[Etiology and clinical analysis of central nervous system infection caused by Coxsackievirus B5 in severe hand, foot and mouth disease in Qingdao City, 2013-2014].

Objective: To illuminate the gene characteristics and clinical characterization of Coxsackievirus B5 (CV-B5) strains isolated from patients with sevre hand, foot and mouth disease (HFMD) in Qingdao city. Methods: A total of 1 844 patients of HFMD were consecutively admitted to Qingdao Women and Children's Hospital from 2013 to 2014. Information of the study population described above was collected retrospectively. The samples were collected from at least 1 site (throat swab, cerebrospinal fluid), which viral nucleic acid extracted and the entire VP1 gene sequences of CV-B5 isolates were amplified and sequenced, then the homology and phylogeny analysis were conducted by MEGA7.0. The prototype Faulkner strain and other VP1 amino acid sequences were derived from the GenBank database. Results: A total of 8 CV-B5 positive cases were obtained, including 4 males and 4 females; 6 severe hospitalized cases and 2 outpatients. The age of 6 hospitalized patients ranged from 3 to 48 months, with a median of 26 months. For the six inpatients, fever, convulsions vomiting, diarrhea and rash were the main clinical manifestation, and all combined with viral encephalitis. Compared with the prototype strain Faulkner, in the VP1 region,the nucleotide and the amino acid homologies was 77.3%-78.8% and 95.5%-97.0% respectively. Five out of the six severe cases with substitution of serine (S) to asparagine (N) at amino acid site 95 in the VP1 region. The sequences of 8 CV-B5 strains were classified into genogroup D. Conclusion: Hand, foot and mouth disease associated with CV-B5 virus infection can result in nervous system involvement and the main complication was viral encephalitis. The CV-B5 strains associated with severe hand, foot and mouth disease had high nucleotide homology and present a certain regional aggregation.

[Analysis of contrast echocardiography for detecting right to left shunt in adults with patent foramen ovale].

Objective: To analyze the contrast echocardiography for detecting right to left shunt in adults with patent foramen ovale(PFO), and study the relationship between PFO and cryptogenic stroke. Methods: Clinical data of forty-six adults patients with PFO diagnosed by transesophageal echocardiography(TEE)from March, 2012 to March, 2017 were retrospectively collected, and the patients were divided to 3 groups according to the direction and brightness of the color Doppler shunts: obvious left-to-right shunt (OLRS, group A), weak left-to-right shunt(WLRS, group B), bi-directional shunt(BDS, group C). A right-to-left shunt (RLS) scale was calculated using the method of 10 ml hand-operated saline for contrast echocardiography. Results: There were seventeen cases in group A, four cases (23.5%) showed RLS at level 1, and thirteen cases (76.5%) showed no RLS; there were twenty cases in group B, and all cases (100%) showed RLS, with five cases (25%) at level 1 and fifteen cases (75%) at level 2-3; there were nine cases in group C, and all cases (100%) showed RLS, with two cases (22.2%) at level 1 and seven cases (77.8%) at level 2-3. Anteroposterior diameter of left atrium of patients with no RLS (4.8 cm±0.6 cm) was significantly larger than that of patients with RLS in contrast echocardiography (3.6 cm±0.5 cm)(P=0.000). Conclusions: OLRS of adults with patent foramen ovale and with larger left atrium have less RLS than WLRS and DLRS with normal left atrium in contrast echocardiography.The possibility of paradoxical embolism in WLRS and DLRS is higher than that in OLRS, which should be taken seriously in clinical practice.

Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use.

[Composition of potassium channels in normal rat coronary smooth muscle cells and activation effects of docosahexaenoic acid].

OBJECTIVE: To investigate the composition of potassium channels in normal rat coronary smooth muscle cells (CASMCs) and the activation effects of docosahexaenoic acid (DHA). METHODS: CASMCs were isolated by enzyme digestion.Effects of different types of potassium channel blockers and/or DHA on potassium channels currents were studied by whole-cell patch clamp technique. RESULTS: Potassium currents were significantly increased with 5 µmol/L DHA perfusion (P<0.05). The current density was increased from (52.80±6.68) pA/pF to (110.09±13.39) pA/pF (P<0.05) after DHA perfusion when the stimulation voltage was 100 mV.Compared with baseline, potassium currents were significantly decreased by various inhibitor perfusion (tetraethylammonium: (49.63±5.75) pA/pF vs. (13.96±2.18) pA/pF; ibritoxin: (50.67±7.89) pA/pF vs. (26.53±4.68) pA/pF; TRAM-34: (52.60±7.02) pA/pF vs. (46.05±7.60) pA/pF; apamin: (51.97±3.83) pA/pF vs. (44.89±5.04) pA/pF; 4-aminopyridine: (51.19±3.44) pA/pF vs. (29.92±2.81) pA/pF; glyburide: (49.67±1.77) pA/pF vs. (49.61±1.87) pA/pF, all P<0.05). In presence of different inhibitors, potassium channel current densities were increased after DHA perfusion except tetraethylammonium (tetraethylammonium: ( 12.79±1.89) pA/pF; ibritoxin: (67.08±5.54) pA/pF; TRAM-34: (117.91±21.79) pA/pF; apamin: (108.33±7.06) pA/pF; 4-aminopyridine: (127.73±20.56) pA/pF; glyburide: (121.53±13.83) pA/pF, all P<0.05 compared with baseline). CONCLUSIONS: Large-conductance calcium-activated potassium channel and voltage-gated potassium channel are the major constituents of potassium channels in CASMCs.DHA can activate potassium channels in CASMCs, mainly the large conductance calcium-activated potassium channel, thus dilate coronary arteries.

[Docosahexaenoic acids activate large conductance calcium-activated potassium channels via phospholipase C- inositol triphosphate- calcium pathway in normal rat coronary smooth muscle cells].

OBJECTIVE: To investigate the mechanisms of docosahexaenoic acids (DHA) on activating large conductance calcium-activated potassium channels (BK channels) in normal rat coronary smooth muscle cells. METHODS: Normal coronary smooth muscle cells were isolated by enzyme digestion from Sprague-Dawley rats. BK currents were recorded by patch clamp in whole cell and single channel configurations, respectively. The effects of DHA on cytosolic calcium concentrations were examined by recording the changes of fluorescence intensity ratios. RESULTS: DHA (1 µmol/L) could activate BK channels. Open probabilities (NP0) of BK channels at test potential 60 mV, and calcium concentrations in external solution at 0, 0.01, 0.1, 1, 3, 10, 50 and 100 µmol/L were 0.002 7±0.000 4, 0.006 0±0.001 4, 0.097 2±0.010 6, 0.137 9±0.032 9, 0.468 7±0.163 7, 2.097 1±0.310 4 and 3.120 4±0.242 7, respectively (P<0.05, n=4). Before DHA perfusion, the fluorescence intensity ratio was 0.51±0.01, and the ratios were 0.53±0.02 and 0.55±0.01 after 0.001 and 0.01 µmol/L DHA perfusion, respectively (P>0.05, n≥5). The ratios were 0.64±0.01, 0.65±0.01, 0.70±0.01, 0.69±0.01, 0.68±0.01 and 0.67±0.02 after 0.1, 0.3, 1, 3, 5 and 10 µmol/L DHA perfusion, respectively, and EC50 was (0.04±0.02) µmol/L(P<0.05, n≥4). They were all higher than that before DHA perfusion. After incubating with phospholipase C (PLC) blocker U73122 and inositol triphosphate (IP3) blocker 2-APB, the ratios were 0.52±0.01 and 0.49±0.02 on the setting of 0.1 µmol/L DHA, respectively. Compared with control group(0.64±0.01), the ratios decreased after incubating with blockers (P<0.05, n≥4). CONCLUSIONS: Docosahexaenoic acids can activate large conductance calcium-activated potassium channels by the pathway of PLC-IP3-Ca(2+) to increase cytosolic calcium concentration in normal coronary smooth muscle cells, dilate the coronary vessels and bestow protective effects on cardiovascular system.

Proapoptotic effects of heme oxygenase-1 inhibitor on Kasumi-1 cells via the ATF4/CHOP/Ire-1α pathway.

We evaluated the effects of down-regulated heme oxygenase (HO)-1 expression on the proliferation of the acute myelocytic leukemia Kasumi-1 cell line by using the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) in combination with daunorubicin (DNR), and evaluated the mechanism. The proliferation rates of cells treated with 10 mg/mL DNR and 10 mM ZnPPIX individually or in combination for different time periods were detected using the MTT assay. The apoptotic outcomes of the blank control, ZnPPIX, DNR, and ZnPPIX groups in combination with the DNR group were detected by flow cytometry. The expression of HO-1, activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α mRNA and proteins were detected by fluorescent quantitative real-time polymerase chain reaction and western blotting, respectively. Combined administration inhibited the cells most potently and time-dependently, decreased the expression of HO-1, and significantly increased the expression of activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α expression levels. The cell apoptotic rates in the blank control, DNR, ZnPPIX, and combined administration groups were 8.32 ± 0.53, 39.16 ± 1.46, 10.46 ± 0.88, and 56.26 ± 2.24%, respectively. Inhibiting HO-1 expression can enhance the damaging effects of DNR on Kasumi-1 cells, providing experimental evidence for the improvement of therapeutic effects on acute myelocytic leukemia in clinical practice.

Percutaneous sclerotherapy of massive macrocystic lymphatic malformations of the face and neck using fibrin glue with OK-432 and bleomycin.

Picibanil (OK-432) and bleomycin have been used as alternative sclerosing agents for lymphatic malformations. This study evaluated the clinical curative effect of sclerotherapy using fibrin glue combined with OK-432 and bleomycin for the treatment of macrocystic lymphatic malformations of the face and neck. Fifteen paediatric patients (6 males; 9 females, aged 13 months to 14 years) who had received percutaneous sclerotherapy for massive macrocystic lymphatic malformations of the face and neck were retrospectively reviewed. Affected regions included the neck, parotid region and parapharynx, mouth floor, face and cheek, and orbital regions. All patients showed preoperative symptoms of space-occupying lesions between 4 cm × 5 cm and 12 cm × 16 cm in size. Fibrin glue with OK-432 and bleomycin was injected under general anaesthesia. All patients received preoperative and follow-up CT scans. Outcomes were assessed by three surgeons. All patients exhibited mid-facial swelling for 3-4 weeks after surgery, but no major complications. Follow-up periods ranged from 8 to 16 months. Eight lesions were completely involuted, five were mostly involuted, and two were partially involuted. Percutaneous sclerotherapy using fibrin glue with OK-432 and bleomycin provided a simple, safe, and reliable alternative treatment for massive macrocystic lymphatic malformations of the face and neck.

Effect of total flavonoid fraction of Astragalus complanatus R. Brown on angiotensin II-induced portal-vein contraction in hypertensive rats.

The objective of the present study was to examine further the underlying mechanism of the antihypertensive effect of the total flavonoid (TF), extracted from the seed of Astragalus complanatus R.Brown. Renovascular hypertension rats (RHR) were established by the two-kidney one clip (2K1C) method. The effect of TF on the contraction of portal vein was studied in an isolated preparation. The response of portal vein to angiotensin II (Ang II) was expressed as a percentage of the 100 mmol/l KCl induced maximum contraction. We took the dose-response curve of portal vein to Ang II (from 10(-9) to 10(-6) mmol/l) as the control and then observed the change of curve after TF and Valsartan (Ang II receptor blocker) administration. Ang II induced a concentration-dependent increase of the contraction amplitude (maximal increase, 46.53+/-5.15% of 100 mmol/l KCl induced contraction at Ang II 10(-6) mmol/l in RHR). The Ang II-induced portal vein contraction was prevented by TF with a concentration related manner (maximal inhibition amplitude from 46.53+/-5.15% to 22.525+/-4.67% of 100 mmol/l KCl contraction at 10(-6)mmol/l Ang II and 3.12 x 10(-1) mg/l TF in RHR). The effect of TF on Ang II-induced portal vein contraction was similar to Valsartan. These results showed that the antihypertensive action of TF was attributed to the dilation of vessels and is related to the blockade of the Ang II receptor.

The use of transgenic animals in the study of diabetic kidney disease.

Diabetic nephropathy is one of the most common diseases leading to fibrosis and end-stage renal disease (ESRD) world wide. Under normal conditions, a delicate equilibrium exists between synthesis, composition, and removal of extracellular matrix (ECM). If this is disturbed, ECM accumulation and fibrosis may result. The fragile balance between synthesis and removal of ECM is crucial for the prognosis of glomerular as well as interstitial pathological processes. Some features may favor ECM accumulation and progression to ESRD (dialysis and transplantation), whereas other elements may favor ECM removal and resolution (recovery). Pathogenetic mechanisms and the cellular sources of ECM in the glomerular basement membrane as well as in the tubulointerstitial space are still under investigation. Among several growth factors, transforming growth factor beta1 (TGF-beta1) plays a major role. We consider the use of living animals necessary for our understanding of the complex biological processes that occur during the development of ESRD. The present review will discuss the glomerular as well as interstitial accumulation of ECM and the use of transgenic animals in studying the pathogenetic mechanisms with special emphasis on diabetic kidney disease and TGF-beta1.

Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors.

RNA primer removal during DNA replication is dependent on ribonucleotide- and structure-specific RNase H and FEN-1 nuclease activities. A specific RNase H involved in this reaction has long been sought. RNase HII is the only open reading frame in Archaeoglobus fulgidus genome, while multiple RNases H exist in eukaryotic cells. Data presented here show that RNase HII from A. fulgidus (aRNase HII) specifically recognizes RNA-DNA junctions and generates products suited for the FEN-1 nuclease, indicating its role in DNA replication. Biochemical characterization of aRNase HII activity in the presence of various divalent metal ions reveals a broad metal tolerance with a preference for Mg(2+) and Mn(2+). Combined mutagenesis, biochemical competitions, and metal-dependent activity assays further clarify the functions of the identified amino acid residues in substrate binding or catalysis, respectively. These experiments also reveal that Asp129 form a second-metal binding site, and thus contribute to activity attenuation.

Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication.

DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. In combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity.

TGF-beta1-induced glomerular disorder is associated with impaired concentrating ability mimicking primary glomerular disease with renal failure in man.

Transforming growth factor-beta1 (TGF-beta1) may play a major role in the pathogenesis of glomerulopathy and end-stage renal disease (ESRD). The aim of this study was to explore the functional consequences of localized overproduction of TGF-beta1 in relation to glomerular ultrastructure and the composition of the extracellular matrix (ECM) in the inner medulla. We used a transgenic mouse with overexpression of TGF-beta1 targeted to the juxtaglomerular apparatus (JGA) by the Ren-1c promoter. The kidney function was evaluated using urine production and metabolite excretion over a 24-hour period, glomerular filtration rate (GFR), and concentrating ability. The glomerular structure was analyzed in terms of volume, ie, the volume of the mesangium per glomerulus (Vv[mes/glom]) and the volume of the matrix per glomerulus (Vv[matrix/glom]), ECM per glomerulus, the area of the filtration surface, and the thickness of the peripheral basement membrane (PBM). Immunohistochemistry or in situ hybridization was used to examine the expression of aquaporin 2 (AQP2), plasminogen activator inhibitor-1 (PAI-1), and the composition of the ECM in the inner medulla. The mice exhibited polyuria, reduced concentrating ability, decreased GFR, and albuminuria paralleled by increased glomerular volume, with increased volume of ECM, decreased filtration surface, and thickening of the PBM being detectable between 1 and 2 months of age. The deposition of glomerular ECM was accompanied by increased levels of PAI-1. As estimated by excretion of Clara cell protein-1 (CC16) and lysozyme, tubular damage occurred only in older mice. Collagen Type I was deposited in the inner medulla in the presence of normal AQP2-expression in the collecting ducts. This study reached the following conclusions: (a) TGF-beta1 reduces the GFR and the glomerular filtration surface, (b) TGF-beta1 induces albuminuria in association with widening of the PBM, (c) expansion of the mesangial volume seems to precede the widening of the PBM, (d) TGF-beta1-induced accumulation of glomerular ECM is partly explained by increased PAI-1 expression, (e) Decreased concentrating ability and polyuria caused by accumulation of ECM in the inner medulla may be an early marker of glomerular diseases associated with increased expression of TGF-beta1 in man.

Improved MCMAC with momentum, neighborhood, and averaged trapezoidal output.

An improved modified cerebellar articulation controller (MCMAC) neural control algorithm with better learning and recall processes using momentum, neighborhood learning, and averaged trapezoidal output, is proposed in this paper. The learning and recall processes of MCMAC are investigated using the characteristic surface of MCMAC and the control action exerted in controlling a continuously variable transmission (CVT). Extensive experimental results demonstrate a significant improvement with reduced training time and an extended range of trained MCMAC cells. The improvement in recall process using the averaged trapezoidal output (MCMAC-ATO) are contrasted against the original MCMAC using the square of the Pearson product moment correlation coefficient. Experimental results show that the new recall process has significantly reduced the fluctuations in the control action of the MCMAC and addressed partially the problem associated with the resolution of the MCMAC memory array.

[Effect of cGMP on calcium-activated potassium channels in primary cultured porcine coronary artery smooth muscle cells].

Guanosine 3',5'-cyclic monophosphate (cGMP) is capable of relaxing vascular smooth muscle through activating calcium-activated potassium channels (KCa channel) in the vascular smooth muscle cell membrane. But regarding the mechanism it is still under debate. In the present work the mechanism of the effect of cGMP on KCa channel in primary cultured porcine coronary artery smooth muscle cells using patch clamp technique was investigated. Experimental results showed that (1) different concentrations of 8-bromo-cGMP (0.25 mmol/L, 0.50 mmol/L, 1.00 mmol/L), serving as a membrane permeable analogue, could activate KCa channels, and (2) 1.00 mmol/L 8-bromo-cGMP could not activate such kind of channels in inside-out patch under the same condition. These results suggested that the activating effect of cGMP on KCa channels was indirect, being mediated by some intracellular process.

[Protective effect of puerarin on acute cerebral ischemia in rats].

Acute incomplete brain ischemia in rats was induced by bilateral carotid artery occlusion(BCAO). BCAO results in severe suppression of EEG and increase of brain water content. Left middle cerebral artery occlusion(MCAO) in rats by electrical coagulation results in increase of brain water content of ipsilateral hemisphere and contralateral hemiparesis. The typical ischemic neuropathological damage emerges in both BCAO and MCAO models. Intraperitoneal injection of puerarin significantly helps improve all these brain ischemic disturbances.

Angiotensin II controls p21ras activity via pp60c-src.

Angiotensin II is the major effector peptide of the renin-angiotensin system, and it exerts its physiologic functions via a G protein-coupled cell surface receptor called AT1. We found that in rat aortic smooth muscle cells, angiotensin II stimulated the formation of Ras-GTP, Ras-Raf-1 complex formation, and the tyrosine phosphorylation of two important Ras GTPase-activating proteins (GAPs), p120 Ras-GAP and p190 Rho-GAP. Electroporation of anti-pp60c-src antibody into cultured, adherent smooth muscle cells blocked the angiotensin II stimulation of Ras-GAP and Rho-GAP tyrosine phosphorylation. In contrast electroporation of antibodies against c-Yes or c-Fyn had no effect. Anti-pp60c-src antibody also blocked angiotensin II-stimulated Ras activation and Ras-Raf-1 complex formation. These data strongly suggest that a G protein-coupled receptor such as the AT1 receptor can activate the Ras protein cascade via the tyrosine kinase pp60c-src.

[Effect of puerarin on cerebral blood flow in dogs].

The hemodynamics of left intra- and extracranial arteries was determined by a pulsatile Doppler flowmeter and a transcranial Doppler system simultaneously in anesthetized dogs. Low doses of puerarin (50mg/kg, iv) did not alter the cerebral perfusion pressure, but reduced the flow velocities of both middle cerebral artery and anterior cerebral artery. The globle cerebral blood flow was enhanced due to dilatation of the intracranial arteries.