-F.Chang, unpublished). A total of 1,054 additional reactions and three shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. Illumina http://www.selleckchem.com/products/Imatinib(STI571).html reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [32]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 199.5 �� coverage of the genome. The final assembly contained 697,305 pyrosequence and 20,331,123 Illumina reads Genome annotation Genes were identified using Prodigal [33] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [34].

The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes – Expert Review (IMG-ER) platform [35]. Genome properties The genome consists of a 4,888,353 bp long chromosome with a GC content of 33.8% (Table 3 and Figure 3). Of the 4,347 genes predicted, 4,285 were protein-coding genes, and 62 RNAs; 122 pseudogenes were also identified. The majority of the protein-coding genes (59.5%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3 Genome Statistics Figure 3 Graphical circular map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew. Table 4 Number of genes associated with the general COG functional categories Insights from genome sequence A closer look on the genome sequence of strain IC166T revealed a set of genes which might be responsible for the yellow-orange color of C. algicola cells by encoding enzymes that are involved in the synthesis of carotenoids. Carotenoids are produced by the action of geranylgeranyl pyrophosphate synthase (Celal_1770), phytoene synthase (Celal_2446), phytoene desaturase (Celal_2447), lycopene cyclase (Celal_1771) and carotene hydroxylase (Celal_2445).

Geranylgeranyl pyrophosphate synthases Cilengitide start the biosynthesis of carotenoids by combining farnesyl pyrophosphate with C5 isoprenoid units to C20-molecules, geranylgeranyl pyrophosphate. The phytoene synthase catalyzes the condensation of two geranylgeranyl pyrophosphate molecules followed by the removal of diphosphate and a proton shift leading to the formation of phytoene.

From outside to the center: http://www.selleckchem.com/products/MLN-2238.html Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew. Table 4 Number of genes associated with the general COG functional categories Insight into the genome sequence Comparative genomics Lacking an available genome sequence of Deferribacter abyssi, the species yielding the highest score, the following comparative analyses were done with D. desulfuricans [14] (GenBank “type”:”entrez-nucleotide”,”attrs”:”text”:”AP011529″,”term_id”:”290753135″,”term_text”:”AP011529″AP011529, “type”:”entrez-nucleotide”,”attrs”:”text”:”AP011530″,”term_id”:”290755253″,”term_text”:”AP011530″AP011530) and Calditerrivibrio nitroreducens (GenBank “type”:”entrez-nucleotide”,”attrs”:”text”:”CP002347″,”term_id”:”312938725″,”term_text”:”CP002347″CP002347, “type”:”entrez-nucleotide”,”attrs”:”text”:”CP002348″,”term_id”:”312940771″,”term_text”:”CP002348″CP002348) [16], the phylogenetically closest organisms for which a genome sequence was available.

The genomes of F. sinusarabici, D. desulfuricans and C. nitroreducens are similar in sizes (2.5 Mb, 2.5 Mb and 2.2 Mb, respectively) and have a similar, quite low G+C content (38%, 30% and 36%, respectively). Whereas F. sinusarabici has no plasmid, D. desulfuricans harbors a 5.9 kb plasmid; C. nitroreducens contains a 30.8 kb megaplasmid. An estimate of the overall similarity between the three genomes was generated with the GGDC-Genome-to-Genome Distance Calculator [40,41].

This system calculates the distances by comparing the genomes to obtain HSPs (high-scoring segment pairs) and inferring distances from a set of formulas (1, HSP length / total length; 2, identities / HSP length; 3, identities / total length). Table 5 shows the results of the pairwise comparison between the three genomes. Table 5 Pairwise comparison of F. sinusarabici, D. desulfuricans and C. nitroreducens using the GGDC-Calculator. The comparison of the F. sinusarabici and D. desulfuricans genomes revealed that 5.9% of the average of both genome lengths are covered with HSPs. The identity within these HSPs was 83.2%, whereas the identity over the whole genome was only 4.9%. Similar results were inferred for F. sinusarabici and C. nitroreducens (Table 5). The genomes of D. desulfuricans and C.

nitroreducens show a significantly higher degree of similarity with 9.9% of the average of both genomes are covered with HSPs of 83.3% Dacomitinib identity. The identity over the whole length of the genomes was 8.3%. These values corroborate the relationship between the three organisms as shown in the 16S rRNA-based phylogenetic tree in Figure 1, as there is no bootstrap support that F. sinusarabici is closer related to either C. nitroreducens or D. desulfuricans. The fraction of shared genes in the three genomes is shown in a Venn diagram (Figure 4).

Bergmann. Like other sessions in the day, it included illustrative hands-on exercises for attendees to try on their laptops. The day was closed with a tutorial by Nicolas Rodriguez on JSBML. Following the tutorial sessions, the HARMONY meeting officially opened with a simultaneous buffet reception and evening poster session. In all, 15 posters were presented. They covered a wide variety of topics��everything from databases that can export data in the various formats discussed at HARMONY, to new software tools and emerging standards, including BioPAX and CellML. Day 2, plenary session on SBGN The first plenary session focused on SBGN. It was chaired by Falk Schreiber and focused on unresolved issues in the 3 SBGN sublanguages: Process Description (SBGN-PD), Entity Relationship (SBGN-ER) and Activity Flow (SBGN-AF). The session started with an overview and history of the SBGN standard from Nicolas Le Nov��re. This was followed by a report on the status of SBGN-PD by Stuart Moodie and discussion of items that were scheduled to be included in the Level 1 Version 2.0 release of SBGN-PD, but which still remained contentious. These problematic topics included the rules of subunit naming, the exact semantics of the ��Empty Set�� glyph, resolution of the community vote about reversible arcs, and the use of the SBGN Unit of Information to describe the cardinality of multimers and the material type of EPNs. A record of these SBGN-PD issues is available online at [91]. The next session of the morning concerned SBGN-ER and was led by Nicolas Le Nov��re. Discussions focused on several issues, particularly the semantics of an ��entity�� and whether different outcome glyphs were required to describe occurrent (e.g. an interaction itself) and continuant (e.g. a complex resulting from the interaction). Huayu Mi then presented the status of SBGN-AF, which was nearing a maintenance release (Level 1 Version 1.1). The major topic of discussion was whether a separate phenotype and perturbing agent activity were required, and whether SBGN-AF needed different glyphs. In addition, the outcome of the vote on how the type of activity (macromolecule, complex etc.) was indicated in AF [92]. Day 3, plenary session on BioPAX The plenary session of the third day was devoted to BioPAX. Gary Bader began with an overview of the planned activities, then introduced the projects and issues that were of interest within the community. The goal was nucleation of interested parties to begin discussions and work. This was followed by the session on specification and data. It was chaired by Emek Demir, who gave an overview of data integration and normalization and discussed progress since his last presentation on the topic during COMBINE 2010. Arman Aksoy then introduced the Patch algorithm and outlined the goal to test data integration at the meeting. He reported that several data providers tested integration with specific data sets.

Annotation of novel genomes is a complex problem [76]. Efforts at automated annotation of molluscan genomic sequences have demonstrated the challenge facing the future selleckchem DAPT secretase annotation of cephalopod genomes. Long branch lengths within the phylum, the taxonomic distances to well annotated animal genomes, and the relatively low quantity of previous molecular and genetic work in the Mollusca will demand the generation of additional resources to assist and train automated gene detection programs. Of primary importance will be the generation of transcript inventories to identify genes, refine gene models, detect start points and intron-exon boundaries, and train automated gene identification algorithms. Transcriptome data such as those from RNAseq are quick and relatively inexpensive to generate, and will be immensely useful.

Systematic sequencing of nervous system tissues and embryonic stages can be combined with relatively early-stage assemblies to generate gene models and exon structures. In addition, pairs of Octopus species (O. vulgaris and O. bimaculoides) and Idiosepius species (I. notoides and I. paradoxus), through comparative sequence analysis, may be critical for annotation. Annotation efforts are labor-intensive but also offer an opportunity to grow the cephalopod research community and attract outside expertise. For example, domain experts of particular gene families or pathways can be recruited to assist in the description of likely protein function.

Bioinformatics researchers interested in the problems of annotation across long phylogenetic distances, the assessment of unique gene families and the evolution of biochemical novelty, and the likely challenges of extensively RNA-edited transcriptomes, will also be enlisted. Finally, annotation provides an outreach opportunity to involve young scientists and K-12 classrooms in cutting-edge scientific discovery on these fascinating organisms. Data sharing plan An important goal of the CephSeq Consortium is to share data rapidly and effectively both within and beyond the Consortium. Data sharing is necessary to foster the broadest possible impact of our sequencing and annotation efforts. This sharing will prove critically important for the cephalopod community. We expect sequence homology within the taxon to be an important foundation for collaboration within the field because cephalopods have evolved many new and unique character features.

Sharing data prior to publication could significantly accelerate cephalopod research. However, data sharing policies must Carfilzomib also recognize that there is significant publication, funding, and career recognition risks involved in making data available before publication: often the first to publish a particular observation garners the most recognition. Broad data-sharing agreements such as the Ft. Lauderdale agreement [77] have already been adopted by the international genomics community, and, most significantly, by many large sequencing centers.

parva strain HT, DSM 21527, download catalog was grown in semisolid DSMZ medium 1113 (Leptospira medium) [43] at 30��C. DNA was isolated from 1-1.5 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with modification st/DL for cell lysis as described in Wu et al. 2009 [41]. DNA is available through the DNA Bank Network [44]. Genome sequencing and assembly The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [45]. Pyrosequencing reads were assembled using the Newbler assembler (Roche).

The initial Newbler assembly consisting of 217 contigs in 1 scaffold was converted into a phrap [46] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (8,018.4 Mb) was assembled with Velvet [47] and the consensus sequences were shredded into 1.5 kb overlapped fake reads (shreds) and assembled together with the 454 data. The 454 draft assembly was based on 200.6 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 21. The Phred/Phrap/Consed software package [46] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [45], Dupfinisher [48], or sequencing cloned bridging PCR fragments with subcloning.

Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 361 additional reactions and 11 shatter library were necessary to close some gaps and to raise the quality of the final contigs. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [49]. The error rate of the final genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 1,722.1 �� coverage of the genome. The final assembly contained 348,698 pyrosequence and 97,925,368 Illumina reads.

Genome annotation Genes were identified using Prodigal [50] as part of the DOE-JGI annotation pipeline [51], followed by a round of manual curation using the JGI GenePRIMP pipeline [52]. The predicted Anacetrapib CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes – Expert Review (IMG-ER) platform [53]. Genome properties The genome statistics are provided in Table 3 and Figure 3.

Sequencing, finishing and annotation were performed by the JGI. A summary of the project PR-171 information is shown in Table 2. Table 2 Genome sequencing project information for Bradyrhizobium sp. strain WSM471. Growth conditions and DNA isolation Bradyrhizobium sp. strain WSM471 was grown to mid logarithmic phase in TY rich medium [24] on a gyratory shaker at 28��C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [25]. Genome sequencing and assembly The genome of Bradyrhizobium sp. WSM471 was generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina [26] and 454 technologies [27]. An Illumina GAii shotgun library which generated 67,039,982 reads totaling 5,095 Mb and 1 paired end 454 library with an average insert size of 5 Kb which generated 397,976 reads totaling 83.

7 Mb of 454 were generated for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [25]. The initial draft assembly contained 236 contigs in 2 scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data was assembled with Velvet, version 1.0.13 [28], and the consensus sequence were computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina Velvet consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS – 4.

24 (High Performance Software, LLC). The software Consed [29-31] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [32], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 327 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The estimated genome size is 7.

8 Mb and the final assembly is Brefeldin_A based on 53.8 Mb of 454 draft data which provides an average 6.9�� coverage of the genome and 4,879.9 Mb of Illumina draft data which provides an average 625.6�� coverage of the genome. Genome annotation Genes were identified using Prodigal [33] as part of the DOE-JGI Annotation pipeline [34] followed by a round of manual curation using the JGI GenePRIMP pipeline [35]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases.

Our hypothesis predicts that as a photosensitizer, LJ001′s antiviral activity should also be dependent on light. Indeed, the antiviral activity of LJ001 was dependent on both its concentration and the time-of-exposure to white light. For example, doubling the time of light exposure achieved the same viral inhibitory selleck chemicals effect at ten-fold lower concentrations (Figure 3C, compare 50 and 500 nM curves). Importantly, LJ001′s antiviral activity was absent when no visible light source was used (Figure 3D). Since LJ001 membrane intercalation is dictated by its lipophilic properties and not the presence of light, this latter observation underscores our previous observations [4] that, at the active concentrations used, membrane insertion itself does not account for the antiviral activity of LJ001.

Finally, to provide independent confirmation of the type II photosensitizing properties of LJ001, we subjected a solution of LJ001 in CD2Cl2 under ambient conditions to flash excitation, and observed the characteristic 1O2 emission in the near-infrared (Figure S5). Figure 3 The antiviral activity of LJ001 is dependent on its ability to generate singlet oxygen (1O2). The effect of LJ001 on the biophysical properties of model versus cellular membranes We propose that after insertion into the viral membrane, light activation of LJ001 triggers the generation of 1O2 that oxidizes the unsaturated chains of fatty acids composing the phospholipids of the viral membrane.

In further support of our model, we showed that LJ001 (and LJ025) efficiently partitions into model lipid membranes mimicking the lipid packing density, fluidity, and composition of viral (HIV-like) or cell (POPC) membranes (Figure 4A and Table S1). Indeed, when lipid membranes were non-limiting (>50-fold molar excess of lipid), over 85% of LJ001 or LJ025 were protected from the water-soluble quencher (acrylamide), and thus, completely buried in the lipid bilayer (Figure S6). 1O2-mediated oxidation of unsaturated phospholipids proceeds by a ��singlet oxygen ene�� reaction, resulting in a cis-to-trans isomerization of a double bond in the unsaturated fatty acids and the presence of a polar group (hydroperoxy- or hydroxy-) in the hydrophobic core of the lipid bilayer (Figure S7, first and second panel).

Cis-to-trans isomerization allows for closer packing of the fatty acid acyl chains in the lipid bilayer, which could result in a tighter positive curvature, while lipid oxidation results in clustering of the oxidized lipids into microdomains, reducing exposure of the polar groups to the hydrophobic acyl chains in the lipid Dacomitinib bilayer core (Figure S7, third and fourth panel) [20]. The latter effectively reduces membrane average fluidity (and/or increases rigidity), as lipid species are now not as freely diffusible.

Nicotine-Induced Analgesia In the tail immersion test, baseline measures revealed an effect of Sex (F(1, 110) = 17.0, p < .001), with females having lower nociceptive thresholds than males, independent of genotype (Figure 4A). Deletion of the ��4 subunit, however, did not affect basal thermal pain sensitivity, with no main effect sellckchem of Genotype and no interaction between Sex and Genotype. Nicotine had a dose-dependent antinociceptive effect in both males (Figure 4B) and females (Figure 4C). A three-way repeated-measures ANOVA revealed a significant effect of Sex (F(1, 110) = 40.9, p < .001), consistent with the sex difference in basal nociception. None of the two- or three-way interactions between Sex and the other factors were significant. Data from each sex were analyzed separately using two-way repeated-measures ANOVAs.

There was a significant main effect of Nicotine in both sexes (males: F(1, 43) = 17.3, p < .001, Figure 4B; females: F(1, 67) = 13.5, p < .001, Figure 4C). A main effect of Genotype (males: F(4, 172) = 48.9, p < .001; females: F(4, 268) = 86.0, p < .001) and a significant interaction between Nicotine and Genotype (males: F(4, 172) = 11.8, p < .001; females: F(4, 268) =10.0, p < .001) were also detected in males and females, indicating that nicotine-induced analgesia was reduced in ��4?/? mice (Figure 4B�CC). Figure 4. (A) Basal nociceptive thresholds in the tail immersion test. Tail withdrawal latencies (mean �� S E M) were measured in na?ve male and female ��4+/+ (black bars) and ��4?/? (white bars) mice. The numbers of ...

A similar pattern of results emerged in the hot plate test (Figure 5). Nicotine increased the latencies to display the first nocifensive response to noxious heat (males, Figure 5A; females, Figure 5D), hindpaw licking or flinching (males, Figure 5B; females, Figure 5E), and jumping (males, Figure 5C; females, Figure 5F). Three-way ANOVAs revealed a significant effect of Sex for all responses (first sign: F(1, 102) = 16.5, p < .001; hindpaw sign: F(1, 102) = 5.2, p < .05; jumping: F(1, 102) = 4.7, p < .05), with females having longer latencies than males. None of the two- or three-way interactions between Sex and the other factors were significant, with the exception of the Sex �� Nicotine interaction for jumping (F(2, 102) = 5.7, p < .05), which reflected the higher sensitivity of females to the antinociceptive effect of nicotine for that sign.

Data from each sex were then analyzed separately using two-way ANOVAs. There was a significant effect of Nicotine on the latency to display first sign (males: F(2, 39) = 15.3, p < .001, Figure 5A; females: F(2, 63) = 52.9, p < .001, Figure 5D), hindpaw sign (males: F(2, 39) = 10.7, p < .001, Figure 5B; females: F(2, 63) = 13.8, GSK-3 p < .001, Figure 5E), and jumping (males: F(2, 39) = 3.9, p = .056, Figure 5C; females: F(2, 63) = 48.0, p < .001, Figure 5F).

In conclusion, the results of the present study show PEG-IFN (40 kDa) administered at a dose of 135 ��g weekly for 48 mo was efficacious but not perfectly tolerable in dialysis patients with HCV infection. COMMENTS Background Our study showed that PEG-IFN (40 kDa) administered at a dose of 135 ��g weekly for 48 mo was efficacious but not perfectly tolerable in dialysis selleck chemicals llc patients with chronic hepatitis C. Research frontiers The present study was carried out in South-east Anatolien/Diyarbakir, Turkey, in hemodialysis patients with chronic hepatitis C. Related publications Yu JW, Wang GQ, Sun LJ, Li XG, Li SC. Predictive value of rapid virological response and early virological response on sustained virological response in hepatitis C virus (HCV) patients treated with pegylated interferon ��-2a and ribavirin.

J Gastroenterol Hepatol. 2007 Jun; 22(6): 832-836. Sporea I, Popescu A, Sirli R, Golea O, Totolici C, Danila M, Vernic C. Pegylated-interferon �� 2a treatment for chronic hepatitis C in patients on chronic hemodialysis. World J Gastroenterol. 2006 Jul 14; 12(26): 4191-4194. Innovations and breakthroughs Many studies published that PEG-IFN treatment is efficacious and tolerable in hemodialysis patients with chronic hepatitis C. In our study we found similar efficacy, but this treatment was not perfectly tolerable in dialysis patients with HCV infection. Peer review Ayaz et al investigated the effects of PEG-IFN for treatment of HCV infection in hemodialysis patients. Eleven of 17 patients had a SVR at wk 24 after end-of-treatment. In 5 patients treatment had to be stopped because of side effects.

The currently available data on this topic are scarce and therefore this is an important study which should be published. Footnotes S- Editor Rivera CA L- Editor Mihm S E- Editor Yin DH
MYH-associated polyposis (MAP) is an autosomal recessive condition (Al-Tassan et al, 2002) associated with the development of multiple adenomas and carcinomas of the colon and rectum, as well as other possible malignancies and extra-colonic manifestations (Enholm et al, 2003; Sampson et al, 2003; Sieber et al, 2003; Gismondi et al, 2004; Nielsen et al, 2005). The syndrome is caused by germline biallelic mutations in MYH, a gene that codes for a DNA glycosylase involved in base excision repair (BER) (Al-Tassan et al, 2002).

MYH mutations have been shown to be ethnically specific, and most (80%) Caucasian individuals with MAP possess the Y165C or G382D mutations (Enholm et al, 2003; Croitoru et al, 2004; Fleischmann et al, 2004; Wang et al, 2004; Farrington et al, 2005; Peterlongo et al, 2005). Italian populations also carry a three base pair deletion (1395delGGA) in exon 14 (Gismondi GSK-3 et al, 2004), whereas non-Caucasian populations harbour a range of different MYH mutations (Jones et al, 2002).

A specific format was followed that included (a) asking about tobacco use status, (b) discussing selleck kinase inhibitor motivations for tobacco reduction, (c) discussing encountered problems, (d) problem-solving difficult situations or compliance issues, and (e) providing support. For individuals wanting to quit after the 8-week period, counseling involved(a) discussing reasons for wanting to quit and potential obstacles; (b) discussing the content of the treatment manual, Tough Enough to Quit Snuff, including preparation for the quit day, identification of high risk situations, and strategies to deal with these situations; and (c) identifying sources of support. Follow-up A follow-up session occurred at Week 12. Subjects were asked to monitor ST and other tobacco use for the prior week using daily diary cards.

They were also asked to provide first morning urine. Participants received $10 per each clinic visit during treatment, $25 per follow-up visit, and $150 bonus for attending all the visits and complying with the study requirements. Statistical analysis Baseline demographics were tabulated for treatment groups using summary statistics. Tins per week, dips per day, and mean concentrations of cotinine and NNAL through Week 12 were calculated with baseline values assumed for missing data. Outcomes were analyzed using an intention-to-treat approach. The primary hypothesis tested was whether the treatment group had different effects on level of ST reduction at the end of 4 and 8 weeks and at the 12-week follow-up.

Analysis of ST use and exposure measures used linear mixed models to investigate whether the two interventions had different effects on ST use reduction (Cnaan, Laird, & Slasor, 1997; Littell, Pendergast, & Natarajan, 2000; Schluchter, 1988). Analyses modeled the repeated measures in reported ST use and concentrations of total cotinine and NNAL at each visit (baseline and Weeks 4, 8, and 12). Covariates included main effects for the discrete time and the treatment group as well as interactions between treatment group and time. Models were fitted using restricted maximum likelihood to estimate covariance structure for within-subject responses. The percentage of each group achieving targeted reduction level at the end of 4 (��50%), 8 (��75%), and 12 (��50%) weeks was calculated and compared using chi-square test.

The analysis of the percentage in each group that achieved the targeted reduction level at the end of 4 (��50%), 8 (��50% and ��75%), and 12 (��50%) weeks compared the success rates for the different treatment groups using a logistic regression model. We examined both ��50% and ��75% reduction at 8 weeks although the targeted reduction was 75% because 50% is the typical outcome of reduction studies. Generalized linear mixed model was used to model the probability of achieving the reduction target at each follow-up visit (Weeks AV-951 4, 8, and 12).