High-throughput transcriptional profiling of perturbations by Panax ginseng saponins and Panax notoginseng saponins using TCM-seq / 药物分析学报

Panax ginseng(PG)and Panax notoginseng(PN)are highly valuable Chinese medicines(CM).Although both CMs have similar active constituents,their clinical applications are clearly different.Over the past decade,RNA sequencing(RNA-seq)analysis has been employed to investigate the molecular mechanisms of extracts or monomers.However,owing to the limited number of samples in standard RNA-seq,few studies have systematically compared the effects of PG and PN spanning multiple conditions at the transcriptomic level.Here,we developed an approach that simultaneously profiles transcriptome changes for multiplexed samples using RNA-seq(TCM-seq),a high-throughput,low-cost workflow to molecularly evaluate CM perturbations.A species-mixing experiment was conducted to illustrate the accuracy of sample multiplexing in TCM-seq.Transcriptomes from repeated samples were used to verify the robustness of TCM-seq.We then focused on the primary active components,Panax notoginseng sa-ponins(PNS)and Panax ginseng saponins(PGS)extracted from PN and PG,respectively.We also char-acterized the transcriptome changes of 10 cell lines,treated with four different doses of PNS and PGS,using TCM-seq to compare the differences in their perturbing effects on genes,functional pathways,gene modules,and molecular networks.The results of transcriptional data analysis showed that the tran-scriptional patterns of various cell lines were significantly distinct.PGS exhibited a stronger regulatory effect on genes involved in cardiovascular disease,whereas PNS resulted in a greater coagulation effect on vascular endothelial cells.This study proposes a paradigm to comprehensively explore the differences in mechanisms of action between CMs based on transcriptome readouts.

A chimeric influenza virus vaccine expressing fusion protein epitopes induces protection from human metapneumovirus challenge in mice.

Human metapneumovirus (HMPV) is a common virus associated with acute respiratory distress syndrome in pediatric patients. There are no HMPV vaccines or therapeutics that have been approved for prevention or treatment. In this study, we constructed a novel recombinant influenza virus carrying partial HMPV fusion protein (HMPV-F), termed rFLU-HMPV/F-NS, utilizing reverse genetics, which contained (HMPV-F) in the background of NS segments of influenza virus A/PuertoRico/8/34(PR8). The morphological characteristics of rFLU-HMPV/F-NS were consistent with the wild-type flu virus. Additionally, immunofluorescence results showed that fusion proteins in the chimeric rFLU-HMPV/F-NS could work well, and the virus could be stably passaged in SPF chicken embryos. Furthermore, intranasal immunization with rFLU-HMPV/F-NS in BALB/c mice induced robust humoral, mucosal and Th1-type dominant cellular immune responses in vivo. More importantly, we discovered that rFLU-HMPV/F-NS afforded significant protective efficacy against the wild-type HMPV and influenza virus challenge, with significantly attenuated pathological changes and reduced viral titers in the lung tissues of immunized mice. Collectively, these findings demonstrated that chimeric recombinant rFLU-HMPV/F-NS as a promising HMPV candidate vaccine has potentials for the development of HMPV vaccine.

Application of white noise therapy on the alleviation of hospital procedural pain of colostomy newborns / 中国实用护理杂志

Objective:To investigate the application value of white noise therapy on the alleviation of procedural pain of colostomy newborns.Methods:By a prospective, randomized and controlled trial, a total of 88 colostomy newborns in Hunan Children′s Hospital from January 2018 to January 2020 divided into experimental group (44 cases) and control group (44 cases) according to the random number table method. The control group received routine nursing; based on thesis, the experimental group played white noise intervention therapy on the basis of routine nursing. The intervention effect was assessed byNeonatal Infant Acute Pain Assessment Scale (NIAPAS), the first crying time and the duration of first crying, the first painful face and the duration of first painful face as well as heart rate and blood oxygen saturation.Results:The first crying time and the duration of first crying, the first painful face and the duration of first painful face were (28.05 ± 7.39) s, (46.18 ± 13.29) s, (32.89 ± 6.79) s, (52.75 ± 10.71) s in the experimental group, significantly shorter than in the control group (35.79 ± 5.81) s, (35.79 ± 5.81) s, (38.64 ± 10.53) s, (59.79 ± 13.52) s, the difference was statistically significant ( t values were 2.71-5.47, all P<0.05). During and after the procedure, the scores of NIAPAS were (6.32 ± 1.62) points, (4.18 ± 1.06) points in the experimental group, significantly lower than that in the control group (7.43 ± 1.78) points, (4.79 ± 1.34) points ( t=3.06, 2.38, both P<0.05); the heart rate were (152.82 ± 13.25) times/min and (147.84 ± 12.37) times/min in the experimental group, significantly lower than in the control group (166.11 ± 13.79) times/min and (155.77 ± 12.84) times/min ( t=4.61, 2.95, both P<0.05); the blood oxygen saturation were 0.979 8 ± 0.009 5 and 0.980 9 ± 0.012 4 in the experimental group, significantly higher than in the control group 0.969 1 ± 0.014 9, 0.972 3 ± 0.017 8, the difference was statistically significant ( t=4.01, 2.65, both P<0.05). Conclusions:White noise therapy can effectively alleviate procedural pain and stabilizing vital signs of colostomy newborns.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patients immunological status, and found dramatic changes in the IGH within the patients immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones during 2-3 weeks of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34 and IGHV4-39 in COVID-19 patients were more abundant than that of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

Virus shedding patterns in nasopharyngeal and fecal specimens of COVID-19 patients

Diagnosis is the key point for confirmation and treatment of COVID-19. we focused on comparative analysis of virus dynamics between the upper respiratory and feces specimens in the COVID-19 patients. A total of 66 upper respiratory swabs, 51 feces, 56 urine and 56 plasma samples were sequentially collected from 23 patients in a designated hospital. The plasma and urine samples were all negative, except for urine samples from two severe cases at the latest available detection point. Conversely, virus was shed in respiratory swabs and feces samples during the diseased period. Ten of 12 (83.3%) cases were positive for feces samples, while 14 of 21 (66.7%) were positive for respiratory samples. In addition, the median duration of virus shedding was 10.0 days (IQR 8.0 to 17.0) in the upper respiratory swabs, but was 22.0 days (IQR 15.5 to 23.5) for the feces. Notably, at 26 days after discharge, case 3 (a 45-year-old) was detected positive again in the feces samples, but appears to be healthy and negative for respiratory swabs. These results indicated that beside respiratory samples, intestinal samples (e.g. feces) should be recommended for diagnosis of COVID-19, especially before a patient discharge and for monitoring the relapse of discharged patients.

Anti-hypertensive Angiotensin II receptor blockers associated to mitigation of disease severity in elderly COVID-19 patients

BackgroundThe novel coronavirus (CoV) severe acute respiratory syndrome (SARS)-CoV-2 outbreak started at the end of 2019 in Wuhan, China, and spread over 100 countries. SARS-CoV-2 uses the membrane protein Angiotensin I converting enzyme 2(ACE2) as a cell entry receptor. Indeed, it was reported that the balance of Renin-Angiotensin System (RAS), regulated by both ACE and ACE2, was altered in COVID-19 patients. It is controversial, however, whether commonly used anti-hypertensive drugs Angiotensin I converting enzyme inhibitor (ACEI) and Angiotensin II receptor blocker (ARB) shall be continued in the confirmed COVID-19 patients. This study was designed to investigate any difference in disease severity between COVID-19 patients with hypertension comorbidity. The included COVID-19 patients used ACEI, ARB, calcium channel blockers (CCB), beta blockers (BB), or thiazide to treat preexisting hypertension prior to the hospital were compared to patients who did not take any of those drugs. MethodsIn this multicentre retrospective study, clinical data of 511 COVID-19 patients were analyzed. Patients were categorized into six sub-groups of hypertension comorbidity based on treatment using one of anti-hypertension drugs (ACEI, ARB, CCB, BB, thiazide), or none. A meta-analysis was performed to evaluate the use of ACEI and ARB associated with pneumonia using published studies. FindingsAmong the elderly (age>65) COVID-19 patients with hypertension comorbidity, the risk of COVID-19-S (severe disease) was significantly decreased in patients who took ARB drugs prior to hospitalization compared to patients who took no drugs (OR=0{middle dot}343, 95% CI 0{middle dot}128-0{middle dot}916, p=0{middle dot}025). The meta-analysis showed that ARB use has positive effects associated with morbidity and mortality of pneumonia. InterpretationElderly (age>65) COVID-19 patients with hypertension comorbidity who are taking ARB anti-hypertension drugs may be less likely to develop severe lung disease compared to patients who take no anti-hypertension drugs. FundingNational Natural Science Foundation of China, Chinese Academy of Medical Sciences Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed for articles published up to March 15, 2020 using keywords "2019-nCoV", "SARS-CoV-2", "novel coronavirus", and COVID-19 AND "ARB", and "angiotensin II receptor blocker" for papers published in both English and Chinese. We found three papers: one from our group, published in Science China Life Science that demonstrated an elevated Angiotensin II level in blood samples from COVID-19 patients; another a perspective article in Chinese recommending ACEI and ARBs as potential remedies for SARS-CoV-2 infections; the third a retrospective study in Chinese identifying no significant difference between ACEI/ARB associated with outcomes in 112 COVID-19 patients with CVD comorbidity. The International society of Hypertension stated on March 16th, 2020: "there are no clinical data in human to show that ACE-inhibitors or ARBs either improve or worsen susceptibility to COVID-19 infection nor do they affect the outcomes of those infected". Added value of this studyWe retrospectively reviewed different types of anti-hypertensive drugs taken by COVID-19 patients with hypertension comorbidity prior to entering the hospital. We discovered that ARB hypertensive drugs were associated with a decreased risk of severe disease in elderly (age>65) COVID-19 patients (OR=0{middle dot}343, 95% CI 0{middle dot}128-0{middle dot}916, p=0{middle dot}025), the first evidence of ARBs association to COVID-19 infections in human. We conducted a meta-analysis in the literature and found that ARB has positive effects associated with morbidity and mortality of pneumonia. Implications of all the available evidenceARB drugs are widely used in the population with hypertension. Treatments with ACEI and ARBs should be continuous according to medical guidelines. RCT trials of ARB associated with morbidity and mortality of SARS-CoV-2 infection are recommended in the future.

Epidemiological and clinical features of COVID-19 patients with and without pneumonia in Beijing, China

BackgroundSARS-CoV-2-caused coronavirus disease (COVID-19) is posing a large casualty. The features of COVID-19 patients with and without pneumonia, SARS-CoV-2 transmissibility in asymptomatic carriers, and factors predicting disease progression remain unknown. MethodsWe collected information on clinical characteristics, exposure history, and laboratory examinations of all laboratory-confirmed COVID-19 patients admitted to PLA General Hospital. Cox regression analysis was applied to identify prognostic factors. The last follow-up was February 18, 2020. ResultsWe characterized 55 consecutive COVID-19 patients. The mean incubation was 8.42 (95% confidence interval [CI], 6.55-10.29) days. The mean SARS-CoV-2-positive duration from first positive test to conversion was 9.71 (95%CI, 8.21-11.22) days. COVID-19 course was approximately 2 weeks. Asymptomatic carriers might transmit SARS-CoV-2. Compared to patients without pneumonia, those with pneumonia were 15 years older and had a higher rate of hypertension, higher frequencies of having a fever and cough, and higher levels of interleukin-6 (14.61 vs. 8.06pg/mL, P=0.040), B lymphocyte proportion (13.0% vs.10.0%, P=0.024), low account (<190/{micro}L) of CD8+ T cells (33.3% vs. 0, P=0.019). Multivariate Cox regression analysis indicated that circulating interleukin-6 and lactate independently predicted COVID-19 progression, with a hazard ratio (95%CI) of 1.052 (1.000-1.107) and 1.082 (1.013-1.155), respectively. During disease course, T lymphocytes were generally lower, neutrophils higher, in pneumonia patients than in pneumonia-free patients. CD8+ lymphocytes did not increase at the 20th days after illness onset. ConclusionThe epidemiological features are important for COVID-19 prophylaxis. Circulating interleukin-6 and lactate are independent prognostic factors. CD8+ T cell exhaustion might be critical in the development of COVID-19.

Oncolytic Activity of a Novel Influenza A Virus Carrying Granulocyte-Macrophage Colony-Stimulating Factor in Hepatocellular Carcinoma.

Oncolytic virotherapy is a promising strategy for the treatment of cancer. Influenza A virus has shown potential as an oncolytic agent. In this study, a recombinant PR8 influenza viral vector, called delNS1-GM-CSF, was generated with a partial deletion in NS and the granulocyte-macrophage colony-stimulating factor (GM-CSF) coding sequence inserted into the influenza nonstructural protein 1 gene. The morphological characteristics of delNS1-GM-CSF were examined. The delNS1-GM-CSF virus replicated well in various cell lines, including MDCK, A549, SMCC7721, and HepG2 cells. Moreover, selective cytotoxicity of the virus was observed in various hepatocellular carcinoma (HCC) cell lines, while no effect was demonstrated in the normal liver cell line LO2, as indicated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet assays. Importantly, using a model based on the growth of HepG2 cells as a xenograft in nude mice, it was found that a reassortant delNS1-GM-CSF virus inhibited tumor growth significantly following intratumoral injection in a dose-dependent manner. Ex vivo results showed that the tumor inhibition efficacy of delNS1-GM-CSF was observed in HCC clinical samples. Taken together, these results are the first to demonstrate that influenza A viruses may have potential as oncolytic virotherapeutic agents against HCC.

Pharmacokinetics of a fusion protein for human acidic fibroblast growth factor and transcriptional activator protein in rat and its penetration across blood-brain barrier / 药学学报

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.

Development and Identification of a Live Attenuated Influenza B Virus Vaccine Candidate / 生物化学与生物物理进展

A cold-adapted (ca), temperature sensitive (ts), live attenuated influenza B virus strain B/Ann Arbor/1/66 was chosen for influenza virus rescue research, in which six internal gene segments, PB1, PB2, PA, NP, M, NS, were fully synthesized and nine amino acid substitutions were artificially alter by human intervention. The resultant B/Ann Arbor/1/66 plasmids were named as pAB121-PB1, pAB122-PB2, pAB123-PA, pAB124-HA, pAB125-NP, pAB126-NA, pAB127-M and pAB128-NS, respectively. A recombinant influenza A virus was previously generated entirely from cloned cDNA. An infectious recombinant influenza B virus was generated here, and designated as rMDV-B, by plasmid-based reverse genetics. The rMDV-B virus contained HA and NA genes from an epidemic influenza B vires strain B/Malaysia/2506/2004 in the background of internal genes derived from influenza B virus strain B/Ann Arbor/1/66. HA titer of rMDV-B in MDCK cells and embryonated chicken eggs ranged from 1 : 64 to 1 : 512. The results may allow an effective live influenza B vaccine to be produced from a single master strain, providing a model for the design of future live human influenza vaccines.

Generation of cold-adapted, attenuated reassortant human influenza A virus with eight-plasmid system / 中华微生物学和免疫学杂志

Objective To set up a technical platform of reverse genetics based on the 8 plasmid.virus rescue system of cold-adapted influenza virus strain. Methods The cold-adapted, temperature sensitive, live attenuated influenza virus strain A/AnnArbor/6/60(H2N2)was chosen as the master donor virus(MDV)for rescue research,and its six internal gene fragments PB2,PB1,PA,NP,M and NS were artificially synthesized. Meanwhile, five amino acid mutations have been introduced as tags. Six fragments were ligated with modified pAD3000 for the construction of rescue plasmid. Six transcription/expression plasmids(pMDV-A-PB2,pMDV-A-PB1,pMDV-A-PA,pMDV-A-NP,pMDV-A-M,and pMDV-A-NS)were obtained, and their sequences were accurate. Results The reassorted virus named as rMDV-A contains HA and NA gene segments derived from PR8 strain along with six gene segments,PB2,PB1,PA,NP,M and NS,from MDV. The COS-1 cells were co-transfected with eight recombinant plasmids. The results showed that a cold-adapted, attenuated reassortant influenza A virus with hemagglutination activity was rescued successfullv bv"6+2" combination of MDV and PR8, and the allanotoic fluid of the injected eggs gave a posigenes of A/AA/6/60 used as backbone has provided experimental materials for further research on the gene function and novel vaccine candidate of cold-adapted, attenuated human influenza virus.

Construction of Attenuated Influenza Virus Vaccines of Vaccines of-2006-2007 / 生物化学与生物物理进展

Six gene segments,PB1,PB2,PA,NP,M and NS,were fully synthesized which derived from the master donor virus (MDV),cold-adapted(ca),temperature sensitive(ts),live attenuated influenza virus strain A/Ann Arbor/6/60(MDV-A).Meanwhile,five amino acid substitutions (PB1-391E,58lG,661T,PB2-265S,NP-34G) were artificially altered by human intervention.HA and NA fragments derived from the 2006-2007 circulating strain A/New Caledonia/20/99 (H1N1).Eight fragments were ligated with modified pAD3000 for rescue plasmid construction.Eiight transcription/expression plasmids were named as pMDV-A-PB2,pMDV-A-PB1,pMDV-A-PA,pMDV-A-NP,pMDV-A-M,pMDV-A-NS,pMDV-A-HA,pMDV-A-NA,respectively.The COS-l cells were co-transfected with eight plasmids representing 6 internal viral backbone of the strain A/AA/6/60 and two plasmids containing the CDNA of the HA and NA segments of the strain A/New Caledonia/20/99 (H1N1),the results showed that cold-adapted,attenuated reassortant influenza A virus Was rescued successfully.Titers of a reassorted influenza A virus in embryonated chicken eggs mnged from 1:29to l:210.The rescue system of six intemal genes used as backbone opens the way for further research on gene function and neotype vaccine candidate of cold-adapted,live attenuated human influenza virus.

DETECTING LOW DENSITY LIPOPROTEIN RECEPTOR MUTANT GENE OF RABBIT BY PCR / 药物分析学报

Objective Watanabe Heritable Hyperlipidaemic (WHHL) rabbits with low density lipoprotein receptor (LDLr) gene mutation have provided unprecedented opportunities for the study of human atherosclerosis, in order to confirm LDL receptor gene status in rabbits, we developed a simple PCR technique to detect LDL mutations in rabbits. Methods Rabbits genomic DNA were extracted from ear biopsy, and amplified by PCR to detect 12 bp deletion mutation in WHHL rabbits. PCR products were directly digested with BglⅠ, and then applied to polyacrylamide gel electrophoresis. Results PCR products from homozygous LDLr +/+ rabbits generated 2 bands of 212 and 94 bp after BglⅠ digestion, LDLr +/- rabbits generated 3 bands (294, 212, and 94 bp), LDLr -/- animals, however, generated only 1 product (294 bp). Conclusion This modified PCR method is simple and reliable.

Construction of Attenuated Influenza Virus Vaccines of 2006～2007 / 生物化学与生物物理进展

Six gene segments, PB1, PB2,PA, NP, M and NS, were fully synthesized which derived from the master donor virus(MDV), cold-adapted(ca),temperature sensitive(ts), live attenuated influenza virus strain A/Ann Arbor/6/60(MDV-A). Meanwhile, five amino acid substitutions (PB1-391E, 581G, 661T, PB2-265S, NP-34G) were artificially altered by human intervention. HA and NA fragments derived from the 2006～2007 circulating strain A/New Caledonia/20/99 (H1N1). Eight fragments were ligated with modified pAD3000 for rescue plasmid construction. Eight transcription/expression plasmids were named as pMDV-A-PB2, pMDV-A-PB1, pMDV-A-PA, pMDV-A-NP, pMDV-A-M, pMDV-A-NS, pMDV-A-HA, pMDV-A-NA, respectively. The COS-1 cells were co-transfected with eight plasmids representing 6 internal viral backbone of the strain A/AA/6/60 and two plasmids containing the cDNA of the HA and NA segments of the strain A/New Caledonia/20/99 (H1N1), the results showed that cold-adapted, attenuated reassortant influenza A virus was rescued successfully. Titers of a reassorted influenza A virus in embryonated chicken eggs ranged from 1∶29 to 1∶210. The rescue system of six internal genes used as backbone opens the way for further research on gene function and neotype vaccine candidate of cold-adapted, live attenuated human influenza virus.