As such, we could not document the long-term health outcome for t

As such, we could not document the long-term health outcome for the infected hatchling(s). A 50-μL sample of the hydrolyzed blood with bacteria was added to 10 mL of Luria–Bertani (LB) broth, incubated at 37 °C overnight on a rocker plate. Bacteria were then subcultured on LB agar plates at 37 °C. Ten colonies were isolated from these plates for subsequent assays of hemolytic activity on human and sheep blood agar plates. Human blood agar plates (5% blood) were prepared by dissolving

Sunitinib 19 g trypticase soy agar in 475 mL of ddH2O in a microwave oven, cooled to 50 °C and then mixing in 25 mL of freshly drawn human blood from a student volunteer (in accord with our IRB Committee) before pouring into sterile Petri dishes. The sheep blood agar plates were purchased from All 10 colonies of the sea turtle bacteria were found to be hemolytic. The 16S RNA genes of three of these were amplified and partially sequenced (methods described below), all yielding essentially identical sequences. It would appear that the hatchling was infected with a single bacterial species. One clone, 2-04LB-Cl-5, was then selected for complete sequencing

as described below. The chemical and growth characteristics of the bacteria were kindly assessed by the US Centers for Disease Control, Washington, DC. To detect any soluble toxins with hemolytic activity, bacteria were grown overnight in an LB broth and 1.5 mL was centrifuged find more at 18 500 g in a microcentrifuge for 4 min. The bacterial supernatant was then filtered through a 0.45-μm filter twice to ensure the removal of all bacteria. Removal

was confirmed through the absence of bacterial growth after incubation of a filtered sample overnight in LB media. Freshly drawn human blood was then diluted 1 : 1 with a sterile isotonic saline and 200 μL was incubated with 10, 50, 100 and 200 μL of the bacterial supernatant or equivalent volumes Progesterone of LB broth. Samples were observed microscopically for lysis after 1, 4, 24 and 48 h. DNA was isolated from bacterial pellets obtained from 10 mL cultures. Bacteria were lysed in 1 mL of DNAzol and DNA isolated according to the manufacturer’s protocol (Invitrogen). The virtually complete rRNA gene sequence was established by sequencing multiple PCR samples run in the forward and reverse directions (four to six runs in each direction) with two sets of previously described universal primer pairs [P0mod (forward) and PC3 (reverse) gene location 18–32 and 787–806, respectively; P3 (forward) and PC5 (reverse) gene location 787–806 and 1487–1507, respectively] (Wilson et al., 1990).

Strain 761M, based on its 16S rRNA gene sequence, was later found

Strain 761M, based on its 16S rRNA gene sequence, was later found to group with the Gammaproteobacteria (Bowman et al., 1995). To the best of our knowledge, the phylogenetic grouping of strain R6 was never determined (although enzymatic analyses suggested its affiliation to Alphaproteobacteria). None of these strains appear to be still extant, making it impossible to repeat these experiments. Two methanotrophs isolated from freshwater lake sediments were also described as being facultative, i.e., able to utilize not

only methane, but also casamino acids, nutrient HM781-36B cost agar, and a variety of organic acids and sugars for carbon and energy (Lynch et al., 1980). However, one of these isolates, Methylobacterium ethanolicum, was

later found by members of the same laboratory to actually consist of a stable syntrophic consortium of two methylotrophs, i.e., a Methylocystis strain capable of utilizing methane, and a Xanthobacter Navitoclax strain capable of utilizing a variety of multicarbon compounds for growth (Lidstrom-O’Connor et al., 1983). Collectively, the inability of putative facultative methanotrophs to grow on methane after growth on multicarbon substrates, the lack of extant strains, and evidence of stable mixed cultures initially originally described as pure methanotrophic strains all cast serious doubts on the possibility of facultative methanotrophy. As a result, research in this area was severely limited for the next 20 years. Efforts to identify novel methanotrophs

significantly regained momentum in the 1990s with the discovery of acidophilic methanotrophs from Sphagnum peat bogs (Dedysh et Sclareol al., 1998a, b). The first characterized acidophilic methanotroph was found to represent a new genus and species within Alphaproteobacteria, Methylocella palustris (Dedysh et al., 2000), and subsequently two further strains of the same genus were isolated, Methylocella silvestris and Methylocella tundrae (Dunfield et al., 2003; Dedysh et al., 2004). All three strains were considered novel methanotrophs as their optimal pH for growth was <6.0. Even more remarkably, all three isolates could only express the sMMO, and not the pMMO. This finding was quite unexpected as it showed that these were the first methanotrophs that did not express pMMO. Initial screens of each isolate showed that they could not grow on sugars or multicarbon substrates, but could grow on methane and methanol, as well as on methylamine to a variable degree, thus they were considered obligate methanotrophs. These methanotrophs, however, were later shown to be facultative as they could utilize not only C1 compounds for growth, but also acetate, pyruvate, succinate, malate, and ethanol (Dedysh et al., 2005 and Table 1).

72 and 074 for the

two sets of models, respectively, and

72 and 0.74 for the

two sets of models, respectively, and significantly lower at 0.55 for genotyping. The two sets of models performed comparably well and significantly outperformed genotyping as predictors of response. The models identified alternative regimens predicted to be effective in almost all cases. It is encouraging that models that do not require a genotype were able to predict responses to common second-line therapies in settings where genotyping is unavailable. “
“Combining noninvasive tests increases diagnostic accuracy for staging liver fibrosis in hepatitis C virus (HCV)-infected Selleck SCH772984 patients, but this strategy remains to be validated in HIV/HCV coinfection. We compared the performances of transient elastography (TE), Fibrotest (FT), the aspartate aminotransferase-to-platelet ratio index (APRI) and two algorithms

combining TE and FT (Castera) or APRI and FT (SAFE) in HIV/HCV coinfection. One hundred and sixteen HIV/HCV-coinfected patients (64% male; median age 44 years) enrolled in two French multicentre studies (the HEPAVIH cohort and FIBROSTIC) for whom TE, FT and APRI data were available were included in the study. Diagnostic accuracies for significant fibrosis (METAVIR F ≥ 2) and cirrhosis (F4) were evaluated by measuring the area under the receiver-operating characteristic curve (AUROC) and calculating percentages of correctly classified (CC) patients, taking liver biopsy as a reference. For GSK2126458 purchase F ≥ 2, both TE and FT (AUROC = 0.87 and 0.85, respectively) had a better diagnostic performance than APRI (AUROC = 0.71; P < 0.005).

Although the percentage of CC patients was others significantly higher with Castera’s algorithm than with SAFE (61.2% vs. 31.9%, respectively; P < 0.0001), this percentage was lower than that for TE (80.2%; P < 0.0001) or FT (73.3%; P < 0.0001) taken separately. For F4, TE (AUROC = 0.92) had a better performance than FT (AUROC = 0.78; P = 0.005) or APRI (AUROC = 0.73; P = 0.025). Although the percentage of CC patients was significantly higher with the SAFE algorithm than with Castera's (76.7% vs. 68.1%, respectively; P < 0.050), it was still lower than that for TE (85.3%; P < 0.033). In HIV/HCV-coinfected patients, TE and FT have a similar diagnostic accuracy for significant fibrosis, whereas for cirrhosis TE has the best accuracy. The use of the SAFE and Castera algorithms does not seem to improve diagnostic performance. "
“We evaluated the efficacy, safety and tolerability of etravirine in paediatric patients vertically infected with HIV-1. A multicentre retrospective study of 23 multidrug-resistant paediatric patients (five children and 18 adolescents) enrolled in the study from 1 September 2007 to 28 February 2010 was carried out. We performed a longitudinal analysis of immunological, virological and clinical data. The median age of the patients was 14.2 years [interquartile range (IQR) 12.5–15.8 years]. At baseline, the median HIV-1 RNA was 29 000 (4.

PCR was carried with template cry2Ab and In-fusion™ primers cry2

PCR was carried with template cry2Ab and In-fusion™ primers. cry2Ab insert was column purified, and concentration was determined. The pET30b+ vector (Novagen) was digested using XhoI and NdeI. Linearized vector was gel extracted, and concentration was measured. selleck chemicals llc Vector was ethanol precipitated and was resuspended in water, 5× In-fusion reaction buffer, BSA, cry2Ab and In-fusion enzyme. Reaction mix was

incubated at 37 °C for 30 min and subsequently incubated at 50 °C for an additional 15 min. A portion of incubated mix was transformed into stellar competent cells (Clontech). DNA was purified from various colonies, and cry2Ab-pET30b+ plasmid DNA sequence was confirmed. Plasmid Map Enhancer for Windows 95 version was utilized to generate cry2Ab-pET30b+ plasmid map (not shown). Cry2Aa was used as a positive Crizotinib control. The source of the cry2Aa gene is described elsewhere (Liang & Dean, 1994). Nine primers (Table 1) were designed to create single-residue, D block mutants (Sigma). PCR analysis was carried out using KOD polymerase (Novabiochem). Site-directed mutagenesis was performed on cry2Ab-pET30b+ recombinant plasmid (Sadeghi et al., 2010). Cry2Ab wild type and mutants were transformed into DH5α Escherichia coli cells. Samples were grown in 5 mL of Luria

broth (LB) supplemented with 30 μg mL−1 of kanamycin overnight (Lin et al., 2008). DNA was purified using Qiagen miniprep kit and sequences were confirmed for each sample. Cry2Ab wild type and mutants were transformed into BL21 Roseetta 2 (DE3) cells and selected for kanamycin and chloramphenicol resistance. LB (5 mL), supplemented with 30 μg mL−1 of kanamycin plus chloramphenicol, was inoculated with Cry2Ab wild type or respective mutant and grown at 37 °C overnight (O/N). Five millilitres of inoculum were added to 500 mL of 2xYT supplemented with 30 μg mL−1 of kanamycin and grown at 37 °C to OD600 nm ranging from 0.5 to 0.7. Culture was induced with 0.1 mM isopropyl-β-D-thio-galactoside (IPTG) at 25 °C O/N

Phosphoglycerate kinase (Li et al., 2011). IPTG-induced bacterial cells were centrifuged (9820 g) at 4 °C. Harvested cells were resuspended in 50 mL of 50 mM Tris bruffer, pH 8. Cells were sonicated at room temperature for 2 min (10 s pulse on : 10 s pulse off ). The final pellet was resuspended in minimal crystal wash II. Protein samples were solubilized for 2 h in 50 mM Na2CO3, pH 10.5. Protoxin samples were separated by 10% SDS-PAGE and stained using Coomassie Brilliant Blue G-250. Densitometric scanning (Personal densitometer SI) was employed to scan Coomassie-stained gel for quantification (Fig. 2). Standard curve was generated by ion-exchange purified protein ranging in concentration from 0 to 40 μg and corresponding volume bands analysed by imagequant.

05) The intracanal medications had similar antibacterial activit

05). The intracanal medications had similar antibacterial activity. Conclusion.  The association of chlorhexidine with calcium hydroxide did not increase the antibacterial activity of the intracanal medication in the treatment

of primary teeth with necrotic pulp with and without furcal/periapical lesion. “
“International Journal of Paediatric Dentistry 2010; 20: 330–335 Objectives.  To assess and compare the oral health status of preschool children with and without cerebral palsy (CP). Methods.  Preschool children with CP (72) were recruited from 23 Special Child Care Centers in Hong Kong. An age (±3 months) and gender matched sample of preschool children from mainstream preschools were recruited as the control group. Dental caries status, gingival health status, tooth EGFR inhibitor wear, developmental defect of enamel, malocclusion, dental trauma and oral mucosal health were assessed and compared between the two groups. Results.  Significant differences in gingival health status were found between children with and without CP (mean plaque index scores, P = 0.001 and mean gingival index scores, P < 0.05).

Tooth wear involving dentine was more prevalent among GS-1101 manufacturer CP children (P < 0.001), as were evidence of anterior open-bite (P < 0.001) and oral mucosal lesions (P < 0.05). Children with and without CP had similar caries experiences (P > 0.05), prevalence of enamel defects (P > 0.05) and dental trauma (P > 0.05). Conclusions.  Differences of oral health status exist among preschool children with and without CP. Preschool children fare worse in terms of gingival health, tooth wear, oral mucosal health and malocclusion. “
“International Journal of Paediatric Dentistry 2011 Background.  Caries in children younger than 72 months is called early childhood caries (ECC). Sixty-six per cent of Chinese children younger than 5 years old have dental decay, and about Temsirolimus ic50 97% of them

are untreated. Aims.  This in vitro study was conducted to evaluate the remineralization effects of the casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) crème on the artificial early enamel lesions of the primary teeth and to assess its caries-prevention efficiency. Design.  Enamel specimens with artificial early lesions were produced and were then randomly divided into Group A: distilled and deionized water, DDW, as negative control; Group B: CPP-ACP crème, test group; Group C: 500 ppm NaF solution, as positive control. The enamel surface microhardness (SMH) was measured before, after demineralization, and 30 days after remineralization. The results were analysed with the SPSS 13.0 software package. The enamel specimens were analysed by the scanning electron microscope. Results.  The CPP-ACP crème increased SMH of the eroded enamel significantly more than 500 ppm NaF solution did. The morphology of the enamel was different in each group. Conclusions.

matrixsciencecom) Molecular weight and pI were calculated based Molecular weight and pI were calculated based on amino acid sequence and compared with gel location. Functional annotation of the identified protein was carried out using the gene ontology (GO) database ( and UniProtKB ( Bacterial sample preparation was same as described for proteomic analysis. Extraction of intracellular metabolites was performed as previously described with slight modifications (Frimmersdorf et al., 2010). Four replicates were used in each group. The compounds were derivatized with methoxyamine hydrochloride and N-methyl-N-trimethylsilyltrifluoracetamide. A set of alkane standards were

added to calculate retention indices. The derivatized extracts were analyzed with a GC-MS-QP-5050A (Shimadzu). Spectral deconvolution, calibration and identification of metabolites

were performed using amdis software from NIST (Natural Institute of Standards and Technology). EPZ-6438 clinical trial Prior to statistical analysis, each compound was normalized by the peak area of the standard (ribitol) and the optical density of each bacterial culture (Strelkov et al., 2004). These relative ratios can be compared directly among different groups without knowledge of the absolute compound concentrations. Hierarchical cluster analysis with Pearson correlation as the distance measure and a one-way PD-0332991 cost anova test was performed with tigr mev 4.7.4. Significant differences in the metabolite level were determined by comparing the P values (P < 0.05). The metabolites

with significant changes were submitted to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( to obtain the compound IDs and then submitted to Metabolite Pathway Enrichment Analysis (MPEA) Analysis ( to determine which metabolic pathways are most likely to be involved with these compounds. P-values were calculated by Monte Carlo simulation. Pseudomonas Silibinin sp. TLC6-6.5-4 is a rod-shaped bacterial strain with an average length of 6.52 ± 1.60 μm when grown in LB broth without copper (Fig. 1b, d and e). However, when the bacterial isolate was exposed to 4 mM copper, about 70% reduction in bacterial cell length was observed. The mean cell length was 1.92 ± 0.38 μm, which is significantly (P < 0.05) shorter than cells grown in LB broth without copper (Fig. 1a, c and e). A comprehensive genome-wide analysis of Pseudomonas sp. TLC6-6.5-4 was performed to identify the genes involved in copper resistance using random transposon mutagenesis. A total of 5023 colonies with transposon insertions were screened for copper-sensitive mutants, which resulted in the identification of three mutants with a decrease in resistance to copper. These mutants were designated CSM1 (Copper Sensitive Mutant), CSM2 and CSM3. Growth of these mutants in the medium without copper was not affected (Fig. 2).

The mean number of months per year was 106 (median

The mean number of months per year was 10.6 (median find more 12). The dependent variable was the number of distinct in-patient bacteraemia/septicaemia episodes in a calendar year. We calculated incidence rates for bacteraemia in each year. Multivariate analyses used the person-year as the unit of analysis, with the number of months of exposure in each year incorporated in the model as an offset. To incorporate the correlation between multiple observations for most patients, we used generalized estimating equations (GEEs), with an exchangeable correlation matrix and robust standard errors clustered on each patient. Because 86–90% of patients with a bacteraemia

episode in a year had only one episode, we used logistic regression to analyse any episode (none vs. one or more) in a year. For comparison, we also report a negative binomial regression Selleck INCB018424 of number of episodes in a year. We first examined each demographic and clinical factor separately. In further multivariate analyses, we used logistic regression to estimate the adjusted odds ratios for age, sex, race, HIV transmission risk factor,

CD4 cell count, HIV-1 RNA, HAART and insurance. To assess trends over time, dummy variables for each year (2001–2008) were included in the model. In all models, the first CD4 cell count and HIV-1 RNA of each calendar year were used. Age, CD4, HIV-1 RNA, insurance and HAART were treated as time-varying covariates. To account for geographic differences

in HIV care, all models were also adjusted for HIV care site. All reported P-values are two-tailed. Statistical analyses were performed using stata 9.0 (Stata Corporation, College Station, TX, USA). We classified bacteraemia episodes on the basis of the type of organism producing the infection, and we examined trends over time in the types of organisms. A subanalysis was performed at one large academic hospital where all ‘bacteraemia, not otherwise specified’ (NOS) cases were evaluated by manual abstraction by searching hospital laboratory records to determine the organism of interest. This large hospital constituted 42% of all bacteraemia NOS cases. Because of Institutional Review Board issues, hand searching was not possible at other participating study sites. Between January 2000 and December 2008, 39 318 patients were followed for a total mafosfamide of 146 289 PY at 10 HIVRN sites. The sample was 71% male, 48% Black and 21% Hispanic, with a median age of 39 years (range 18–94 years) (Table 2). The major HIV risk factors included MSM (36%), HET (34%) and IDU (22%). During the study period, 57% of the patients received HAART. Most patients had Medicaid (32%) or were uninsured (27%). The median CD4 count was 321 cells/μL and the median HIV-1 RNA was 7760 copies/mL. During the study period, 2025 episodes of bacteraemia occurred, for an incidence rate of 13.8 events per 1000 PY. A total of 1538 patients (3.9% of 39 318) had one or more episodes.

Routine genotyping to identify HIV-resistant CBUs could create a

Routine genotyping to identify HIV-resistant CBUs could create a bank of CB-derived stem/progenitor cells with which to treat HIV infection. HIV depletes the cells of the immune system, allowing rare Saracatinib opportunistic infections to thrive and eventually leading to the death of the individual. The virus displays selective tropism

towards the monocyte/macrophage lineage and T lymphocytes (T cells). As the viral infection depletes the host of T cells, the patient advances to the condition known as AIDS, and the decline in the number of T cells marks this progression. The viral envelope contains two major glycoproteins required for the fusion of the viral and cell membranes: glycoprotein 120 (gp120) and glycoprotein 41 (gp41). gp120 binds to a cell membrane receptor known as cluster of differentiation 4 (CD4) and a co-receptor expressed by T cells [1–3]. This binding induces a conformational change allowing the gp41 fusion peptide to interact with the lipid bilayer and form membrane pores used to transfer viral contents inside the cell for replication. The different HIV-1 strains can be distinguished by the co-receptor used to infect cells. The predominant strain found early during HIV infection, the chemokine (C-C motif) receptor 5 (CCR5)-tropic (R5) Nutlin 3a strain, infects cells expressing the CCR5 co-receptor

[4–6], while the chemokine (C-X-C motif) receptor 4 (CXCR4)-tropic (X4) strain binds to the CXCR4 co-receptor. There are also dual-tropic strains that use both CCR5 and CXCR4 co-receptors. Without a functional CCR5 co-receptor the HIV-1 R5 strain is incapable of infecting T cells [7,8]. Currently, the treatment for HIV-infected individuals is highly active antiretroviral therapy (HAART). Although effective, this therapy requires a daily regimen consisting of several pills. If the patient discontinues therapy, viral Monoiodotyrosine reservoirs in the gut-associated lymphoid tissue and in haematopoietic progenitor cells can restore the levels

of HIV and trigger T-lymphopenia [9]. Interestingly, there are individuals who are resistant to HIV-1 infection as a result of a common 32-bp deletion in both copies of the CCR5 gene [10–12]. The 32-bp deletion (Δ32) results in an early termination of translation, which produces a nonfunctional protein. CCR5 and CXCR4 are both chemokine receptors that HIV-1 uses in conjunction with CD4 to infect T cells [4,5,13]. Individuals who inherit one CCR5Δ32 allele are partially resistant to HIV infection [14]. Recently, Hutter et al. (2009) showed that long-term remission of HIV disease could be achieved in a patient with acute myeloid leukaemia following an allogeneic bone marrow transplant from a CCR5Δ32/Δ32 donor [15]. These findings demonstrate that CCR5Δ32/Δ32 donor bone marrow has the potential to be used in stem cell therapy for HIV infection.

Five randomly selected plantlets per treatment were collected at

Five randomly selected plantlets per treatment were collected at 6, 24 or 48 h postinoculation. The root

material was weighed before being homogenized in 1 mL of sterile phosphate buffer. Selleck Palbociclib Serial dilutions were then inoculated onto sterile MMAB plates placed to incubate at 28 °C. Colonization was estimated as CFU g−1 of fresh root material. A one-tailed t-test assuming equal variances and P < 0.05 (Microsoft Excel) was used to assess statistical significance of differences in attachment and colonization between the strains. Attachment of A. brasilense to glass or PVLC surfaces was first analyzed under different growth and incubation conditions. Attachment to glass was not significant irrespective of the growth conditions or the incubation time (data not shown). This was confirmed by AFM (Supporting Information, Fig. S1) and topographic analysis of the surfaces (Fig. S2), suggesting that the physical surface properties of glass do KPT-330 concentration not facilitate attachment

for A. brasilense. Growth conditions mediating surface attachment (biofilm) in A. brasilense were thus subsequently analyzed only on PVLC surfaces. Attachment was found to increase when the experiments were conducted under low aeration (i.e. nonshaking) conditions with cells transferred from culture in a rich medium (TY) to a minimal media (data not shown). No significant effect of varying the concentrations of either phosphorous or potassium, found to increase attachment in other bacterial species (Danhorn & Fuqua, 2007) could be detected (data not shown). When biofilm formation was monitored in media lacking nitrogen or containing

relatively low concentrations (1 mM) of NH4Cl or NaNO3, surface adherence for all strains was greater compared with higher concentrations (10 mM) of NH4Cl C59 datasheet or NaNO3, respectively (Table 2). Biofilm formation was the greatest for all strains with low concentrations of sodium nitrate. Differences seen initially between strains remained unchanged over time, although overall biofilm formation was increased at day 7 (Table 2). Nutritional conditions were previously shown to be powerful modulators of the attachment of various bacterial species to surfaces but specific effects of nitrogen availability on attachment have been seldom noted (O’Toole et al., 2000; Rinaudi et al., 2006; Danhorn & Fuqua, 2007). Compared with the parental strain Sp7 and regardless of the incubation conditions, the AB101 and AB102 strains showed a consistent greater attachment to PVLC surfaces. Different attachment abilities detected for these strains was apparent early (day 1) suggesting that the initial surface attachment step was affected (Table 2).

While an analogue of DHOCTO had been detected in an earlier study

While an analogue of DHOCTO had been detected in an earlier study on deoxycholate degradation by another Pseudomonas sp. (Leppik, 1983), a structure similar to THOCDO has, to our knowledge, not been described in any study on bacterial degradation of bile salts. Within the shoulder tailing off from the DHOCTO peak (Fig. 4), LC-MS analysis revealed an ion [M+H]+ with m/z 403.24, which could be the monoene derivative of DHOCTO. With the transposon mutant strain R1, we

also observed the transient accumulation of the monoene derivative of DHOPDC in the culture supernatants (Birkenmaier et al., 2007). The compound P3, which formed the second largest peak in HPLC analysis, coeluted with and had a UV spectrum very similar to that of the previously identified Δ1,4-3-ketocholate (X) from culture supernatants of the transposon mutant strain R1 (Birkenmaier et al., 2007). To characterize the mutant strain Chol1-KO[skt] further, it was tested for growth with intermediates of cholate degradation. Strain Chol1-KO[skt] could grow with DHADD (VIII) and THSATD (IX). Importantly, strain Chol1-KO[skt] could also grow with DHOPDC (XIII) that was provided with filter-sterilized supernatants of a culture of the acad mutant strain R1 that had been grown with succinate in the presence

of cholate as described previously (Birkenmaier et al., 2007). The growth of strain Chol1-KO[skt] with DHOPDC clearly showed that the skt-gene must be responsible for a reaction step preceding the formation of DHOPDC. The accumulation of DHOCTO and THOCDO supports this conclusion because progestogen antagonist both compounds could have arisen from hydrolyzed CoA-esters III and IV that are presumptive intermediates of β-oxidation of the acyl side chain of cholate (Fig. 1). Thus, the accumulation of DHOCTO and THOCDO indicates that at least the first

two steps of β-oxidation starting from Δ1,4-3-ketocholyl-CoA (II) could be catalyzed in the skt mutant. This narrowed the probable function of the skt-encoded protein down to being either a 3-hydroxy-acyl-CoA dehydrogenase or a β-ketothiolase. A closer analysis of the predicted protein reveals that Skt and its orthologs in other cholate-degrading bacteria (Fig. 2) have similarities to the β-ketothiolase domain Anidulafungin (LY303366) of eukaryotic sterol carrier protein SCP-x (Stolowich et al., 2002). SCP-x, which is also referred to as a nonspecific lipid transfer protein, is a fusion protein with a smaller C-terminal and a larger N-terminal domain. While the C-terminal domain (also called the SCP-2 domain) is responsible for intracellular targeting and the uptake of sterols, the N-terminal domain has 3-ketoacyl-CoA-thiolase activity for branched-chain-acyl-CoA esters. Interestingly, SCP-x is also responsible for the final step of cholate biosynthesis in mammals (Kannenberg et al., 1999; Russell, 2003).