Characterization of a fatal feline panleukopenia virus derived from giant panda with broad cell tropism and zoonotic potential

Represented by feline panleukopenia virus (FPV) and canine parvovirus (CPV), the species carnivore protoparvovirus 1 has a worldwide distribution through continuous ci13rculation in companion animals such as cats and dogs. Subsequently, both FPV and CPV had engaged in host-to-host transfer to other wild animal hosts of the order Carnivora. In the present study, we emphasized the significance of cross-species transmission of parvoviruses with the isolation and characterization of an FPV from giant panda displaying severe and fatal symptoms. The isolated virus, designated pFPV-sc, displayed similar morphology as FPV, while phylogenetic analysis indicated that the nucleotide sequence of pFPV-sc clades with Chinese FPV isolates. Despite pFPV-sc is seemingly an outcome of a spillover infection event from domestic cats to giant pandas, our study also provided serological evidence that FPV or other parvoviruses closely related to FPV could be already prevalent in giant pandas in 2011. Initiation of host transfer of pFPV-sc is likely with association to giant panda transferrin receptor (TfR), as TfR of giant panda shares high homology with feline TfR. Strikingly, our data also indicate that pFPV-sc can infect cell lines of other mammal species, including humans. To sum up, observations from this study shall promote future research of cross-host transmission and antiviral intervention of Carnivore protoparvovirus 1, and necessitate surveillance studies in thus far unacknowledged potential reservoirs

The molecular diversity of transcriptional factor TfoX is a determinant in natural transformation in Glaesserella parasuis

Natural transformation is a mechanism by which a particular bacterial species takes up foreign DNA and integrates it into its genome. The swine pathogen Glaesserella parasuis (G. parasuis) is a naturally transformable bacterium. The regulation of competence, however, is not fully understood. In this study, the natural transformability of 99 strains was investigated. Only 44% of the strains were transformable under laboratory conditions. Through a high-resolution melting curve and phylogenetic analysis, we found that genetic differences in the core regulator of natural transformation, the tfoX gene, leads to two distinct natural transformation phenotypes. In the absence of the tfoX gene, the highly transformable strain SC1401 lost its natural transformability. In addition, when the SC1401 tfoX gene was replaced by the tfoX of SH0165, which has no natural transformability, competence was also lost. These results suggest that TfoX is a core regulator of natural transformation in G. parasuis, and that differences in tfoX can be used as a molecular indicator of natural transformability. Transcriptomic and proteomic analyses of the SC1401 wildtype strain, and a tfoX gene deletion strain showed that differential gene expression and protein synthesis is mainly centered on pathways related to glucose metabolism. The results suggest that tfoX may mediate natural transformation by regulating the metabolism of carbon sources. Our study provides evidence that tfoX plays an important role in the natural transformation of G. parasuis

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Coronavirus S and HE proteins:a two‐component system for dynamic virion‐sialoglycan interactions: Implications for embecovirus cross‐species transmission, host adaptation and host exclusivity

Coronaviruses (CoVs) are notorious for crossing host species barriers. Their emergence upon zoonotic introduction poses a significant threat to public health as poignantly illustrated by the ongoing SARS-CoV-2 pandemic. Thus, there is an evident need to understand the conditions that facilitate or hamper coronaviruses species jumping. Of four established human CoVs, HCoV-OC43 and HCoV-HKU1 use 9-O-Ac-Sia for attachment and both emerged from the same minor clade of embecoviruses. They also differ from all other coronaviruses in that they possess two types of envelope proteins: the receptor binding fusion protein S, and the hemagglutinin-esterase protein (HE) which has receptor-destroying activity. In chapter 2, we provide evidence that, in contrast to the HEs of animal corona- and toroviruses, the HEs of OC43 and HKU1 gradually lost their lectin function during circulation in the human population. We show that the HE lectin domain is a regulator of HE esterase activity and enhances the destruction of clustered glycotopes. Loss of HE lectin function, apparently in combination with the size difference between S and HE, altered virion-associated receptor-destroying activity: it was strongly reduced, became fully dependent on receptor-binding by S and became selective for certain receptor populations. In chapter 3, we took a comparative structural analysis approach in combination with automated ligand docking and mutational analysis to identify the RBS in the β1CoV S protein. We show that this site is not only present in the S proteins of BCoV, HCoV-OC43 and PHEV, but also conserved and functional in HCoV-HKU1. We provide direct evidence that the RBS is essential for virus infectivity by vesicular stomatitis virus pseudotyping. In chapter 4, we extended our findings by engaging in a collaborative study to structurally identify the RBS of HCoV-OC43. A holostructure was determined by cryo-electron microscopy of OC43 S in complex with 9-O-acetylated sialic acid at 2.8 Å resolution. Moreover, we were first to analyze the binding kinetics of the S RBS to 9-O-Ac-Sia in monovalent 1:1 binding assays using biolayer interferometry and synthetic sialosides as ligand. We showed that binding is highly dynamic with association and release occurring within tenths of seconds. In chapter 5, we developed a reverse genetics system for BCoV and, replaying OC43 evolution, used the system to abolish the HE RBS. We show that loss of HE lectin function consistently selects for second-site mutations in the S RBS that dramatically reduce S binding affinity. In some mutants, bivalent binding of S1A-Fc fusion proteins to the clustered sialoglycans of bovine submaxillary mucin was reduced by four orders of magnitude. We present data to demonstrate that HE and S are in functional balance and co-evolve, with selection not only for optimal balance between attachment and receptor-destruction, but also for maximal virion avidity within the given constraints. The data lead us to conclude that the acquisition of an HE gene has set the embecoviruses on an evolutionary path that selected for mechanisms of virus-glycan interactions that are unique among coronaviruses, but remarkably similar to those seen in influenza A viruses

Coronavirus S and HE proteins:a two‐component system for dynamic virion‐sialoglycan interactions: Implications for embecovirus cross‐species transmission, host adaptation and host exclusivity

Coronaviruses (CoVs) are notorious for crossing host species barriers. Their emergence upon zoonotic introduction poses a significant threat to public health as poignantly illustrated by the ongoing SARS-CoV-2 pandemic. Thus, there is an evident need to understand the conditions that facilitate or hamper coronaviruses species jumping. Of four established human CoVs, HCoV-OC43 and HCoV-HKU1 use 9-O-Ac-Sia for attachment and both emerged from the same minor clade of embecoviruses. They also differ from all other coronaviruses in that they possess two types of envelope proteins: the receptor binding fusion protein S, and the hemagglutinin-esterase protein (HE) which has receptor-destroying activity. In chapter 2, we provide evidence that, in contrast to the HEs of animal corona- and toroviruses, the HEs of OC43 and HKU1 gradually lost their lectin function during circulation in the human population. We show that the HE lectin domain is a regulator of HE esterase activity and enhances the destruction of clustered glycotopes. Loss of HE lectin function, apparently in combination with the size difference between S and HE, altered virion-associated receptor-destroying activity: it was strongly reduced, became fully dependent on receptor-binding by S and became selective for certain receptor populations. In chapter 3, we took a comparative structural analysis approach in combination with automated ligand docking and mutational analysis to identify the RBS in the β1CoV S protein. We show that this site is not only present in the S proteins of BCoV, HCoV-OC43 and PHEV, but also conserved and functional in HCoV-HKU1. We provide direct evidence that the RBS is essential for virus infectivity by vesicular stomatitis virus pseudotyping. In chapter 4, we extended our findings by engaging in a collaborative study to structurally identify the RBS of HCoV-OC43. A holostructure was determined by cryo-electron microscopy of OC43 S in complex with 9-O-acetylated sialic acid at 2.8 Å resolution. Moreover, we were first to analyze the binding kinetics of the S RBS to 9-O-Ac-Sia in monovalent 1:1 binding assays using biolayer interferometry and synthetic sialosides as ligand. We showed that binding is highly dynamic with association and release occurring within tenths of seconds. In chapter 5, we developed a reverse genetics system for BCoV and, replaying OC43 evolution, used the system to abolish the HE RBS. We show that loss of HE lectin function consistently selects for second-site mutations in the S RBS that dramatically reduce S binding affinity. In some mutants, bivalent binding of S1A-Fc fusion proteins to the clustered sialoglycans of bovine submaxillary mucin was reduced by four orders of magnitude. We present data to demonstrate that HE and S are in functional balance and co-evolve, with selection not only for optimal balance between attachment and receptor-destruction, but also for maximal virion avidity within the given constraints. The data lead us to conclude that the acquisition of an HE gene has set the embecoviruses on an evolutionary path that selected for mechanisms of virus-glycan interactions that are unique among coronaviruses, but remarkably similar to those seen in influenza A viruses

Effects of different conformations of polylysine on the anti-tumor efficacy of methotrexate nanoparticles

Drug delivery systems require that carrier materials have good biocompatibility, degradability, and constructability. Poly(amino acids), a substance with a distinctive secondary structure, not only have the basic features of the carrier materials but also have several reactive functional groups in the side chain, which can be employed as drug carriers to deliver anticancer drugs. The conformation of isomers of drug carriers has some influence on the preparation, morphology, and efficacy of nanoparticles. In this study, two isomers of polylysine, including ε-polylysine (ε-PL) and α-polylysine (α-PL), were used as drug carriers to entrap methotrexate (MTX) and construct nano-drug delivery systems. ε-PL/MTX nanoparticles with the morphology of helical nanorods presented a small particle size (115.0 nm), and relative high drug loading content (57.8 %). The anticancer effect of ε-PL/MTX nanoparticles was 1.3-fold and 2.6-fold stronger than that of α-PL/MTX nanoparticles in vivo and in vitro, respectively. ε-PL is an ideal drug carrier with potential clinical application prospects

Machine learning integrations develop an antigen-presenting-cells and T-Cells-Infiltration derived LncRNA signature for improving clinical outcomes in hepatocellular carcinoma

Abstract As a highly heterogeneous cancer, the prognostic stratification and personalized management of hepatocellular carcinoma (HCC) are still challenging. Recently, Antigen-presenting-cells (APCs) and T-cells-infiltration (TCI) have been reported to be implicated in modifying immunology in HCC. Nevertheless, the clinical value of APCs and TCI-related long non-coding RNAs (LncRNAs) in the clinical outcomes and precision treatment of HCC is still obscure. In this study, a total of 805 HCC patients were enrolled from three public datasets and an external clinical cohort. 5 machine learning (ML) algorithms were transformed into 15 kinds of ML integrations, which was used to construct the preliminary APC-TCI related LncRNA signature (ATLS). According to the criterion with the largest average C-index in the validation sets, the optimal ML integration was selected to construct the optimal ATLS. By incorporating several vital clinical characteristics and molecular features for comparison, ATLS was demonstrated to have a relatively more significantly superior predictive capacity. Additionally, it was found that the patients with high ATLS score had dismal prognosis, relatively high frequency of tumor mutation, remarkable immune activation, high expression levels of T cell proliferation regulators and anti-PD-L1 response as well as extraordinary sensitivity to Oxaliplatin/Fluorouracil/Lenvatinib. In conclusion, ATLS may serve as a robust and powerful biomarker for improving the clinical outcomes and precision treatment of HCC

Exploration and validation of a novel signature of seven necroptosis-related genes to improve the clinical outcome of hepatocellular carcinoma

Abstract Necroptosis has been reported to be involved in cancer progression and associated with cancer prognosis. However, the prognostic values of necroptosis-related genes (NRGs) in hepatocellular carcinoma (HCC) remain largely unknown. This study aimed to build a signature on the basis of NRGs to evaluate the prognosis of HCC patients. In this study, using bioinformatic analyses of transcriptome sequencing data of HCC (n = 370) from The Cancer Genome Atlas (TCGA) database, 63 differentially expressed NRGs between HCC and adjacent normal tissues were determined. 24 differentially expressed NRGs were found to be related with overall survival (OS). Seven optimum NRGs, determined using Lasso regression and multivariate Cox regression analysis, were used to construct a new prognostic risk signature for predicting the prognosis of HCC patients. Then survival status scatter plots and survival curves demonstrated that the prognosis of patients with high-Riskscore was worse. The prognostic value of this 7-NRG signature was validated by the International Cancer Genome Consortium (ICGC) cohort and a local cohort (Wenzhou, China). Notably, Riskscore was defined as an independent risk factor for HCC prognosis using multivariate cox regression analysis. Immune infiltration analysis suggested that higher macrophage infiltration was found in patients in the high-risk group. Finally, enhanced 7 NRGs were found in HCC tissues by immunohistochemistry. In conclusion, a novel 7-NRG prognostic risk signature is generated, which contributes to the prediction in the prognosis of HCC patients for the clinicians

Synthetic O-Acetylated Sialosides and their Acetamido-deoxy Analogues as Probes for Coronaviral Hemagglutinin-esterase Recognition

O-Acetylation is a common modification of sialic acids that can occur at carbons 4-, 7-, 8-, and/or 9. Acetylated sialosides are employed as receptors by several betacoronaviruses and toroviruses, and by influenza C and D viruses. The molecular basis by which these viruses recognize specific O-acetylated sialosides is poorly understood, and it is unknown how viruses have evolved to recognize specific O-acetylated sialosides expressed by their host. Here, we describe a chemoenzymatic approach that can readily provide sialoglycan analogues in which acetyl esters at C4 and/or C7 are replaced by stabilizing acetamide moieties. The analogues and their natural counterparts were used to examine the ligand requirements of the lectin domain of coronaviral hemagglutinin-esterases (HEs). It revealed that HEs from viruses targeting different host species exhibit different requirements for O-acetylation. It also showed that ester-to-amide perturbation results in decreased or loss of binding. STD NMR and molecular modeling of the complexes of the HE of BCoV with the acetamido analogues and natural counterparts revealed that binding is governed by the complementarity between the acetyl moieties of the sialosides and the hydrophobic patches of the lectin. The precise spatial arrangement of these elements is important, and an ester-to-amide perturbation results in substantial loss of binding. Molecular Dynamics simulations with HEs from coronaviruses infecting other species indicate that these viruses have adapted their HE specificity by the incorporation of hydrophobic or hydrophilic elements to modulate acetyl ester recognition