Thus, in the case that the population structure can be described by the clonal replacement model, most mutations are lost and only one mutation can become established leading to one selective sweep at a time; therefore, the population is assumed to be homogeneous except

during the periods when the beneficial mutant is sweeping through the population. The second theory is called clonal interference (Fig. 1b) or sometimes called one-by-one clonal interference because GDC-0449 chemical structure it is assumed that only one mutation can become fixed at a time. This occurs when mutations are established faster than the rate of fixations, multiple beneficial mutants can coexist and compete against each other until the one with the greatest fitness Selleckchem AC220 advantage outcompetes all the other

genotypes and become the next founding genotype for subsequent evolution. The population is thus heterogeneous except immediately after the complete sweep by the fittest mutant. This theory focuses on the competition between mutations with different fitness effects (Gerrish & Lenski, 1998; Orr, 2000; Gerrish, 2001; Kim & Stephan, 2003; Campos & de Oliveira, 2004; Wilke, 2004) and assumes that mutations cannot be stacked in the same genetic background before the fixation of the most-fit mutation. However, the size of a typical laboratory microbial population is large enough to support multiple beneficial mutations occurring in same lineage before the first mutation in that lineage can fix (Desai & Fisher, 2007), which is the basis of the third theory: the ‘multiple-mutation’ model (Desai et al., 2007) (Fig. 1c). Multiple theoretical and experimental studies in other organisms have indirectly suggested the importance of this multiple-mutation effect (Yedid & Bell, 2001;

Shaver et al., 2002; Bachtrog & Gordo, 2004). A study using Saccharomyces cerevisiae evolving under carbon source limitation showed experimental support for this theory (Desai et al., 2007). Therefore, depending on the size of the population, the rate of mutation, time required for the establishment of a beneficial mutation, the fitness distribution of the mutations, and other important factors, evolution dynamics Tyrosine-protein kinase BLK in C. albicans during long-term exposure to antifungal agents may be described by one, or combinations, of the theories mentioned above. Because without exact genotype information, it is difficult to differentiate between the one-by-one clonal interference model and the multiple-mutation model, we will use the general term clonal interference to describe a heterogeneous evolving population structure. In the seminal paper on C. albicans adaptive evolution during antifungal drug exposure, Cowen et al. (2000) evolved 12 parallel populations, six in the absence and six in the presence of fluconazole for 330 generations, and isolated clones throughout the course of the evolution.

Port doctors and health officers must be aware that ciguatera fish poisoning is a risk for seafarers traveling in tropical and subtropical areas. Stocking food from safe sources only, adequate training of ship cooks, and informing sailors about the risk of fishing in endemic areas are needed to prevent disease occurrence in seafarers in international traffic. The authors thank Dr rer. nat. Guido Westhoff, this website Leiter des Tropen-Aquarium Hagenbeck in Hamburg, Germany for identification of

the suspicious fish, and Dr Anja These, Nationales Referenzlabor für Marine Biotoxine, Bundesinstitut für Risikobewertung, Berlin for toxin analysis (National Reference Laboratory for the Monitoring of Marine Biotoxins at the Federal Institute for Risk Assessment in Berlin). The authors state they have no conflicts of interest to declare. “
“Dengue virus ( DENV) nonstructural protein 1 ( NS1) has been used as a novel diagnostic marker during the early phase of DENV infection. Presence of NS1 antigen was examined using 336 serum samples

obtained from 276 travelers returning to Japan from Asia, Central and South America, Pacific Islands, and Africa with dengue. Assay specificity was evaluated using 148 non-dengue samples. Positive rates among four DENV serotypes were 68%–89%. NS1 antigen VEGFR inhibitor positive rates were at similar levels between primary infection and secondary infection. NS1 antigen positive rates were 88%–96% on days 1–5, 75%–100% on days 6–10, and 36–60% on ≥day 11. Positive rates using real-time polymerase chain reaction (RT-PCR) were over 70% on days 1–5, but decreased thereafter. The results indicate that NS1 antigen positive rates were higher than those of RT-PCR during longer period of early phase in DENV infection. Thus, NS1 antigen ELISA is a useful

tool for confirming DENV infection in international travelers, when it is used in combination with anti-DENV IgM ELISA. Dengue virus (DENV) infection is a major health problem in tropical and subtropical regions. The disease is estimated to affect 50 million people annually worldwide.[1] It has been suggested that the spread of dengue epidemics in the present decade Resveratrol has been caused by increased international travel and urbanization.[2-4] Recently, DENV transmission has been documented in previously nonendemic areas, including Nepal, Bhutan, and France.[5-7] The number of imported dengue cases has also increased in nonendemic countries such as Japan, where there was more than a twofold increase in DENV cases from 92 in 2009 to 245 in 2010.[8] Infection with any of the four DENV serotypes causes a range of symptoms: from mild undifferentiated fever to the more severe and sometimes fatal, dengue hemorrhagic fever and dengue shock syndrome.[9-11] No specific therapeutics are available to treat the disease. Early disease confirmation is essential for clinical management as some patients’ symptoms change from mild to severe disease in a short period of time.

Candida species cause approximately 11% of all bloodstream infections (reviewed in MacCallum, 2010), with C. albicans generally the most frequently isolated fungal species. It should be noted,

however, that in some geographical areas and in certain patient groups, other Candida species are more commonly isolated (reviewed in MacCallum, 2010). This frequent isolation of C. albicans is partly due to the fact that this species is the most common commensal, but may also be a reflection of the greater virulence of this species (Arendrup et al., 2002). In general, isolates obtained Olaparib from blood samples are identical, or highly similar, to those obtained from commensal sites of the same individuals, suggesting endogenous origins of infection (Bougnoux

et al., 2006; Odds et al., 2006; Miranda et al., 2009). One of the major problems with clinical systemic Candida infection is the difficulty in the diagnosis of infection. Bloodstream Candida infections tend to present clinically with nonspecific symptoms, similar to those seen with systemic bacterial infections. This can lead to delays in the initiation of effective antifungal therapy, as antifungals may HIF-1 pathway not be administered until the patient fails to respond to antibacterials. These delays contribute to the high mortality rates (>40%) associated with Candida bloodstream infection (Morrell et al., 2005), which can be further compounded by intrinsic or acquired antifungal IMP dehydrogenase drug resistance of Candida species (Sanglard & Odds, 2002; Ostrosky-Zeichner et al., 2003). Because of the problems in the diagnosis of human infection, models of systemic Candida infection are essential for our understanding of disease initiation and progression, and also to allow the development and evaluation of novel, more effective,

diagnostics and therapies. In recent years, minihosts (e.g. Drosophila melanogaster, Caenorhabditis elegans and Galleria mellonella larvae; reviewed in Chamilos et al., 2007) have been used to study aspects of Candida disseminated infection; however, it is only in mammalian hosts that fungal disease can be fully studied. Although larger mammals, such as piglets, rabbits, guinea-pigs and rats, can be used to investigate candidiasis, the majority of studies have been carried out in mice. This is mainly due to economic factors, ease of handling, the availability of knockout mouse strains and other reagents for analyses of host responses and the availability of well-characterized, reproducible infection models. This review discusses murine models of systemic Candida infection, their contribution to our understanding of these infections and their use to evaluate diagnostics and therapies. Murine models of disseminated Candida infection fall into two main categories: the intravenous infection model and the gastrointestinal colonization and dissemination model. This review focuses mainly on C.

We transiently expressed HopF1 in bean leaves using BPMV vector-mediation. After 2 weeks of infection, new fully expanded leaves with high transcription of HopF1 (Fig. 1a) were inoculated with flg22

peptide derived from flagellin of P. syringae species to activate PTI responses. Expressed HopF1 significantly suppressed flg22-induced ROS production (Fig. 1b), flg22-induced callose deposition (Fig. 1c) and flg22-induced kinase activation (Fig. 1d). Also, expression of HopF1 contributed to the bacterial growth of a nonpathogenic strain of Psp race 6 (hrpL−) (Fig. 1e). Overall, the results indicated that HopF1 displays selleck chemical its virulence through inhibiting bean PTI responses. HopF2 had been confirmed to target RIN4 in Arabidopsis. Therefore, whether HopF1 targeted RIN4 orthologs of bean was examined. Two RIN4 orthologs, PvRIN4a (TC20682) and PvRIN4b (TC26404), were registered in the common bean expressed sequence tags (ESTs) database (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=bean; Chen et al., 2010). Amino acid sequence alignment showed that PvRIN4a and PvRIN4b share 41.1% and 38.2% identity, respectively, with AtRIN4, and the two bean RIN4 orthologs share 58.3% identity with each other. The two orthologs contain a highly conserved AvrB binding site (BBS) and AvrRpt2 cleavage

sites (RCS1 and RCS2) (Fig. S1) (Kim et al., 2005; Desveaux et al., 2007). The interaction between HopF1 and the two PvRIN4 proteins was tested with a yeast two-hybrid (Y2H) assay. HopF1 was expressed as a GAL4-activating domain (AD)-fusion protein (AD-HopF1), and PvRIN4a and PvRIN4b were expressed as GAL4-binding I-BET-762 mw domain (BD)-fusion Ribonuclease T1 proteins (BD-RIN4a/b). Y2H assay detected specific

interactions between HopF1 and both PvRIN4a and PvRIN4b (Fig. 2a). Interaction in plant cells between HopF1 and PvRIN4 proteins was confirmed by coimmunoprecipitation assay. Arabidopsis protoplasts was prepared and transfected with HA-tagged PvRIN4a or PvRIN4b alone or in combination with FLAG-tagged HopF1. Following gene expression overnight, total protein extract was immunoprecipitated with anti-FLAG antibody, and the presence of PvRIN4-HA was then detected in the immunocomplex. The results showed that PvRIN4a-HA and PvRIN4b-HA were detected in the immunocomplex from protein extracts of HopF1-FLAG and PvRIN4-HA coexpression, but not when PvRIN4a-HA and PvRIN4b-HA were expressed alone, indicating specific interactions between HopF1 and PvRIN4 orthologs (Fig. 2b). AtRIN4 negatively regulates PTI in Arabidopsis (Kim et al., 2005). The effects of PvRIN4 on bean PTI was tested here through detection of flg22-induced callose deposition on bean leaves silencing PvRIN4a and/or PvRIN4b. Silencing PvRIN4 was carried out with the BPMV-based vector. RT-PCR showed that PvRIN4 expression was almost completely abolished in new fully expanded leaves 3 weeks after infection with PvRIN4 silence vectors, but not with BPMV empty vector (Fig. 3a).

One limitation of our study may be that the population of cases was highly heterogeneous, particularly

in terms of history of antiretroviral therapy. The cases were diagnosed between 1988 and 2007, i.e. before and during the cART era. We tried to minimize this effect by matching cases and controls for click here time period as well as for CD4 cell counts, in order that cases and controls should be similar in terms of antiretroviral regimens received. Another limitation of this study is that results of EBER staining were not available and therefore some lymphoma cases may have been EBER negative and not EBV-driven NHL. This may have minimized the predictive value of high EBV loads in PBMCs for progression to systemic lymphoma in our study. However, this potential

bias does not invalidate our finding that a high EBV load in PBMCs was associated with an increased risk of developing systemic B lymphoma. Finally, one could argue that EBV load may only be a surrogate marker for immunosuppression rather than an independent marker for the risk of occurrence of B systemic lymphoma. However, a high level of EBV DNA in PBMCs remained significantly associated with a higher risk of subsequent progression to systemic B lymphoma, after adjustment for CD4 cell count at sample date or for CD4 cell count nadir. Immune reconstitution is probably the main explanation for the lower Janus kinase (JAK) incidence of ARL following the widespread use Alectinib of cART. Nevertheless, some treated patients with satisfactory immune recovery (CD4 cell count > 350 cells/μL) still develop systemic B lymphoma [7, 27]. This underlines the need to identify additional risk factors for lymphoma in HIV-infected patients. Gasser et al. demonstrated that a lack of EBV-specific CD4 T-cell immunity was associated with

the occurrence of PBL irrespective of CD4 cell count [28]. Different groups reported that uncontrolled HIV replication during cART, assessed by HIV cumulative viraemia, was predictive of the development of AIDs-related lymphoma independently of the CD4 cell count, but the underlying mechanisms of this association remain unclear [6, 27, 29]. Recently, Bohlius et al. reported that, among patients under cART, those who had experienced decreasing CD4 cell counts despite suppression of HIV-1 replication were at a higher risk of developing Hodgkin lymphoma [30]. Jaffe et al. reported that, in untreated patients, initially low and further decreasing CD4 cell counts within 12 months before the diagnosis were predictive of both NHL and Kaposi sarcoma [31]. High EBV DNA blood loads have been reported in up to 20% of asymptomatic HIV carriers and high viral loads persisted over time in more than 80% of this subset of patients [32].

Any queries (other than missing material) should be directed to the corresponding author for the article. “
“In this study, we describe the characterization, cloning, expression and purification of the lysin A gene of the mycobacteriophage TM4. The gene TM4_gp29 (gp29) is a 1644-bp gene that codes for a 58.6-kDa protein and contains peptidoglycan

recognition protein, Zn-binding and amidase catalytic domains. The gene was cloned into Escherichia coli using the ‘His-Tag’ pQE60 vector. After BI 6727 mw affinity chromatography-mediated purification, the protein was concentrated and visualized using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Evidence of peptidoglycan-degrading activity was observed initially by a

chloroform assay and later by conventional zymogram analysis. Mycobacteria cause a wide spectrum PF-02341066 mw of diseases in humans and animals. In particular, Mycobacterium tuberculosis and Mycobacterium leprae are significant pathogens. Mycobacteriophages were first isolated in 1946 from samples of soil and leaf mould (Gardner & Weiser, 1947) and were able to infect fast-growing saprophytic mycobacteria such as Mycobacterium smegmatis. Because of the growing scarcity of effective antimycobacterial agents, phages and their products are of interest in the context of new antimicrobial agents. Lysin proteins have become a focus of phage research in recent years (Borysowski et al., 2006; Jagusztyn-Krynicka & Wyszyńska, 2008; Courchesne et al., 2009; O’Flaherty et al., 2009; Wang & Lu, 2009; Fenton et al., 2010; Fischetti, 2010). They have evolved to lyse the host from the inside out, but can also cause lysis of cells when applied externally. The heterologous production of recombinant lysins has been achieved in a number of genera and the antimicrobial potential of these proteins has been well documented. Examples include Streptococcus equi (Hoopes et al., 2009), Staphylococcus aureus SB-3CT (Obeso et al., 2008) (including multidrug-resistant strains) (Rashel et al., 2007),

Bacillus anthracis (Kikkawa et al., 2008), Streptococcus pneumoniae (Grandgirard et al., 2008) (including β-lactam-resistant strains) (Rodríguez-Cerrato et al., 2007), antibiotic-resistant Enterococci (Yoong et al., 2004) and Clostridium difficile (Mayer et al., 2008). To date, 75 mycobacteriophage genomes sequences are available in GenBank; however, little has been published on their lysis genes, and apart from a more general study of lysin B by Payne et al. (2009), most of the recently published literature focuses on the mycobacteriophage Ms6 (Gil et al., 2008, 2010; Catalao et al., 2010). The first description of the lysis region within a mycobacteriophage (Ms6) was recorded by Garcia et al. (2002). The protein encoded by Ms6 ORF2 induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. Therefore, ORF2 was designated as Ms6 lysin A. In a later study by Hatfull et al.

Except within the thalamus, very few labeled cells co-stained for the inhibitory neuronal marker GAD67. Immunofluorescence staining in sections from mice injected at P3 demonstrated that the majority of cells transduced by AAV8 at this age were S100β-positive astrocytes (n = 3, Fig. 5K). These data indicate that the timing of intraventricular AAV8 injection can

strongly influence both the overall transduction efficiency as well as cell-type specificity. This unique property of AAV8 expands GSK1120212 molecular weight the potential repertoire for AAV targeting based on infection time, and provides a novel approach to astrocyte-specific transgene delivery. One advantage of viral-mediated gene transfer is that the viral titer can be MS-275 chemical structure easily adjusted to alter the transduction efficiency. We tested whether viral dilution could be reliably harnessed to generate controllable transgene mosaicism, and at what dilutions different serotypes were effective. We prepared serial dilutions of AAV8-YFP and AAV1-YFP from ~1010 to ~108 particles/μL; 2 μL of each dilution was bilaterally injected into the lateral ventricles of

P0 pups (n = 5–8 for each condition). Dilution of both AAV8 and AAV1 reduced the transduction efficiency throughout the brain, with far less fluorescent protein expression at 109 particles/μL than at 1010 particles/μL (Fig. 6). Dilution of AAV8 resulted in progressively fewer neurons being transduced at 109 particles/μL than at 1010 particles/μL, and fewer still at 108 particles/μL than at 109 particles/μL. However, the spread of AAV8 infection was essentially identical among the different dilutions. In contrast, the spread of transduction with AAV1 declined sharply at the first 10-fold dilution to 109 particles/μL (Fig. 6A). To directly compare the transduction efficiency of AAV8 with AAV1, we co-injected the two

serotypes at the Phenylethanolamine N-methyltransferase same titer (109 particles/μL, n = 4 per condition). As when injected alone at these titers, AAV8 transduced neurons throughout the brain, whereas transduction by AAV1 was largely restricted to the choroid plexus (Fig. 6B). The strong transduction of the ventricular epithelia suggests that high-affinity binding of AAV1 to these cells left little virus free to enter the rest of the brain. Next, we optimised the viral titers needed to attain reliable high- and low-density expression with each serotype based on serial dilution of each preparation. High-density neuronal transduction was consistently achieved by intraventricular injection of 4.0 × 109–2.0 × 1010 particles/hemisphere of AAV8 or 4.0 × 1010 particles/hemisphere of AAV1. Injection of virus at these concentrations left only a small population of wild-type cells surrounded by a field of transduced neighbors, ideal for studying the cell-extrinsic effects of a virally-delivered transgene. A complementary transduction pattern was attained by low-titer injections using 4.0 × 107 particles/hemisphere of AAV8 or 2.

The authors would like to thank Patricia Schlagenhauf and Koen Van Herck for their support to develop the standardized Rapamycin purchase questionnaire used in this survey. The authors state they have no conflicts of interest to declare. “
“Objective. Scarce data are available on the occurrence of ailments and diseases in children during travel. We studied the characteristics and frequencies of ailments in children aged 0 to 18 years and their parents during traveling. Methods. A prospective observational study on ailments reported by children and parents traveling to (sub)tropical countries was conducted.

The ailments were semi-quantitatively graded as mild, moderate, or severe; ailments were expressed as ailment rates per personmonth of travel. Results. A ZD1839 research buy total of 152 children and 47 parents kept track of their ailments for a total of 497 and 154 weeks,

respectively. The children reported a mean ailment rate of 7.0 (5.6–8.4) ailments per personmonth of travel; 17.4% of the ailments were graded as moderate and 1.4% as severe. The parents reported a mean ailment rate of 4.4 (3.1–5.7); 10.8% of the ailments were graded as moderate and 5.5% as severe. Skin problems like insect bites, sunburn and itch, and abdominal complaints like diarrhea were frequently reported ailments in both

children and parents. Children in the age category 12 to 18 years showed a significantly higher ailment rate of 11.2 (6.8–14.1) than their parents. Conclusions. Skin problems and abdominal problems like diarrhea are frequently reported ailments in children and their parents and show a high tendency to recur during travel. The majority of these ailments are mild but occasionally interfere with planned activities. Children in the age group 12 to 18 years are at a greater risk of developing ailments during a stay in a (sub)tropical country and they should be actively informed about the health risks of traveling to the tropics. Increasingly, children filipin travel with their parents to (sub)tropical destinations. The great variety of destinations and reasons for traveling illustrate that traveling to (sub)tropical countries has become common practice.1 Research shows that at least one third of the adults traveling to a (sub)tropical country becomes subjectively ill.2 In contrast, only scarce data are available on the prevalence of ailments in children traveling to (sub)tropical countries, while they have special needs and vulnerabilities and are believed to be more susceptible to diseases.3 The health risks of traveling to a (sub)tropical destination are not exactly known for children.

For routine monitoring purposes, viral load testing should be performed on plasma. The viral load assays can be adapted to perform well in other compartments including cerebrospinal selleck compound fluid (CSF) and seminal plasma. However, routine monitoring of viral load in compartments other than plasma is not currently recommended because of undemonstrated clinical utility or practicality (IV). Testing of CSF collected from patients with neurological

disease should be considered, especially in patients with suppressed plasma viral load (III). Using sensitive testing methods in research settings, HIV-1 RNA can be detected in plasma in a large proportion of patients receiving standard ART regimens and showing a viral load stably below 50 copies/mL for many years [1-10]. This residual viraemia is not generally associated with the emergence of drug resistance or low antiretroviral drug levels in plasma [8, 11, 12], and is not responsive to short-term intensification with efavirenz, ritonavir-boosted atazanavir, ritonavir-boosted lopinavir, enfuvirtide or raltegravir

Selleck NVP-BKM120 [8-10]. These findings are shedding new light on the significance of low-level viraemia detected by routine viral load assays during ART, while falling short of providing clear guidance for its management in patients receiving standard ART regimens. As a consequence of technical fluctuation around the cut-off level of quantification, routine viral load assays are more likely to report low-level viraemia above 50 copies/mL in treated patients who have a level of residual viraemia just below the assay cut-off (e.g. around 30 copies/mL), as seen in some patients [8]. The detection of this residual viraemia is likely to be technically inconsistent, leading to the phenomenon of viral load ‘blips’. Viral load ‘blips’ are defined as transient rises in viral load to levels above the lower detectable limit of the assay [13]. Although currently there

is no consensus definition, in practice a blip is considered to be a single viral load measurement of 50–1000 copies/mL preceded Bumetanide and followed by a measurement of fewer than 50 copies/mL. It is controversial whether blips are associated with an increased risk of virological failure, although most studies show that isolated blips are of little clinical significance [14-17]. The scenario is different, however, for patients with two or more consecutive measurements above 50 copies/mL [17] and possibly for patients with frequent blips, as these are more likely to experience virological rebound above 400 copies/mL. These patients may benefit from intervention to review expected drug potency, adherence and tolerability, and drug resistance, and modifications of therapy should be considered in line with treatment guidelines [18].

Some studies revealed attentional impairments in both early and advanced PD (e.g. Brown & Marsden, 1988; Yamada

et al., 1990; Hodgson et al., 1999; Muslimovic et al., 2005; Allcock et al., 2009; Zhou et al., 2012), whereas others did not do so (e.g. Rafal et al., 1984; Lee et al., 1999; Kingstone et al., 2002; Cristinzio et al., 2012). Dopaminergic signals in the striatum and its interaction with the prefrontal cortex would be especially critical in the regulation and integration of higher-level processes, such as attention and cognitive control (Cools, 2011). The first aim of the present study was to examine how dopamine participates in the regulation of attentional 3-Methyladenine manufacturer boost by the investigation of patients with PD before and after the administration of dopaminergic medications. We hypothesized that patients with PD receiving dopamine agonists would improve scene recognition performance when scenes are presented with rewarded target letters. Second, we studied the relationship between attentional boost and traditional components of attention (alerting, orienting, executive). Third, we explored the relationship between changes in clinical symptom and psychological trait (motor symptoms, depression, impulsivity) and attentional boost before and after dopamine agonist therapy. Finally, selleck screening library we assessed

a separate group of patients with PD receiving L-DOPA medication to test the reproducibility of the results and to examine whether the observed effects are specific for dopamine agonists or not. In the first sample, we recruited 26 newly diagnosed, drug-naive patients with PD and 25 control individuals (acquaintances of hospital staff and non-biological family members of patients matched for age, gender, education and IQ; Table 1). After baseline testing in an unmedicated state, patients received dopamine

agonist therapy and were followed-up for 12 weeks [pramipexole: n = 10, mean dose at follow-up: 4.5 mg/day, range 3.0–6.5 mg/day; ropinirole: n = 10, mean dose at follow-up: 6.0 mg/day, range: 2.5–7.5 mg/day; rotigotine: n = 6; 6 mg/24 h; levodopa equivalent dose (LED): 250 mg/day; Tomlinson et al., 2010]. After http://www.selleck.co.jp/products/Neratinib(HKI-272).html the 12-week follow-up period, participants were re-evaluated. In the second sample, we included 15 patients with recent-onset PD receiving L-DOPA monotherapy and 15 matched healthy controls (Table 2). We assessed the second sample only once. The diagnosis of PD was based on the UK Parkinson’s Disease Society Brain Bank Clinical Diagnostic Criteria (Hughes et al., 1992). All participants gave written informed consent prior to their participation. All procedures were approved by the Human Investigation Review Board (protocol number: 2697/2011) in accordance with the declaration of Helsinki (1964). 1.0 : 4 1.5 : 13 2 : 9 1.0 : 1 1.