In order to assess in which FOR the BOLD activity associated with

In order to assess in which FOR the BOLD activity associated with covert search in the anterior insula and the SEF was modulated, we calculated the percentage signal change for the ROIs in these two areas in both hemispheres (see Supporting

Information Fig. S1). These ROIs were defined by comparing covert search with the control condition (see ‘Materials and methods’). In all four ROIs, covert search seemed to evoke larger higher BOLD responses than the control condition (see Supporting Information Fig. S1). However, one-way anova across the different search conditions did not yield a significant modulation of the signal change (P > 0.1 for all MI-503 mouse four ROIs). Hence, the search related BOLD response in both the anterior insula and the SEF does not encode the FOR in which covert search operates. We tried to identify the FOR for covert serial search by studying the dependence of cortical BOLD activity, evoked by visual search, on eye-gaze position. Our key observation was that specific parts of the IPS and the right FEF showed a higher BOLD response during covert search to eye-centred contralateral locations, independent of

eye position. In other words, objects singled out in a search array by covert serial search are represented in an eye-centred or retinal coordinate system. However, compared with the left IPS, this effect was weaker in the right pIPS. Early and later Dabrafenib in vivo visual regions similarly exhibited stronger responses for covert

search directed to contralateral eye-centred locations, as expected from their known retinotopic organization. The anterior insula and the SEF did not show the above-mentioned eye-centred modulation of their search related response. Although not very likely, we admit that with the paradigm used we cannot exclude that a modification related to an effector such as the hand or the head could have had a modulatory effect on the clearly eye-centred BOLD responses, which we observed. However, in our paradigm the non-eye-centred search array location did not have any influence on the results, so we think that it is unlikely, though in principle possible to expect different results by changing the head or body position in our experiment. In the following discussion we will first address the question: can our findings based on BOLD responses be reconciled with single-unit studies before on covert visual search? We will then discuss how the evidence for eye-centred coding of covert search provided by our study fits with previous fMRI studies that addressed the reference frame for the encoding of covert as well as overt shifts of attention, i.e. saccades. This comparison seems pertinent, given the fact that overt and covert shifts of attention are tightly coupled and, moreover, usually assumed to share most of their cortical (Rizzolatti et al., 1987; Corbetta et al., 1998) and subcortical (Ignashchenkova et al., 2004) substrates.

Of these, 35 patients (32%) experienced a problem related to a ch

Of these, 35 patients (32%) experienced a problem related to a chronic condition. In comparison, 24 (22%) patients experienced an acute infection. Sixty percent of patients

were nonadherent to medications during travel. An average increase in diastolic blood pressure of 3.6 mmHg among patients with hypertension was the only statistically significant change in a chronic disease marker when values before and after travel were compared. Subgroup analysis revealed that travel to Africa and nonadherence to medications were also associated with worsening blood pressure control, and patients traveling to Africa experienced a decrease in body 5-Fluoracil purchase mass index. This study identified a high proportion of problems related to chronic conditions experienced during VFR travel, while pre-travel appointments tended to focus on infectious disease prevention. A greater emphasis on medication adherence and chronic disease management during VFR travel is also needed MLN0128 molecular weight during pre-travel preparations. International tourist arrivals were estimated to reach 1 billion for the first time in 2012, with nearly half

of all traveler arrivals in emerging economies.[1] In 2011, 46% of individuals traveling internationally by air from the United States were visiting friends and relatives (VFR) travelers.[2] Although the definition of VFR travelers varies throughout the literature, this term mafosfamide generally refers to immigrants currently residing in high-income countries returning to their homelands for a temporary visit, particularly when there is a gradient of epidemiologic risk between home and destination.[3] VFR travelers are generally considered to have higher travel-related health risks than tourists and business travelers. They typically have longer durations of travel, have more intimate contact with the host population, and travel to regions of the world with higher prevalence of communicable disease. They generally live and share meals with local hosts, with potentially greater exposure to unsafe food, water, and vector-borne diseases.

VFR travelers have been consistently found to experience an increased burden of travel-related infectious diseases including malaria, viral hepatitis, typhoid fever, and sexually transmitted infections relative to tourists and business travelers.[4-10] Unfortunately, VFR travelers may underestimate their travel-associated health risks and may be less likely to seek pre-travel health advice or be appropriately vaccinated prior to travel.[4-7, 9, 10] While the available literature demonstrates that VFR travelers have increased risk of travel-related infectious diseases relative to other travelers, little is known about the impact of VFR travel on chronic disease. Pre-travel health consultations often emphasize diarrhea prevention and treatment, vaccine-preventable diseases, and malaria prophylaxis.

We transiently expressed HopF1 in bean leaves using BPMV vector-m

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 learn more 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 PD-1 antibody inhibitor domain (BD)-fusion PtdIns(3,4)P2 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).

In patients dually infected with HIV-1 and HIV-2, HIV-1 may be co

In patients dually infected with HIV-1 and HIV-2, HIV-1 may be considered the dominant virus; however, careful consideration should be given when choosing treatment

for dual-infected patients to ensure activity against both viruses and to reduce the risk of drug resistance developing [47]. A small data series suggests that treatment of dual infection in this way can be effective [47,74,75]. Therapy should consist of two NRTIs and one AP24534 mouse or more PIs. World Health Organization guidelines suggest that three NRTIs may also be effective [76]; however, recent data from an observational study in Europe [77] showed an inferior CD4 cell response when treatment with three NRTIs was compared with a PI-based regimen, and therefore the preferred recommendation in this guideline is for treatment consisting of a combination of classes. Once therapy has been started, HIV-2 viral load should be periodically monitored. Patients treated successfully have so far been treated mainly with two NRTIs plus lopinavir/ritonavir 17-AAG or indinavir/ritonavir [35,36,62,74]. A good first-line regimen would be tenofovir/emtricitabine/boosted lopinavir, for which there are published data proving efficacy with a response rate of 60% out to 96 weeks, based on CD4 and HIV-2 RNA composite endpoints [62]. Truvada and

saquinavir (particularly with the development of V47A on failure of lopinavir) or darunavir in combination with raltegravir should be the preferred second-line therapy (see Table 2). It is important to note that there are few data on the outcome of second-line treatment in HIV-2 infection. Recent data, on two highly treatment-experienced patients only, showed a combination, selected based on RT and protease genotyping, of abacavir, tenofovir, darunavir and raltegravir to be very effective; however, this needs

to be evaluated in higher numbers of patients longer term [70]. There are not many NRTI choices available for second- and third-line therapy. Tenofovir or zidovudine must be used as the NRTI backbone with lamivudine or FTC in spite of the fact that an M184V mutation may be Nintedanib cell line present. The final choice will depend on whether Q151M and/or K65R has developed on treatment failure. The choice should ultimately be based on the genotypic resistance report, but one should always bear in mind that the interpretations of HIV-2 mutations are based on a few clinical cases and in vitro studies, and not on randomized controlled trials. The clinical efficacy of CCR5 inhibitors is still unknown, but they can be considered as part of a third-line regimen. It is unclear whether double-boosted PI regimens would be more efficacious, but at least for HIV-1 it has been shown that darunavir outperforms double-boosted PI regimens. Therefore, the current recommendation would be to use darunavir.

In a reciprocal manner, adipocytes and their precursors interact

In a reciprocal manner, adipocytes and their precursors interact with the immune system through the release of various cytokines, potentially linking fat and inflammation [2]. Interleukin-17A (IL-17A) is a recently discovered cytokine produced primarily in T-helper 17 (Th17) cells which play a role in a variety of inflammatory conditions [3] and HIV infection [4]. In adipose tissue, IL-17A is

an important regulator of adipogenesis in murine models, and in vitro it acts on preadipocytes and adipocytes to inhibit adipogenesis [5, 6]. However, the relevance of IL-17 to human obesity remains to be established. The pathway regulating the association between IL-17A and obesity remains controversial, and the association between Th17 cells and adipose tissue inflammation remains to be determined. There are no data on the role of IL-17A Navitoclax in adipogenesis or obesity in HIV-1-infected subjects. The aim of the study was to assess the correlation between IL-7A plasma level and visceral obesity in HIV-1-infected patients. Eighty-four patients between 18 and 70 years of age with a chronic HIV-1 infection, who had been

on highly active antiretroviral therapy (HAART) for more than 6 months, were consecutively recruited. An in-depth assessment was performed, including HIV disease history, duration of HAART and infection, viral load, metabolic parameters, BMI, abdominal waist circumference, smoking status and blood Protein Tyrosine Kinase inhibitor pressure. Subjects were excluded from participating if they had any of the following clinical conditions: active AIDS-defining illness, active drug abuse or alcohol abuse. HIV-1-infected patients were divided into two groups. The first group comprised patients with a diagnosis of visceral obesity. The second group included patients for whom a diagnosis of visceral obesity had been excluded. Forty-six subjects (23 with visceral obesity and 23 without) Tyrosine-protein kinase BLK negative for HIV infection were also selected to match HIV-positive patients in terms of age range and gender distribution as a control

group. The diagnosis of central obesity was confirmed by measurement of visceral fat thickness based on ultrasound measurement of the PRFD/BMI ratio according to previously published data [7-9]. For ultrasound measurement, a Logiq 5 ultrasound scanner (General Electric Medical Systems, Wallingford, CT) equipped with a 3.75-MHz convex probe was used. Sonographic evaluation of visceral obesity was performed by a single trained sonographer blinded to the patients’ data. For each subject, an aliquot of serum sample was collected and stored at −80°C. Serum IL-17 was measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Abingdon, UK) in duplicate, adding 100 μL of serum per well following the manufacturer’s recommendations.

In a reciprocal manner, adipocytes and their precursors interact

In a reciprocal manner, adipocytes and their precursors interact with the immune system through the release of various cytokines, potentially linking fat and inflammation [2]. Interleukin-17A (IL-17A) is a recently discovered cytokine produced primarily in T-helper 17 (Th17) cells which play a role in a variety of inflammatory conditions [3] and HIV infection [4]. In adipose tissue, IL-17A is

an important regulator of adipogenesis in murine models, and in vitro it acts on preadipocytes and adipocytes to inhibit adipogenesis [5, 6]. However, the relevance of IL-17 to human obesity remains to be established. The pathway regulating the association between IL-17A and obesity remains controversial, and the association between Th17 cells and adipose tissue inflammation remains to be determined. There are no data on the role of IL-17A click here in adipogenesis or obesity in HIV-1-infected subjects. The aim of the study was to assess the correlation between IL-7A plasma level and visceral obesity in HIV-1-infected patients. Eighty-four patients between 18 and 70 years of age with a chronic HIV-1 infection, who had been

on highly active antiretroviral therapy (HAART) for more than 6 months, were consecutively recruited. An in-depth assessment was performed, including HIV disease history, duration of HAART and infection, viral load, metabolic parameters, BMI, abdominal waist circumference, smoking status and blood Z-VAD-FMK solubility dmso pressure. Subjects were excluded from participating if they had any of the following clinical conditions: active AIDS-defining illness, active drug abuse or alcohol abuse. HIV-1-infected patients were divided into two groups. The first group comprised patients with a diagnosis of visceral obesity. The second group included patients for whom a diagnosis of visceral obesity had been excluded. Forty-six subjects (23 with visceral obesity and 23 without) Paclitaxel clinical trial negative for HIV infection were also selected to match HIV-positive patients in terms of age range and gender distribution as a control

group. The diagnosis of central obesity was confirmed by measurement of visceral fat thickness based on ultrasound measurement of the PRFD/BMI ratio according to previously published data [7-9]. For ultrasound measurement, a Logiq 5 ultrasound scanner (General Electric Medical Systems, Wallingford, CT) equipped with a 3.75-MHz convex probe was used. Sonographic evaluation of visceral obesity was performed by a single trained sonographer blinded to the patients’ data. For each subject, an aliquot of serum sample was collected and stored at −80°C. Serum IL-17 was measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Abingdon, UK) in duplicate, adding 100 μL of serum per well following the manufacturer’s recommendations.

When concentrations of morin exceeded 225 μM, biofilm biomass was

When concentrations of morin exceeded 225 μM, biofilm biomass was reduced by over 50%,

compared to the untreated control (Fig. 1) which was found to be statistically significant (P < 0.001). The reduction in biofilm biomass corresponded to a reduction in viable biofilm cells, from 3.2 × 107 CFU mL−1 (0 μM morin) to between 1.2 and 1.6 × 107 CFU mL−1 (225–300 μM morin). The effect of morin on aggregation of S. pyogenes was investigated using 0, 200, 225, 250, 275 and 300 μM morin. Aggregation was monitored over a period of 120 min; optical density was recorded at 30-min intervals (A650 nm). Morin facilitated bacterial aggregation, and the amount of aggregation was dose dependent (Fig. 2). Table 1 shows the percentage difference in aggregation between treated and untreated

samples. The extent of bacterial aggregation is demonstrated in Fig. 3, where a dense aggregate of cells was deposited Selleck Ibrutinib in the cuvette following treatment with 275 and 300 μM morin for 120 min (Fig. 3b and c, respectively). The TVC of these aggregated cells was determined, and treated cells showed a 14.6- and 18.3-fold decrease (275 and 300 μM morin, respectively) from 2.2 × 108 CFU mL−1 (0 μM morin) to 1.5 × 107 CFU mL−1 (275 μM morin) and 1.2 × 107 CFU mL−1 (300 μM morin). Statistical analysis (anova, minitab v14) demonstrated that following 10-min incubation of the test organism with 250, 275 and 300 μM morin, and aggregation was significantly higher (P < 0.05) than PRKD3 in the untreated culture. Cells treated with 200 and 225 μM did not show a significant increase (P > 0.05) PLX4032 datasheet over the same period of time, but after 20-min incubation at all concentrations, aggregation was significantly increased when compared to the untreated control. Streptoccocal biofilms are associated with persistant infections (Costerton et al., 1999; Donlan, 2001) and are known to exhibit antibiotic resistance (Baldassarri et al., 2006). Flavonols inhibit bacterial growth and have been demonstrated to possess an ‘anti-plaque’ activity, disrupting both the growth and adhesion of Streptococcus mutans (Duarte et al.,

2006; Prabu et al., 2006; Shure et al., 2006; Gregoire et al., 2007; Escaich, 2010). This study demonstrated that the flavonol morin significantly decreased biofilm biomass (P < 0.001) at concentrations of 225 μM and above resulting in up to 65% reductions. The data presented here also demonstrated that morin facilitated rapid, statistically significant (P < 0.05) aggregation of planktonic S. pyogenes in a dose-dependent manner. Streptococcus pyogenes are known to form cellular aggregates ordinarily over time; however, morin appeared to enhance this process (Frick et al., 2000; Collado et al., 2008; Maddocks et al., 2011). Numerous host proteins, including the salivary glycoprotein gp340, are known to facilitate the rapid aggregation of streptococci and as such these are regarded as being components of the innate immune response (Golub et al.

This step was repeated, and the filters were then inverted and ce

This step was repeated, and the filters were then inverted and centrifuged (at 1000 g and 37 °C for 3 min) to remove excess water. Patient plasma (500 μL) was then injected and the devices centrifuged (at 1500 g and 37 °C for 60 min). The resultant ultrafiltrate (∼170 μL per sample) was retained for drug analysis. The percentage recovery of LPV using this technique was assessed using drug-free ultrafiltrate Rapamycin clinical trial spiked with

14C-LPV, and was [mean (standard deviation)] 69% (± 4.1%) and constant over a range of LPV concentrations (1000, 5000, 10 000 and 15 000 ng/mL); thus no correction to unbound concentrations was applied, consistent with other plasma protein-binding studies [22–27]. All demographic and clinical

characteristics are given as the median (range). LPV and RTV trough concentrations (Ctrough) are expressed in terms of the geometric mean with 95% confidence intervals (CIs). Inter-subject variation in plasma concentrations was estimated using a coefficient of variation, expressed as a percentage [%CV=(standard deviation/mean) × 100]. The fraction of unbound LPV in plasma (fu), expressed as a percentage, was determined by: fu%=(unbound Ctrough/total Ctrough) × 100. The minimum effective concentration (MEC) for LPV was defined as 1000 ng/mL [28]. In addition, a predefined cut-off for nonadherence was proposed based on data from a healthy volunteer study assessing the decline in LPV over 72 h after drug cessation click here [29]. For an LPV/r twice daily regimen, LPV plasma concentrations were approximately (geometric mean; n=16) 384 ng/mL in the case of a single missed dose (24 h post drug cessation) and<10 ng/mL following two or more missed doses

(36–48 h post drug cessation). Thus we assumed plasma concentrations of <384 ng/mL to be indicative of noncompliance and requiring further verification by study personnel and excluded these values from subsequent statistical analyses. Although there are reported differences in antiretroviral for concentrations between healthy subjects and HIV-infected patients, no such relationship has been demonstrated for LPV/r [30], and hence in the current analysis comparison of the two populations was considered justifiable. Differences in pharmacokinetic data antepartum vs. postpartum were assessed independently using a one-way analysis of variance (anova), with a Bonferroni correction to test for multiple comparisons. Normality of data was assessed using a Shapiro–Wilk test, and non-normally distributed data were log-transformed. Additionally, patients with matched third trimester and postpartum samples were compared by means of a paired t-test. All statistics were performed and analysed using Arcus Quickstat (version 1.1©1997; Biomedical Software, Statsdirect Ltd, Cheshire, UK). P-values are two-sided at the 0.05 significance level.

The cyanobacterium Nostoc punctiforme ATCC 29133 (N punctiforme)

The cyanobacterium Nostoc punctiforme ATCC 29133 (N. punctiforme) harbours two enzymes directly involved in production and consumption of molecular hydrogen: a nitrogenase and an uptake hydrogenase (Tamagnini

et al., 2002, 2007). The nitrogenase enzyme produces H2 as a byproduct during fixation of atmospheric N2 (Rees & Howard, 2000). Nitrogenases are oxygen sensitive, and in cyanobacteria of the genus Nostoc the enzyme is localized to heterocysts, specialized cells with a microaerobic environment due to the lack of O2-evolving activity of photosystem II, a high respiration rate, and a thick glycolipid envelope layer that reduces the flux of O2 (Flores & Herrero, 2010). The uptake hydrogenase catalyses the reoxidation of H2 formed by the nitrogenase, and thus recaptures GDC-0941 molecular weight the electrons from H2. The presence of the uptake hydrogenase is tightly connected to nitrogen fixation and all filamentous N2-fixing cyanobacteria contain an uptake hydrogenase (Ludwig et al., 2006). Upon deprivation of combined nitrogen, approximately every 10th–20th cell, evenly distributed in a filament of LY294002 in vitro Nostoc, differentiates into a heterocyst. During the heterocyst development, the transcription of the nif genes, encoding the nitrogenase, and the hup genes,

encoding the uptake hydrogenase, take place (Elhai & Wolk, 1990; Axelsson et al., 1999; Holmqvist, 2010). The uptake hydrogenase in cyanobacteria consists of a small (HupS) and a large (HupL) subunit, encoded by hupS and hupL, respectively. hupS and hupL are located in an operon, and transcribed as a single unit (Lindberg et al., 2000). The cellular

localization of the uptake hydrogenase in N2-fixing heterocyst-forming cyanobacteria is still not definite. Early work showed that in an aerobically grown culture of Nostoc PCC 7120, the activity of uptake hydrogenase is localized solely to the heterocysts (Peterson & Wolk, 1978; Houchins & Burris, 1981). This is in agreement with recent immunolocalization investigations where HupL was solely detected in the heterocysts of Nostoc PCC 7120 (Seabra et al., 2009). In Nostoc Rucaparib order PCC 7120, the expression of the hupSL is controlled by the removal of an excision element in hupL during heterocyst differentiation, which allows for a functional transcript only in the heterocyst (Carrasco et al., 1995, 2005). However, in N. punctiforme, no such rearrangement takes place (Oxelfelt et al., 1998) and immunolocalization studies have reported that HupL may be present in both heterocysts and vegetative cells (Lindblad & Sellstedt, 1990; Seabra et al., 2009). Nonetheless, as determined by investigation of the promoter activity of hupSL with a promoter-green fluorescent protein (GFP) construct, the transcription of hupSL takes place solely in the heterocysts (Holmqvist et al., 2009). The subcellular localization of the uptake hydrogenase is not fully resolved.

Sera from 42 patients allergic to A alternata (18 female and 24

Sera from 42 patients allergic to A. alternata (18 female and 24 male; mean age, 20.5 years; age range, 10–33 years) and 17 control subjects (11 female and six male; mean age, 32.3 years; age range, 14–70 years) were included in the study. Diagnosis of A. alternata allergy was based on a clinical history of recurrent rhinitis (four patients), asthma (four patients), rhinoconjunctivitis (12 patients), rhinitis and asthma (12 patients), rhinoconjunctivitis and asthma (10 patients); a positive cutaneous response to a commercial A. alternata extract (Bial-Arístegui, Bilbao, Spain); and specific IgE to A. alternata extract > 0.35 IU mL−1 according ImmunoCAP (Thermo-Fisher,

Uppsala, Sweden). Eight SAHA HDAC nmr healthy subjects and nine allergic individuals AZD0530 chemical structure sensitized to different allergenic sources unrelated

to A. alternata, as demonstrated by negative SPT responses and lack of A. alternata-specific IgE, were used as controls. Standard molecular genetic techniques were used (Sambrook et al., 1989). For Southern blotting, genomic DNA was prepared from recombinant yeasts as previously described (Barth & Gaillardin, 1996). Afterwards, DNA was digested, separated on a 0.8% agarose gel, and transferred onto Hybond-N+ nylon membranes (GE-Healthcare, Little Chalfont, Buck, UK). Probes were labeled with [32P]-dCTP using the MegaPrime Kit (GE-Healthcare). The autosomal vector pMM4 was used to express the Alt a 1 allergen. The YlMETII promoter was obtained from plasmid pSG70 (García, 1993; Domínguez et al., 2003) and cloned between the EcoRI-BamHI restriction sites of the pBluescript-SK. The Alt a 1-coding gene sequence (Asturias et al., 2003) Epothilone B (EPO906, Patupilone) was cloned after this promoter into the BamHI site, resulting in the pMMR2 vector. As the insertion of the target gene between yeast promoter and terminator sequences produces more efficient expression of heterologous genes in Y. lipolytica (Franke et al., 1998), the YlSTE7 terminator was amplified using specific primers and cloned into the SpeI and XbaI-restriction sites of pMMR2, resulting in the pMMR3 plasmid. This plasmid

was digested with ClaI and XbaI and the 1.8 kb-fragment containing the fusion of the YlMTPII promoter-Alt a 1-YlSTE7-terminator was purified and inserted into the pINA240 plasmid (Barth & Gaillardin, 1996), giving rise to pMMR4. The correct construction was verified by sequencing. The construction of the integrative plasmid pMMR10 was performed as follows. To create the pMMR10 plasmid, the 1.8-kb ClaI-SphI fragment from pMMR4 carrying the YlMTPII promoter-Alt a 1-YlSTE7-terminator fusion was cloned into the pINA62 plasmid. The correct construction was verified by sequencing. Plasmid maps of pMMR4 and pMMR10 and sequences of the specific primers used for YlSTE7 terminator amplification are available as Supporting Information (Fig. S1, Table S1). nAlt a 1 was purified from A. alternata CBS 603.78 spent culture medium after 3 weeks of static growth in Czapeck broth at 25 °C.