-V semiconductor layers are introduced on top of the 300-nm-thick SOI layer through low temperature, plasma-assisted direct wafer-bonding and etched to form a III-V ridge waveguide on silicon as the gain section. The optical VIA is formed by tapering the III-V and the beneath SOI in the same direction with a length of 50 µm for efficient coupling of light down to the 600 nm wide silicon nanophotonic waveguide or vice versa. Fabrication details and specification characterizations of this heterogeneous III-V/Si Fabry-Perot (FP) laser are given. The fabricated FP laser shows a continuous-wave lasing with a threshold current of 65 mA at room temperature, and the slope efficiency from single facet is 144 mW/A. The maximal single facet emitting power is about 4.5 mW at a current of 100 mA, and the side-mode suppression ratio is ∼30 dB. This new heterogeneously integrated III-V/Si laser structure demonstrated enables more complex laser configuration with a sub-system on-chip for various applications.