SARS-CoV-2 minor variant genomes at the start of the pandemic contained markers of VoCs

SARS-CoV-2 emerged through limited zoonotic spillovers and was predicted to have constrained sequence diversity. The dominant consensus and minor variant genomes were determined from the earliest samples associated with the Huanan market and the start of the pandemic. The sequence data confirmed that the dominant consensus genomes shared very close homology. However, there were minor variant genomes present in each sample, which encompassed synonymous and non-synonymous changes. Fusion sequences characteristic of defective RNAs were identified that could be linked between patients. Several substitutions (but not deletions) associated with much later variants of concern (VoCs) were already present as minor variant genomes. This suggests it may be possible to predict futures variants at the start of a pandemic by examining where variability in sequence occurs.

Prognostic value of several immune and inflammatory indices after curative-intent resection for intrahepatic cholangiocarcinoma / 临床肝胆病杂志

Objective To investigate the value of different immune and inflammatory indices in predicting the survival outcome of patients with intrahepatic cholangiocarcinoma (ICC) after curative-intent resection. Methods A retrospective analysis was performed for the case data of 122 patients with ICC who underwent curative-intent resection in Affiliated Hospital of Weifang Medical University and Tianjin Medical University Cancer Institute and Hospital from January 2012 to December 2017 to analyze the correlation of neutrophil-lymphocyte ratio (NLR), lymphocyte-monocyte ratio (LMR), systemic immune-inflammation index (SII), prognostic inflammation index (PII), inflammation score (IS), and systemic inflammation score (SIS) with the disease-free survival (DFS) and overall survival of ICC patients after surgery, and the value of the above indices in predicting prognosis was evaluated. The chi-square test or the Fisher's exact test was used for comparison of categorical data between groups. The Kaplan-Meier method was used to plot survival curves, and the Log-rank test was used for comparison between groups; the Cox regression model was used for univariate and multivariate analyses, and hazard ratio ( HR ) and 95% confidence interval [ CI ] were calculated. Results The univariate survival analysis showed that NLR ( HR =2.212, P =0.004), LMR ( HR =0.403, P =0.012), PII ( HR =3.013, P < 0.001), prognostic nutritional index (PNI) ( HR =0.530, P =0.019), IS ( HR =1.809, P =0.001), SII ( HR =2.107, P =0.002), and SIS ( HR =2.225, P < 0.001) were predictive factors for postoperative DFS of patients with ICC, and NLR ( HR =2.416, P =0.009), LMR ( HR =0.297, P =0.008), PII ( HR =3.288, P < 0.001), PNI ( HR =0.292, P =0.003), IS ( HR =2.048, P =0.002), SII ( HR =1.839, P =0.049), and SIS ( HR =2.335, P < 0.001) were predictive factors for postoperative OS of patients with ICC. The multivariate survival analysis showed that high levels of PII ( HR =2.146, P =0.035) and SIS ( HR =2.511, P < 0.001) were independent influencing factors for postoperative DFS of ICC patients, and high levels of PII ( HR =2.981, P =0.009), PNI ( HR =0.261, P =0.002), and SIS ( HR =2.294, P =0.010) were independent influencing factors for postoperative OS. The patients with a high level of PII tended to have advanced tumor T stage ( χ 2 =8.777, P =0.003) and M stage ( P =0.029), and the patients with high-grade SIS tended to have advanced N stage ( χ 2 =9.985, P =0.030) and M stage ( χ 2 =8.574, P =0.012). Conclusion Among the various inflammation indices, PII and SIS are recommended for preoperative stratification and prediction of the outcome of ICC patients after curative-intent resection.

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

Distinct immune responses in patients infected with influenza or SARS-CoV-2, and in COVID-19 survivors, characterised by transcriptomic and cellular abundance differences in blood.

BackgroundThe worldwide pandemic caused by SARS-CoV-2 has claimed millions of lives and has had a profound effect on global life. Understanding the pathogenicity of the virus and the bodys response to infection is crucial in improving patient management, prognosis, and therapeutic strategies. To address this, we performed functional transcriptomic profiling to better understand the generic and specific effects of SARS-CoV-2 infection. MethodsWhole blood RNA sequencing was used to profile a well characterised cohort of patients hospitalised with COVID-19, during the first wave of the pandemic prior to the availability of approved COVID-19 treatments and who went on to survive or die of COVID-19, and patients hospitalised with influenza virus infection between 2017 and 2019. Clinical parameters between patient groups were compared, and several bioinformatic tools were used to assess differences in transcript abundances and cellular composition. ResultsThe analyses revealed contrasting innate and adaptive immune programmes, with transcripts and cell subsets associated with the innate immune response elevated in patients with influenza, and those involved in the adaptive immune response elevated in patients with COVID-19. Topological analysis identified additional gene signatures that differentiated patients with COVID-19 from patients with influenza, including insulin resistance, mitochondrial oxidative stress and interferon signalling. An efficient adaptive immune response was furthermore associated with patient survival, while an inflammatory response predicted death in patients with COVID-19. A potential prognostic signature was found based on a selection of transcript abundances, associated with circulating immunoglobulins, nucleosome assembly, cytokine production and T cell activation, in the blood transcriptome of COVID-19 patients, upon admission to hospital, which can be used to stratify patients likely to survive or die. ConclusionsThe results identified distinct immunological signatures between SARS-CoV-2 and influenza, prognostic of disease progression and indicative of different targeted therapies. The altered transcript abundances associated with COVID-19 survivors can be used to predict more severe outcomes in patients with COVID-19.

Sequence analysis of SARS-CoV-2 in nasopharyngeal samples from patients with COVID-19 illustrates population variation and diverse phenotypes, placing the in vitro growth properties of B.1.1.7 and B.1.351 lineage viruses in context.

New variants of SARS-CoV-2 are continuing to emerge and dominate the regional and global sequence landscapes. Several variants have been labelled as Variants of Concern (VOCs) because of perceptions or evidence that these may have a transmission advantage, increased risk of morbidly and/or mortality or immune evasion in the context of prior infection or vaccination. Placing the VOCs in context and also the underlying variability of SARS-CoV-2 is essential in understanding virus evolution and selection pressures. Sequences of SARS-CoV-2 in nasopharyngeal swabs from hospitalised patients in the UK were determined and virus isolated. The data indicated the virus existed as a population with a consensus level and non-synonymous changes at a minor variant. For example, viruses containing the nsp12 P323L variation from the Wuhan reference sequence, contained minor variants at the position including P and F and other amino acids. These populations were generally preserved when isolates were amplified in cell culture. In order to place VOCs B.1.1.7 (the UK Kent variant) and B.1.351 (the South African variant) in context their growth was compared to a spread of other clinical isolates. The data indicated that the growth in cell culture of the B.1.1.7 VOC was no different from other variants, suggesting that its apparent transmission advantage was not down to replicating more quickly. Growth of B.1.351 was towards the higher end of the variants. Overall, the study suggested that studying the biology of SARS-CoV-2 is complicated by population dynamics and that these need to be considered with new variants. ImportanceSARS-CoV-2 is the causative agent of COVID-19. The virus has spread across the planet causing a global pandemic. In common with other coronaviruses, SARS-CoV-2 genetic material (genomes) can become quite diverse as a consequence of replicating inside cells. This has given rise to multiple variants from the original virus that infected humans. These variants may have different properties and in the context of a widespread vaccination program may render vaccines less ineffective. Our research confirms the degree of genetic diversity of SARS-CoV-2 in patients. By isolating viruses from these patients, we show that there is a 100-fold range in growth of even normal variants. Interestingly, by comparing this to the pattern seen with two Variants of Concern (UK and South African variants), we show that at least in cells the ability of the B.1.1.7 variant to grow is not substantially different to many of the previous variants.

Sequential infection with influenza A virus followed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to more severe disease and encephalitis in a mouse model of COVID-19.

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2, a recently emerged coronavirus that has rapidly caused a pandemic. Coalescence of a second wave of this virus with seasonal respiratory viruses, particularly influenza virus is a possible global health concern. To investigate this, transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) were first infected with IAV followed by SARS-CoV-2. The host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 only. Infection of mice with each individual virus resulted in a disease phenotype compared to control mice. Although SARS-CoV-2 RNA synthesis appeared significantly reduced in the sequentially infected mice, these mice had a more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to singly infected or control mice. The sequential infection also exacerbated the extrapulmonary manifestations associated with SARS-CoV-2. This included a more severe encephalitis. Taken together, the data suggest that the concept of twinfection is deleterious and mitigation steps should be instituted as part of a comprehensive public health response to the COVID-19 pandemic.

Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19

COVID-19 is a complex disease phenotype where the underlying microbiome could influence morbidity and mortality. Amplicon and metagenomic MinION based sequencing was used to rapidly (within 8 hours) identify SARS-CoV-2 and assess the microbiome in nasopharyngeal swabs obtained from patients with COVID-19 by the ISARIC 4C consortium.

The relationship between vimentin protein expression in endothelial cells and contrast-enhanced ultrasound characters in VETC (+ ) hepatocellular carcinoma / 中华肿瘤杂志

Objective@#To detect the possible molecular mechanisms of the formation of vessels that encapsulated tumor clusters (VETC) and identify the relationship between vimentin protein expression in endothelial cells and contrast-enhanced ultrasound characters in VETC (+ ) hepatocellular carcinoma (HCC).@*Methods@#A total of 64 paraffin embedded HCC tissue samples were collected, all of which the tumor diameters were between 2 cm and 5 cm measured by the preoperative ultrasound. Immunohistochemistry staining for CD34 was used to detect the formation of VETC and the expressions of angiopoietin-2 (Ang-2) and vimentin were also determined. Human umbilical vein endothelial cells (HUVECs) were treated with 150 ng/ml recombinant human Ang-2 protein (rhAng-2) at various times and the protein expression of vimentin was detected by western blot assay. The contrast-enhanced ultrasound characters were also analyzed in both VETC (+ ) and VETC (-) HCC.@*Results@#Tumor clusters encapsulated by vessels to form cobweb-like networks, which were identified as VETC phenotype, were observed in 27 HCC tissues (42.18%). In VETC (+ ) HCC tissues, Ang-2 was overexpressed in tumor cells and endothelial cells while vimentin was only upregulated in endothelial cells. With the treatment of 150 ng/ml rhAng-2 protein, the expression of vimentin in HUVECs was 0.878±0.102 and 0.918±0.092 at 12 h and 36 h, significantly upregulated when compared to the 0.322±0.061 at 6 h (P<0.01). In contrast-enhanced ultrasound, a crack and tendon-like filling character was observed in VETC (+ ) HCC during the arterial-phase, while the large scale and diffuse-like filling character was observed in VETC (-) HCC. The filling time of unit diameter in VETC (+ ) HCC was (3.95±0.22)s, significantly longer than (2.28±0.27)s of VETC (-) HCC (P<0.01).@*Conclusions@#The overexpressions of Ang-2 and vimentin are positively correlated with the formation of VETC and considered as potential therapeutic targets of VETC (+ ) HCC. The crack and tendon-like filling characters in arterial-phase of contrast-enhanced ultrasound indicates the VETC (+ ) HCC.

Research progress on tumor-vessel associated extravascular tumor metastasis / 中华消化外科杂志

The occurrence of malignant tunors is increasing annually around the world.In every year,8.2 million patients died of malignant tumors in which about 90％ patients died of malignant tumors associated multiple organs metastases.Inhibiting tumor metastasis is the key event to prolong the survival time of patients.With the further research,tumor-vessel associated extravascular tumor metastasis is playing an increasingly important role in the clinical application.This paper summarizes the new developments of tumor-vessel associated extravascular tumor metastasis to provide possible ideas for the therapy of malignant tumors.

Hypoxia inducible factor-2 alpha regulating hypoxia-induced angiogenesis via angiopoietin-2 pathway / 中华消化外科杂志

Objective To investigate the mechanisms of hypoxia inducible factor-2 alpha (HIF-2a) regulating human umbilical vein endothelial cells (HUVECs) under hypoxic conditions.Methods The experimental study was adopted.(1) HUVECs in logarithmic growth phase were taken:HUVECs without any disposals as control group,HUVECs with shRNA transfection control as shRNA control group,HUVECs with HIF-2α shRNA transfection as HIF-2α shRNA group and HUVECs with HIF-2α shRNA transfection then added rhAng-2 as HIF-2α ± rh-Ang-2 group.(2) Western blot testing:the expressions of Ang-2 and HIF-2α proteins in HUVECs were cultured under hypoxia conditions at 0,2,4,8,12,16,20 hours,and the levels of which were detected in the control group,shRNA control group and HIF-2α shRNA group.(3) Enzyme-linked immunosorbent assay(ELISA):the level of Ang-2 protein in supernatant of HUVECs was detected in the control group,shRNA control group and HIF-2α shRNA group.(4)The amounts of endothelial cell tubes in HUVECs among the 4 groups were detected by tube formation experimental testing.(5) Transwell method was performed to detect the amounts of cells migration in HUVECs and hepatoma cells SMMC-7721 migration intervened by supernatant of HUVECs among the 4 groups.Measurement data with normal distribution were presented as x ± s,repeated measurement data were analyzed by the repeated measures ANOVA,comparison among groups and pairwise comparison were conducted respectively by the one-way ANOVA and Dunnett's test.Results (1) Western blot test:the expression levels of Ang-2 and HIF-2α proteins in HUVECs under hypoxia conditions at 0,2,4,8,12,16,20 hours were 0.110 ±0.011,0.120 ±0.020,0.210 ±0.070,0.410 ±0.100,0.520 ± 0.090,0.790±0.130 1.010 ±0.220 and 0.180 ±0.090,0.410 ±0.070,0.470 ±0.110,0.470 ±0.070,0.580 ± 0.120,0.690 ± 0.140,0.920 ± 0.130,respectively,and which were increased after culturing under hypoxia conditions and had an ascending tendency as the hypoxia time extended,with statistically significant differences (F =403.550,3 265.587,P ＜ 0.05).The expression levels of Ang-2 and HIF-2α proteins in the control group,shRNA control group and HIF-2α shRNA group were 1.030 ±0.180,1.070 ±0.120,0.210 ± 0.070,and 0.940 ± 0.110,0.930 ± 0.190,0.170 ± 0.021,respectively,showing statistically significant differences (F =290.242,26.688,P ＜ 0.05).(2) The results of ELISA:the expression levels of Ang-2 in the control group,shRNA control group and HIF-2α shRNA group were (433.2 ±9.7)ng/L,(438.3 ± 2.6)ng/L,(114.6 ± 4.2) ng/L,with a statistically significant difference (F =2 642.180,P ＜ 0.05).(3) The results of tube formation experiments:the number of endothelial cell tubes in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α ± rh-Ang-2 group were 48.3 ± 2.5,47.4 ± 3.1,19.7 ± 1.5 and 38.3 ± 2.1,respectively,with a statistically significant difference (F =148.196,P ＜ 0.05).(4) The results of Transwell method:① the number of HUVECs migration in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α + rh-Ang-2 group were 140.3-± 3.5,142.7 ± 2.1,42.7 ± 3.1 and 78.1 ± 4.2,respectively,showing a statistically significant differences (F =212.205,P ＜ 0.05).②The results of Transwell method:the number of SMMC-7721 cells migration after intervening using four different supernatant in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α ± rh-Ang-2 group were 106.7 ± 5.5,102.7 ± 6.6,63.0 ± 3.3 and 96.7 ± 2.1,respectively,showing a statistically significant difference (F =55.122,P ＜ 0.05).Conclusion HIF-2a could not only affect HUVECs formation but also promote SMMC-7721 cells migration via regulating Ang-2 expression.

Effects of arsenic trioxide on the methylation of TMS1 gene in K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect the methylation status of TMS1 gene and its demethylation by arsenic trioxide (As₂O₃) in K562 cells.</p><p><b>METHODS</b>K562 cells were treated with different concentrations of As₂O₃ for 48 hours. Methylation-specific PCR (MSP) was used to determine the methylation status of TMS1. RT-PCR and Western blot were used to detect the levels of TMS1 mRNA and protein. TMS1 associated apoptosis proteins Bcl-2/Bax were also analyzed by Western blot. Apoptosis were evaluated by flow cytometry using Annexin V/propium iodide (PI) double staining.</p><p><b>RESULTS</b>TMS1 gene was completely methylated in K562 cells and the levels of TMS1 mRNA and protein were low (0.01±0.01, 0.09±0.02), which could be reversed (mRNA: 0.72±0.04; protein: 1.30±0.06; P<0.01) by 2 μmol/L As2O3 via overt demethylation of TMS1 gene. Apoptosis in experiment group (12.24±1.06) was significantly higher than that in control group (2.05±0.16, P<0.05). In experiment group, the down-expression of antiapoptotic protein Bcl-2 and up-expression of pro-apoptotic protein Bax led to an obvious decline ratio of Bcl-2/Bax (0.56±0.12), as compared to the control group (1.94±0.14, P<0.01).</p><p><b>CONCLUSION</b>As₂O₃ could up-regulate TMS1 gene expression by reversing its hypermethylation and induced apoptosis by down-regulation of Bcl-2/Bax ratio in K562 cells.</p>

Sleep apnea syndrome and stroke / 国际脑血管病杂志

Sleep apnea syndrome (SAS) is an independent risk factor for atherosclerosis and stroke. Studies have shown that the incidence of SAS increases significantly after stroke.This article reviews the mechanism of atherosclerosis caused by SAS and the characteristics and research progress of sleep apnea after stroke.