Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didnt identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2s integration into host genome is lacking. One-Sentence SummarySARS-CoV-2 does not integrate into host genome through whole genome sequencing.

Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants of concern (VOC), such as Delta (B.1.617.2) and Omicron (B.1.1.529), has continued to drive the worldwide pandemic. Therefore, there is a high demand for vaccines with enhanced efficacy, high thermostability, superior design flexibility, and fast manufacturing speed. Here, we report a circular RNA (circRNA) vaccine that encodes the trimeric RBD of SARS-CoV-2 Spike protein. Without the need of nucleotide modification, 5-capping or 3-polyadenylation, circRNA could be rapidly produced via in vitro transcription and is highly thermostable whether stored in naked or lipid-nanoparticle (LNP)-encapsulated format. LNP-encapsulated circRNARBD elicited potent neutralizing antibodies and T cell responses, providing robust protection against Beta (B.1.351) and native viruses in mice and rhesus macaques, respectively. Notably, circRNA vaccine enabled higher and more durable antigen production than 1m{Psi}-modified mRNA vaccine, eliciting a higher proportion of neutralizing antibodies and stronger Th1-biased immune responses. Importantly, we found that circRNARBD-Omicron vaccine induced effective neutralizing antibodies against only Omicron but not Delta variant. By contrast, circRNARBD-Delta could elicit high level of neutralizing antibodies against both Delta and Omicron. Following two doses of either native- or Delta-specific vaccination, circRNARBD-Delta, but not Omicron or Beta vaccines, could effectively boost the neutralizing antibodies against both Delta and Omicron variants. These results suggest that circRNARBD-Delta is a favorable choice for vaccination to provide a broad-spectrum protection against the current variants of concern of SARS-CoV-2.

Effects of brain-specific knockout of Sirt6 gene on cerebral cortex in aged mice / 中华行为医学与脑科学杂志

Objective:To study the effect of senescence gene silent information regulator 6 (Sirt6) knockout on the brain of aged mice.Methods:Sirt6-flox transgenic mice were constructed, and the mouse brain tissue was specifically knocked out by Emx1-Cre tool mice.According to genotyping, 11 wild-type mice were selected as control group(WT group) and 10 Sirt6 gene konckout mice were selected as conditional knockout group(cKO group). Body size and body weight of the aged mice were measured and cerebral cortex thickness was measured by HE staining.Brain neurogenesis was analyzed with EdU markers.The expression of RNA-binding protein HuR and apoptosis-related protein Caspase-3 were detected by Western blot.Meanwhile, histone acetylation levels in the cortex were detected.Results:Sirt6 brain tissue-specific knocked out mice were successfully constructed.Compared with the brain tissue area((2.07±0.22) cm 2)and cortical thickness ((970.56±80.91) μm) of WT mice in the 12-month-old group, the brain tissue area ((1.61±0.14)cm 2) and cortical thickness ((822.88±53.94) μm) in Sirt6 cKO group were smaller, and the differences were statistically significant (both P<0.05). EdU incorporation into nerve cells showed that the number of EdU incorporation into periventricular nerve cells in cKO group was lower ((4.75±1.48)) than that in WT group ((10.29±1.93)). The difference was statistically significant ( P<0.001). In the experiment of 17 months age group, mice in cKO group were smaller in body size, lower in body weight ((29.00±1.08) g) and smaller in brain area ((1.54±0.55)cm 2)compared with WT group in body size, body weight ((35.25±4.17) g) and brain tissue area ((1.98±0.18) cm 2)(both P<0.05). The expression of Caspase-3 and HuR in cortical proteins of these two age groups decreased( t=2.95, 5.38, both P<0.05), and the expression of H3K9ac and H3K56ac increased( t=3.53, 2.78, both P<0.05), but the expression of Sirt1 homologous gene remained unchanged( t=1.26, P>0.05). Conclusion:The specific deletion of Sirt6 in brain tissue can lead to the decrease of brain neurogenesis in aged mice, and the aggravation of aging and the increase of apoptosis, which may be the reason for the thinning of cerebral cortex and brain tissue atrophy.The molecular mechanism is speculated to be related to the increase of acetylation level after Sirt6 knockout

Transferrin receptor is another receptor for SARS-CoV-2 entry

Angiotensin-converting enzyme 2 (ACE2) has been suggested as a receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry to cause coronavirus disease 2019 (COVID-19). However, no ACE2 inhibitors have shown definite beneficiaries for COVID-19 patients, applying the presence of another receptor for SARS-CoV-2 entry. Here we show that ACE2 knockout dose not completely block virus entry, while TfR directly interacts with virus Spike protein to mediate virus entry and SARS-CoV-2 can infect mice with over-expressed humanized transferrin receptor (TfR) and without humanized ACE2. TfR-virus co-localization is found both on the membranes and in the cytoplasma, suggesting SARS-CoV-2 transporting by TfR, the iron-transporting receptor shuttling between cell membranes and cytoplasma. Interfering TfR-Spike interaction blocks virus entry to exert significant anti-viral effects. Anti-TfR antibody (EC50 ~16.6 nM) shows promising anti-viral effects in mouse model. Collectively, this report indicates that TfR is another receptor for SARS-CoV-2 entry and a promising anti-COVID-19 target.

Susceptibility of tree shrew to SARS-CoV-2 infection

Since SARS-CoV-2 became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and COVID-19 were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature (above 39{degrees} C) particular in female animals during infection. Low levels of virus shedding and replication in tissues occurred in all three age groups, each of which showed his own characteristics. Histopathological examine revealed that pulmonary abnormalities were mild but the main changes although slight lesions were also observed in other tissues. In summary, tree shrew is not susceptible to SARS-CoV-2 infection and may not be a suitable animal for COVID-19 related researches.

Comparison of SARS-CoV-2 infections among 3 species of non-human primates

COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta, 6 Macaca fascicularis) and New world monkeys (6 Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis, the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. One Sentence SummaryM. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus.

The Pathogenicity of 2019 Novel Coronavirus in hACE2 Transgenic Mice

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) caused the Corona Virus Disease 2019 (COVID-19) cases in China has become a public health emergency of international concern (PHEIC). Based on angiotensin converting enzyme 2 (ACE2) as cell entry receptor of SARS-CoV, we used the hACE2 transgenic mice infected with SARS-CoV-2 to study the pathogenicity of the virus. Weight loss and virus replication in lung were observed in hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes and monocytes in alveolar interstitium, and accumulation of macrophages in alveolar cavities. Viral antigens were observed in the bronchial epithelial cells, alveolar macrophages and alveolar epithelia. The phenomenon was not found in wild type mice with SARS-CoV-2 infection. The pathogenicity of SARS-CoV-2 in hACE2 mice was clarified and the Kochs postulates were fulfilled as well, and the mouse model may facilitate the development of therapeutics and vaccines against SARS-CoV-2.

NS4A protein of Zika virus influences the neuronal migration of mouse cortex / 基础医学与临床

Objective To determine the effect of NS3 and NS4A proteins of Zika virus on the neuronal migration in vivo.Methods To identify the coding sequence of NS3 and NS4A,the genome of Zika SZ01 was sequenced by rapid amplification of cDNA ends (RACE) and reverse-transcription PCR,then NS3 and NS4A was constructed in pCIG vector fused with Flag-tag to express these proteins.And then these plasmids was transfected into the embryo brain of E13.5 mice by in utero electroporation,the distribution of the cells which express these proteins in the cortex was detected by Flag,eGFP and TBR1 fluorescence in E18.5 mice through immunohistochemistry so as to assess the influence of viral proteins on the neuronal migration of mouse cortex.Results 1) Sequence results showed that the amino acid sequence of NS4A is consistent with NCBI data,while NS3 has 1 amino acid mutant.2) As the fluorescence of Flag and eGFP can co-localization,the eGFP fluorescence signal marks the cells that have expressed these virus proteins in cortex.3) TBR1 fluorescence shows the distribution of the cells that express NS4A in vivo are significantly different from pCIG control and NS3 (P＜0.001).Conclusions The NS4A protein of Zika virus may affect the neuronal migration in vivo.

Comparison of the clinical efficacy between obesity and non-obesity with lumbar spinal stenosis and instability by transforaminal lumbar interbody fusion / 重庆医学

Objective To observe the clinic efficacy of open transforaminal lumbar interbody fusion (TLIF) compared with minimally invasive operation in treating lumbar spinal stenosis and instability among obese and non‐obese patients .Methods A ret‐rospective analysis was performed in these cases of mono‐segmental lumbar spinal stenosis and instability between January 2011 and January 2013 .Perioperative index ,clinical efficacy ,and imaging results were observed and compared between different groups .Re‐sults Thirty‐four obese cases and 105 non‐obese cases were divided into two groups ,including conventional posterior open TLIF and minimally invasive TLIF operation ,to compare the results .Perioperative indexes of obese patients were more than non‐obese patients undergone open TLIF operation way and there was significant difference(P0 .05) .No cases of slippage or breakage of implants were found among all these patients after 6 months of follow up .Postoperative VAS and ODI among these four groups were better than before(P0 .05);undergoing minimally invasive postoperative VAS in obese group and in non‐obese group ,there was not significant difference(P>0 .05) .Conclusion Therefore ,obese may be risk factor in treating lumbar spinal stenosis and instability .

Novel progression of GROβin basic research and cancer / 基础医学与临床

GROβis a member of the CXC chemokine superfamily.It plays an important role in inflammation and wound healing process.As extensive research continued, researchers realized that the gro gene was one of onco-genes.And its expression product, GROβ, was also found to be very important in angiogenesis, tumorigeness, me-tastasis, and interaction between tumor and immune cells.

Minimally invasive versus open pedicle screw fixation for repair of thoracolumbar fractures / 中国组织工程研究

BACKGROUND:Recently, minimal y invasive techniques obtained more attention. Some new minimal y invasive methods have been used in the treatment of spine fracture and provide new chal enges for conventional open surgery. OBJECTIVE:To discuss the clinical efficacy of conventional posterior open pedicle screw fixation versus minimal y invasive operation (using Mast Quadrant System and Sextant percutaneous pedicle screw fixation) for treating single-segment thoracolumbar fractures without neurological damages. METHODS:A total of 94 cases of single-segment thoracolumbar fracture without neurological damages, who were treated in Department of Spine Surgery, Liuzhou Worker’s Hospital in China from January 2012 to January 2013, were enrol ed in this study. According to patients’ conditions and wil ing, they were divided into open fixation group, Quadrant fixation group and percutaneous Sextant fixation group. Perioperative index, clinical efficacy, and imaging results were observed and compared among different groups. RESULTS AND CONCLUSION:Intraoperative blood loss, incision length and length of stay were better in the Quadrant fixation group and percutaneous Sextant fixation group than in the posterior open fixation group (P0.05). Postoperative Visual Analog Scale scores and Oswestry Disability Index were better in the two minimal y invasive groups than in the conventional open fixation group (P<0.05). These results suggested that compared with conventional open operation, minimal y invasive operation (Mast Quadrant System and Sextant percutaneous pedicle screw fixation) in the treatment of thoracolumbar fractures not only can achieve similar imaging result, but has smal incision, less blood loss, quick recovery, high safety, and obtains good clinical therapeutic outcomes. In the case of strict surgical indications, minimal y invasive method is an ideal choice in treating thoracolumbar fractures without neurological damages.

Correlates of health-care seeking behavior in patients with irritable bowel syndrome / 中华全科医师杂志

Objective To explore correlates of health-care seeking behavior in patients with irritable bowel syndrome (IBS).Methods Four thousand permanent residents were recruited from eight urban communities and rural villages in Guangzhou and Huizhou, Guangdong province during 2009 by cluster stratified sampling for face-to-face questionnaire survey, including symptoms of bowel disease,behavior of seeking for health-care, demographic characteristics, coping style, life events and medical history.IBS was identified based on the Rome Ⅱ Criteria.Patient with IBS were divided into two groups,one seeking health-care at hospitals or clinics and the other non-seeking health-care.Univariate and multivariate logistic regression analysis was used to compare difference between the two groups and explore its related factors.Results A total of 237 IBS patients were identified based on the Rome Ⅱ Criteria, 53 of them (22.4% ) had sought health-care due to their symptoms.Results of multivariate logistic regression analysis showed that preference in seeking for health-care, abdominal pain lasting for more than one hour in each episode and extra-gastrointestinal symptoms were main factors related to their seeking for health-care,adjusted for age and gender, with odds ratios (ORs) of 1.81 (95% CI: 1.27 -2.58), 1.41 (95% CI:1.01 - 5.14 ) and 2.14 ( 95% CI: 1.06 - 4.33 ), respectively.Conclusions Extra-gastrointestinal symptoms and abdominal pain lasting for more than one hour in each episode correlate their health-care seeking behavior in patients with IBS, as well as their preferences in seeking for health-care.

Screen of human single chain antibody against N protein of SARS-CoV / 基础医学与临床

Objective To prepare the single chain antibody against N protein of SARS-CoV. Methods With N protein of SARS-CoV expressed in E.coli as antigen, we obtained the single chain antibody against N protein by screening the phage display library of human single chain antibodies. Results The anti-N protein antibody didn’t cross-interacte with BSA and the short peptide containing 6 histidines. The specific interaction between the antibody and N protein was inhibited by the anti-N protein monoclone antibody from immunized mice. ConclusionThe single chain antibody we got is specific to N protein of SARS-CoV,it can be a candidate antibody for fast detection of N protein of SARS-CoV and SARS virus particles in clinical trial study of SARS pathogenesis.

Proteomic approaches to post-translational modification by ubiquitin family proteins / 基础医学与临床

Post-translational modification by ubiquitin and ubiquitin-like modifiers(Ubls) is one of the most important mechanisms regulating a wide range of cellular processes in eukaryotes.Previous research showed that,through covalently modification by ubiquitin or ubls,the substrate proteins can be regulated in many different ways like stability,subcellular localization,enzymatic activity,protein-protein interaction and so on.Therefore,we believe,that ubiquitin and ubls play very important roles in cellular and biological processes by modifying plenty of proteins.To better understand the ubiquitin and ubls system,proteomic approaches have been developed to purify and identify more protein substrates.Large-scale idendification of ubiquitin/ubls-modification sites by mass spectrometry is particularly important for understanding the molecular mechanism and function of ubiquitin/ubls modification.Upto the present,more and more scientists are getting interested and participating in proteomics research of ubiquitin/Ubl modifications.This review summarizes the rencent results in this field.

Tandem affinity purification technique: an extraordinary valuable tool for purifying protein complexes / 基础医学与临床

It's a multi-protein complex and not single protein that accomplish most of cellular activity.Therefore,to identify and analyze the composition of protein complex is necessary for studying the function of proteins.Tandem affinity purification technique(TAP) enables the effective purification of complex under physiological conditions without knowledge of the complex composition and function.Now,it has been adapted to analyze the composition of the protein complexes in yeast.Combined with mass spectrometry,TAP can identify the interacting proteins of target protein and offer great help for opening protein interacting network and function out.

NSPc1 is an essential factor for HeLa cells' proliferation / 基础医学与临床

Objective To study the effect of PcG member NSPc1 on proliferation of HeLa cells.Methods Using bioinfomatic analysis to design the siRNA sequence to knockdown NSPc1.Detecting the expression level of NSPc1 in HeLa cell line using semi-quantitative RT-PCR,Real-time PCR and Western blot after transfection of the designed siRNA.Transient transfecting pSUPER-NSPc1 into Hela cells and performing BrdU incorporation assay.Establishing NSPc1 stably knockdown cell line,comparing proliferation abilities with the control cells.Results(1)The designed siRNA did efficiently knockdown the expression of NSPc1;(2)Transient knockdown of NSPc1 could repress BrdU incorporation;(3) The established NSPc1-knockdown cell lines had a significantly lower proliferation rate than that of control cells.Conclusion The expression of NSPc1 is necessary for the normal proliferation of HeLa cells.The NSPc1 stably knockdown cell pool is a useful model for further study of pathway related to NSPc1.

Preparation and identification of monoclonal antibody against human SUMO1 / 基础医学与临床

Objective To express the human recombinant SUMO1 protein and prepare monoclonal antibody(mAb) against it.Methods The recombinant expression plasmid pET32a-HIS-SUMO1 was made and transformed into E.coli(BL21),then the recombinant fusion protein HIS-SUMO1 was expressed and purified.The BALB/c mice were immuned with pure protein HIS-SUMO1 as antigen.Monoclonal antibody against SUMO1 was prepared with standard hybridoma technology.The hybridoma cell lines were obtained by ELISA and Western blot screening procedure,the isotype of the mAbs were further identified by immune-double diffusion.Ascites were collected from one propagated hybridoma cell line and mAbs were purified by using the Kit of Millipore.The valence of mAb was detected by Western Blot.Results The recombinant protein HIS-SUMO1 is expressed and purified.Three hybfidmas producing antibodies against SUMO1 were obtained,the isotypes of three mAbs are IgG1,Western blot showed that the antibodies were specific for SUMO1.The antibody purified from the ascites has better specificity.Conclusion The SUMO1 mAb prepared by using recombinant SUMO1 protein as antigen can be used for detectingthe protein sumoylation.

Cloning and characterization of human ubiquitin binding enzyme 2 cDNA / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern.</p><p><b>METHODS</b>According to the sequence of human EST, which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2), primers were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple-tissue Northern blot.</p><p><b>RESULTS</b>Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified. Both containing the ubiquitin conjugating domain, the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence. They belong to a highly conserved and widely expressed E2 enzyme family. Northern blot shows that they are expressed exclusively in adult human heart, placenta, and pancreas but no transcripts can be detected in brain, lung, liver, skeletal muscle or kidney.</p><p><b>CONCLUSIONS</b>The gene encoding human ubiquitin binding enzyme is expressed under temporal control. As a key enzyme in the degradation of proteins, ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post-translation.</p>

Gastric sensorimotor function in patients with functional dyspepsia / 中华消化杂志

0.05). Conclusions Gastric hypersensitivity, impaired proximal gastric accommodation and delayed gastric emptying may be important but independent pathophysiological factors of FD. Different pathophysiological factors can coexist in one patient with FD.