Abstract:Objective To identify key amino acid variations of major proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by biophysical methods. Methods Through amino acid sequence alignment, classification of variant amino acid residues, three-dimensional structure reconstruction of proteins, and electrostatic interaction analysis of amino acid residues, the key amino acid variations of major proteins from SARS-CoV-2 was analyzed with RaTG13, the bat coronavirus with the highest homology, as the reference. Results At least ten amino acid variations that affect the possible electrostatic interactions were identified in RNA-dependent RNA polymerase (RdRp), exoribonuclease (ExoN), uridylate-specific endoribonuclease (NendoU), and spike (S) protein from SARS-CoV-2. These variations may affect the spatial conformation and biological functions of the proteins. Conclusion The key amino acid variations of the major proteins from SARS-CoV-2 have been preliminarily identified, providing clues for understanding the genetic, pathogenic and epidemiological characteristics of the virus.

Abstract:Objective To investigate the efficacy of neutralizing antibodies induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) and spike (S) protein S1 subunit. Methods The SARS-CoV-2 RBD and mouse immunoglobulin G1 (IgG1) Fc fragment (mFc) fusion protein expression plasmid pVRC-RBD-mFc was constructed and transfected into human embryonic kidney 293T cells. The RBD-mFc fusion protein in the cell supernatants was detected by Western blotting. The effect of RBD-mFc in cell supernatants and CHO recombinant S1-human IgG1 Fc (S1-hFc) fusion protein on SARS-CoV-2 infection was detected by microneutralization test. BALB/c mice were immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein via intramuscular injection. Anti-S1 IgG antibodies in mouse sera were detected by enzyme-linked immunosorbent assay (ELISA), and the virus neutralization activity of mouse sera was detected by microneutralization test. Results The RBD-mFc fusion protein could be detected in the culture supernatants of 293T cells transfected with the plasmid pVRC-RBD-mFc, the concentrated supernatants and the S1-hFc fusion protein could inhibit SARS-CoV-2 infection on Vero E6 cells in a concentration-dependent manner. Anti-S1 IgG antibodies could be detected in the sera of mice immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein, and the sera of both groups could neutralize SARS-CoV-2 infection. The serum antibody titers and virus neutralization activity of S1-hFc fusion protein immunized mice were significantly higher than those of plasmid pVRC-RBD-mFc immunized mice (both P<0.01). Conclusion Both SARS-CoV-2 RBD and S1 subunit may be used as effective vaccine antigens. Compared with DNA vaccine, recombinant subunit vaccine can induce neutralizing antibody more effectively.

Abstract:Objective To express and purify the recombinant nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and prepare antiserum from immunized mice. Methods The prokaryotic plasmid pET28a-N containing SARS-CoV-2 N gene was transformed into Escherichia coli BL21 (DE3). The expression of recombinant SARS-CoV-2 N protein was induced by isopropyl-β-D-thiogalactopyranoside. The Ni-NTA affinity chromatography column was used to purify the recombinant SARS-CoV-2 N protein, and antiserum was obtained from the BALB/c mice immunized with recombinant SARS-CoV-2 N protein combined with manganese adjuvant through intramuscular and subcutaneous injections. The reactions of recombinant SARS-CoV-2 N protein with SARS-CoV-2 N monoclonal antibodies and severe acute respiratory syndrome coronavirus (SARS-CoV) N polyclonal antibodies were detected by Western blotting. The reaction of mouse antiserum with the recombinant SARS-CoV-2 N protein expressed in the cells transfected with eukaryotic expression plasmid was examined by indirect immunofluorescence assay. Results The recombinant SARS-CoV-2 N protein was successfully induced and expressed as a soluble protein with a molecular weight of about 55 000. High concentration of purified protein was obtained. The results of Western blotting showed that the recombinant SARS-CoV-2 N protein could be specifically recognized by the SARS-CoV-2 N monoclonal antibodies and the SARS-CoV N polyclonal antibodies. The prepared mouse antiserum could also correctly recognize the recombinant SARS-CoV-2 N protein expressed in mammalian cells by indirect immunofluorescence assay. Conclusion Recombinant SARS-CoV-2 N protein has been successfully expressed and purified from the prokaryotic expression system, and mouse antiserum has been prepared, which lays a foundation for establishing a rapid SARS-CoV-2 diagnostic tool and further studying the function of SARS-CoV-2 N protein.

Abstract:Objective To explore the diagnosis and treatment process and experience of emergency interventional ultrasound in our hospital under the coronavirus disease 2019 (COVID-19) epidemic situation, so as to provide reference for the safe and effective implementation of interventional ultrasound during the epidemic. Methods This study summarized and reviewd the experience of interventional ultrasound for the diagnosis and treatment of three emergency patients in the department of ultrasound of our hospital during the COVID-19 epidemic, focusing on the screening of severe COVID-19 patients, the selection of indications for interventional puncture, the perioperative protection and postoperative management. Results According to epidemiological screening, patient 1 had close contact with the confirmed COVID-19 patient. Chest CT showed diffused interstitial exudation in the lower lobes of both lungs, and the patient was seen as a suspected case after consultation with the hospital expert group, who had retropharyngeal space infection on admission and needed to remove the infection focus quickly. COVID-19 was excluded in patient 2, who was diagnosed as acute episode of calculous incarcerated cholecystitis on admission, requiring rapid drainage of bile to relieve gallbladder obstruction. Patient 3 was excluded from COVID-19 and diagnosed as acute pericardial tamponade on admission, requiring rapid drainage of pericardial effusion. All 3 patients underwent strict COVID-19 screening procedures to identify the indications of interventional puncture. During the operation, tertiary protective measures were adopted for patient 1, and primary protective measures were adopted for the other two patients. Three severe patients were successfully treated by interventional ultrasound, with short average time, less bleeding and improved clinical symptoms. Conclusion Interventional ultrasound is minimally invasive, convenient and efficient, making it suitable for the emergency treatment during the COVID-19 epidemic. It can be used as an alternative to some emergency surgery, or provide a safe window period for confirmed or suspected severe COVID-19 patients. Our experience may provide a reference for the safe and effective practice of interventional ultrasound during the COVID-19 epidemic.

Abstract:Objective To explore the rule of traditional Chinese medicine (TCM) syndrome differentiation of coronavirus disease 2019 (COVID-19) patients. Methods The symptoms of 756 cases with COVID-19 in Guanggu Branch of Maternity and Child Healthcare Hospital of Hubei Province were collected by cross sectional survey. The incidence rates of the symptoms were recorded by frequency method at different courses of the disease:prodromal stage (onset), middle stage (7-30 days), and later stage (>30 days). The common symptoms (incidence rate>5.0%) were analyzed by systematic clustering. With expert experience, the rule of TCM syndrome differentiation of COVID-19 patients was summarized. Results Fever (52.25%, 395 cases), cough (43.25%, 327 cases), asthenia (27.25%, 206 cases), chest distress (26.72%, 202 cases), asthma (17.59%, 133 cases) and expectoration (5.03%, 38 cases) were the most common symptoms in the prodromal stage (756 cases) of the disease, which were clustered into one category except expectoration, indicating the pathogenesis of both lung and body surface suppressed by dampness. In the middle stage (383 cases), the 19 common symptoms including greasy fur (64.49%, 247 cases), yellow fur (43.86%, 168 cases), thick fur (40.21%, 154 cases), cough (34.73%, 133 cases), red tongue (32.38%, 124 cases), poor stool (25.85%, 99 cases), asthma (25.33%, 97 cases), asthenia (25.07%, 96 cases), poor appetite (23.76%, 91 cases), bitterness of mouth (14.36%, 55 cases), dry fur (12.01%, 46 cases), purple tongue (12.01%, 46 cases), perspiration (11.49%, 44 cases), constipation (10.18%, 39 cases), white phlegm (8.62%, 33 cases), insomnia (7.31%, 28 cases), nausea (7.05%, 27 cases), diarrhea (6.79%, 26 cases) and yellow phlegm (6.27%, 24 cases), were clustered into three groups, indicating the pathogenesis of damp-heat accumulation, obstruction of lung and spleen by dampness, and dryness due to dampness-heat. In the later stage (373 cases), the 13 common symptoms including greasy fur (50.94%, 190 cases), asthenia (39.41%, 147 cases), cough (37.80%, 141 cases), red tongue (33.78%, 126 cases), asthma (32.17%, 120 cases), perspiration (23.86%, 89 cases), dry mouth (22.79%, 85 cases), poor appetite (20.11%, 75 cases), poor stool (19.30%, 72 cases), bitterness of mouth (15.01%, 56 cases), white phlegm (10.72%, 40 cases), palpitation (8.31%, 31 cases) and little fur (8.04%, 30 cases), were clustered into two groups, indicating the pathogenesis of deficiency of Qi and Yin with residual dampness, and deficiency of lung Qi and spleen Qi with residual dampness. Conclusion The TCM syndromes of COVID-19 patients in different stages have its own typical characteristics, with a regular change from exterior to interior, from dampness to dampness-heat and from excess to deficiency.

Abstract:Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic and led to a serious impact on public health and economic development. This paper summarizes the source of the first isolated and identified SARS-CoV-2 samples and the problems present in the etiology detection of virus RNA. The necessity and limitation of bronchoscopy use in the diagnosis of COVID-19 were discussed and the occupation protection measures for bronchoscopy in COVID-19 were emphasized. It is important to accelerate the development of new disposable protective devices for the bronchoscopic examination.

Abstract:As the outbreak of coronavirus disease 2019 (COVID-19) continues, hospitals in Wuhan have invested great efforts to combat the epidemic. The risk of nosocomial infection and cross infection should not be ignored, and the treatment of non-COVID-19 patients was challenged. According to the characteristics of COVID-19, our hospital has established a transitional ward, enforced the screening and exclusion procedures of COVID-19. We also scientifically formulated and smoothly operated various programs and procedures for the treatment of emergency and critical patients, so that non-COVID-19 patients can be treated in time, ensuring medical safety and nursing quality, also effectively avoiding the spread of COVID-19 in non-isolated areas of the hospital, which has played a very important role at the outbreak stage of the epidemic.

Abstract:Objective To explore the effect of avasimibe in preventing cholangiocarcinoma induced by thioacetamide, a chemical carcinogen. Methods Male SD rats were given drinking water containing 300 mg/L thioacetamide and stopped drinking after 3 months, and then were divided into two groups (n=5):control group and avasimibe intervention group. The control group was given normal drinking water, while the avasimibe intervention group was given drinking water containing avasimibe (30 mg/kg) by gavage. After 3 months of intervention, all rats were sacrificed and liver lesions were observed by hematoxylin-eosin (H-E) staining. The expression of aldo-keto reductase family 1 member C1 (AKR1C1) and Ki-67 in liver lesions of rats was detected by immunohistochemical staining. Meanwhile, different concentrations (0, 10, 20 μmol/L) of avasimibe were used to treat the cholangiocarcinoma cell line QBC939, the cell proliferation ability was detected by cell counting kit 8 (CCK-8) assay, and the expression of AKR1C1 in QBC939 cells was detected by qRT-PCR and Western blotting. Results The results of H-E staining showed that all rats in the control group developed visible tumors under the microscope, and the tumor formation rate was 100% (5/5), while only one rat in the avasimibe intervention group developed tumors, with a 20% (1/5) tumor formation rate. The results of immunohistochemistry showed that the expression levels of AKR1C1 and Ki-67 in liver lesions of rats in the avasimibe intervention group were significantly lower than those in the control group (P<0.05, P<0.01). The results of CCK-8 assay showed that avasimibe significantly inhibited the proliferation of QBC939 cells. The results of qRT-PCR and Western blotting showed that the expression levels of AKR1C1 mRNA and protein in QBC939 cells after the intervention of avasimibe were significantly decreased compared with those of the control group (both P<0.01). Conclusion Avasimibe can interrupt the occurrence of intrahepatic cholangiocarcinoma caused by chemical carcinogens and is a potential drug for preventing cholangiocarcinoma. AKR1C1 might be a potential therapeutic target of cholangiocarcinoma.

Abstract:Objective To construct human embryonic stem cell (hESC) line with forkhead box G1 (FOXG1) gene knockout by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology, and to investigate the role of FOXG1 gene in the early neural induction of hESCs. Methods Two guide RNAs (gRNAs) were transfected to induce FOXG1 gene large fragment knockout in hESCs by CRISPR/Cas9 gene editing technology. FOXG1 gene knockout hESCs were confirmed by monoclonal screening, sequencing and Western blotting analysis. The expression of the key markers including paired box 6 (PAX6), sex-determining region Y-box 2 (SOX2) and orthodenticle homeobox 2 (OTX2) was detected by immunofluorescence staining and qRT-PCR in the early process of neural induction before and after FOXG1 gene knockout. Results hESCs with FOXG1 gene large fragment knockout were successfully obtained by CRISPR/Cas9 gene editing technology. The results of immunofluorescence staining and qRT-PCR suggested that FOXG1 deletion did not significantly influence the expression of PAX6, SOX2 and OTX2 during neural induction. Conclusion FOXG1 gene large fragment knockout in hESCs can be quickly induced by a pair of gRNAs co-transfection. FOXG1 deletion has no significant impacts on neural induction of hESCs.

Abstract:Objective To establish a construction method for luciferase-microRNA (miRNA) sponge adenovirus, and to verify the binding efficiency of the expression product of the adenovirus and miRNA. Methods DNA fragments that can be partly complementary to miRNA-126 were chemically synthesized. The miRNA-126 sponge fragments were amplified by PCR, and cloned into pMD-18T vector and the 3'-end non-coding region of psiCheck2 plasmid; hRluc-miRNA-126 sponge was further cloned into Ad-Track plasmid. After linearization by Pme Ⅰ, Ad-Track-hRluc-miRNA-126 sponge plasmid was transformed into competent Escherichia coli strains BJ5183 to homologous recombinate with Ad-Easy plasmid; recombinant Ad-Easy-hRluc-miRNA-126 sponge plasmid was identified by Pac Ⅰ, and then transfected into 293 cells to produce adenovirus. Adenovirus was used to infect human umbilical vein endothelial cells (HUVECs), and the effect of miRNA-126 on cell migration ability was detected by scratch test. Results The miRNA-126 sponge had a good complementary base-pairing relationship with miRNA-126; double luciferase experiment showed that miRNA-126 could directly act on hRluc-miRNA-126 sponge. Cloning hRluc-miRNA-126 sponge into Ad-Track plasmid made it possible to synchronously monitor the efficiency of cellular transfection and miRNA inhibition. Ad-Easy-hRluc-miRNA-126 sponge plasmid was successfully established by homologous recombination of Ad-Track and Ad-Easy, identified by Pac Ⅰ digestion with 4.5 kb fragments. When transfected into 293 cells, luciferase-miRNA sponge adenovirus was obtained 7 days later. Overexpression of miRNA-126 inhibited the expression of hRluc-miRNA-126 sponge (P<0.01). Infection with miRNA-126 sponge adenovirus inhibited the migration of HUVECs (P<0.05). Conclusion Luciferase-miRNA sponge adenovirus has been successfully established. MiRNA-126 has a high binding efficiency with the expressed product of the adenovirus.

Abstract:Objective To clarify the advantages and disadvantages of domestic precision radiotherapy equipment by comparing the clinical efficacies of domestic and imported precision radiotherapy equipments in the treatment of lymphoma. Methods We investigated the treatments of lymphoma in hospitals of different levels using domestic and imported medical linear accelerators and divided them into domestic and imported groups. The data of patients in the two groups were retrospectively analyzed, and the radiotherapy equipment brand, short-term efficacy, acute toxicity, dosimetric parameters of organs at risk and the costs of radiation therapy were compared between the two groups. Results A total of 101 cases receiving radiation therapy for lymphoma in 10 hospitals were collected, including 77 cases in the imported group and 24 cases in the domestic group. The overall response rates were high in both groups, being 88.3% (68/77) in the imported group and 87.5% (21/24) in the domestic group, with no significant difference (P=1.00). The incidences of hematological toxicity (>grade 2) were low in both groups, being 1.3% (1/77) vs 8.3% (2/24), with no significant difference (P=0.14). Subgroup analysis was performed on the dosimetric parameters of organs at risk and acute toxicity of the two groups for extranodal nasal type NK/T cell lymphoma, and there were no significant differences in the maximal dose (D_max) of the lens, D_max of the optic nerve, or mean dose (D_mean) of the parotid gland between the two groups (all P>0.05). However, there was an increasing trend in the domestic group compared with the imported group for D_mean of bilateral parotid gland (left:[2 306.53±1 119.66] cGy vs[1 279.44±1 026.95] cGy, P=0.16; right:[2 328.35±1 009.76] cGy vs[1 303.79±1 116.79]cGy, P=0.17). The incidence of grade 2 xerostomia in the domestic group was higher than that in the imported group, but the difference was not statistically significant (50.0%[2/4] vs 14.3%[1/7], P>0.05). The domestic group had significantly lower radiotherapy cost ([26 743.9±8 061.2] yuan vs[42 428.7±14 744.7] yuan) and total hospitalization cost ([36 702.1±12 225.8] yuan vs[50 192.7±15 494.4] yuan) than the imported group (both P<0.01). Conclusion There is no significant difference in the short-term efficacy of lymphoma treatment between the domestic radiotherapy equipment and the imported radiotherapy equipment, while the treatment cost of the domestic radiotherapy equipment is relatively low. However, the dosimetric parameters of organs at risk of imported radiotherapy equipment is lower and the acute toxicity is less.

Abstract:Objective To investigate the relationship between the clinical invasive interventions during hospitalization and the admission indicators within 24 hours with death within 28-day after admission in sepsis patients. Methods The clinical data of 112 sepsis patients who were admitted to the First Medical Center of Chinese PLA General Hospital from Jan. 2016 to Dec. 2018 were analyzed retrospectively. Four clinical invasive interventions (mechanical ventilation, tracheal intubation, continuous renal replacement therapy[CRRT] and deep vein catheterization) and admission indicators (including demographic characteristics, vital signs and laboratory indexes) that may have an impact on the death of sepsis patients were selected during hospitalization, and the 28-day survival of patients was taken as the outcome variable. The above parameters were compared between the survival group (n=81) and the death group (n=31), and the independent risk factors of sepsis death within 28-day after admission were determined by multivariate logistic regression analysis. Results There were statistically significant differences in age, the sequential organ failure assessment (SOFA) score, acute physiology and chronic health evaluationⅡ (APACHEⅡ) score, IL-6, procalcitonin, aspartate aminotransferase, CRRT and deep vein catheterization between the two groups (all P<0.05). The results of the multivariate logistic regression analysis revealed that high IL-6 level (odds ratio[OR]=1.001, P=0.011), low procalcitonin level (OR=0.966, P=0.005) and CRRT (OR=6.846, P=0.002) were independent risk factors of death within 28-day after admission in sepsis patients. Conclusion Sepsis patients with increased IL-6 level, decreased procalcitonin level and CRRT during hospitalization have a higher risk of death within 28-day after admission. Close observation and dynamic monitoring of changes in IL-6, procalcitonin and renal function will be beneficial for early identification of septic patients who might have adverse clinical outcomes. The proportions of essential invasive interventions such as mechanical ventilation, tracheal intubation, CRRT, and deep venous catheterization are higher in dead patients, so clinicians need to carefully evaluate and accurately grasp the balance point.

Abstract:Objective To evaluate the correlation of long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) with disease risk and severity of bronchial asthma as well as plasma microRNA (miRNA)-125a expression in child patients. Methods Seventy children with asthma exacerbation, 70 children with asthma remission and 70 matched healthy controls were consecutively enrolled in this study. Laboratory parameters and lung function indexes of the participants were recorded. LncRNA ANRIL and miRNA-125a expression levels in plasma were determined using qRT-PCR. Tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and IL-17 levels in plasma were determined using enzyme-linked immunosorbent assay (ELISA). Results LncRNA ANRIL expression was the highest in the asthma exacerbation children, followed by asthma remission children and healthy controls. There were significant differences in the expression of lncRNA ANRIL among the three groups (all P<0.01). Receiver operating characteristic curves revealed that lncRNA ANRIL could well differentiate the participants in the three groups. In the children with asthma exacerbation, lncRNA ANRIL expression was associated with disease severity (P=0.001), positively associated with the levels of TNF-α, IL-1β, IL-6 and IL-17 (all P<0.05), while negatively associated with forced expiratory volume in one second (FEV₁)/forced vital capacity (FVC) and FEV₁ as percentage of predicted (FEV₁%Pred) value (both P<0.05). LncRNA ANRIL expression was also positively associated with the levels of TNF-α, IL-1β and IL-17 in the asthma remission children and IL-6 level in healthy controls (all P<0.05). LncRNA ANRIL expression was negatively associated with miRNA-125a expression in all the participants (all P<0.05). Besides, miRNA-125a expression was positively correlated with FEV₁/FVC and FEV₁%Pred value in the children with asthma exacerbetion (both P<0.001). Conclusion LncRNA ANRIL may serve as a novel biomarker for predicting asthma, asthma acute exacerbation and severity, and inflammation level. It may participate in the development and progression of asthma in children via targeting miRNA-125a.

Abstract:Objective To detect the changes of T lymphocyte subset levels in peripheral blood of acute myeloid leukemia (AML) patients and explore the clinical significance. Methods Total of 68 newly diagnosed AML patients and 76 healthy candidates (healthy controls) were selected for the study. Prognostic risk stratification of AML patients was stratified according to Chinese guidelines for diagnosis and treatment of adult AML (not acute promyelocytic leukemia) (2017). The enrolled patients were divided into high-risk group (20 cases) and non-high-risk group (48 cases). The curative effect of AML patients was evaluated after standard chemotherapy. The flow cytometry technique was used to detect T lymphocyte subset levels in peripheral blood of the AML patients and the healthy controls. Results Proportions of CD3⁺, CD4⁺ T cells as well as CD4⁺/CD8⁺ ratio in AML group were significantly lower than those in healthy controls (all P<0.05), but the proportion of regulatory T (Treg) cells was significantly higher than that in healthy controls (P<0.05). The proportions of CD3⁺, CD4⁺ T cells and CD4⁺/CD8⁺ ratio in high-risk patients were significantly lower than those in non-high-risk patients (all P<0.05). The proportion of Treg cells in high-risk patients was significantly higher than that in non-high-risk patients (P<0.05). The proportions of CD3⁺, CD4⁺ T cells and CD4⁺/CD8⁺ ratio in patients who achieved complete remission (CR) (n=41) were significantly higher than those in patients without CR (n=27) (all P<0.05). However, the proportion of Treg cells in AML-CR patients was significantly lower than that in patients without CR (P<0.05). Conclusion Observation of T lymphocyte subsets and CD4⁺/CD8⁺ ratio can benefit to disease monitoring of patients with AML.

Abstract:Blast wave can stimulate the sympathetic and parasympathetic nervous system, leading to the changes of autonomic nervous function. It can also damage the hypothalamus and pituitary, and activate the hypothalamic-pituitary-target gland axis and the locus ceruleus-sympathetic-adrenal medulla axis, resulting in the changes of endocrine level. In addition, it can activate inflammatory cells, synthesize and release different inflammatory mediators, resulting in the changes of inflammatory response. This paper reviews the changes and interactions of autonomic nervous function, endocrine level and inflammatory response in blast wave injury, and further explains the injury mechanism of blase wave, providing references for the treatment of blast wave injury.

Abstract:Novel oral anticoagulants (NOACs) play an important role in the prevention and treatment of cardiovascular and cerebrovascular thrombosis. Different individuals have different pharmacokinetic parameters in vivo after taking the same dose of NOACs, which may be related to gene polymorphisms of transporters and metabolic enzymes involved in the in vivo process of NOACs. Compared with drug transporters, gene polymorphisms of drug metabolizing enzymes have a greater impact on the pharmacokinetic properties of NOACs, but there have been few related studies up to now. In this paper we summarized the effects of gene polymorphisms on the pharmacokinetic properties of NOACs.

Abstract:Objective To design and establish a new method for simultaneous determination of hepatitis B virus (HBV) pregenomic RNA (pgRNA) and DNA from nucleic acid extracts. Methods We established a duplex fluorescence quantitative PCR system to determine HBV pgRNA and DNA. DNA gel electrophoresis and quantitative PCR were used to test the specificity and sensitivity. We tested the feasibility and accuracy by determining the HBV pgRNA and DNA in HepG2.2.15 cells and the culture supernatants. Results The established duplex fluorescence quantitative PCR system has a good specificity and sensitivity. When it was used to determine cell culture supernatants with different dilution ratios, the dilution ratios and results were well correlated. However, this method was more suitable for the determination of HBV pgRNA and DNA in cell culture supernatants, rather than cell samples. Conclusion Our method can avoid inaccuracy of HBV RNA determination caused by HBV DNA contaminant in nucleic acid extracts, and realize simultaneous detection of HBV pgRNA and DNA in one PCR reaction, which greatly improves the determination efficiency and has potential clinical application value.

Abstract:Objective To investigate the dietary and nutritional status of submarine crew, and to provide guides for reasonable diet and reference for seting new food standards for military personal. Methods During submarine docking, 100 submarine crew were selected as subjects. A total of 100 questionnaires were sent out and 96 valid questionnaires were collected, with an effective rate of 96.0%. The dietary survey was carried out for 4 consecutive days by weighing method. The daily energy consumption of the crew was measured by 24-hour life observation method, meanwhile the body mass index (BMI) and body fat rate were also measured. The common nutritional deficiency symptoms and suggestions on food supply were evaluated by the questionnaire. Results The survey showed that the food intakes, such as cereals, soybean, vegetables, poultry, milk, fish and shrimp, were insufficient, while the intakes of livestock meat and vegetable oil were above the military standard. Their daily energy intakes were higher than the military standard requirements and could meet the energy consumption; the protein and fat energy supply ratios were too high, while that of carbohydrate was too low. The intakes of sodium, iron, phosphorus and vitamin E were all over the standard, while the intakes of iodine, vitamin A and B vitamins were insufficient. Physical investigation showed that overweight and obesity was 46.8% (36/77), and slightly higher and high body fat rate was 48.1% (37/77). In addition, the incidences of nutritional deficiency symptoms, such as dry eyes, bleeding gums, and mouth and tongue ulcers were 26.0% (25/96), 22.9% (22/96) and 19.8% (19/96), respectively. Fruits, dairy products, beef and mutton were the top three recommended food to be increased for the crew's food supply. Conclusion The submarine crew have an unreasonable dietary structure, inappropriate energy supply of the three major nutrients, and unbalanced nutrient intakes. It is necessary to adjust the dietary structure and improve the diet pattern so as to promote the crew's health.

Abstract:Objective To explore the relationship between lipid metabolism and bone mineral density (BMD) in postmenopausal women with type 2 diabetes mellitus (T2DM) in Xinjiang. Methods A total of 136 postmenopausal women in Xinjiang were included and divided into four groups:normal glucose tolerance with normal bone mass group (control group, 26 cases), normal glucose tolerance with abnormal bone mass group (abnormal bone mass group, 28 cases), T2DM with normal bone mass group (T2DM group, 27 cases), and T2DM with abnormal bone mass group (55 cases). The age, height and other baseline data of each group were recorded. Blood calcium, blood phosphorus, alkaline phosphatase (ALP), triacylglycerol, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by Roche automatic biochemical analyzer. The BMD of L₁-L₄, femoral neck and hip joint were measured by dual energy X-ray. Results The age and menopause years of T2DM with abnormal bone mass group were significantly higher than those of the control group (both Z=-2.54, both P<0.05). There were no significant difference in baseline indexes between abnormal bone mass group or T2DM group and control group (all P>0.05). The fasting blood glucose (Z=-4.08, -5.87, both P<0.01) and hemoglobin (Z=-4.59, -6.33, both P<0.01) levels in T2DM group and T2DM with abnormal bone mass group were higher than those in the control group; the lipid metabolism index triacylglycerol in T2DM with abnormal bone mass group was lower than that in the control group (Z=-2.01, P<0.05). The BMD of L₁-L₄ (Z=-6.23, -6.84, both P<0.01), femoral neck (Z=-5.32, -4.30, both P<0.01), and hip joint (Z=-5.44, -5.00, both P<0.01) in abnormal bone mass group and T2DM with abnormal bone mass group were lower than those in the control group. After adjusting the influence of age and menopause years, the results of partial correlation analysis showed that L₁-L₄, femoral neck and hip joint BMD were positively correlated with body mass index (r=0.209, 0.623, 0.265, P=0.015, 0.002, 0.002) and triacylglycerol (r=0.298, 0.233, 0.273, P<0.001, P=0.007, P=0.001), femoral neck and hip joint BMD were negatively correlated with HDL-C (r=-0.191, -0.200, P=0.027, 0.021) in 82 postmenopausal women with T2DM. Conclusion The elevated HDL-C and decreased triacylglycerol are associated with decreased BMD, which may promote the occurrence of osteoporosis in postmenopausal women with T2DM in Xinjiang.

Abstract:患者，男，53岁。因“胸闷不适1月余”入院。患者1月前无明显诱因开始出现活动后胸闷，伴咳嗽，无咳痰、胸痛及咯血，无低热、盗汗等结核中毒症状。既往体健，否认结核、心血管病史，曾于钢厂工作数十年。于2019年06月13日至我院查胸部增强CT示：右肺上叶团片病灶，增强部分强化，双肺多发结节；双侧肺门、纵隔淋巴结增多、增大，部分钙化（见图1）。各项常规检查（血常规、凝血、肝肾功能、电解质）、心电图及心脏彩超均未见明显异常；结核感染T细胞检测试验（T-SPOT）阴性。 为明确病变性质，于2019年07月12日行CT引导下经皮肺穿刺活检术。嘱患者仰卧位于CT检查床上，常规平扫，选定在平静呼吸过程中病灶的重叠层面为穿刺层面，穿刺层面与定位栅的交点为穿刺点（右侧锁骨旁线线第3肋间隙），常规消毒铺巾局麻后，垂直穿刺平面进针约10cm，CT证实针尖在病灶内（见图2），用BARD活检枪取1.5cm长组织送病检。术中患者未诉特殊不适。操作结束后，患者有轻微咳嗽，未见咳痰及咳血，随即坐起过程中患者突发意识丧失、呼吸急促、瞳孔扩大、牙关紧闭，心电监护提示室速，立即给予肾上腺素静推、心肺复苏、电除颤、气管插管等处理后，其生命体征未见明显好转，血氧饱和度进行性下降，最终患者颈动脉搏动、瞳孔对光反射消失，心电图呈直线，宣布临床死亡。