Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Conjugation is a major form of horizontal gene transfer, contributing to bacterial evolution and the acquisition of new traits. During conjugation, a donor cell transfers DNA to a recipient through a specialized DNA translocation channel classified as a type IV secretion system (T4SS). Here, we focused on the T4SS of ICEBs1, an integrative and conjugative element in Bacillus subtilis. ConE, encoded by ICEBs1, is a member of the VirB4 family of ATPases, the most conserved component of T4SSs. ConE is required for conjugation and localizes to the cell membrane, predominantly at the cell poles. In addition to Walker A and B boxes, VirB4 homologs have conserved ATPase motifs C, D, and E. Here, we created alanine substitutions in five conserved residues within or near ATPase motifs in ConE. Mutations in all five residues drastically decreased conjugation frequency but did not affect ConE protein levels or localization, indicating that an intact ATPase domain is critical for DNA transfer. Purified ConE is largely monomeric with some oligomers and lacks enzymatic activity, suggesting that ATP hydrolysis may be regulated or require special solution conditions. Finally, we investigated which ICEBs1 T4SS components interact with ConE using a bacterial two-hybrid assay. ConE interacts with itself, ConB, and ConQ, but these interactions are not required to stabilize ConE protein levels and largely do not depend on conserved residues within the ATPase motifs of ConE. The structure-function characterization of ConE provides more insight into this conserved component shared by all T4SSs. IMPORTANCE Conjugation is a major form of horizontal gene transfer and involves the transfer of DNA from one bacterium to another through the conjugation machinery. Conjugation contributes to bacterial evolution by disseminating genes involved in antibiotic resistance, metabolism, and virulence. Here, we characterized ConE, a protein component of the conjugation machinery of the conjugative element ICEBs1 of the bacterium Bacillus subtilis. We found that mutations in the conserved ATPase motifs of ConE disrupt mating but do not alter ConE localization, self-interaction, or levels. We also explored which conjugation proteins ConE interacts with and whether these interactions contribute to stabilizing ConE. Our work contributes to the understanding of the conjugative machinery of Gram-positive bacteria.

Penile neurovascular structure revisited: immunohistochemical studies with three-dimensional reconstruction.

Penile erection is a neurovascular phenomenon that requires well coordinated and functional interaction between penile vascular and nervous systems. In order to provide a useful tool to examine pathologic changes in the erectile tissue, mainly focusing on penile neurovascular dysfunction, we established the technique to determine the differential distribution of endothelial cells, smooth muscle cells, pericytes, and nerve fibers in the mouse penis using immunohistochemical staining with three-dimensional reconstruction. Immunofluorescent staining of penile tissue was performed with antibodies against CD31 (an endothelial cell marker), smooth muscle α -actin (SMA, a smooth muscle cell marker), NG2 (a pericyte marker), or ßIII-tubulin (a neuronal marker). We reconstructed three-dimensional images of penile vascular or neurovascular system from stacks of two-dimensional images, which allows volume rendering and provides reliable anatomic information. CD31-positive endothelial cells, SMA-positive smooth muscle cells, and NG2-positive pericytes were evenly distributed and composed sinusoidal or venous wall. However, the endothelial layer of the cavernous artery or dorsal artery was mainly covered with smooth muscle cells and rarely associated with pericytes. The reconstructed three-dimensional images clearly visualized typical wavy appearance of nerve fibers that evenly innervate to cavernous sinusoids, cavernous artery, dorsal vein, and dorsal artery. We observed a significant decrease in CD31-positive endothelial cells, NG2-positive pericytes, and ßIII-tubulin-positive nerve fibers in the penis of diabetic mice compared with those in normal condition. Our protocol for immunofluorescent staining with three-dimensional reconstruction will allow a better understanding of the penile neurovascular anatomy and may constitute a standard technique to determine the efficacy of candidate therapeutics targeting therapeutic angiogenesis or neural regeneration.

Calorie restriction reverses age-related alteration of cavernous neurovascular structure in the rat.

Calorie restriction (CR) refers to a reduction of calorie intake without compromising essential nutrients to avoid malnutrition. CR has been established as a non-genetic method of altering longevity and attenuating biological changes associated with aging. Aging is also an important risk factor for erectile dysfunction. The aim of this study was to examine whether CR diet can reverse the age-related alterations of erectile tissue in the aged rat. Four groups of rats were used: young rats (7 months) + ad libitum, aged rats (22 months) + ad libitum, young rats + CR diet, and aged rats + CR diet. The ad libitum group had free access to both food and water, and CR groups were fed 60% of the food intake of their ad libitum littermates, starting from 6 weeks before sacrifice. The penis was harvested and stained with antibodies to von Willebrand factor, smooth muscle α-actin, platelet-derived growth factor receptor-ß, phospho-eNOS, nNOS, and neurofilament. We also performed Masson trichrome staining and TUNEL assay. The blood samples were collected for the measurement of serum total testosterone level. The contents of endothelial cells, smooth muscle cells, pericytes, and neuronal cells as well as serum testosterone levels were significantly lower in the penis of aged rats than in their young littermates. CR significantly restored cavernous endothelial cells, smooth muscle cells, pericytes, and neuronal cell contents and decreased cavernous endothelial cell apoptosis and fibrosis in both young and aged rats. CR also increased serum testosterone level in aged rats, but not in young rats. CR successfully improved age-related derangements in penile neurovascular structures and hormonal disturbance. Along with a variety of lifestyle modifications, our study gave us a scientific rationale for CR as a non-pharmaceutical strategy to reprogram damaged erectile tissue toward neurovascular repair in aged men.

Establishment of in vitro model of erectile dysfunction for the study of high-glucose-induced angiopathy and neuropathy.

Penile erection requires complex interaction between vascular endothelial cells, smooth muscle cells, pericytes, and autonomic nerves. Diabetes mellitus is one of the most common causes of erectile dysfunction (ED) and multiple pathogenic factors, such as cavernous angiopathy and autonomic neuropathy, are associated with diabetic ED. Although a variety of animal models of diabetic ED play an important role in understanding pathophysiologic mechanisms of diabetes-induced ED, these animal models have limitations for addressing the exact cellular or molecular mechanisms involved in ED. Therefore, we established an in vitro model of ED for the study of high-glucose-induced angiopathy and neuropathy. We successfully isolated and cultivated mouse cavernous endothelial cells (MCECs) and mouse cavernous pericytes (MCPs). The cells were exposed to the normal-glucose (5 mmoL) or high-glucose (30 mmoL) condition for 48 h. In vitro matrigel assay revealed impairments in tube formation in primary cultured MCECs or MCPs exposed to high-glucose condition. To study cellular interaction between MCECs and MCPs, co-culture systems including indirect contact, indirect non-contact, and direct mixed co-culture system, were established. We observed impaired tube formation and increased permeability in MCECs-MCPs co-culture exposed to high-glucose condition. To evaluate the effect of high-glucose on neurite sprouting, the mouse major pelvic ganglion (MPG) tissue was harvested and cultivated in matrigel. Neurite outgrowth and nNOS-positive nerve fibers were significantly lower in MPG tissues exposed to the high-glucose condition than in the tissues exposed to the normal-glucose condition. We believe that in vitro model of ED will aid us to understand the role of each cellular component in the pathogenesis of diabetic ED, and also be a useful tool for determining the efficacy of candidate therapeutics targeting vascular or neuronal function. This model would present a new avenue for drug discovery and development of novel therapeutic modalities for erectile dysfunction.

Zika virus: no cases in the Eastern Mediterranean Region but concerns remain.

Following the WHO declaration on 1 February 2016 of a Public Health Emergency of International Concern (PHEIC) with regard to clusters of microcephaly and neurological disorders potentially associated with Zika virus, the WHO Regional Office for the Eastern Mediterranean conducted three rounds of emergency meetings to address enhancing preparedness actions in the Region. The meetings provided up-to-date information on the current situation and agreed on a set of actions for the countries to undertake to enhance their preparedness and response capacities to Zika virus infection and its complications. The most urgent action is to enhance both epidemiological and entomological surveillance between now and the coming rainy seasons in countries with known presence of Aedes mosquitoes. Zika virus like other vector-borne diseases poses a particular challenge to the countries because of their complex nature which requires multidisciplinary competencies and strong rapid interaction among committed sectors. WHO is working closely with partners and countries to ensure the optimum support is provided to the countries to reduce the risk of this newly emerged health threat.

Zika virus: no cases in the Eastern Mediterranean Region but concerns remain

Following the WHO declaration on 1 February 2016 of a Public Health Emergency of International Concern [PHEIC] with regard to clusters of microcephaly and neurological disorders potentially associated with Zika virus, the WHO Regional Office for the Eastern Mediterranean conducted three rounds of emergency meetings to address enhancing preparedness actions in the Region. The meetings provided up-to-date information on the current situation and agreed on a set of actions for the countries to undertake to enhance their preparedness and response capacities to Zika virus infection and its complications. The most urgent action is to enhance both epidemiological and entomological surveillance between now and the coming rainy seasons in countries with known presence of Aedes mosquitoes. Zika virus like other vector-borne diseases poses a particular challenge to the countries because of their complex nature which requires multidisciplinary competencies and strong rapid interaction among committed sectors. WHO is working closely with partners and countries to ensure the optimum support is provided to the countries to reduce the risk of this newly emerged health threat

A la suite de la déclaration de l'OMS le 1[er] février 2016 faisant état d'une urgence de santé publique de portée internationale, eu égard à l'existence de groupes de cas de microcéphalie et de troubles neurologiques potentiellement associés à la maladie à virus Zika, le Bureau régional de l'OMS de la Méditerranée orientale a conduit trois cycles de réunions d'urgence en vue d'améliorer les mesures de préparation dans la Région. Les réunions ont fourni des informations actualisées de la situation actuelle et ont permis de convenir d'un ensemble d'actions à entreprendre par les pays afin d'améliorer leurs capacités de préparation et de réponse face à l'infection à virus Zika et ses complications. L'action la plus urgente consiste à améliorer la surveillance épidémiologique et entomologique à partir d'aujourd'hui et jusqu'à la prochaine saison des pluies dans les pays où la présence de moustiques Aedes est établie. La maladie à virus Zika, comme toutes les maladies à transmission vectorielle, constitue un défi pour les pays du fait de sa nature complexe qui nécessite des compétences multidisciplinaires et une interaction forte et rapide entre les secteurs impliqués. L'OMS travaille en étroite collaboration avec ses partenaires et les pays afin de garantir que le meilleur soutien soit apporté aux pays en vue de la réduction du risque de cette nouvelle menace sanitaire émergente

Effect of soil pH on as hyperaccumulation capacity in fern species, Pityrogramma calomelanos.

Arsenic uptake by hyperaccumulator plant species depends on many different environmental factors. Soil pH is one of the most important factors due to its combined effect on both chemical and biological processes. In greenhouse experiment, the effect of pH (within the pH range 3.6 - 8.9) on As uptake as well as biomass of Pityrogramma calomelanos was evaluated. The plants were grown in mining soil containing 645.6 mg As kg(-1) for 14 weeks. Within this time, the plant biomass growth was 3.78 - 8.64 g d. wt. per plant and the removal amounted 6.3-18.4 mg As per plant. Translocation factor (ratio of As in fronds to roots) of the fern was 3.6 - 9.7, indicating its potential in phytoremediation of As contaminated soil. Influence of pH on As bioavailability was visible as the available As concentration was higher in acidic soil compared to alkaline soil. Furthermore, it was found that As accumulation by Pityrogramma calomelanos was optimum in the soil of pH 3.6. Nevertheless, the results of this study demonstrate that remediation of As-contaminated mining soils, by this fern, can be improved by changing the soil pH from 4.6 to 6.8.

Hematopoietic SCT activity in Asia: a report from the Asia-Pacific Blood and Marrow Transplantation Group.

The hematopoietic SCT (HSCT) activity in nine Asian countries/regions was surveyed to overview the current situation. Data of 58 113 HSCTs (allogeneic: 63% vs autologous: 37%) performed between 1986 and 2006 by 432 transplant teams were collected. The number of HSCTs has been increasing in the past two decades in most countries/regions. The increase in allogeneic HSCTs is greater than in autologous HSCTs. The proportion of unrelated donors among allogeneic HSCTs in 2006 varied widely from <1% (Iran and Vietnam) to 62% (Japan). The use of each stem cell source, that is, BM, PBSC, cord blood and others (including co-infusion of BM and PBSC), also varied widely (36, 58, 0.1 and 6% in HSCT from related donors, respectively, and 53, 11, 35 and 1% in HSCT from unrelated donors, respectively). HSCTs have been continuously increasing for all indications except for chronic myelogenous leukemia and solid tumors. Hemoglobinopathy is a common indication among non-malignant diseases in many Asian countries/regions except for China, Japan and Korea. This survey clearly shows the recent progress of HSCTs in Asia and also some differences in donor and stem cell selection and disease application among countries/regions.

Multiple norovirus outbreaks among workplace canteen users in Finland, July 2006.

Multiple gastroenteritis outbreaks occurred between 25 and 31 July 2006 in 10 workplace canteens in south-western Finland. One vegetable processing plant provided raw vegetables to all the canteens. We conducted cohort studies in the three most visited canteens and environmental investigations in the kitchens and the plant. Patients' stools, food, water and environmental samples were tested for enteric bacteria and viruses. Of the three canteens, 150/273 respondents (response rate 82%) had gastroenteritis. Consumption of mixed raw vegetables was significantly associated with the illness but no single vegetable explains the outbreak. An identical norovirus GII.1 genotype was detected from all genotyped patient samples. Water, food, and environmental samples were negative for norovirus. The facilities had appropriate hygienic conditions and no staff member had gastroenteritis prior to the outbreak. Tracing back the vegetables to the farm level proved unsuccessful. This was the largest foodborne norovirus outbreak in Finland.

Immune responses to pertussis antigens eight years after booster immunization with acellular vaccines in adults.

Pertussis-specific antibody and cell-mediated immune (CMI) responses were studied in adults 8 years after booster immunization with either a bicomponent (pertussis toxin and filamentous hemagglutinin) or a monocomponent (pertactin) acellular vaccine and in age-matched healthy controls. The levels of vaccine-induced antibodies were also compared between the serum samples collected before, 1 month, 4 years, and 8 years after immunization. Over the follow-up period, geometric mean values (GMV) of antibodies to the vaccine antigens decreased in both groups of vaccinees. However, the 8-year postimmunization GMV were 3-20 times higher than preimmunization GMV (all P values <0.01). Moreover, both antibody and CMI responses to the vaccine antigens were significantly higher in the vaccinees than in the controls (all P<0.01 for antibody; all P<0.001 for CMI responses). The results show that antibody and CMI responses induced by acellular pertussis vaccines can persist for up to 8 years after booster immunization of adults primed with whole-cell vaccine.

Acellular vaccines containing reduced quantities of pertussis antigens as a booster in adolescents.

OBJECTIVE: To evaluate the immunogenicity and reactogenicity of an acellular pertussis vaccine (pa) either formulated with diphtheria and tetanus toxoids (dTpa) or administered consecutively with a licensed tetanus and diphtheria vaccine (Td) as a 5th dose in adolescents. METHODS: A total of 510 healthy children 10 to 13 years of age were assigned randomly, using a single-blind design, to receive either the dTpa vaccine or the Td vaccine with the pa vaccine 1 month later. The quantities of 3 pertussis antigens (pertussis toxin, filamentous hemagglutinin, and pertactin) in the dTpa and the pa vaccines were one third of those of the Infanrix vaccine (SmithKline Beecham Biologicals, Rixensart, Beligium) licensed for use in infants. For enzyme-linked immunosorbent assay measurement of serum immunoglobulin G antibodies and proliferation assay of peripheral blood mononuclear cells, blood samples were obtained before and 1 month after immunization. Local and systemic reactions were recorded on diary cards for 15 days after immunization. RESULTS: After immunization with dTpa or pa, significant and comparable rises in geometric mean values of antibodies (12- to 46-fold) and proliferations (8- to 18-fold) to each of the pertussis antigens were noted. After immunization with dTpa or Td, significant rises in geometric mean values of antidiphtheria and antitetanus antibodies (35- to 76-fold) were noted, and all subjects had values of these antibodies >/=.1 international units/mL. The dTpa and pa vaccines were at least as well tolerated as the licensed Td vaccine. CONCLUSIONS: Booster immunization of adolescents with an acellular vaccine containing reduced quantities of pertussis antigens in addition to diphtheria and tetanus toxoids induces good responses in both arms of the immune system without an increase in adverse reactions.

Cell-mediated immune responses to antigens of Bordetella pertussis and protection against pertussis in school children.

BACKGROUND: Increasing evidence suggests that cell-mediated immunity (CMI) is involved in immune response against Bordetella pertussis. However, there are practically no studies evaluating the significance of pertussis-specific CMI in relation to protection against clinical pertussis. METHODS: An outbreak of pertussis was studied prospectively in 13-year-old pupils in a rural school. B. pertussis infection was diagnosed by culture, microagglutination and enzyme immunoassay serology with the use of pertussis toxin, filamentous hemagglutinin and pertactin as antigens. Pertussis-specific CMI responses were assessed by in vitro proliferation assay of peripheral blood mononuclear cells. RESULTS: At the initial sampling 7 of 22 children had symptoms suggestive of pertussis and 15 were asymptomatic. Of the latter 3 remained healthy, 8 were later confirmed to have had asymptomatic infection, 3 developed laboratory-confirmed pertussis and 1 developed cough without laboratory evidence of pertussis. Initial in vitro proliferations of peripheral blood mononuclear cells induced by pertussis toxin, filamentous hemagglutinin and/or pertactin were positive in all 3 healthy children, in 6 of 8 children who had asymptomatic infection, but in none of the 3 children who later developed pertussis. Although some children who remained healthy had high values of antibodies, no clear association was found between initial serum antibody values and clinical outcome. CONCLUSIONS: These preliminary data suggest that CMI may have an important role in protection against clinical pertussis but do not exclude a role for antibodies. Furthermore the results stress a multifactorial nature of the immune protection against B. pertussis.

Antibody and cell-mediated immune responses to booster immunization with a new acellular pertussis vaccine in school children.

235 healthy 10-12 years old school children were randomly immunized with either a booster dose of diphtheria-tetanus-acellular pertussis (dTap) or diphtheria-tetanus (dT) vaccine. For this booster immunization designed for school children and adults, the quantities of Bordetella pertussis antigens in the dTap vaccine had been reduced to one third of those of the Infanrix vaccine (SmithKline Beecham) commonly used for infants. IgG antibodies and cell-mediated immune (CMI) responses to pertussis toxin (PT), pertactin (PRN) and filamentous hemagglutinin (FHA) were assessed by an enzyme immunosorbent assay and in vitro proliferation of peripheral blood mononuclear cells, respectively. Before immunization, 55%, 80% and 99% of children had detectable serum IgG antibodies to PT, PRN and FHA, whereas CMI response was found in 35%, 27% and 50% of children, respectively. After immunization, a 20-30-fold increase in geometric mean level (GML) of antibodies to the pertussis antigens occurred and CMI response to PT, PRN and FHA was seen in 88%, 94% and 100% of children, respectively. Adverse reactions following the immunization were rare. The results show that booster immunization with an acellular pertussis vaccine with reduced concentrations of antigens induces both antibody and CMI responses and support further studies of this pertussis vaccine in school children.

Cytokine mRNA expression and proliferative responses induced by pertussis toxin, filamentous hemagglutinin, and pertactin of Bordetella pertussis in the peripheral blood mononuclear cells of infected and immunized schoolchildren and adults.

Pertussis infection is increasingly recognized in older children and adults, indicating the need of booster immunizations in these age groups. We investigated the induction of pertussis-specific immunity in schoolchildren and adults after booster immunization and natural infection. The expression of mRNA of gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 in the peripheral blood mononuclear cells (PBMCs) was assayed by reverse transcription-PCR. The PBMCs of 17 children immunized with one dose of an acellular vaccine containing pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) significantly proliferated in vitro after stimulation with the vaccine antigens. The PBMCs of seven infected individuals markedly proliferated in the presence of PT and FHA, but the cells of only two of these subjects responded to PRN. At least one of the antigens induced mRNA for IL-4 and/or IL-5 in the cells of 93% of tested vaccinees and patients, and FHA induced IFN-gamma mRNA in the cells of two-thirds of them. Expression of mRNA for IFN-gamma correlated with the production of the cytokine protein. Anti-FHA immunoglobulin G antibodies significantly correlated with FHA-induced proliferative responses both before and after immunization. These results show that booster immunization with acellular pertussis vaccine induces both antibody- and cell-mediated immune responses in schoolchildren. Further, booster immunization and natural infection seem to induce the expression of mRNA of T-helper 1 (Th1) and Th2 type cytokines in similar manners. This observation supports the use of acellular pertussis vaccines for booster immunizations of older children, adolescents, and adults.

Islet cell and glutamic acid decarboxylase antibodies and heat-shock protein 65 responses in children with newly diagnosed insulin-dependent diabetes mellitus.

Islet cell antibodies (ICA) were detected in 66% and glutamic acid decarboxylase (GAD) antibodies in 64% of children (n = 47) with newly diagnosed insulin-dependent diabetes mellitus (IDDM). Fifteen percent of the patients had neither GAD nor ICA antibodies. Responses to mycobacterial heat-shock protein 65 (Hsp65) were detected in all patients. There was a significant correlation between anti-GAD antibodies and proliferation of peripheral blood mononuclear cells to Hsp65, and between ICA and antibodies to Hsp65.