Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Animals ; Antibodies, Viral ; biosynthesis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Genetic Vectors ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H9N2 Subtype ; Plasmids ; Rabbits ; Viral Proteins ; immunology

BACKGROUND: The effective therapy of artificial liver for severe hepatitis needs an absorbing material which possesses strong adsorptive property, high adsorption rate and good blood compatibility.OBJECTIVE: To study the adsorptive property of eight novel adsorbents for plasma bilirubin and cytokines in severe hepatitis patients. DESIGN: A controlled observation.SETTINGS: Graduate School of Tianjin Medical University, Tianjin Haihe Hospital, Tianjin Third Central Hospital and the Institute of Polymer Chemistry in Nankai University.PARTICIPANTS: All plasma was collected from 30 severe hepatitis patients hospitalized in Tianjin Third Central Hospital from November 2004 to November 2005. Informed consent was obtained from each patients. This experiment was approved by the hospital ethical committee. All the patients were divided into two groups at random: group 1 (n=10) and group 2 (n=20). The level of total bilirubin (TBiL) before therapy in two groups was (377.3±147.5) μmol/L and (327.6±140.1) μmol/L, respectively.METHODS: ①Adsorbents: Chitosan (Qingdao Lizhong Chitosan Factory, Shandong) with relative molecular weight 97 000 and de-acetyl grade 85%; Adsorbents No.1-3 were prepared by using 1%, 3%, 5% polyethyleneglycol (relative molecular weight 600) as porogenic agent. Adsorbent No.4 was aminated crosslinked chitosan microspheres; Adsorbent No.5 was divinyl-benzene crosslinked macroporous polystyrene microspheres; Adsorbent No.6 was post-crosslinked macroporous divinyl-benzene styrene copolymer microspheres; Adsorbents No.7 and 8 were chitosan wrapped adsorbent No.5 and 6.②Detection: Step 1: 3 mL plasma collected from each severe hepatitis patient in group 1 was absorbed with 1 mL of 8 kinds of adsorbents. Levels of plasma TBiL, direct bilirubin (DBiL) and indirect bilirubin (IBiL) before and after adsorption were determined by using the vanadate oxidation method to analyze the average adsorption capacity so as to screen the adsorbents with the better adsorptive properties. Step 2: 3 mL plasma collected from each severe hepatitis patient in group 2 was absorbed with 1 mL of two adsorbents selected from the step 1. The levels of bilirubin, interleukin-6 and tumor necrosis factor-α before and after the adsorption were analyzed by ELISA method.MAIN OUTCOME MEASURES: The levels of bilirubin and cytokines before and after the adsorption were determined.RESULTS: ①The data in the first step experiment showed that after No.4 and No.5 adsorbents were used, the level of plasma TBiL, DBiL and IBiL were significantly decreased (P < 0.01); no differences were found for other six kinds of adsorbents (P > 0.05).②The data in the second step experiment showed that the average levels of plasma TBiL, DBiL, IBiL, interleukin-6 and tumor necrosis factor-α were remarkably reduced after using adsorbents No.4 and No.5 (P < 0.01). Compared with adsorbent No.5, there were significant decrements for adsorbents No.4 (P < 0.01).CONCLUSION: Aminated chitosan microsphere has significant effects on adsorbing bilirubin and cytokines in plasma of severe hepatitis patients in vitro.

Objective To investigate the value of spleen stiffness measured by transient elastography (FibroScan,FS) for diagnosing esophageal-gastric varices in liver cirrhosis patients.Methods A total of 259 cirrhotic patients in Tianjin Third Central Hospital from Apr 2011 to Apr 2012,and 30 healthy controls were enrolled.All the patients and controls were evaluated for spleen and liver stiffness by FS and 201 cirrhotic patients also underwent gastroscopy for the diagnosis of esophageal-gastric varices.By using gastroscopy as the gold standard,the receiver operating characteristic (ROC) curves of three parameters including spleen stiffness,liver stiffness and platelet/thickness of spleen were delineated for different disease stage.The areas under curves (AUC) were used to evaluate the value of these parameters in the diagnosis of esophageal-gastric varices.Results The spleen and liver stiffness values in cirrhotic patients were (44.64 ± 22.27) kPa and (24.27 ±18.89) kPa,respectively,while those in healthy controls were (20.94± 14.78) kPa and (6.12±5.77) kPa,respectively,which were both lower than cirrhotic patients (P＜0.05).The stiffness values of liver and spleen both increased with higher Child-Pugh scores.And the liver stiffness values were different among groups (F=0.068,P =0.000),while the spleen stiffness values in patients with Child-Pugh A and B were different from that in patients with Child-Pugh C (P＜0.05).In patients with moderate or serious esophageal-gastric varices,the spleen and liver stiffness values were significantly higher.The ROC curve analysis showed that the AUC of spleen stiffness,liver stiffness and platelet/thickness of spleen in the patients with moderate to serious esophageal-gastric varices were 0.918,0.749 and 0.743,respectively.The corresponding optimal cut-off values were 50.7 kPa,20.1 kPa and 1.65.The AUC,sensitivity and specificity of spleen stiffness were all higher than liver stiffness and platelet/thickness of spleen.Conclusion Spleen stiffness measured by transient elastography is a valuable parameter for non-invasive diagnosis of esophageal-gastric varices in cirrhotic patients.

OBJECTTo study the significance of the ultra-high power microscope in the examination of vaginal discharge.

METHODSBy the ACT-2000 ultra-high power microscope system and Olympus CX21 microscope, the vaginal discharge of 1,100 gynaecology out-patients was examined respectively.

RESULTSThe positive rate of mould in the patients was 11.55% by CX21 and was 20.27% by ACT-2000, respectively. The positive rate of trichomonas vaginalis was 2.55% by CX21 and 3.0% by ACT-2000, respectively. The clue cell was detected in 11.27% of the patients by ACT-2000, but no such cell reported by CX21. Totally, positive results were obtained in 14.09% of the patients by CX21 and 32.55% by ACT-2000.

CONCLUSIONBy using the ultra-high power microscope, the positive result can be increased obviously in the examination of vaginal discharge. It is very important in clinical practices.

Adult ; Female ; Humans ; Microscopy ; methods ; Microscopy, Electron ; methods ; Middle Aged ; Vaginal Discharge ; pathology ; Young Adult

OBJECTIVETo evaluate the repairing methods of bulky tissue defect of forearm with flaps.

METHODSTwenty-one burned patients with wounds in the forearm were enrolled in this study. The injury causes were high-voltage electricity, hot press or crush injury. After local debridement, the forearm defects were repaired with pedicled complex flaps, latissimus dorsi musculocutaneous island flaps or large thoraco-abdominal flaps immediately.

RESULTSAll the flaps survived very well with satisfactory results except for 1 patient in whom local ischemic necrosis and sub-flap infection at the distal end of the flap occurred.

CONCLUSIONEarly debridement followed by skin flaps with pedicles or musculocutaneous flaps transfer could be simple, safe and reliable treatment strategies in the management of bulky tissue defects of the forearm due to burn, electric injury, or other devastating injuries.

Adolescent ; Adult ; Child ; Female ; Forearm Injuries ; surgery ; Humans ; Male ; Middle Aged ; Pectoralis Muscles ; transplantation ; Skin Transplantation ; methods ; Surgical Flaps ; Time Factors ; Wound Healing

Objective:To summarize the clinical characteristics of children with dystonia 28 (DYT28) caused by KMT2B gene variations so as to improve clinicians′ understanding of the disease. Methods:The clinical manifestations, treatment and gene variation data of 11 children with DYT28 caused by KMT2B gene variations were retrospectively collected and analyzed.The subjects were recruited from the Department of Neurology, Beijing Children′s Hospital, Capital Medical University from March 2018 to January 2021.The patients were followed up. Results:There were 8 males and 3 females.The age at onset was ranging from 1 month to 6 years without inducement.Eight cases were gene-ralized dystonia and 3 cases were multifocal dystonia.The initial symptoms of 7 cases were unilateral or bilateral lower limbs tiptoeing.Four cases presented dysarthria, retching or swallowing difficulties at onset.As the disease progressed, all the cases had laryngeal dystonia, 10 cases had lower limbs dystonia, and 8 cases had upper limbs dystonia.Six cases were complicated with other dyskinesia symptoms.Ten cases had varying degrees of short stature, microcephalus, micrognathia, musculoskeletal abnormalities, intellectual disability, endocrinopathies and sleep difficulties.The brain magnetic resonance imaging showed abnormal in only 1 case.Eleven KMT2B gene pathogenic variants were found, including 8 frameshift variants, 1 in-frame variant and 2 missense variants.Four variants were novel.Eleven cases were followed up at the age of 1 year and 7 months to 17 years and 9 months.One case wasn′t given therapy.The dystonia in 3 cases was mildly improved after medication.Dysfunction of urination and defecation was disappeared in 1 case after medication.The symptom of 6 cases had no improvement after drug therapy.Among the above 6 cases, 5 drug refractory cases had deep brain stimulation, and their dystonia symptoms are all obviously improved; 2 cases had normal control of urination and defecation after deep brain stimulation.The motor scores in the Burke-Fahn-Marsden dystonia rating scale were improved by 55.8%-90.7%, and the disability scores were improved by 14.8%-69.6%. Conclusions:DYT28 caused by KMT2B gene variations is one of the most common and early-onset genetic dystonia in children.The dystonia symptom progresses from local parts to the whole body, prominently involving laryngeal muscles and lower limbs.Control of urination and defecation requires attention.Patients with mild dystonia symptoms can be effectively treated by drugs.However, patients with severe dystonia symptoms were drug refractory, and their dystonia symptoms can be effectively improved by deep brain stimulation.