Of these, 34 withdrew consent, 7 were lost to follow-up, 2 reported scheduling difficulties, and 1 subject (assigned to 2,700 mg/day) discontinued because of an adverse effect (diarrhea). Dropout rates did not differ significantly by treatment group (p = .91). Of the 44 participants discontinuing during MEK162 ARRY-162 the medication phase, 6 (1, 1, and 4 receiving placebo; 1,800; and 2,700 mg/day, respectively) did not attend any study visits after starting medication. Among the remaining participants, there were 4 (2, 2, 0), 7 (4, 2, 1), 6 (2, 3, 1), 8 (2, 5, 1), 5 (1, 0, 4), 3 (1, 0, 2), 4 (2, 0, 2), and 1 (0, 1, 0) whose last attended visit was after 1, 2, 3, 4, 5, 6, 8, and 10 weeks of medication, respectively. Of those who discontinued during the medication phase, 91% (40/44) reported smoking at the last study visit they attended (15/15 vs.

12/14 vs. 13/15 for placebo; 1,800; and 2,700 mg/day, respectively; Fisher��s exact test p = .44). Adverse effects Adverse events considered possibly, probably, or definitely related to study drug did not differ significantly across groups. The specific adverse events and number of subjects reporting these events in each dose group (placebo; 1,800; and 2,700 mg/day) included dizziness (1, 4, 4), decreased concentration (0, 3, 0), edema (1, 2, 0), sleep disturbance (3, 0, 0), ataxia (0, 1, 1), dry mouth (0, 2, 0), fatigue (0, 2, 0), headache (1, 1, 0), nausea (1, 1, 0), rash (0, 1, 1), hand tremors (0, 0, 1), increased energy (0, 1, 0), and polyuria (0, 0, 1). Adequacy of blinding and perceived efficacy Of the 36 subjects who completed the medication phase, 34 completed an exit survey.

The percentage of subjects who correctly guessed their treatment assignment on this survey was similar across groups (5/11 for placebo; 6/12 for 1,800 mg/day; and 7/11 for 2,700 mg/day; p = .91). The mean perceived helpfulness of medication did not differ significantly across groups (4.3 �� 2.8 vs. 4.2 �� 3.4 vs. 2.8 �� 2.1 for placebo; 1,800; and 2,700 mg/day, respectively; p = .41). Discussion No meaningful differences in smoking abstinence outcomes between treatment groups were detected. Compared with baseline, all treatment groups significantly reduced the number of cigarettes smoked per day, but smoking reduction did not differ between treatment groups. Participant dropout rates were notably high with more than one half of subjects in each study arm discontinuing prior to the end of the medication phase.

Our ability to interpret the negative study findings is compromised by the high dropout rate. Two possible reasons for the high dropout rate are difficulty with the three Batimastat times daily dosing regimen and lack of efficacy. In support of the former hypothesis, five participants (14% of those who completed exit surveys) noted that they had difficulty adhering to the three times daily dosing regimen.

All these issues may have potentially biased our results in 2 selleck chemical Belinostat opposite directions. On one hand we might have overestimated the number of cases, as one or more of suspect cases might actually be prevalent cases who lost anti-HBcAg/anti-HBsAg through severe immunosuppression (false negative) and eventually tested positive after immune reconstitution (potential misclassification). On the other end, we might have underestimated the actual number of cases, in fact, additional case(s) belonging to the reported cluster or even whole additional cluster(s) might have passed thorough unrecognized because of the large number of patients with no information about HBV status (potential misclassification). The results of the inferential study (case-control) might be biased because we included only patients for whom we could define the pre-admission HBV sero-status (potential selection bias).

However this is quite unlikely as there is no reason for a systematic difference between exposure to risk factors and availability of pre-admission HBV sero-status. Given the small number of cases (low inferential power) we could not carry out a multivariate analysis and define other potential risk factor associated with the transmission. Despite the above limitations this study provided, for the first time, molecular evidence to relate the use of one multi-patient lancing device to one HBV infection cluster. This evidence strongly supports the need to remove all shared pricking devices from HCS in order to avoid the occurrence of similar events. Footnotes Competing Interests: The authors have declared that no competing interests exist.

Funding: This study was funded by grants from the Italian Ministry of Health (��Ricerca Corrente IRCCS Lazzaro Spallanzani��) and the Regional Reference Centre for Healthcare-related infections (Centro di Riferimento per le Infezioni associate alle Pratiche Assistenziali della Regione Lazio �C CRIPA-Lazio). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Hepatitis C virus (HCV) is a hepatotropic, positive-strand RNA member of the Flaviviridae family [1]. Approximately 170 million people worldwide are infected with HCV. A notable feature of the virus is its tendency to cause chronic infection. Only a minority of infected patients is able to eradicate the virus, and up to 80% of the patients become chronic carriers.

The molecular mechanisms leading to viral persistency and its ability to evade intracellular host defenses are multi-fold and only partly understood [2]. Currently, there is no effective vaccine against HCV and AV-951 anti-viral therapy has limited efficacy [3]. Similar to other positive-strand RNA viruses, HCV replication involves the formation of a membrane associated replication complex [4].

Model 2 tested the adjusted relationship between menthol use status and continuous smoking abstinence by including age, race/ethnicity, partner status, income, kinase inhibitor Pazopanib education, the number of prequit cigarettes smoked per day, and the time to the first cigarette of the day. Model 3 tested the interaction of menthol use status and race/ethnicity by additionally including the interaction term in the adjusted model. All models additionally controlled for treatment group and time. Secondary analyses were also conducted to ensure that any effects resulting from the primary analyses were resilient to the inclusion of the 27 participants who declined to provide data on their income. This was accomplished by assigning those with missing income data to a ��refused to answer�� income category so that their data would be maintained in adjusted analyses (see Ko et al.

, 2010; Quach et al., 2011 for precedent) and re-running the analyses described above. Results Participant Characteristics Participants (N = 244) were racially/ethnically diverse (33% African American, 31% Latina, 36% White) women with an average age of 25 years (Table 1). The sample was almost evenly divided by menthol use status (menthol users n = 123, non-menthol users n = 121). Participant characteristics by menthol use status are in Table 1. Menthol users were significantly younger than non-menthol users and smoked fewer cigarettes per day. There were also significant differences between menthol and non-menthol users by race/ethnicity, partner status, income, and educational achievement. Table 1.

Participant Characteristics by Menthol Use Status Primary Analyses Menthol users had nonsignificantly lower rates of continuous abstinence than non-menthol users at both follow-up points. Longitudinal analyses indicated that menthol cigarette use did not significantly predict continuous abstinence from smoking through 26 weeks postpartum in analyses adjusted for treatment group and time (Model 1: �� = ?.24, SE = .23; ��2 = 1.05; p = .31; n [number of observations] = 331) or in analyses adjusted for age, race/ethnicity, partner status, income, and education, treatment group, cigarettes smoked per day, time to the first cigarette of the day, and time (Model 2: �� = ?.32, SE = .30; ��2 = 1.12; p = .29; n = 297). However, the menthol use status by race/ethnicity interaction was significant (Model 3: �� = ?.59, SE = .34; ��2 = 7.93; p = .02; n = 297). Follow-up racial/ethnic specific subgroup analyses indicated that menthol use status predicted abstinence among White women in adjusted analyses (�� = ?1.62, SE = .76; ��2 = 4.49; p = .03; Brefeldin_A n = 108, odds ratio = .19 [.04?.89]). White menthol users were less likely to maintain continuous abstinence than White non-menthol users.

, 2009). In short, the SSU rRNA gene was amplified from 10-ng DNA with the T7prom-Bact-27-for selleck chemicals Pazopanib and Uni-1492-rev primers followed by in vitro transcription and labeling with Cy3 and Cy5, respectively. The Cy3/Cy5-labeled target mixes were fragmented and hybridized on the arrays at 62.5��C for 16h in a rotation oven (Agilent Technologies, Amstelveen, The Netherlands). After washing and drying, the slides were scanned. Data was extracted from the microarray images using the Agilent Feature Extraction software, versions 7.5�C9.1 (http://www.agilent.com) and subsequently, the microarray data normalization, and further analyzed using a set of R-based scripts (http://r-project.org) in combination with a custom-designed relational database that runs under the MySQL database management system (http://www.

mysql.com;Rajili?-Stojanovi? et al., 2009). Hierarchical clustering of probe profiles was carried out using the Euclidian distance and Ward’s minimum variance method. These small intestinal microbiota composition profiles generated for the samples obtained from healthy subjects were compared with the ileostoma effluent profiles that were already present in the database (Booijink et al., 2010). Multivariate statistical software Canoco 4.5 for windows (Leps and Smilauer, 2003;Biometrix, Plant Research International, Wageningen, The Netherlands) was used to perform the principle component analysis on log-transformed HITChip probe signal-intensity profiles. High molecular weight DNA isolation from ileostoma effluent samples From the ileostoma effluent samples, 30�C40g were used for high molecular weight DNA isolation as described by Berridge et al.

(1998) with modifications. Effluent samples were resuspended as portions of 10g in 20ml 1 �� phosphate-buffered saline containing 10�C20 glassbeads of 2mm and vortexed for 3min. After centrifugation at 700g for 1min to remove large-sized debris, the supernatant was centrifuged at 3000g for 10min. The pellet was resuspended in 5ml 1 �� phosphate-buffered saline with 2% w/v polyvinylpyrrolidone (PVP), incubated at room temperature for 5min and centrifuged at 3000g for 10min. After washing with 1 �� phosphate-buffered saline, the pellet was resuspended in 3ml lysis buffer (50mM glucose, 50mM Tris-HCl (pH 8.0), 100mM EDTA, 2% w/v PVP, 150mM NaCl, 0.2mgml?1 RNAse A (Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.

02mgml?1 mutanolysin (Sigma-Aldrich) and 1mgml?1 lysozyme (Sigma-Aldrich)), and incubated at 37��C Brefeldin_A for 2h. Following addition of 360��l 10% w/v SDS and 160��l of a 10mgml?1 Proteinase K (Sigma-Aldrich) solution, the samples were incubated at 45��C for 2h. After addition of 600��l 5M NaCl and 480��l CTAB/NaCl solution (10% Cetyl trimethylammonium bromide in 0.7M NaCl), samples were incubated at 65��C for 10min.

7% decline in cigarette consumption in 2003. The recent increase in cigarette price/tax variation across and within U.S. states provided an incentive for smokers to look for lower cost cigarette sources. Hyland, Bauer, et al. (2005) found that 34% of heavy adult smokers try to reduce their cost of smoking by getting cigarettes from cheaper sources. This is particularly true selleck chemicals for those smokers who lived within 40 miles of a state border or an Indian reservation that sold cigarettes with a lower excise tax. Living in an area with higher average cigarette prices increases the probability of using discount and/or generic cigarettes (Hyland, Bauer, et al., 2005). The use of these cheaper cigarettes is primarily concentrated among high-intensity and low -ncome smokers (Cummings, Hyland, Lewit, & Shopland, 1997).

One study found that an access to low-taxed cigarettes inhibited quit attempts and possibly quit rates (Hyland, Higbee, et al., 2005). Hyland et al. (2006) studied the use of low/untaxed cigarettes in Australia, Canada, the United Kingdom, and the United States using the survey data employed in this paper. The differences across the countries were attributed to the affordability of cigarettes and the availability of cheaper cigarette sources. Using a low/untaxed source was positively related to age, income, level of education, and to being White and/or speaking English. The association between higher income and buying cigarettes from low/untaxed sources is consistent with a U.S. study (Hyland, Bauer, et al., 2005) that indicates that a minimum set of resources is needed to purchase low/untaxed cigarettes.

Smoking intensity was not a significant predictor of purchasing low/untaxed cigarettes, but those who reported purchasing from a low/untaxed source at Wave 1 were less likely to make a quit attempt at Wave 2 of the survey (Hyland et al., 2006). To summarize, the impact of cigarette prices on smoking cessation has been studied primarily in the United States, and only two studies have examined the impact of cigarette price choices on smoking cessation. Apart from one industry-sponsored study, there is no research evidence on the impact of hypothetical price increase on adult smoking behavior. No study has linked smokers�� expectation about their quit intentions to future quit intentions and quit behavior.

The ITC data from four countries with very different cigarette markets allows us to examine how a proposed future price increase might differentially impact contemplation to stop smoking. This is an important issue since the availability of cigarettes with a wide range of prices may inhibit cessation (Hyland, AV-951 Higbee, et al., 2005; Hyland et al., 2006). Data and Methods This study uses data from the ITC surveys conducted in the United Sates, Canada, the United Kingdom, and Australia among a nationally representative cohort of adult smokers.

39, p < .01; see Figure 1). Gender was also associated our with tobacco use among khat users. More than two thirds of men reported that they consume tobacco daily while only one third of women said they smoke regularly (��2 = 11.6, p < .01; see Table 1). Within these habitual users, cigarette smoking was more prevalent in men relative to women (��2 = 41.0, p < .001) while waterpipe smoking was more common among women as compared with men (��2 = 22.2, p < .001). Reported consumption of cigarettes per day was greater in men than in women (F(1, 100) = 69.1, p < .001). Tobacco product used during a khat session was different between men and women; relative to their counterparts, the number of cigarettes smoked was greater in men (F(1, 99) = 52.0, p < .001), whereas the number of waterpipe heads smoked was higher among women (F(1, 71) = 51.

8, p < .001). The majority (55%) of men cited that they smoked their first cigarette after breakfast, while 87% of women said that they used it only when they were chewing khat (��2 = 34.9, p < .001). Men were more likely to smoke before they went to bed than women (��2 = 12.2, p < .001). Both men and women mentioned that they had thought about quitting smoking (90%) and had tried quitting smoking in the past (66%). Figure 1. Differences in hours of khat use per typical session between khat-only and concurrent users. Correlational analysis showed associations between tobacco and khat use. Reported number of cigarettes smoked during a typical khat session was inversely related to age of onset of khat use (r = ?.29, p < .

01) but was positively linked to number of hours spent chewing khat per session (r = .26, p < .01), number of times of khat sessions per week (r = .41, p < .001), and number of years of khat use (r = .22, p < .05; see Figure 2). Similar results were obtained between the number of cigarettes per day and frequency of khat use (hours per day, times per week, duration in years; ps < .05). Figure 2. Associations between khat-related variables (age of onset, hours per typical session, times per week, duration in years) and the number of cigarettes consumed during a khat session. Years of smoking was positively correlated with the number of khat use episodes per week (r = .30, p < .01) and number of years of khat use (r = .53, p < .001). Furthermore, gender was associated with the link between tobacco and khat consumption. There was a gender �� smoking status (daily or occasional use) interaction in the number of khat AV-951 sessions per week (F(1, 98) = 18.35, p < .001). One-way ANOVAs conducted separately by gender with Bonferroni corrections indicated that while daily smoking was related to an increase in khat use among women (F(1, 36) = 25.71, p < .

g., low-increasing from low-stable) LB42708? or converged toward a similar smoking pattern by Y4 (e.g., high-decreasing and low-stable). Figure 1. Trajectories of cigarette smoking during 4 years of college (n = 1,253). Means and SE bars of number of smoking days in the past month depicted at each timepoint. The percentages shown in the figure are unweighted. After statistically weighting the sample … BIC values, parameter estimates, and probabilities of trajectory group membership are available as Supplementary Tables 1�C3. In summary, significant changes in smoking frequency occurred within all but the stable nonsmokers. Mean probability of membership in each of the five groups ranged from .91 to .99.

Pairwise comparisons of trajectory slopes indicated that the trajectory shapes were significantly different from each other, with the exception that the stable nonsmokers�� trajectory shape was not significantly different from that of the low-stable or high-stable groups (results available upon request). Comparisons of Smoking Trajectory Group Means Between-group comparisons of smoking trajectory intercepts at Y1 and Y4 illustrate the divergence and convergence of certain trajectories. High-decreasers initially (Y1) smoked somewhat less than high-stable smokers (Figure 1; 14.3 vs. 18.2, p < .001), but by Y4, they were smoking only slightly more than the low-stable group (2.6 vs. 1.7, p < .001). Conversely, low-increasers initially (Y1) smoked no more frequently than the low-stable group (1.3 vs. 1.2 days, p = .44) and subsequently diverged, yet never approached the high-stable mean, even in Y4 (15.

5 vs. 23.8, p < .001). All remaining comparisons were statistically significant at p < .003. Comparison of Y1 Characteristics of Smoking Trajectory Groups Bivariate comparisons revealed significant differences between smoking trajectory groups on both demographic and Y1 substance use characteristics (Table 1). Both males and Whites were slightly over-represented in all four smoking trajectory groups (relative to stable nonsmokers). No differences in age or neighborhood income were observed. Stable nonsmokers also had less alcohol involvement as measured by past-year drinking, typical alcohol consumption, and alcohol dependence, although some comparisons did not achieve statistical significance. Comparing the low-stable and low-increasing groups, Y1 smoking patterns were similar in terms of time since first cigarette (2.7 vs. 2.9 years), past-month smoking (49.5% vs. 41.0%), number of cigarettes per day (1.5 vs. 1.5), and their alcohol involvement were also similar. The high-decreasing and high-stable groups were not significantly different, although high-stable smokers had been smoking somewhat longer (4.1 vs. 3.5 years) and more heavily Entinostat (4.7 vs. 3.

Studies carried out www.selleckchem.com/products/CP-690550.html thus far indicate that the Flotac technique detects human helminth infections with a higher sensitivity than the Kato-Katz technique (20, 21, 39). Recently, a study from southern Italy��designed to assess the prevalence of parasitic infections among immigrants��showed that the Flotac technique is able to diagnose not only helminths but also intestinal protozoon infections (16); hence, further studies are warranted to compare the diagnostic accuracy of Flotac with those of currently more widely used techniques. The objective of the study presented here was to validate the Flotac-400 dual technique for the diagnosis of human intestinal protozoon infections in stool samples obtained from an area in sub-Saharan Africa where they are highly endemic.

Hence, we compared the diagnostic accuracy of the Flotac-400 dual technique and the FECT, which is broadly considered the diagnostic standard technique for the identification of intestinal protozoon species infections in humans. Stool samples were obtained from a random sample of 108 individuals from C?te d’Ivoire and were subjected to the Flotac-400 dual technique, using two different FSs, and the FECT, adhering to standard protocols. MATERIALS AND METHODS Study area and population. The study was carried out between May and September 2009 and consisted of a field and a laboratory component. In May and June 2009, stool samples were collected in L��l��bl��, a rural setting of south-central C?te d’Ivoire, located approximately 160 km north of the country’s economic capital Abidjan.

Our study was embedded in a cross-sectional epidemiologic baseline survey assessing people’s infection status with helminths and other parasites in the recently established health demographic surveillance system in Taabo (Taabo HDSS). In late 2008, a population database was created in the Taabo HDSS, which provides demographic, health, and socioeconomic data on approximately 38,000 inhabitants. In order to obtain baseline epidemiologic data in L��l��bl��, the existing Taabo HDSS database was used to draw up a computer-generated random list of 351 individuals as a representation for the whole Taabo HDSS population in the village, thus including children and female and male adults of all age groups. Ethical considerations.

The study was Cilengitide approved by the institutional research commissions of the Swiss Tropical and Public Health Institute (Basel, Switzerland) and the Centre Suisse de Recherches Scientifiques en C?te d’Ivoire (Abidjan, C?te d’Ivoire). Ethical approval was granted by the ethics committee of Basel (EKBB, reference number 316/08) and C?te d’Ivoire, and a research authorization for the first author of this paper (S. L. Becker) was provided by the Ministry of Higher Education and Scientific Research in C?te d’Ivoire (authorization number: 124/MESRS/DGRSIT/YKS/sac).

It is unclear why the FTND item ��Can��t refrain from smoking�� showed www.selleckchem.com/products/BIBW2992.html the strongest association with rs6474412, but this could be related to its significant correlation with CPD (r = .59). The strongest association with rs6474412 was with the WISDM ��Tolerance�� scale, which comprises items, such as ��I consider myself a heavy smoker,�� again affirming that rs16969968 and rs6474412 are associated with overlapping domains of nicotine dependence characterized by heavy smoking. Before the biological mechanisms are clarified for CHRNA5 and CHRNB3, the possibility of having distinct but related phenotypes with these two variants cannot be ruled out. The third variant, rs3733829 in the EGLN2 gene about 40 kb from the 3�� end of CYP2A6, showed a modest association with nicotine dependence.

When the genetic association was modest, we were unable to differentiate the degree of association based on the different phenotypic definitions. Our data do not support the previously reported association with rs1329650 on chromosome 10. Most nicotine dependence phenotypes showed no evidence of association (p > .50). Though DSM-IV nicotine dependence demonstrated a weak association, the effect was in the opposite direction from what was previously reported; thus, our results must be interpreted as nonreplication (TAG, 2010). Based on the allele frequency and our sample size, our study has sufficient power to detect an association with OR of 1.14 or higher (Gauderman & Morrison, 2006). There are several limitations to our study.

Our sample selection for cases and controls was based on extreme FTND scores, so a comparison Brefeldin_A of FTND results with the other nicotine dependence phenotypes cannot be directly made. We purposely examined the strength of genetic associations within FTND measures as one set of analyses. In a separate analysis, we examined the level of genetic association among other nicotine dependence phenotypes (DSM-IV, DSM-V, NDSS, and WISDM). In order to adjust for the ascertainment bias that was built into our study design, we performed additional analyses correcting for the bias (Lin & Zeng, 2009) and obtained similar results. Second, caution is needed in interpreting these intertwined clinical constructs. There is moderate to high correlation among the examined phenotypes and subphenotypes. As a consequence, the pattern of significant findings can be somewhat misleading. For instance, the DSM ��Craving�� item has a significant stepwise association with rs16969968, but the WISDM ��Craving�� item did not (instead, the WISDM ��Loss of Control�� subscale was significant in the stepwise tests). In fact, both craving measures are similarly highly associated with rs16969968 as indicated by their CIs (Table 4), suggesting no real inconsistency.

Blots were probed at 4��C overnight with primary antibodies in 5% milk/TBST. The antibodies used for Western blotting were CD133, CD29, Musashi-1, ABCG2, TERT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA). Two-DE and silver-staining Cultured SW1116 cells were harvested, washed with PBS, and lysed in a lysis selleck Pazopanib buffer containing 8 mol/L urea, 4% CHAPS, 40 mmol/L Tris, 65 mmol/L DTT, 2% Bio-Lytes+ and centrifuged at 25 000 �� g for 1 h at 4��C. Protein concentrations were measured by a modified Bradford assay. All samples were stored at -80��C for electrophoresis. Two-DE was performed using the PROTEAN IEF and PROTEAN xi II systems (Bio-Rad, Hercules, CA, USA). Total protein (80 mg) was run in an IEF system using a 17 cm pH 3 – 10 ReadyStrip (Bio-Rad, Hercules, CA, USA).

The total Vh was 47 000-52 000. Following IEF separation, gel strips were equilibrated with buffer I containing 6 mol/L urea, 30% glycerol, 2% SDS, 1% DTT, followed by buffer II (DTT was replaced with 2.5% IAA), each for 15 min. The equilibrated gel strips were placed on top of a 12% T slab gel and sealed with 0.5% agarose. SDS-PAGE was performed for 30 min at a constant current of 10 mA and then at 25 mA until bromophenol blue reached the bottom of gels. The separated proteins were visualized with silver diamine-staining. For preparative 2-DE, 400 ��g of total proteins was separated as described above. The total Vh of IEF was 90 000-120 000. Proteins were detected with modified silver-staining compatible with MS analysis.

Experiments (from cell culture to 2-DE) were performed in triplicate. Image acquisition and statistical analysis Silver-stained 2-DE gels were scanned at an optical resolution of 84.7 ��m perpixel using a GS-710 imaging densitometer (Bio-Rad, Hercules, CA, USA). Digitized images were analyzed with the PD Quest 7.1 software package (Bio-Rad, Hercules, CA, USA). Following spot detection, a matchset including the three batches of SW1116 cells was built. A reference gel was selected from one of the SW1116 gels, and unmatched protein spots of the member gels were automatically added to the reference gel. The raw quantity of each spot in a member gel was divided by the total quantity of valid spots in the gel. Quantitative analysis of SW1116 gels was performed by Student��s t-test.

Protein identification Protein spots were excised from gels, destained and washed until the gels became clear. The spots were kept in 0.2 mol/L NH4HCO3 for 20 min, dried by lyophilization, and digested overnight Anacetrapib in 12.5 ng/mL trypsin in 0.1 mol/L NH4HCO3. Peptides were extracted three times with 50% ACN (Fisher, Fair Lawn, New Jersey, USA), 0.1% TFA (Merck, Schuchardt, Hohenbrunn, Germany) and dried in vacuum. The peptide mixture was dissolved in 0.1% TFA and desalted using a C18 ZipTip (Millipore, Bedford, MA). The eluted peptides in 0.1% TFA/50% ACN mixed with an equal volume of 0.