Study on early markers of death in patients with COVID-19.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) was first discovered in December 2019, and since then rapidly spread worldwide. Our study aimed to investigate the early indicators of death in patients suffering from severe and critical COVID-19. PATIENTS AND METHODS: A retrospective cohort study was conducted on patients with severe and critical COVID-19, admitted to the Seventh Hospital of Wuhan. Clinical information was collected from electronic medical records according to standardized data collection tables. Patients were divided into non-survival and survival groups based on the disease outcome. Using univariate and multivariate logistic regression analysis, and calculating odds ratios (OR) and 95% confidence intervals (CI), independent risk factors for death in severe and critically ill COVID-19 patients were identified. RESULTS: The median age of 162 patients (57.4% males) was 67.5 years old. Patients in the non-survival group had significantly higher white blood cell count, decreased lymphocyte count, anemia and thrombocytopenia compared to patients in the survival group (p < 0.05). A 28-day mortality rate of the study cohort was 31.5%. Multivariate logistic regression analysis showed that underlying heart disease, lymphocyte count < 1.0 × 109/L, glomerular filtration rate < 66, lactate > 2.2 mmol/L, higher Sequential Organ Failure Assessment (SOFA) score, lower oxygenation index (OR 1.748; 95% CI 1.024-2.984; p=0.041) and higher "multi-lobar infiltration, hypo lymphocytosis, bacterial co-infection, smoking history, hypertension and age" (MuLBSTA) score (OR 1.601; 95% CI 1.062-2.415; p=0.025) were risk factors associated with death in patients with severe and critical COVID-19. CONCLUSIONS: Underlying heart disease, lymphocyte count, glomerular filtration rate, lactate, oxygenation index, SOFA score, and MuLBSTA score were associated with the risk of death in severe and critical COVID-19 patients.

[Role of allergen immunotherapy in the prevention of allergic asthma].

The prevalence of allergic asthma is still increasing, which affects the quality of life of patients, threatens their lives, and brings enormous social and economic burden. Allergen immunotherapy (AIT) is the only treatment that can alter the progression of the "Atopic March". It has been widely used in the treatment of allergic rhinitis and conjunctivitis, and its role and effect in the treatment of allergic asthma have been gradually recognized. A few studies have shown that AIT may have a preventive effect on the development and progression of allergic asthma. In this article, the definition of tertiary prevention of allergic asthma is described, and the respective role of AIT in primary, secondary and tertiary prevention of allergic asthma is summarized and analyzed. The aim of this article is to provide evidence for the prevention and control of allergic asthma.

[Brief analysis on the current status of medical education and specialty of allergy in China and abroad and the strategies for the development of allergology in China].

The increase in the prevalence of allergic diseases has brought a substantial medical, social and economic burden. The development of allergology is relatively lag behind the allergy prevalence in China. Both the numbers of allergy specialty and allergist are scarce and thus the diagnosis and treatment of allergic disease does not meet the needs of allergy patients. This article summarizes the status of medical education and specialty development of allergology in China and abroad. In addition, the key strategies for promoting the development of allergy education and specialty were discussed, including undergraduate and graduate education of allergology, the orientation of allergy specialty and related specialty/subspecialty, the integration of allergology into the standardized residents training system, training and certification of allergists, and multidisciplinary diagnosis and treatment model.

[Cytotoxicity and underlying mechanism of evodiamine in HepG2 cells].

OBJECTIVE: To investigate evodiamine (EVO)-induced hepatotoxicity and the underlying mechanism. METHODS: HepG2 cells were treated with EVO (0.04-25 µmol/L) for different time intervals, and the cell survival rate was examined by cell counting kit-8 (CCK-8) method. After HepG2 cells were treated with EVO (0.2, 1 and 5 µmol/L) for 48 h, the alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities and total bilirubin (TBIL) content of supernatant were detected. A multifunctional microplate reader was used to detect the intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in HepG2 cells to evaluate the level of cell lipid peroxidation damage. The interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins were simulated by molecular docking. The HepG2 cells were stained by mitochondrial membrane potential (MMP) fluorescent probe (JC-10) and annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI), and MMP and apoptosis in HepG2 cells were detected by flow cytometry. The protein expression levels of caspase-9, caspase-3, bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were detected by Western blot. RESULTS: The cell survival rate was significantly reduced after the HepG2 cells were exposed to EVO (0.04-25 µmol/L) in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) of the HepG2 cells treated with EVO for 24, 48 and 72 h were 85.3, 6.6 and 4.7 µmol/L, respectively. After exposure to EVO (0.2, 1 and 5 µmol/L) for 48 h, the ALT, AST, LDH, ALP activities and TBIL content in the HepG2 cell culture supernatant, and the MDA content in the cells were increased, and SOD enzyme activity was decreased. Molecular docking results showed that EVO interacted with apoptosis-associated proteins (caspase-9 and caspase-3) better. JC-10 and Annexin V-FITC/PI staining assays demonstrated that EVO could decrease MMP and promote apoptosis in the HepG2 cells. Western blot results indicated that the protein expressions of cleaved caspase-9 and cleaved caspase-3 were upregulated in the HepG2 cell treated with EVO for 48 h. In contrast, the protein expressions of pro-caspase-3, BSEP and MRP2 were downregulated. CONCLUSION: These results suggested that 0.2, 1 and 5 µmol/L EVO had the potential hepatotoxicity, and the possible mechanism involved lipid peroxidation damage, cell apoptosis, and cholestasis.

[Analysis of incidence and risk factors for postoperative venous thromboembolism in patients with stageâ a non-small-cell lung cancer].

Objective: To analyze the incidence and risk factors for postoperative venous thromboembolism(VTE) in patients with stage Ⅰa non-small-cell lung cancer(NSCLC), so as to find evidence for further research of prophylactic anticoagulation. Methods: A total of 132 patients with stage Ⅰa NSCLC, 42 males and 90 females aged from 26 to 79 years with an average of (57±10) years, were retrospectively included in this study. All of them underwent surgical treatment at the Department of Thoracic Surgery of Beijing Chaoyang Hospital Affiliated with Capital Medical University from January 2017 to October 2020. A lower extremity venous ultrasound was performed before and after the operation. Participants were divided into VTE group (n=11) or non-VTE group (n=121) according to whether or not VTE occurred after operation. The surgical conditions, test indicators, imaging information, pathology information were compared between the two groups. Logistic regression analysis was performed to test the associations of VET with putative risks factors in which significant differences were observed. The independent risk factors of VET were determined by this way. Results: Postoperative VTE occurred in 11 cases (8.3%), including 10 cases (90.9%) of deep vein thrombosis (DVT) of lower limbs and 1 case (9.1%) of DVT complicated with pulmonary embolism (PE). The mean age of Patients in the VTE group was older than that in non-VTE Group ((65±9) years vs (57±10) years, P=0.009). On the fifth day after operation, patients in both groups had significantly higher D-dimer level compared with that before operation (3.18(1.55, 5.15) vs 1.54(1.09, 2.57); 2.66(1.17, 4.65) vs 1.34(0.78, 2.04))(both P<0.05). The value of neuron-specific enolase (NSE) and the number of lymph nodes removed during the operation in the VTE group were significantly higher than those in the non-VTE group ((21.54±12.37) vs (14.72±5.75); (19.7±8.2) vs (13.0±7.9)) (both P<0.05). There was no statistically significant difference in the approach of surgery, imaging features (tumor location, vascular cluster signs, etc.), and pathological information (pathological types, etc.) (all P>0.05). The logistic regression analysis showed that the number of lymph nodes removed during the operation was an independent risk factor related to the occurrence of VTE (OR=1.306, 95%CI:1.000-1.600,P<0.05). Conclusions: The incidence of postoperative VTE in patients with stage Ⅰa NSCLC is approximately 8.3%. The number of lymph nodes removed during the operation may be an independent risk factor for postoperative VTE in patients with stage Ⅰa NSCLC.

[Correlation analysis between serum ferritin level and liver damage in acute stage of dengue fever].

Objective: To investigate the correlation between serum ferritin (SF) level and liver damage in the acute stage of dengue fever. Methods: A retrospective study was conducted to analyze 171 cases diagnosed with dengue fever as dengue fever group and 130 healthy patients as control group in Hangzhou 3A grade hospital from July to December 2017. Clinical data, SF and liver function related indicators were collected from both groups: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) to analyze the correlation between liver damage and SF in patients with dengue fever. Results: ALT, AST, and SF levels were significantly higher in the dengue fever group than those in the healthy control group (Z = 11.553, 15.054 and 15.163, P < 0.001). SF levels were higher in the dengue fever combined with liver damage group than those without the liver damage group (z = 6.930, P < 0.001). However, there was no statistically significant differences in age, gender, peak body temperature, and history of liver disease (P > 0.05). In addition, Spearman's correlation analysis showed that SF was positively correlated with ALT, AST, and TBIL (r = 0.464, 0.531 and 0.315, P < 0.001). Among dengue patients with different SF levels, there were significant difference in ALT, AST levels and incidence of liver damage (H = 14.240 and 17.584, χ(2) = 49.547, P < 0.001). Patients with higher SF levels had higher ALT, AST levels and incidence of liver damage. Binary logistic regression analysis showed that hyperferritinemia (SF≥500 ng/ml) was the risk factor for dengue fever combined with liver damage (OR = 8.120, P < 0.001). Furthermore, ROC curve analysis showed that the AUC for SF to judge dengue fever combined liver damage was 0.846 (95% CI: 0.785-0.908), and the sensitivity and specificity when the SF cut-off value was 1 506 ng/ml were 74.8% and 83.3%. Conclusion: There is a certain correlation between the SF level and the degree of liver damage in acute stage of dengue fever patients, and hyperferritinemia is a risk factor for dengue fever combined with liver damage.

[Study of effects of micheliolide on ovarian cancer cell lines and its mechanism].

Objective: To study the roles of micheliolide on ovarian cancer cells. Methods: Firstly, human ovarian cancer cell lines HeyA8, SKOV3 and A2780/DDP were treated with different concentration of micheliolide(0.25, 0.5, 1, 2.5, 5, 10, 20, 50 µmol/L)for 72 hours, then methyl thiazolyl tetrazolium(MTT)assay was used wo detect the growth of the human ovarian cancer cell lines and the stongest inhibited cell line were selected for the following test. Secondly, after HeyA8 cell line was treated with different concentration(5, 10, 20 µmol/L)of micheliolide for 24 hours, the HeyA8 cell apoptosis was measured byflow cytometry. Thirdly, the expression of RelA mRNA in HeyA8 cell was detected through real-time PCR, the expressions of nuclear factor κB(NF-κB)signal pathway related protein RelA and the activited cysteinyl aspartate specific proteinase(caspase-9)were detected by western blot analysis. Results: (1)The growth of HeyA8, SKOV3 and A2780/DDP cells were all significantly inhibited after being treated with different concentration of micheliolide for 72 hours and the roles of inhibition were all concentration dependant(P<0.05). The half maximal inhibitory concentration(IC50)of HeyA8, SKOV3 and A2780/DDP were(9.8±2.2),(12.0±2.1)and(12.8±1.8)µmol/L, respectively. We chose HeyA8 cell to do the following expreriments because of its best inhibited effect.(2)After HeyA8 cell was treated with micheliolide of different concentrations, as the concentration increased(20 and 0 µmol/L, for example), the apoptosis rate of HeyA8 cell raised from(7.2±1.0)% to(17.4±1.1)%, the percentage of survived cells reduce from(92.8 ± 1.3)% to(82.6 ± 1.4)%, and the relative mRNA level of RelA decreased from 1.00 ± 0.13 to 0.18 ± 0.00(P<0.01); furthermore, the expression of RelA protein was weaken and the activited caspase-9 protein expression was increased gradually. Conclusions: Micheliolide plays a significantly inhibited role in HeyA8, SKOV3 and A2780/DDP cells. The inhibited role of micheliolide inovarian cancer cells might through inhibiting nuclear factor-kappa B(NF-кB)signaling pathway, and inducing the expression of activited caspase-9 protein to promoting apoptosis of HeyA8 cell.

[Study on the application effect of Once Vex(GM)(-)(CSF) and adriamycin in the interventional therapy of malignant tumor in clinical curative effect].

OBJECTIVE: To investigate the application effect of oncolytic herpes simplex virus Once Vex(GM-CSF) and adriamycin in malignant tumor. METHODS: A total of 102 cases of malignant tumor were analyzed retrospectively from April 2013 to April 2015 of Cancer Treatment Center of Affiliated Hospital of Beihua University, and these cases were randomly divided into trial group (n=51) and control group (n=51). The control group was treated with adriamycin and the trial group patients received conditionally replicating adenoviruses on the basis of the control group.The adenovirus proliferation was analyzed via quantitative PCR to investigate the malignant tumor cell inhibitory action in two groups. RESULTS: Treatment group total effective rate (96.1%, 49/51) was significantly higher than the control group (78.4%, 40/51) (χ(2)=7.845, P=0.014). There were statistically significant difference of the killing capability between trial group (86.3%, 44/51) and control group (23.5%, 12/51) (χ(2)=7.859, P=0.013). Tumor shrinkage rate of trial group(76.5%, 39/51) was superior to that of the control group(19.6%, 10/51), the difference was statistically significant (χ(2)=7.654, P=0.019). There was statistically significant difference of the decrease of tumor marker CA125 between trial group (74.5%, 38/51) and control group (23.5%, 12/51) (χ(2)=7.342, P=0.023). CONCLUSION: Once Vex(GM-CSF) can effectively kill the tumor cells in the treatment of malignant tumor, and shrink the tumor significantly.

Observation of a spontaneous particle-transport barrier in the HL-2A tokamak.

Using the profile analysis, the density perturbation transport analysis, and the Doppler reflectometry measurement, for the first time a spontaneous and steady-state particle-transport barrier has been evidenced in the Ohmic plasmas in the HL-2A tokamak with no externally applied momentum or particle input except the gas puffing. A threshold in density has been found for the observation of the barrier. The particle diffusivity is well-like, and the convection is found to be inward outside the well and outward inside the well. The formation of the barrier coincides with the transition between the trapped electron mode and the ion temperature gradient driven mode.

A method of particle transport study using supersonic molecular beam injection and microwave reflectometry on HL-2A tokamak.

A method of the particle transport study using supersonic molecular beam injection (SMBI) and microwave reflectometry is reported in this paper. Experimental results confirm that pulsed SMBI is a good perturbation source with deeper penetration and better localization than the standard gas puffing. The local density modulation is induced using the pulsed SMBI and the perturbation density is measured by the microwave reflectometry. Using Fourier transform analysis for the local density perturbation, radial profiles of the amplitude and phase of the density modulation can be obtained. The experimental results in HL-2A show that the particle injected by SMBI is located at about r/a=0.65-0.75. The position of the main particle source can be determined through three aspects: the minimum of the phase of the first harmonic of the Fourier transform of the modulated density measured by microwave reflectometry; the H(a) intensity profile and the local density increase ratio. The maximum of the amplitude of the first harmonic shifts often inward relative to the particle source location, which indicates clearly there is an inward particle pinch in this area. Good agreement has been found between the experimental results and the simulation using analytical transport model. The particle diffusivity D and the particle convection velocity V have been obtained by doing this simulation. The sensitivity in the transport coefficients of the amplitude and the phase of the density modulation has been discussed.

Attenuating pregnane X receptor (PXR) activation: a molecular modelling approach.

Recent studies have demonstrated that the pregnane X receptor (PXR) is a key regulator of cytochromes P450 3A (e.g. CYP3A4 in human) gene expression. As a result, activation of PXR may lead to CYP3A4 protein over-expression. Because induction of CYP3A4 could result in clinically important drug drug interactions, there has been a great interest in reducing the possibility of PXR activation by drug candidates in drug-discovery programmes. In order to provide structural insight for attenuating drug candidate-mediated PXR activation, we used a docking approach to study the structure activity relationship for PXR activators. Based on our docking models, it is proposed that introducing polar groups to the end of an activator should reduce its human PXR (hPXR) activity via destabilizing interactions in the hydrophobic areas of the PXR ligand-binding pocket. A number of analogues that incorporate these structural features then were designed and synthesized, and they exhibited significantly lower hPXR activation in a transactivation assay and decreased CYP3A4 induction in a human hepatocytes-based assay. In addition, an example in which attenuating hPXR activation was achieved by sterically destabilizing the helices 11 and 12 of the receptor is presented.

1H NMR structural analysis of human ghrelin and its six truncated analogs.

Human ghrelin, the first recognized natural ligand of growth hormone secretagogue growth hormone secretagogue receptors (GHS-Rs) (M. Kojima, H. Hosada, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa, Nature, 1999, Vol. 402, pp. 656-660), consists of 28 amino acids of which Ser3 is modified by n-octanoylation. This new peptide hormone has been implicated not only in regulation of the GH secretion but also in regulation of food intake. The discovery of ghrelin opens up more opportunities to study the relationship of ghrelin with metabolic diseases. Until now, only mass spectometry analysis has been reported on the structure of ghrelin. NMR analysis is a suitable way to study if any tertiary structure of unbound ghrelin is present in solution. NMR studies were carried out on human ghrelin and its five truncated analogs. The full-length ghrelin and its fragments exhibited random coil behavior in aqueous solution. Additional studies were carried out on the shortest active segment of human ghrelin, which consists of the first five amino acids of the ghrelin sequence (M. A. Bednarek, S. D. Feighner, S.-S. Pong, K. K. McKee, D. L. Hreniuk, M. V. Silva, V. A. Warrem, A. D. Howard, L. H. Y. Van der Ploeg, and J. V. Heck, Journal of Medical Chemistry, 2000, Vol. 43, pp. 4370-4376), to compare the spectral features with their counterparts in the full-length ghrelin. The NMR data showed behavior similar to ghrelin except for two additional nuclear Overhauser effects (NOEs) between the Phe4 NH and the protons of the beta-methylene of Ser3. CD on human ghrelin and its short active analog in water were indicative of random coil peptides. Molecular modeling based on NMR data was carried out to probe which structural features were similar to growth hormone-releasing peptide-6 (GHRP-6), a hexapeptide that binds to GHS-R releasing GH and stimulating food intake. Modeling suggested some similarities, but they were not of a nature to account for binding properties of these compounds.

Binding of correolide to the K(v)1.3 potassium channel: characterization of the binding domain by site-directed mutagenesis.

Correolide is a novel immunosuppressant that inhibits the voltage-gated potassium channel K(v)1.3 [Felix et al. (1999) Biochemistry 38, 4922-4930]. [(3)H]Dihydrocorreolide (diTC) binds with high affinity to membranes expressing homotetrameric K(v)1.3 channels, and high affinity diTC binding can be conferred to the diTC-insensitive channel, K(v)3.2, after substitution of three nonconserved residues in S(5) and S(6) with the corresponding amino acids present in K(v)1.3 [Hanner et al. (1999) J. Biol. Chem. 274, 25237-25244]. Site-directed mutagenesis along S(5) and S(6) of K(v)1.3 was employed to identify those residues that contribute to high affinity binding of diTC. Binding of monoiodotyrosine-HgTX(1)A19Y/Y37F ([(125)I]HgTX(1)A19Y/Y37F) in the external vestibule of the channel was used to characterize each mutant for both tetrameric channel formation and levels of channel expression. Substitutions at Leu(346) and Leu(353) in S(5), and Ala(413), Val(417), Ala(421), Pro(423), and Val(424) in S(6), cause the most dramatic effect on diTC binding to K(v)1.3. Some of the critical residues in S(6) appear to be present in a region of the protein that alters its conformation during channel gating. Molecular modeling of the S(5)-S(6) region of K(v)1.3 using the X-ray coordinates of the KcsA channel, and other experimental constraints, yield a template that can be used to dock diTC in the channel. DiTC appears to bind in the water-filled cavity below the selectivity filter to a hydrophobic pocket contributed by the side chains of specific residues. High affinity binding is predicted to be determined by the complementary shape between the bowl-shape of the cavity and the shape of the ligand. The conformational change that occurs in this region of the protein during channel gating may explain the state-dependent interaction of diTC with K(v)1.3.

Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-beta-lactamase.

IMP-1 metallo-beta-lactamase (class B) is a plasmid-borne zinc metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics, including carbapenems, rendering them ineffective. Because IMP-1 has been found in several clinically important carbapenem-resistant pathogens, there is a need for inhibitors of this enzyme that could protect broad spectrum antibiotics such as imipenem from hydrolysis and thus extend their utility. We have identified a series of 2,3-(S,S)-disubstituted succinic acids that are potent inhibitors of IMP-1. Determination of high resolution crystal structures and molecular modeling of succinic acid inhibitor complexes with IMP-1 has allowed an understanding of the potency, stereochemistry, and structure-activity relationships of these inhibitors.

Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to humans.

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.

Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.