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

Objective To explore the influence of type 2 diabetes mellitus combined with subclinical hypothyroidism on diabetic vascular complications.Methods One hundred and two patients with type 2 diabetes mellitus were selected.The serum free triiodothyronine (FT3),free thyroxine (FT4),thyroid stimulating hormone (TSH),anti-thyroid peroxidase antibody (TPO-Ab),thyroglobulin antibody (TG-Ab) levels were measured by chemiluminescence method.The patients were divided into type 2 diabctes mellitus combined with subclinical hypothyroidism group (47 cases) and type 2 diabetes mellitus with normal thyroid function group (55 cases) according to the thyroid function.The glycated hemoglobin (HbA1c),total cholesterol (TC),low-density lipoprotein cholesterol (LDL-C),triacylglycerol (TG),high-density lipoprotein cholesterol (HDL-C),urea nitrogen,creatinine and albumin levels were measured.The estimated glomerular filtration rate (eGFR) was calculated according to the formula of modification of diet in renal disease (MDRD).The presence of diabetic retinopathy was examined by fundus examination,and the presence of lower limb artery lesions was measured by vascular ultrasound.All indicators were compared between 2 groups.Results There were no statistical differences in age,disease course,HbA1c,body mass index (BMI),TC,TG,HDL-C,LDL-C,incidence of lower limb artery lesions and incidence of diabetic retinopathy between 2 groups (P＞ 0.05).TheeGRF in type 2 diabetes mellitus combined with subclinical hypothyroidism group was significantly lower than that in type 2 diabetes mellitus with normal thyroid function group:(83.74 ± 21.55) ml/(min· 1.73 m2) vs.(115.02 ± 12.29) ml/(min· 1.73 m2),and there was statistical difference (t =4.274,P ＜ 0.01).The incidence of diabetic nephropathy in type 2 diabetes mellitus combined with subclinical hypothyroidism group was significanlty higher than that in type 2 diabetes mellitus with normal thyroid function group:48.9％ (23/47) vs.23.6％(13/55),and there was statistical difference (x2 =7.103,P＜ 0.01).Logistic regression analysis showed that subclinical hypothyroidism was a risk factor for diabetic nephropathy (OR =0.524,95％ CI 0.12-0.93,P ＜ 0.05),but it was not the risk factor for diabetic retinopathy (OR =0.618,95％ CI0.19-2.16,P =0.475) and lower limb artery lesions (OR =0.485,95％ CI 0.32-2.13,P =0.689).Conclusion Subclinical hypothyroidism in patients with type 2 diabetes mellitus has no obvious effect on lower limb arterial complications and diabetic retinopathy,but may increase the risk of diabetic nephropathy.

Background To establish the ideal animal model of retinitis pigmentosa (RP) is very important for onward relevant study.Previous research determined that N-methyl-N-nitrosourea (MNU) can selectively damage photoreceptors via intravenous injection in mammal.However,whether MNU can be used to create an RP model needs to be investigated.Objective This experiment was designed to evaluate the toxic effect of MNU on photoreceptor cells of cats.Methods MNU was injected into 20 2-year-old cats via femoral vein and randomized into 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg and 40mg/kg MNU groups,and equal amount of normal saline solution was used in the same way in 4 normal cats as the control group.The activity,pupil size and light reflex were observed after injection of MNU.The cats were sacrificed and eyeballs were enucleated for histological examination to evaluate the structural and morphological changes of photoreceptors at 24 hours,72 hours,7 days and 14 days after the administration of MNU.This experimental study complied with the Statement for the Use of Animals in Ophthalmic and Vision Research.Results Dilated pupil and inertia of light reaction were found in experimental cats on the 7th days in the various groups.In 24 hours after MNU injection,the damage of photoreceptors was primarily characterized by pyknosis and disorder.In 72 hours after MNU injection,attenuation of the outer nuclear layer and disruption of cells were seen.Loss of photoreceptors and disappearance of the outer nuclear layer were observed on the 7th and 14th day.The extent of retinal photoreceptor cell damage was dependent on the dose of MNU.Conclusion MNU can selectively induce serious damage of the photoreceptor cells in cats retina in a time- and dose-dependent manner.

Objective To quantify the expression levels of calpain 1 and caspase-3 in lichen planus (LP) lesions and their relationship with the apoptosis in keratinocytes.Methods Biopsy samples were obtained from the lesions of 20 patients with LP and normal skin of 10 healthy controls,and embedded in paraffin.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was used to evaluate the apoptosis in keratinocytes,and immunohistochemical staining to detect the expressions of calpain 1 and caspase-3,in these tissue specimens.Data were processed by SPSS 13.0 software.Comparison analysis was carried out by t test for apoptosis index,and by rank sum test for the expressions of calpain 1 and caspase-3.Spearman correlation analysis was conducted to evaluate the relationship between these parameters.Results The apoptosis index of keratinocytes was higher in LP lesions than in the normal skin (67.59 ± 13.50 vs.28.26 ± 7.56,t =8.52,P ＜ 0.01).Significantly increased expressions of calpain 1 and caspase-3 were observed in the epidermis of LP lesions compared with the normal skin (T =78.00 and 77.00,respectively,both P ＜ 0.01).The expressions of both calpain 1 and caspase-3 were positively correlated with apoptosis index of keratinocytes (r =0.71 and 0.74,respectively,both P ＜ 0.01).Conclusions The expressions of calpain 1 and caspase-3 are upregulated in LP lesions,which may be closely associated with the accelerated apoptosis in keratinocytes.

Animals ; Epilepsy ; immunology ; metabolism ; Hippocampus ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; immunology ; metabolism

Objective To study the role of cell density in the tyrosol production and morphology for Candida albicans biofilms. Methods C. albicans SC5314 and clinical isolates were propagated in yeast peptone dextrose (YPD) medium. Cells were collected by centrifugation and washed twice in sterile phosphate-buffered saline (PBS) before this study, then resuspended in RPMI 1640 supplemented with L-glutamine and adjusted to a desired concentration of 5 × 10~6 cells/ml, 5×10~5 cells/ml, 5 × 10~4 cells/ml, 5 × 10~3 cells/ml after counting with a hematocytometer. Standardized C. albicans cells were prepared as above description and 2000 μl of this standardized cell suspension was dispensed into the wells, then C. albicans biofilms were formed on the bottom of the polystyrene wells. In this study, tyrosol synthesized by SC5314 and clinical isolates of C. albicans biofilm was quantified by high performance liquid chromatography (HPLC). The effects of tyrosol on morphology of C. albicans biofilms were investigated by scanning electron microscopy (SEM). Results Tyrosol production of C. albicans biofilms was affected by cell densities. At lower inoculation size(5 μ 10~3 cells/ml), there was too less tyrosol production to be detected at the early stage of the biofilms formation. At higher inoculation size (5 μ10~6 cells/ml), tyrosol can be detectable at the early stage or at the mature stage of biofilms formation. There was a sharp increase in tyrosol concentration at 24 h, while there was a decrease in tyrosol concentration after that time from the strains when the strains were at an inoculation size of 5 × 10~6 cells/ml and 5 × 10~5 cells/ml. Cell densities affected the morphology formation of the C. albicans biofilms. At the early stage of the biofilms formation, C. albicans grew less germ tube at lower cell densities than that at the higher cell densities. With the mature of the biofilms, C. albicarts grew more hyphae at higher cell densities than that at the lower cell densities. All these above showed that cell densities played an important role in the propagation for the C. albi-cans in the biofilm formation. Conclusion Cell density play an important role in the formation of the C. albi-cans biofilms and the production of the tyrosol.

Objective:To investigate the role of platelet-derived growth factor(PDGF)-AB on angiogenensis in aged mice.Methods:Cardiac microvascular endothelial cells(CMEC)from the hearts of young adult and old mice were cultured.Expression of PDGF-BB and PDGF-AA was detected by RT-PCR.The migration of CMEC was determined with ChemoTX Chamber.Ear angiogenesis model was made in mice.Blood flow in neo-microvessels and collateral vessels was measured with Laser Doppler.Then biotin-labeled Dextran-lysine was injected into the mice through cardiac puncture to label vessels.Ears were cut and immunohistochemistry was carried out by ABC method.Results:Both PDGF-BB and PDGF-AA were highly ex-pressed in CMEC of young adult mice;expression of PDGF-BB was not detected in aged mice.PDGF-AB reverted the levels of PDGF-BB in aged CMEC to the levels of young adult mice.Migration rate of CMEC in aged mice was significantly increased after stimulation by PDGF-AB(P

Objective To study the method and therapeutic results of intra-arterial interventional treatment (selective intra-arterial fibrinolysis) in acute cerebral infarction(ACl).Methods A total of 106 patients with ACI are divided into two groups:selective intra-arterial fibrinolytic therapy group(SIAF),intra-venously thrombolysis group(IVF);SIAF is selective entered into the obliterative artery with improved Seldinger's technich,pumped urokinase(UK) in a dose of 500 000 i.u.in NS.60ml within 40 minutes;arteriography is done all the time to know if the obliterative artery is unobstruct- ed,if it remains obstructe,then pumps 300 000 i.u.of UK,as long as it unobstructed,the treatment is finished at once; IVF is injected 1000 000 i.u.of UK in NS.150 mL by intravenous drip within 40 minutes,others like SIAF.Results The artery is disobliteration after thrombolysis,48 patients in SIAF,30 patients in IVF,P

Objective To explore the clinical significance of neutrophils/lymphocyte(NLR)and platelet/lymphocyte ratioof (PLR)peripheral blood in patients with primary biliary cirrhosis.Methods Retrospectively analyzed 62 patients with PBC and three subgroup patients according to Child-pugh,who were hospitalized in Shanghai Changhai Hospital and Shanghai Changzheng Hospital from January 2010 to July 2015.Enrolled 59 healthy controls.Baseline data and laboratory indicators were extracted,and the correlation between PLR and NLR and disease activity were analyzed.The changes of NLR in 3 pa-tients with PBC were analyzed.3 patients were followed up and their NLRs were recorded before and after therapy.Results Compared with healthy controls,NLR was significantly increased in PBC patients (P<0.05),and not PLR (P>0.05). NLR was positively correlated with TBIL and Mayo risk (R=0.35,0.30,P<0.05),and not ALT,AST or ALP (P>0.05).After therapy,NLRs were significantly reduced in three PBC patients (P<0.05).Conclusion NLR may be a useful biomarker for assessing clinical therapy in PBC patients.

Objective To assess the efficacy and safety of ketogenic diet(KD) on refractory epilepsy in children.Methods KD treatment was designed to observe the effects for 12 weeks.Totally 22 children with 16 boys and 6 girls were enrolled in the study.The epileptic syndromes included infantile spasms(13 cases),Lennox-Gastaut syndrome(4 cases),Dravet syndrome(2 cases),and the unclassified(3 cases).The KD was prepared according to the modified Johns Hopkins regimen.Urinary ketones were measured every day to ensure that ketosis state and parents′ diaries were kept to find out when it started to work and the change of seizure frequency.Effects of KD was evaluated by Engel standard.Blood chemistry was done at baseline,4 weeks and 12 weeks to analyze the effects of KD on metabolism.Side effects were monitored and treated.Results All cases completed the KD regimen for at least 2 weeks,19 cases for at least 4 weeks,and 10 cases for at least 12 weeks.Sixteen out of 22 children experienced the seizure reduction within 3 weeks(1-15 d),especially in the first week,and seizure free appeared within 5 weeks(1-32 d) in 8 cases.Overall,the diet achieved the seizure-free in 36.4%(8/22 cases) and an over 90% of seizure frequency reduction in 22.7%(5/22 cases).The efficacy of KD seemed not correlated with the sex,age,etiopathogenisis,and syndromes and so on.Blood chemistry suggested a normal range of glucose level at 4 weeks,though higher than that at the baseline.The blood triglyceride and total cholesterol level at 12 weeks increased strikingly,even beyond the normal range compared with the baseline.The side effects mainly including transient gastrointestinal symptoms and metabolic disturbances were mostly tolerable.Conclusions KD is probably a feasible therapy on refractory epilepsy in children,with quick and high efficacy and few side effects.