However, the outcome of HIV patients with HL has dramatically improved after the introduction of HAART; the CR rate, OS and disease-free survival (DFS) approach those seen in the general population [17–19]. The diagnosis of HL, as that of any other lymphoid malignancy, should be based on a tissue sample biopsy, rather than on a cytological sample. Samples should be stained for CD20, CD3, CD15, CD30, BCL-2 and LMP-1 proteins. Following the confirmation of diagnosis, patients should undergo a series of investigations

(which include blood tests, whole body FDG-PET/CT scan and unilateral bone marrow biopsy) to assess the extension of the disease (see Table 10.1). Whereas a bone marrow biopsy is not necessary in all HIV-negative patients with HL, the higher proportion of bone marrow involvement in the HIV population [9,15] makes it mandatory. The above-mentioned investigations allow staging of the disease CHIR99021 according to the Ann Arbor classification/Cotswolds modification [20] (see Table 10.2). A prognostic score, which predicts both freedom from progression (FFP) and OS, has been defined for HIV-negative patients with advanced HL at diagnosis [21] (see

Table 10.3). The applicability of the International Prognostic Score (IPS) in HIV patients was reported in a series of patients treated with Stanford V chemotherapy, in which Akt inhibitor the IPS was the only variable predictive for OS in the multivariate analysis. The IPS also predicted for FFP and CR rate [22]. Other prognostic markers that have been reported to have an impact Ureohydrolase on the outcome of HIV-HL patients include some predictive factors related to characteristics of the lymphoma, such as age, stage and responsiveness to therapy [12,23] and others associated with the HIV infection and/or its treatment [12,16,23–25]. Histological subtypes have

been associated with prognosis in the HIV population in some studies [24] but not in others [23]. Despite the reduction in the incidence of ADMs since the advent of HAART, several large cohort studies have shown no fall in incidence rates of HL pre- and post-HAART [26–28], with some studies even showing increased incidence rates of HL immediately post HAART initiation [29]. The relationship between the incidence of HL and CD4 cell counts is complex. HL occurs most commonly at CD4 cell counts below 200 cells/μL [17,30]. However, there is ongoing risk of developing HL while on HAART despite an adequate CD4 cell count [26–28,30,31]. Furthermore, HL incidence rates are actually higher in the first few months after starting HAART [30–32]. Several cohort studies have also shown that drops in the CD4 cell count or CD4:CD8 ratio in the year prior to HL diagnosis may herald the advent of disease [27,28]. In contrast, viral load has not been shown to relate to incidence rates [26,30,31].

All stool samples were negative for enterovirus and poliovirus. One of the liquid samples analyzed was positive for Coxsackievirus type B5. The Global Polio Eradication Initiative was launched in 1988. Ten years ago all seemed to be

going well with poliomyelitis eradication. The number of polio cases globally had dropped by 99% from an estimated 350,000 in 1988 to fewer than 500 in 2001.1 However, polio is still endemic in four countries (Afghanistan, India, INCB018424 clinical trial Nigeria, and Pakistan) and many previously polio-free countries experienced outbreaks following importation of indigenous wild poliovirus (WPV). In 2010, 1,349 WPV cases were reported, with a large outbreak in Tajikistan. This was the first outbreak of polio since the WHO European Region was certified polio-free in 2002.2 In Italy, there is an increased and continuous inflow of refugees from countries where poliomyelitis is still present, and this may represent a risk of the WPV strains being

introduced. The Italian region of Puglia (Southern Italy) can be deemed a “border region” because, due to its geographic position, it has to face daily arrivals of refugees. In recent months, following Ku-0059436 mouse the wars that have arisen in many countries in the region of North Africa, the flow of refugees seeking political asylum in Italy has intensified. The increased and continuous flow of refugees from countries where poliomyelitis is still present may represent a risk of the WPV strains being introduced in Italy. The aim of this study was to evaluate the presence of WPV or sabin-like poliovirus in stool samples taken from migrants housed in the Accommodation Center in Bari Palese (Puglia, Italy) and liquid waste taken from the sewage systems of the migrant housing units. Two surveys were carried out in September 2008 and March 2011, respectively, during which stool samples were collected from migrants on a voluntary basis. Following the explanation of the study given by cultural mediators and the signing of informed

consent written in the participant’s native language, containers to collect stools Dichloromethane dehalogenase were distributed from the Accommodation Center to migrants present. The migrants were invited to hand their fecal samples in to the center’s outpatient clinic the following morning. A total of 76 stool samples were collected in the survey conducted in 2008, and 76 stool samples were collected in 2011, respectively, of which 11 samples (5 in the 2008 survey and 6 in 2011) belonged to female subjects. The mean age of participants was 20.8 ± 5.5 y for the survey conducted in 2008 and 23.5 ± 6.3 y in 2011. Table 1 shows the subdivision of samples according to the migrant’s country of origin; 30% of migrants analyzed came from countries where polio is still endemic (Afghanistan, Nigeria, and Pakistan). Fecal samples were analyzed for the presence of enterovirus by nested-polymerase chain reaction (PCR).

The treatment of Xcc cultures with 50 mM H2O2 for 30 min resulted in approximately 10% survival (Fig. 2). The addition of CuSO4 (100 μM) to the H2O2 killing mixture was highly lethal to cells and reduced the per cent survival to 0.05% (Fig. 2). The synergistic

effect of CuSO4 and H2O2 was abolished when a Cu chelator (200 μM bathocuproine sulphonate) was added to the cell suspension before the combined treatment of CuSO4 and H2O2 (Fig. 2). This observation suggests the possibility that an elevated level of Cu ions could react with H2O2 to produce hydroxyl radicals, which lead to increased cell death. This speculation was supported by experiments in which the addition of hydroxyl scavengers DMSO (0.4 M) and glycerol (1.0 M) to bacterial cultures, before treatment with CuSO4 and H2O2, significantly protected bacterial cells from the killing effects (Fig. 2). We Bafilomycin A1 molecular weight then determined whether lipid peroxidation contributes to CuSO4 and H2O2 toxicity. The ability of α-tocopherol (1 mM) to reduce the lethal effects of CuSO4 and H2O2 treatment was tested. As illustrated in Fig. 2, α-tocopherol was unable to alleviate CuSO4 and H2O2 Angiogenesis inhibitor killing. The evidence indicates that Cu ions potentiate H2O2 toxicity in a manner different

from tBOOH. While lipid peroxidation is a major factor responsible for the Cu ion-mediated enhancement of tBOOH toxicity, hydroxyl radicals likely account for Cu ion-dependent H2O2 toxicity. Alkyl hydroperoxide reductase, encoded by ahpC, is a member of the peroxiredoxin enzyme family. AhpC not only plays a role in the detoxification of organic hydroperoxides by converting them to their corresponding alcohols, but the enzyme is also necessary for the degradation of endogenously generated H2O2 due to its much lower kcat/Km Ribose-5-phosphate isomerase compared with catalase (Seaver & Imlay, 2001). Thus, the ahpC mutant accumulates intracellular H2O2 and organic

hydroperoxides produced as byproducts of normal aerobic metabolism (Seaver & Imlay, 2001; Charoenlap et al., 2005; Wang et al., 2006). If Cu toxicity is partly due to the stimulation of oxidative stress production, we would expect that the Cu resistance level in the ahpC mutant might be altered. An Xcc ahpC mutant was constructed using the pKNOCK system (Alexeyev, 1999). The ahpC mutant was more sensitive to tBOOH killing treatment than the wild-type Xcc (data not shown). The Cu resistance of the ahpC mutant was measured using a killing assay (Sukchawalit et al., 2005), and the results showed that the mutant was more than 10-fold more sensitive to CuSO4 (1 mM) than the wild-type Xcc (Fig. 3). The ectopic expression of ahpC from the expression plasmid, pAhpC, complemented the CuSO4-sensitive phenotype of the ahpC mutant (Fig. 3, ahpC/pAhpC). The lack of a functional ahpC rendered Xcc vulnerable to elevated levels of CuSO4.

, 2010). Therefore, the Treponema group may be one of the core members of the rumen bacterial community. The proportion of T. bryantii

was about 2% in the Treponema group (0.02% vs. 1.05%), indicating that the uncultured Treponema were more abundant than cultured representatives. Analysis of the Treponema 16S rRNA gene libraries supported this finding. Although a single sequence was identified as T. zioleckii in the present study, no 16S rRNA gene sequence having 97% or more similarity with T. sacchrophilum and T. zioleckii was reported in previous studies (Whitford et al., 1998; Tajima et al., 1999; Koike et al., 2003). Therefore, T. sacchrophilum and T. zioleckii appear to be minor bacterial species in the rumen. Sequence analysis of 16S TGF-beta inhibitor rRNA gene clone libraries constructed in this study for rumen Treponema revealed the presence of phylogenetically diverse and previously undetected OTUs of the rumen Treponema community. The DGGE data further showed diverse bands in the animals fed alfalfa and orchardgrass hay. This finding corresponded to the diversity analysis of the libraries, which showed higher Shannon index diversity values for the hay diets.

A plausible explanation for this finding LBH589 manufacturer would be that more diverse members of Treponema are involved in the degradation of hay diets. Considering the higher percentage (91.1%) of Good’s Cyclooxygenase (COX) coverage for the combined library, our library was comprehensive and likely represented the majority of Treponema in the sheep rumen. It has been suggested that a group-specific clone library approach could identify more diverse members in the target group than a universal library analysis (Hayashi et al., 2006). In human gut studies, attempts to recover diverse members of Bacteroides spp. by increasing the size of libraries constructed by universal primers did not result in a higher diversity of Bacteroides (Li et al., 2008). Preferential PCR

amplification of certain groups of rumen microorganisms has been suggested as a possible reason for the difficulty in detecting a particular group with universal primers (Tajima et al., 2001), and this may explain the low level detection of Treponema sequences in previous studies (Whitford et al., 1998; Tajima et al., 1999; Ozutsumi et al., 2005). Therefore, the group-specific clone library approach that we followed in this study proved useful to obtain a comprehensive description of the diversity of Treponema in the rumen. Phylogenetic analysis of the Treponema 16S rRNA gene sequences showed a closer phylogeny of clones retrieved from a particular diet. In the phylogenetic tree, clade I was mainly comprised of clones (58.4% of the overall concentrate clones) associated with concentrate feeding; while clade II predominantly consisted of Treponema clones (87.3% of the overall hay clones) associated with hay feeding.

The ROC curve analysis showed the accuracy of EBV load in PBMC1 for predicting progression to B lymphoma: the area under the ROC curve was

0.72 (95% CI 0.58–085; http://www.selleckchem.com/Androgen-Receptor.html P = 0.0014). The optimal cut-off threshold of EBV load that yielded the maximal sensitivity and specificity for predicting the development of B lymphoma was determined as 3.2 log10 copies/106 PBMCs. The sensitivity and specificity obtained with this cut-off value were 75 and 65%, respectively. Setting the cut-off level at 2.8 log10 copies/106 PBMCs allowed a higher sensitivity (85%) to be obtained, but at the price of a substantial decrease in specificity (40.5%). Having an EBV DNA load > 3.2 log10 copies/106 PBMCs was associated with an adjusted OR of 4.82 (95% CI 1.33; 17.46) for progression to B lymphoma within the next 10 months. EBV load in PBMCs had, as expected, poor accuracy

for identifying patients at risk for developing brain lymphoma (area under ROC curve 0.57; 95% CI 0.38; 0.76; P = 0.5). In this study, high levels of EBV DNA in PBMCs were predictive of subsequent progression to systemic B lymphoma when measured within 1 year before the diagnosis of lymphoma. One previous study failed to demonstrate such Selleck Erlotinib an association between EBV DNA level and the development of ARL [16]. This could be explained by the fact that, in this previous study, patients and controls were not matched for CD4 cell count and also by the fact that a limited number of cases (n = 9) and controls (n = 12) were tested. As expected, we found no association between EBV DNA levels in PBMCs and/or

in serum and the development of cerebral lymphoma. The lack of sensitivity of EBV PCR in blood Methocarbamol for the diagnosis of cerebral lymphoma in patients with AIDS was demonstrated by Fan et al. [19], although there was a correlation between PBL and high levels of EBV DNA in cerebrospinal fluid [20]. This is likely to be attributable to the fact that PBLs are very compartmentalized tumours with low or no systemic dissemination. This result, however, has no major implications in clinical practice, as the incidence of PBL has dramatically fallen following the introduction of cART, PBL now representing less than 10% of all ARLs [7]. Indeed, no case of PBL was reported after 1995 in our cohorts. In this study, EBV DNA was quantified in stored samples (i.e. mainly cryopreserved PBMCs and serum samples). The choice of biological material used for EBV DNA quantification, i.e. PBMCs, whole blood, plasma or serum, has been a matter for debate and there is currently no consensus on which is the best material for EBV load measurement in EBV-associated lymphoproliferative disorders [21, 22]. In these settings, whole blood or PBMCs more frequently contained amplifiable EBV DNA than did plasma or serum [15, 23-25].

The ROC curve analysis showed the accuracy of EBV load in PBMC1 for predicting progression to B lymphoma: the area under the ROC curve was

0.72 (95% CI 0.58–085; find more P = 0.0014). The optimal cut-off threshold of EBV load that yielded the maximal sensitivity and specificity for predicting the development of B lymphoma was determined as 3.2 log10 copies/106 PBMCs. The sensitivity and specificity obtained with this cut-off value were 75 and 65%, respectively. Setting the cut-off level at 2.8 log10 copies/106 PBMCs allowed a higher sensitivity (85%) to be obtained, but at the price of a substantial decrease in specificity (40.5%). Having an EBV DNA load > 3.2 log10 copies/106 PBMCs was associated with an adjusted OR of 4.82 (95% CI 1.33; 17.46) for progression to B lymphoma within the next 10 months. EBV load in PBMCs had, as expected, poor accuracy

for identifying patients at risk for developing brain lymphoma (area under ROC curve 0.57; 95% CI 0.38; 0.76; P = 0.5). In this study, high levels of EBV DNA in PBMCs were predictive of subsequent progression to systemic B lymphoma when measured within 1 year before the diagnosis of lymphoma. One previous study failed to demonstrate such buy TSA HDAC an association between EBV DNA level and the development of ARL [16]. This could be explained by the fact that, in this previous study, patients and controls were not matched for CD4 cell count and also by the fact that a limited number of cases (n = 9) and controls (n = 12) were tested. As expected, we found no association between EBV DNA levels in PBMCs and/or

in serum and the development of cerebral lymphoma. The lack of sensitivity of EBV PCR in blood however for the diagnosis of cerebral lymphoma in patients with AIDS was demonstrated by Fan et al. [19], although there was a correlation between PBL and high levels of EBV DNA in cerebrospinal fluid [20]. This is likely to be attributable to the fact that PBLs are very compartmentalized tumours with low or no systemic dissemination. This result, however, has no major implications in clinical practice, as the incidence of PBL has dramatically fallen following the introduction of cART, PBL now representing less than 10% of all ARLs [7]. Indeed, no case of PBL was reported after 1995 in our cohorts. In this study, EBV DNA was quantified in stored samples (i.e. mainly cryopreserved PBMCs and serum samples). The choice of biological material used for EBV DNA quantification, i.e. PBMCs, whole blood, plasma or serum, has been a matter for debate and there is currently no consensus on which is the best material for EBV load measurement in EBV-associated lymphoproliferative disorders [21, 22]. In these settings, whole blood or PBMCs more frequently contained amplifiable EBV DNA than did plasma or serum [15, 23-25].

The ROC curve analysis showed the accuracy of EBV load in PBMC1 for predicting progression to B lymphoma: the area under the ROC curve was

0.72 (95% CI 0.58–085; click here P = 0.0014). The optimal cut-off threshold of EBV load that yielded the maximal sensitivity and specificity for predicting the development of B lymphoma was determined as 3.2 log10 copies/106 PBMCs. The sensitivity and specificity obtained with this cut-off value were 75 and 65%, respectively. Setting the cut-off level at 2.8 log10 copies/106 PBMCs allowed a higher sensitivity (85%) to be obtained, but at the price of a substantial decrease in specificity (40.5%). Having an EBV DNA load > 3.2 log10 copies/106 PBMCs was associated with an adjusted OR of 4.82 (95% CI 1.33; 17.46) for progression to B lymphoma within the next 10 months. EBV load in PBMCs had, as expected, poor accuracy

for identifying patients at risk for developing brain lymphoma (area under ROC curve 0.57; 95% CI 0.38; 0.76; P = 0.5). In this study, high levels of EBV DNA in PBMCs were predictive of subsequent progression to systemic B lymphoma when measured within 1 year before the diagnosis of lymphoma. One previous study failed to demonstrate such IDO inhibitor an association between EBV DNA level and the development of ARL [16]. This could be explained by the fact that, in this previous study, patients and controls were not matched for CD4 cell count and also by the fact that a limited number of cases (n = 9) and controls (n = 12) were tested. As expected, we found no association between EBV DNA levels in PBMCs and/or

in serum and the development of cerebral lymphoma. The lack of sensitivity of EBV PCR in blood Ribonucleotide reductase for the diagnosis of cerebral lymphoma in patients with AIDS was demonstrated by Fan et al. [19], although there was a correlation between PBL and high levels of EBV DNA in cerebrospinal fluid [20]. This is likely to be attributable to the fact that PBLs are very compartmentalized tumours with low or no systemic dissemination. This result, however, has no major implications in clinical practice, as the incidence of PBL has dramatically fallen following the introduction of cART, PBL now representing less than 10% of all ARLs [7]. Indeed, no case of PBL was reported after 1995 in our cohorts. In this study, EBV DNA was quantified in stored samples (i.e. mainly cryopreserved PBMCs and serum samples). The choice of biological material used for EBV DNA quantification, i.e. PBMCs, whole blood, plasma or serum, has been a matter for debate and there is currently no consensus on which is the best material for EBV load measurement in EBV-associated lymphoproliferative disorders [21, 22]. In these settings, whole blood or PBMCs more frequently contained amplifiable EBV DNA than did plasma or serum [15, 23-25].

A qualitative approach was adopted on the basis of being well-suited to exploring the range and depth of participants’ perspectives.2 Following institutional ethical approval, in-depth digitally recorded interviews were conducted with 18 staff (9 pharmacists, 8 HLCs and 1 technician) from HLPs in Staffordshire. The sample included participants

from HLPs of different check details types (e.g. independents and branches of multiple chains) and locations to represent a broad range of views. Participants were recruited by sending an invitation letter to HLPs followed by telephone contact. The interview guide was developed from the objectives of the study and a review of the literature. Key topics included reasons for choosing to become a HLP, experiences of the process of their pharmacy achieving HLP status and experiences of providing public health services from their HLP. Interviews were transcribed verbatim and analysed using framework analysis.2

Reported reasons for pharmacies becoming HLPs were business-related, professional standing-related or altruistic. Business-related reasons included viewing HLP status as RG7204 order the ‘way forward’, an opportunity to ‘set ourselves aside from non-HLPs’, but also concerns of not being commissioned to provide future enhanced services if they did not become a HLP. Professional standing-related reasons included increased local recognition for health service provision, whilst altruistic reasons included ‘giving something back to the local community’. Participants reported that the HLC training had increased their confidence in talking to customers Acyl CoA dehydrogenase about sensitive lifestyle issues, but had been time consuming. Other barriers included training sufficient members of staff. Some participants also reported receiving little support. The time to

achieve accreditation ranged from 4 to 12 months. All participants seemed enthusiastic about the HLP initiative and most reported increases in the services provided and service users, especially of the smoking cessation service. Service users’ feedback was reported as being generally positive, although participants commonly also reported most customers appearing unaware of the pharmacy’s HLP status. Participants gave examples of new contacts established with local organisations providing health promotion, but reported observing little evidence of GP surgeries signposting patients to HLP services. Reported difficulties included time constraints, increased workload and cost. Several participants reported that the initiative might benefit from greater local publicity of the HLP brand and more synchronisation of health promotion campaign activity between HLPs. The findings suggest that the initiative has been beneficial for HLP customers and staff, despite difficulties in gaining accreditation and providing services.

Moreover, we observed upregulation of sae in the agr mutant and upregulation

of agr in the sae mutant compared with the isogenic Newman strain, suggesting that the Agr and Sae may be inhibiting each other. The SigB mutation did not affect ssl5 and ssl8 expression, but they were downregulated in the agr/sigB double mutant, indicating that SigB probably acts synergistically with Agr in their upregulation. Staphylococcus aureus is a significant human pathogen capable of causing a variety of diseases ranging from mild skin and soft tissue infections to bacteremia, pneumonia, endocarditis, and osteomyelitis Everolimus mw (Lowy, 1998). The ability of S. aureus to cause a wide range of infections is partly due to the expression of a wide array of virulence factors including, but not limited to, cell wall-associated adhesions, clumping factors, exotoxins, and secreted proteins such as staphylococcal superantigen-like (SSL) proteins (Lowy, 1998; Dinges et al., 2000; Williams et al., 2000; Fitzgerald et al., 2003). The SSL proteins are encoded by a cluster of 11 ssl genes located on S. aureus pathogenicity island-2 (Fitzgerald et al., 2003). These proteins have limited sequence homology to the enterotoxins and toxic shock syndrome toxin 1 and thus represent

a novel family of exotoxin-like selleck chemicals proteins (Williams et al., 2000). The overall order of ssl genes on an S. aureus chromosome is conserved, and allelic forms of individual ssl genes have been identified in different strains. The sequence homology

for individual ssl genes ranges from 85% to 100% in different strains. However, 11 ssl genes within a strain have sequence homology from 36% to 67%, suggesting possible selective pressures encountered during infection (Kuroda et al., 2001; Smyth et al., 2007). Every strain of S. aureus examined so far carries a cluster of at least seven of the 11 ssl genes, suggesting that they probably have distinct and possibly nonredundant Selleckchem Abiraterone functions (Arcus et al., 2002; Fitzgerald et al., 2003; Smyth et al., 2007). Expression studies of a family of ssl genes in COL, an early methicillin-resistant S. aureus (MRSA) strain, showed that they are upregulated during the stationary phase like other exotoxin genes (Fitzgerald et al., 2003). SSL5 and SSL11 show high structural homology with the chemotaxis inhibitory protein of S. aureus and have been shown to interfere with the interaction between P-selectin glycoprotein ligand-1 and P-selectin, suggesting that S. aureus uses SSL proteins to prevent neutrophil recruitment towards the site of infection (Bestebroer et al., 2007; Chung et al., 2007). The same binding site was also found in SSL2, SSL3, SSL4, and SSL6 (Baker et al., 2007). SSL7 and SSL9 interact with two separate cell surface ligands of human antigen-presenting cells (monocytes and dendritic cells), leading to internalization by these cells, and may thus play a role in the modulation of host immunity against S.

coli CC118λpir (Manoil & Beckwith, 1985). After verification by sequencing, they were transferred to E. coli SM10λpir (Miller & Mekalanos, 1988) for mating. EDL933 NalR (spontaneous mutation) and the respective SM10λpir plus the modified pMRS101 were mixed and plated on LB-agar (24 h, 30 °C). Cells were resuspended again and plated on LB-agar with 30 μg mL−1 streptomycin and 20 μg mL−1 nalidic acid. Correct plasmid integration after a first cross-over was checked by PCR. Second cross-over

events, resulting in plasmid loss, either restore wild type or create the mutation. Thus, bacteria were grown without selection to OD600 nm = 0.8 and plated on LB-agar without Fulvestrant datasheet NaCl plus 10% sucrose for sacB-counter

selection. Desired mutants were identified using PCR. Biofilm experiments were conducted according to Domka et al. (2007). A culture grown in M9 minimal medium (Sambrook & Russel 2001) was diluted to OD600 nm = 0.05. Flat-bottom wells of a microtiter plate (Greiner Bio One, Germany) were filled with 100 μL and incubated 24 or 48 h without shaking at 30 °C or 37 °C. OD600 nm was measured (Victor3). The planktonic cells were removed, and each well was carefully washed with water. Staining was achieved using 135 μL 0.1% crystal violet (20 min, RT). After washing thrice with water and air drying, the stain was solubilized in 95% ethanol, transferred to a new plate and the absorbance at 600 nm was measured Rapamycin order (Victor3). The mean was calculated (10 wells, three biological replicates) after

subtracting zero controls (medium only). Amplicons of htgA and yaaW were cloned into pBAD/Myc-His C (Invitrogen). EHEC with plasmids (sequenced for verification) were grown in LB with 100 μg mL−1 ampicillin and induced with 0.2% arabinose. Proteins were purified according to QIAexpress® Ni-NTA Fast-Start kit under denaturing conditions (Qiagen). For this, the bacteria were sonicated in the provided lysis buffer. For SDS-PAGE (15%), Laemmli-buffer was added, and the sample denatured for 5 min at 95 °C. PageRuler Protein Ladder (Fermentas) was used as marker. After electrophoresis, Mannose-binding protein-associated serine protease the proteins were electroblotted (20 min, 120 mA) to an activated PVDF membrane (Amersham). Subsequently, the membrane was blocked, incubated with mouse-anti-human c-myc-antibodies (BD Biosciences), washed, incubated with alkaline phosphatase anti-mouse chimera antibodies (Dianova, Hamburg), washed again, equilibrated and incubated in buffer supplemented with BCIP/NBT. Metabolites were profiled using Ion cyclotron resonance Fourier transform Mass spectrometry (ICR-FT/MS) on a Bruker solariX with a 12-T magnet (Bruker Daltonics, Bremen). Three biological replicate cultures of wild type, ΔhtgA, and ΔyaaW were grown shaking in 1 : 2-diluted LB to OD600 nm = 1. Cultures were vacuum filtered using HVLP filters (0.45 μm; Millipore).