Anisotropic Growth of Covalent Inorganic Complexes to Nanoplatelets.

Colloidal II-VI semiconductor nanoplatelets (NPLs) provide a new platform in material science due to their unique growth mode and advanced optical properties. However, in contrast to the rapid development of zinc blend structured NPLs, studies on the formation of wurtzite (WZ) NPLs have been limited to the lamellar assembly of specific magic-sized nanoclusters (MSCs). Therefore, the study of new precursors is important for enriching the synthesis strategy, improving the study of two-dimensional (2D) nanocrystal growth mechanisms, and constructing complex nanostructures. Here, we demonstrated that covalent inorganic complexes (CICs), as novel functional intermediates, can be directly used to form NPLs without involving MSCs. Using in situ absorption spectra, we demonstrated that the evolution followed a pseudo-first-order kinetics (kobs = 0.02 min-1 (t1/2 = 34.7 min)). Several types of binary WZ NPLs, including CdSe, CdS, CdTe, and ZnS, have been directly prepared based on this mechanism through the anisotropic growth of CICs. In addition, CICs can also be used to prepare Mn-doped CdSe NPLs. The present study not only affords new precursors for the synthesis of WZ NPLs but also advances our understanding of the synthesis mechanism of nanocrystals.

SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike-mediated cell-to-cell fusion.

Revealing the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and cell-to-cell spread might provide insights for understanding the underlying mechanisms of viral pathogenesis, tropism, and virulence. The signaling pathways involved in SARS-CoV-2 entry and viral spike-mediated cell-to-cell fusion remain elusive. In the current study, we found that macropinocytosis inhibitors significantly suppressed SARS-CoV-2 infection at both the entry and viral spike-mediated cell-to-cell fusion steps. We demonstrated that SARS-CoV-2 entry required the small GTPase Rac1 and its effector kinase p21-activated kinase 1 by dominant-negative and RNAi assays in human embryonic kidney 293T-angiotensin-converting enzyme 2 cells and that the serine protease transmembrane serine protease 2 reversed the decrease in SARS-CoV-2 entry caused by the macropinocytosis inhibitors. Moreover, in the cell-to-cell fusion assay, we confirmed that macropinocytosis inhibitors significantly decreased viral spike-mediated cell-to-cell fusion. Overall, we provided evidence that SARS-CoV-2 utilizes a macropinocytosis pathway to enter target cells and to efficiently promote viral spike-mediated cell-to-cell fusion.

Evaluating angiotensin-converting enzyme 2-mediated SARS-CoV-2 entry across species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex.

Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway.

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the roles of porcine APN (pAPN) during PDCoV infection of nonsusceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter nonsusceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly colocalized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with the potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters nonsusceptible cells but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate antiviral drugs.

Unnatural Amino Acid-Based Ionic Liquid Enables Oral Treatment of Nonsense Mutation Disease in Mice.

This investigation addresses the challenge of suboptimal unnatural amino acid (UAA) utilization in the site-specific suppression of nonsense mutations through genetic code expansion, which is crucial for protein restoration and precise property tailoring. A facile and economical oral liquid formulation is developed by converting UAAs into ionic liquids, significantly enhancing their bioavailability and tissue accumulation. Empirical data reveal a 10-fold increase in bioavailability and up to a 13-fold rise in focal tissue accumulation, alongside marked improvements in UAA incorporation efficiency. A 4-week oral administration in mdx mice, a model for Duchenne muscular dystrophy (DMD), demonstrates the formulation's unprecedented therapeutic potential, with up to 40% dystrophin expression restoration and 75% recovery of normal fiber functions, surpassing existing treatments and exhibiting substantial long-term safety. This study presents a potent oral dosage form that dramatically improves UAA incorporation into target proteins in vivo, offering a significant advance in the treatment of nonsense mutation-mediated disorders and holding considerable promise for clinical translation.

The N-glycosylation at positions 652 and 661 of viral spike protein negatively modulates porcine deltacoronavirus entry.

N-glycosylation is a highly conserved glycan modification that plays crucial roles in various physiological processes, including protein folding, trafficking, and signal transduction. Porcine deltacoronavirus (PDCoV) poses a newly emerging threat to the global porcine industry. The spike protein of PDCoV exhibits a high level of N-glycosylation; however, its role in viral infection remains poorly understood. In this study, we applied a lentivirus-based entry reporter system to investigate the role of N-glycosylation on the viral spike protein during PDCoV entry stage. Our findings demonstrate that N-glycosylation at positions 652 and 661 of the viral spike protein significantly reduces the infectivity of PDCoV pseudotyped virus. Overall, our results unveil a novel function of N-glycosylation in PDCoV infection, highlighting its potential for facilitating the development of antiviral strategies.

Attenuating RNA Viruses with Expanded Genetic Codes to Evoke Adjustable Immune Response in PylRS-tRNACUAPyl Transgenic Mice.

Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a prototypical RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. After NAEK supplementation, EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and protected their neonatal offspring from lethal challenges similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNACUAPyl pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.

Investigating the change in gene expression profile of blood mononuclear cells post-laparoscopic sleeve gastrectomy in Chinese obese patients.

Background: Laparoscopic sleeve gastrectomy (LSG) is a sustainable technique that effectively treats morbid obesity. However, the molecular mechanisms underlying the improvement of metabolic health following this process warrants more investigation. This study investigates LSG-related molecules and uses bulk RNA-sequencing high-throughput analysis to unravel their regulatory mechanisms. Methods: Peripheral blood mononuclear cells (PBMC) were collected from ten obese patients with BMI ≥ 32.5 kg/m2 in the Department of General Surgery of Kunming First People's Hospital. After LSG, patients were followed up for one month, and blood samples were retaken. Blood samples from ten patients before and after LSG and bulk RNA-Seq data were analyzed in this study. LSG-associated gene expression was detected by weighted gene coexpression network analysis (WGCNA) and differential analysis. Subsequently, essential signature genes were identified using logistic least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) algorithms. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and single-sample gene set enrichment analysis (ssGSEA) were utilized to reveal the potential functions of the target genes. Furthermore, the Pearson correlation of signature genes with leptin and lipocalin was also explored. Finally, we constructed a robust endogenous RNA (ceRNA) network based on miRWalk and starBase databases. Results: We identified 18 overlapping genes from 91 hub genes, and 165 differentially expressed mRNAs (DE-mRNA), which were revealed to be significantly associated with immune cells, immune response, inflammatory response, lipid storage, and localization upon functional enrichment analysis. Three signature genes, IRF1, NFKBIA, and YRDC, were identified from the 18 overlapping genes by LASSO and SVM-REF algorithms. The logistic regression model based on the three signature genes highlighted how robustly they discriminated between samples. ssGSEA indicated these genes to be involved in lipid metabolism and degradation pathways. Moreover, leptin levels were significantly reduced in patients undergoing LSG, and NFKBIA significantly negatively correlated with leptin. Finally, we identified how the long non-coding RNA (lncRNA) ATP2B1-AS1 regulated the expression of the signature genes by competitively binding to six microRNAs (miRNAs), which were hsa-miR-6509-5p, hsa-miR-330-5P, hsa-miR-154-5P, hsa-miR-145-5P, hsa-miR4726-5P and hsa-miR-134-5P. Conclusion: This study identified three critical regulatory genes significantly differentiated between patients before and after LSG treatment and highlighted their potentially crucial role after bariatric surgery. This provides novel insights to increase our understanding of the underlying mechanisms of weight loss and associated metabolic improvement after bariatric surgery.

Rational incorporation of any unnatural amino acid into proteins by machine learning on existing experimental proofs.

The unnatural amino acid (UAA) incorporation technique through genetic code expansion has been extensively used in protein engineering for the last two decades. Mutations into UAAs offer more dimensions to tune protein structures and functions. However, the huge library of optional UAAs and various circumstances of mutation sites on different proteins urge rational UAA incorporations guided by artificial intelligence. Here we collected existing experimental proofs of UAA-incorporated proteins in literature and established a database of known UAA substitution sites. By program designing and machine learning on the database, we showed that UAA incorporations into proteins are predictable by the observed evolutional, steric and physiochemical factors. Based on the predicted probability of successful UAA substitutions, we tested the model performance using literature-reported and freshly-designed experimental proofs, and demonstrated its potential in screening UAA-incorporated proteins. This work expands structure-based computational biology and virtual screening to UAA-incorporated proteins, and offers a useful tool to automate the rational design of proteins with any UAA.

An Integrative Analysis of the Immune Features of Inactivated SARS-CoV-2 Vaccine (CoronaVac).

Currently, an inactivated vaccine has been widely used with encouraging results as a prophylactic agent against COVID-19 infection, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. However, in vitro SARS-CoV-2 vaccine-specific immune features remain elusive, hindering the promotion of a third dose of the vaccine. Here, we present a detailed in vitro immune cellular response and large-scale multi-omics analysis for peripheral blood mononuclear cells (PBMCs) from participants vaccinated with CoronaVac (Sinovac Life Sciences, Beijing, China) and recovered participants from COVID-19. The mean titers of SARS-CoV-2 serum-neutralizing antibodies were significantly increased after the boosting immunization (Day 45) compared to the unimmunized state. We observed that type-1 helper T cells (Th1) tended to dominate after the first dose of vaccine, while humoral immune responses became dominant after the second dose due to the activation of type-2 helper T cell (Th2), memory B cells, and plasmablasts. T follicular helper cells (Tfh) involved in antibody production were activated after the first dose and were maintained for the observed time points. Single-cell RNA sequencing of PBMCs revealed specific changes in cell compositions and gene expression in immunized participants. Multi-omics analysis also demonstrated that CoronaVac-specific serum proteins, plasma metabolites, and plasma lipid changes were skewed to those changes in convalescent patients. Collectively, we provide a comprehensive understanding of CoronaVac-specific in vitro immune features.

Optimizing eRF1 to Enable the Genetic Encoding of Three Distinct Noncanonical Amino Acids in Mammalian Cells.

Site-specific incorporation of distinct noncanonical amino acids (ncAAs) into proteins via genetic code expansion in mammalian cells represents a new avenue for protein engineering. Reassigning three TAGs with the same ncAA in mammalian cells has previously been achieved using translational machinery. However, simultaneous recoding of three nonsense codons with distinct ncAAs in mammalian cells remains a challenge due to low incorporation efficiencies. Here, three optimized aaRS/tRNA pairs (i.e., the E. coli-derived tyrosyl (EcTyr)/tRNAUUA , E. coli-derived leucyl (EcLeu)/tRNACUA , and Methanosarcina mazei pyrrolysyl (MmPyl)/tRNAUCA pairs) are screened for ncAA incorporation. Furthermore, introduced combinations of eukaryotic release factor 1 (eRF1) mutants (E55R, E55D, N129D, and Y125F) significantly improve the encoding efficiency of the three premature stop codons' sites from 0.78% to 11.6%. Thus, site-specific incorporation of three distinct ncAAs into a single protein is achieved in this study. This work markedly expands the potential for multiple site-specific protein modifications within mammalian cells, thereby facilitating new in vivo applications.

Trypsin promotes porcine deltacoronavirus mediating cell-to-cell fusion in a cell type-dependent manner.

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.

Pseudorabies Virus UL24 Abrogates Tumor Necrosis Factor Alpha-Induced NF-κB Activation by Degrading P65.

The transcription factor NF-κB plays a critical role in diverse biological processes. The NF-κB pathway can be activated by incoming pathogens and then stimulates both innate and adaptive immunity. However, many viruses have evolved corresponding strategies to balance NF-κB activation to benefit their replication. Pseudorabies virus (PRV) is an economically important pathogen that belongs to the alphaherpesvirus group. There is little information about PRV infection and NF-κB regulation. This study demonstrates for the first time that the UL24 protein could abrogate tumor necrosis factor alpha (TNF-α)-mediated NF-κB activation. An overexpression assay indicated that UL24 inhibits this pathway at or downstream of P65. Furthermore, co-immunoprecipitation analysis demonstrated that UL24 selectively interacts with P65. We demonstrated that UL24 could significantly degrade P65 by the proteasome pathway. For the first time, PRV UL24 was shown to play an important role in NF-κB evasion during PRV infection. This study expands our understanding that PRV can utilize its encoded protein UL24 to evade NF-κB signaling.

[Study on association of interleukin-1 receptor antagonist(RA) gene + 2018 locus mutation with silicosis].

OBJECTIVE: To approach whether + 2018 locus (T-- C) mutation within the interleukin- receptor antagonist (IL-1RA) gene promoter region are associated with susceptibility to silicosis in Southwest Chinese Han population. METHODS: 75 cases of silicosis and 137 silica-exposed workers(being employed) were selected as subjects.5ml peripheral vein blood was drawn from each subject. By real-time quantitative polymerase chain reaction and sequencing techniques, IL-1RA gene mutation of all subjects were analyzed. RESULTS: The frequency of IL-1RA + 2018C among the cases with silicosis was higher than that among the silica-exposed workers (controls) (28.0% versus 15.0% , Fisher' s exact P < 0.01 ). The frequency of allelic C was higher in cases with phase I silicosis than in controls(31.8% versus 15.0%, Fisher's exact P < 0.01 ). The frequency of allelic C was higher in cases with phase I and Ill silicosis than in controls, but which statistic difference being not significant(22.6% versus 15.0%, Fisher's exact P < 0.01). After adjusted for confounding factors, the frequency of IL-1RA + 2018C among silicoses and its subgroups was still higher than among controls, which statistic difference being significant (Fisher' s exact P < 0.01). CONCLUSION: IL-1RA gene polymorphisms played an important role in making the Han population of Southwest China susceptible to silicosis. IL-1RA might be took as a gene to be selected for studying the susceptibility of silicosis.

[On the association of tumor necrosis factor-alpha gene polymorphisms with the susceptibility to silicosis].

OBJECTIVE: To find out whether -308 and -238 locus (G --> A) mutation within the tumor necrosis factor-alpha gene (TNF-alpha) promoter region are associated with susceptibility to silicosis in the Han population of southwest China. METHODS: Governed by the principles of voluntatiness and cooperation, 75 patients with silicosis and 137 control with silica-exposure but without silicosis were recruited, and additionally, 140 elderly patients with silicosis and 135 healthy elderly (retired) controls were recruited in this case-control study. 5 ml peripheral vein blood was drawn from each subject. By means of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) and sequencing techniques, TNF-alpha gene polymorphisms of all subjects were analyzed. RESULTS: The frequencies of TNF-alpha -308A and -238A in the 75 patients with silicosis were higher than those in the 137 controls (P < 0.01). After being adjusted for confounding factors, the -308A and the -238A were still associated with the presence of silicosis (P < 0.01). But the frequency of TNF-alpha -308A in the 140 elderly patients was significantly lower than that in the controls (P < 0.001). CONCLUSIONS: TNF-alpha gene -308 and -238 locus (G --> A) mutation might be related to the occurrence of silicosis and the severity of pulmonary fibrosis in silicosis among the Han population of southwest China, and TNF2 (-308A) allele might increase the risk of the disease.

Investigation of delta-aminolevulinic acid dehydratase polymorphism affecting hematopoietic, hepatic and renal toxicity from lead in Han subjects of southwestern China.

UNLABELLED: This study is to explore the effect of ALAD polymorphism on hematopoietic, hepatic and renal toxicity from lead in occupational exposure workers. METHODS: We conducted a cross-sectional study on 156 workers with occupational exposure to lead between 2002 and 2007. The results of laboratory examinations were analyzed. RESULTS: The authors found that workers with the ALAD 1-1 genotype were associated with higher blood lead level than those with the ALADl-2 genotype. Blood and urine lead levels were much higher in storage battery workers than in cable workers. The urine ALA and blood ZPP levels in workers with the ALAD 1-1 genotype were higher than those with the ALADl-2 genotype. The serum Cr level in workers with the ALADl-1 genotype was much higher than those with the ALADl-2 genotype especially in higher lead exposure level. CONCLUSIONS: The ALAD-2 protein might modify the kinetics of lead in blood at a relatively higher blood lead level and protect against hematopoietic, hepatic and renal toxicity from lead. Urine ALA, blood ZPP and serum Cr levels might be considered as effective biological monitoring partners of lead induced hematopoietic and renal toxicology.

Effects of delta-aminolevulinic acid dehydratase polymorphisms on susceptibility to lead in Han subjects from southwestern China.

This study is to determine the distribution of the delta-aminolevulinic acid dehydratase (ALAD) polymorphism among Han subjects of the Chinese population and to study whether the polymorphism in the ALAD gene modifies the toxicity of lead in lead-exposed workers. For this purpose we conducted a cross-sectional study on 156 Chinese workers who were exposed to lead in lead-acid battery and electric-flex manufacturing plants. The authors found that the allele frequencies of ALAD1 and ALAD2 were 0.9679 and 0.0321, respectively. Workers with the ALAD 1-1 genotype were associated with higher blood lead levels than those with the ALAD 1-2 genotype. Blood and urine lead levels were much higher in storage battery workers than in cable workers. The self-conscious symptom survey showed that the incidences of debilitation, amnesia and dreaminess were much higher in those had more than five years of tenure or contact with lead on the job within the ALAD 1-1 genotype subgroup. Laboratory examinations showed that serum iron and zinc levels in workers' with the ALAD 1-2 genotype were higher than those with the ALAD 1-1 genotype, especially in storage-battery workers. Correlation analysis indicated that the blood lead level negatively correlated with serum calcium, iron and zinc level. The data of this study suggest that the ALAD gene polymorphism and serum ion levels may modify the kinetics of lead in blood. Therefore, the authors recommend that an adequate intake of dietary calcium, iron, and zinc or the calcium, iron, and zinc supplementation should be prescribed to Chinese lead exposed workers.