[A clinical analysis of 32 patients with carotid web].

Carotid web is a rare risk factor of ischemic stroke. A total of 32 (0.54%) patients with carotid web were finally diagnosed in 5 943 patients who underwent carotid computerized tomography angiography (CTA) in two hospitals. Only one patient received carotid endarterectomy that pathological findings were fibrous tissue hyperplasia of vascular wall with mucinous degeneration. Stent implantation was administrated in two cases. Among 13 asymptomatic patients, the observational follow-up period was (20.9±12.4) months without strokes. Carotid web is a rare aberration. Asymptomatic patients with carotid web are usually silent. Large sized cohort and long-term follow-up are further needed.

Bacteriophages as Biological Control Agents of Enteric Bacteria Contaminating Edible Oysters.

Bacterial contamination on seafood resulting from unhygienic food-handling practices causes foodborne diseases and significant revenue losses. Moreover, control measures are complicated by a high prevalence of antibiotic-resistant bacteria. Alternative measures such as the phage therapy, therefore, is considered as an environmental and consumer-friendly biological control strategy for controlling such bacterial contamination. In this study, we determined the effectiveness of a bacteriophage cocktail in controlling E. coli strains [JM 109, ATCC 13706 and the, extended spectrum beta-lactamase resistant strain (ATCC BAA 196)] and S. enterica subsp. enterica (ATCC 13311) as single and combined contaminants of the edible oysters. Five different E. coli-specific phages (belonging to the Siphoviridae family) and a Salmonella phage (belonging to the Tectiviridae family) were successfully isolated from sewage water samples taken from a local sewage treatment plan in the Sunshine Coast region of Australia. Phage treatments applied to the pathogens when they were presented on the oysters as either single or combined hosts, resulted in significant decrease of the number of these bacteria on edible oysters. Results obtained indicated that bacteriophages could have beneficial applications in oyster-processing plants in controlling pathogenic bacterial infestations. This study thus contributes towards ongoing international efforts into the effective use of bacteriophages for biological control purposes.

The localization and function of p38α mitogen-activated protein kinase in rat oocytes.

P38α mitogen-activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p-p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p-p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p-p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 µM SB203580 blocked p-p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 µM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.

DNA barcoding and phylogenetic relationships in Timaliidae.

The Timaliidae, a diverse family of oscine passerine birds, has long been a subject of debate regarding its phylogeny. The mitochondrial cytochrome c oxidase subunit I (COI) gene has been used as a powerful marker for identification and phylogenetic studies of animal species. In the present study, we analyzed the COI barcodes of 71 species from 21 genera belonging to the family Timaliidae. Every bird species possessed a barcode distinct from that of other bird species. Kimura two-parameter (K2P) distances were calculated between barcodes. The average genetic distance between species was 18 times higher than the average genetic distance within species. The neighbor-joining method was used to construct a phylogenetic tree and all the species could be discriminated by their distinct clades within the phylogenetic tree. The results indicate that some currently recognized babbler genera might not be monophyletic, with the COI gene data supporting the hypothesis of polyphyly for Garrulax, Alcippe, and Minla. Thus, DNA barcoding is an effective molecular tool for Timaliidae species identification and phylogenetic inference.

Lack of clinical significance of the ImmuKnow(TM)-Cylex assay for the detection of cellular immune function in patients with renal cell carcinoma.

This study aimed to explore the clinical value of the CD4(+) T cell ATP levels in patients with renal cell carcinoma through the application of the ImmuKnow(TM)-Cylex(®) assay. We recruited 104 patients with renal cancer who had undergone surgery at Fuzhou General Hospital from March 2009 to June 2012, and were subsequently treated by dendritic cell and cytokine-induced killer cell bio-therapy or interferon-α therapy. The changes in CD4(+) T cell ATP levels were detected at the perioperative period and at 10 days, 1 month, 3 months, and 1 year after the surgery using the ImmuKnow assay. In addition, the differences in ATP levels in different therapy groups were compared and the prognosis conditions were analyzed. Our results demonstrated that no significant difference in the ATP levels occurred at different time points; furthermore, there were no obviously different ATP levels between the different therapy groups, and the ATP levels were found to have no clinical significance for the assessment of renal cancer prognosis. Overall, this study suggested that CD4(+) T cell ATP levels as detected by the ImmuKnow assay have no obvious clinical value in patients with renal cancer.

Increased expression of RRM2 by human papillomavirus E7 oncoprotein promotes angiogenesis in cervical cancer.

BACKGROUND: The purpose of this study was to confirm that RRM2 as a novel target of HPVE7 involved in cervical cancer angiogenesis. METHODS: Gene expression was analysed by RT-qPCR, western blot and immunohistochemistry in cervical cancer tissue and cell lines. Luciferase reporter assay was used to determine the activities of various RRM2 promoters. Secreted VEGF was measured by ELISA. RRM2-mediated capillary tube formation induced by HPVE7 in cervical cancer cells were evaluated using human umbilical vein endothelial cells in vitro. ROS induced by RRM2 in cercal cancer cells was confirmed by flow cytometry. The growth of cervical cancer cell overexpression RRM2 was examined by nude mouse xenograft. RESULTS: RRM2 as a novel downstream target for HPVE7 was upregulated by it at the transcriptional level through the E7-pRb interaction and binding of E2F to the RRM2 promoter region. Immunohistochemical analysis showed that the level of RRM2 positively correlated with the HPVE7 level in human cervical cancer. Functionally, overexpression of RRM2 enhanced the expression of HIF-1α and VEGF via activation of the ERK1/2 signalling pathway in cervical cancer cells, and significantly associated with increased microvessel densities in cervical cancer tissues. In vitro, HPVE7 stimulated RRM2-dependent capillary tube formation by HUVECs, and RRM2-enhanced angiogenesis was VEGF dependent. RRM2-activated ERK1/2 pathway was mediated through production of ROS. In the xenograft mouse model, overexpression of RRM2 in cervical cancer cells enhanced tumour growth as well as microvessel densities. CONCLUSION: HPVE7 induces upregulation of RRM2, which then promotes cervical carcinogenesis via ROS-ERK1/2-HIF-1α-VEGF-induced angiogenesis. Thus, the inhibition of RRM2 activity may be a novel therapeutic strategy for human cervical cancer.

DNA barcoding and evolutionary relationships of the Phasianidae family in China.

A DNA barcode is a short sequence of standardized genomic region that is specific to a species. According to studies of bird species, the 694-bp sequence of the mitochondrial gene encoding cytochrome c oxidase 1 (COI) is extremely useful for species identification and phylogeny. In the present study, we analyzed the COI barcodes of 31 species from 18 genera belonging to the Phasianidae family in China. Kimura two-parameter (K2P) distances were calculated between barcodes. We found that the average genetic distance between congeneric species was 24 times higher compared to the average genetic distance within species. Each bird species had a barcode that was distinct to all other bird species. The neighbor-joining method was used to construct a phylogenetic tree, which grouped all of the genera into 2 divergent clades. In conclusion, DNA barcoding is an effective molecular tool for Phasianidae species identification and phylogenetic inference.

Thinning of large mammalian cells for cryo-TEM characterization by cryo-FIB milling.

Focused ion beam milling at cryogenic temperatures (cryo-FIB) is a valuable tool that can be used to thin vitreous biological specimens for subsequent imaging and analysis by cryo-transmission electron microscopy (cryo-TEM) in a frozen-hydrated state. This technique offers the potential benefit of eliminating the mechanical artefacts that are typically found with cryo-ultramicrotomy. However, due to the additional complexity in transferring samples in and out of the FIB, contamination and devitrification of the amorphous ice is commonly encountered. To address these problems, we have designed a sample cryo-shuttle that directly and specifically accepts Polara TEM cartridges to simplify the transfer process between FIB and TEM. We optimized several parameters in the cryo-FIB and cryo-TEM processes using the quality of the samples' ice as an indicator and demonstrated high-quality milling with large mammalian cells. By comparing the results from HeLa cells to those from Escherichia coli cells, we discuss some of the artefacts and challenges we have encountered using this technique.

Short-Range Structural Connections Are More Severely Damaged in Early-Stage MS.

BACKGROUND AND PURPOSE: Long-range connections are more severely damaged and relevant for cognition in long-standing MS. However, the evolution of such coordinated network damage in patients with MS is unclear. We investigated whether short- and long-range structural connections sustained equal damage in early-stage MS. MATERIALS AND METHODS: Sixteen patients with early-stage MS and 17 healthy controls were scanned by high-resolution, multishell diffusion imaging on 7T MR imaging and assessed cognitively. We investigated macrostructural properties in short- and long-range fibers and of microstructural metrics derived from 2 quantitative diffusion MR imaging models: DTI and neurite orientation dispersion and density imaging. RESULTS: Patients had significant WM integrity damage-that is, higher radial diffusivity and a lower intracellular volume fraction in the focal WM lesions. Compared with the healthy controls, the patients had noticeable microstructure changes in both short- and long-range fibers, including increased radial diffusivity, mean diffusivity, and axial diffusivity. Z scores further indicated greater damage in the short-range fibers than in the long-range fibers. CONCLUSIONS: Our findings demonstrate that more severe demyelination preceding axonal degeneration occurs in short-range connections but not in long-range connections in early-stage MS, suggesting the possibility that there are cortical lesions that are undetectable by current MR imaging.

The soybean TGA transcription factor GmTGA13 plays important roles in the response to salinity stress.

Soybean (Glycine max L.) is an important oil, food and economic crop in the world. High salinity severely affects the growth and yield of soybean. Overexpressing a specific anti-retroviral transcription factor by biotechnology is an effective way to cultivate new stress-tolerant varieties of soybean. TGA transcription factor is a subfamily of bZIP and plays an important role in abiotic stress responses. A TGA subfamily gene GmTGA13 was cloned and the gene expression, subcellular localization and transcriptional activity were measured. Through the Ag. tumefaciens mediated flower dip method and the Ag. rhizogenes mediated transformation of soybean hairy roots, the transgenic Arabidopsis and the 'combination' soybean plants of overexpressing GmTGA13 were obtained. The two types of transgenic plants were treated with salt stress respectively, and the related physiological indexes were determined. Furthermore, the expression levels of five abiotic stress responsive genes were analyzed in GmTGA13 overexpression hairy roots. GmTGA13 gene was highly expressed in roots and significantly induced by saline stress in soybean. GmTGA13 encoded a nuclear localization protein and had transcriptional activation activity. Overexpression of GmTGA13 enhanced the saline stress tolerance of transgenic Arabidopsis and the 'combination' soybean plants. Furthermore, overexpression of GmTGA13 enhanced the expression of the stress responsive genes in transgenic soybean hairy roots. In conclusion, overexpression of GmTGA13 is beneficial to the absorption of K+ and Ca2+ by the cell, thereby regulating the ion homeostasis in the cell balance. GmTGA13 enhanced salt resistance of plants by regulating the expression of many stress-responsive genes.

The regulation of CIRBP by transforming growth factor beta during heat shock-induced testicular injury.

BACKGROUND: Cold-inducible RNA-binding protein (CIRBP) is associated with cell stress. However, its upstream regulatory factors are still largely unknown. OBJECTIVES: This study investigated whether CIRBP expression was regulated by transforming growth factor beta (TGF-ß) during the process of heat-induced testicular damage. MATERIALS AND METHODS: Ten male adult ICR mice were allocated to heat treatment (scrotal hyperthermia at 43 °C for 30 min, n = 5) and control group (n = 5); CIRBP and TGF-ß1, TGF-ß2, and TGF-ß3 expression levels in the testis in mRNA and protein were analyzed. Then, we conducted in vivo and in vitro studies to investigate the regulatory effects of TGF-ß on CIRBP. In the in vivo study, male adult ICR mice were subjected to testicular hyperthermia followed by a local testicular injection of TGF-ß antagonist (non-selective TGF-ß I/II receptor inhibitor, 5 µg or 10 µg). In the in vitro study, GC2-spd cells were cultured under 43 °C for 30 min or with different TGF-ß isoforms (10 ng/mL), and CIRBP expression levels in the testis and GC2-spd cells were analyzed 24 and 48 h, respectively, after treatment. RESULTS: As a result, heat treatment significantly downregulated the relative CIRBP mRNA and protein expression (p = 0.006 and 0.011), and significantly upregulated TGF-ß2 and TGF-ß3 expression levels (p = 0.022 and 0.04, for mRNA, and p = 0.001 for both protein levels). Local testicular injection of 10 µg TGF-ß antagonist significantly attenuated heat-induced histological damage to the testes and CIRBP downregulation (p = 0.038). Furthermore, TGF-ß2 and TGF-ß3 significantly downregulated CIRBP mRNA and protein expression in GC2-spd cells (all p < 0.01), exerting a similar effect to heat treatment. DISCUSSION AND CONCLUSION: Our in vivo and in vitro experiments demonstrated that heat-induced CIRBP downregulation in the testes was mediated by the upregulation of TGF-ß. Further studies are needed to clarify the molecular mechanisms underlying these processes.

Silk Route to the Acceptance and Re-Implementation of Bacteriophage Therapy-Part II.

This perspective paper follows up on earlier communications on bacteriophage therapy that we wrote as a multidisciplinary and intercontinental expert-panel when we first met at a bacteriophage conference hosted by the Eliava Institute in Tbilisi, Georgia in 2015. In the context of a society that is confronted with an ever-increasing number of antibiotic-resistant bacteria, we build on the previously made recommendations and specifically address how the Nagoya Protocol might impact the further development of bacteriophage therapy. By reviewing a number of recently conducted case studies with bacteriophages involving patients with bacterial infections that could no longer be successfully treated by regular antibiotic therapy, we again stress the urgency and significance of the development of international guidelines and frameworks that might facilitate the legal and effective application of bacteriophage therapy by physicians and the receiving patients. Additionally, we list and comment on several recently started and ongoing clinical studies, including highly desired double-blind placebo-controlled randomized clinical trials. We conclude with an outlook on how recently developed DNA editing technologies are expected to further control and enhance the efficient application of bacteriophages.

New DNA-based PCR approaches for rapid real-time detection and prevention of group B streptococcal infections in newborns and pregnant women.

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis, and maternal infection. Although the pathogenesis of GBS infection is not well understood, several virulence factors have been identified. Two prevention strategies have been proposed: chemoprophylaxis and immunoprophylaxis. Implementation of selective intrapartum chemoprophylaxis on the basis of either screening or risk assessment has led to a substantial decrease in the morbidity and mortality of GBS disease in both mothers and infants. Penicillin remains the antibiotic of choice with no reported resistant GBS so far, whereas resistance of 10-20% of GBS to erythromycin and clindamycin has been reported in North America. Chemoprophylaxis based on screening requires optimal detection methods for GBS, which involve selective broth culture of combined vaginal and anal samples. Other conventional methods are useful for rapid identification of heavily colonised women, but are unreliable for the detection of light GBS colonisation because of poor sensitivity. GBS-specific polymerase chain reaction (PCR) assays using real-time PCR coupled with fluorescence-labelling technology offer powerful tools for sensitive and specific, yet rapid (less than 1 h), detection of GBS directly from clinical specimens at the time of delivery. The application of these assays to the current prevention strategies will simplify the prevention practice and rationalise the use of antibiotics. Immunoprophylaxis relies on the development of new vaccines against GBS, and active research is being conducted in this area.

Molecular methods for rapid detection of group B streptococci.

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis. Implementation of selective intrapartum chemoprophylaxis based on either a screening-based approach or a risk-based approach has led to a substantial decrease in the morbidity and mortality of GBS disease. Current 'gold-standard' detection methods for GBS are selective broth cultures of combined vaginal and anal specimens collected at 35-37 week's gestation. Rapid immunological detection methods, including latex agglutination test, enzyme immunoassay and optical immunoassay, as well as hybridization-based test, are available. These methods are useful in rapid identification of heavily colonized women, but are unable to detect light GBS colonization due to poor sensitivity. Recent development of real-time PCR and fluorescence labeling technologies has provided new detection platforms for bacterial identification. GBS-specific PCR assays using these new technologies offer promising tools for sensitive and specific detection of GBS directly from clinical specimens. The application of these assays in the current prevention strategy will simplify the prevention practice and rationalize antibiotic use.

Small scale sandwich enzyme immunoassay for human chorionic gonadotropin using a new micro-transferable plastic bead as solid phase.

This paper describes a highly sensitive small scale enzyme immunoassay for human chorionic gonadotropin (hCG) in pregnancy urine using a microtransferable plastic bead (MTPB) as solid phase. Results of the present method for determination of 40 samples of pregnancy urine correlated well with those of the commercial radioimmunoassay and a better precision was achieved. This solid phase method may be widely applied to the immunoassay of small volumes.

Anti-human platelet tetraspanin (CD9) monoclonal antibodies induce platelet integrin alphaIIbbeta3 activation in a Fc receptor-independent fashion.

OBJECTIVE: This study characterized the activation of platelet integrin alphaIIbbeta3 induced by two anti-human platelet tetraspanin monoclonal antibodies (mAbs), HI117 and SJ9A4. METHODS: Using 125I-labeled human fibrinogen(Fg), specific Fg binding to human platelets induced by HI117 and SJ9A4 was measured as indication of activation of platelet integrin alphaIIbbeta3 by the two mAbs. RESULTS: HI117 and SJ9A4 (10 microg/ml and 20 microg/ml) induced evident specific Fg binding to human platelets, suggesting that the two mAbs evoked activation of platelet integrin alphaIIbbeta3. Further study indicated that HI117 and SJ9A4 induced integrin alphaIIbbeta3 activation independent of platelet Fc-receptors, and that HI117 and SJ9A4-induced integrin alphaIIbbeta3 activation was inhibited by sphingosing, aspirin, apyrase, and/or PGI2. CONCLUSION: The anti-platelet tetraspanin (CD9) mAbs, HI117 and SJ9A4, can induce platelet integrin alphaIIbbeta3 activation independent of Fc-receptor. Three signaling pathways, i.e. thromboxane, secreted ADP, and cAMP pathways may be involved in the process, with protein kinase C activation presumably being the common step of the three pathways.

[Role of basolateral amygdaloid nuclei in sleep and wakeful state regulation].

The role of basolateral amygdaloid nuclei (BLN) in sleep and wakeful state (W) was investigated with polysomnography. Electrical lesion of bilateral BLN increased slow wave sleep (SWS) and paradoxical sleep (PS), but decreased W. Bilateral injection of kainic acid (KA), which selectively destroyed the soma of neurons, resulted in biphasic effects. At d 1 after KA, insomnia occurred. At d 4, SWS increased while W decreased, but PS did not change. Unilateral injection of NO donor sodium nitroferricyanide (SNP) caused an enhancement of W and a decrease of SWS. Injection of nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) led to opposite effects. L-arginine, a precursor of NO, had no direct effect on sleep and W, but blocked the effects of L-NNA. The results suggest that BLN plays an important role in regulating sleep and wakeful state. Destruction of BLN increases SWS, and NO in BLN has W-promoting and SWS-inhibiting effects, which may be mediated by activation of neurons in BLN.

Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce.

Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 +/- 2 degrees C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (-)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.

Ethylene production by suspension-cultured pear fruit cells as related to their senescence.

Suspension-cultured pear fruit cells produce low levels of ethylene during growth and division in auxin containing medium. When deprived of auxin, division gradually ceases and ethylene production falls to barely discernible levels. However, notable ethylene production can now be induced by indoleacetic acid, CuCl(2), or 1-aminocyclopropane-1-carboxylic acid. If the auxin-deprived cells are transferred to ;aging' medium that lacks auxin but contains 0.4 molar mannitol, inducible ethylene production increases several-fold reaching levels of 40 to 60 nanoliters/10(6) cells per hour. Maximum inducible ethylene productivity is attained at varying times (1-6 days) after transfer to aging medium and appears to be temporally related to cell survival, i.e. the time of subsequent cell death. It is argued that auxin depletion initiates senescence which, in turn, leads to a transient increase in inducible ethylene production and eventual death. The limitations and potentials of the suspension-cultured pear cells as a system for the study of cellular senescence are discussed.

Electrostatic interactions play a critical role in Mycobacterium tuberculosis Hsp16.3 binding of substrate proteins.

Mycobacterium tuberculosis Hsp16.3, a member of a small heat shock protein family, has chaperone-like activity in vitro and suppresses thermally or chemically induced aggregation of proteins. The nature of the interactions between Hsp16.3 and the denatured substrate proteins was investigated. A dramatic enhancement of chaperone-like activity of Hsp16.3 upon increasing temperature was accompanied by decreased ANS-detectable surface hydrophobicity. Hsp16.3 exhibited significantly enhanced chaperone-like activity after preincubation at 100 degrees C with almost unchanged surface hydrophobicity. The interaction between Hsp16.3 and dithiothreitol-treated insulin B chains was markedly weakened in the presence of NaCl but greatly enhanced by the addition of a low-polarity alcohol, accompanied by significantly increased and decreased surface hydrophobicity, respectively. A working model for Hsp16.3 binding to its substrate proteins is proposed.