difficile infection should be treated with metronidazole with consideration of vancomycin for fulminant disease, relapsing disease or non-responsive infection (category IV recommendation), following the recommendations for treatment in HIV-seronegative

populations outlined in Department of Health guidelines [50]. Therapy Proteasome inhibitor review is indicated for C. difficile infection regardless of the CD4 cell count. Acute bacterial diarrhoea in HIV-seropositive individuals with CD4 counts >200 cells/μL usually does not require treatment, but should be treated when the CD4 count is <200 cells/μL (category IV recommendation). 4.4.1.4 Impact of HAART. Trimethoprim-sulphamethoxazole (TMP-SMX, co-trimoxazole) reduced the incidence of infectious diarrhoea

in the pre-HAART era [51]. Retrospective studies suggest that introduction of antiretroviral therapy, including zidovudine monotherapy, has been more effective than targeted antimicrobial prophylaxis in preventing recurrence Tyrosine Kinase Inhibitor Library screening of nontyphoidal salmonella [52], and that duration of antimicrobial prophylaxis, with agents such as fluoroquinolones need not exceed 30 days in patients established on HAART [53]. The incidence of bacterial diarrhoea declined steadily after the introduction of HAART [28], therefore HAART is the mainstay of preventing bacterial diarrhoea (category III recommendation). 4.4.2.1 Background and epidemiology. Cytomegalovirus (CMV) is a member of the herpes family of viruses, usually acquired during

childhood. CMV infection remains dormant unless an individual becomes immunosuppressed, when reactivation of latent infection may occur [54,55]. In the pre-HAART era, retinitis was the most common presentation of CMV [56], followed by gastrointestinal disease (see Lenvatinib Table 4.2 for a list potential clinical manifestations of CMV in the GI tract). Most of the data about incidence of CMV were obtained from populations with retinitis. The majority of affected individuals had CD4 counts <100 cells/μL, with 80% of episodes occurring in those with CD4 counts <50 cells/μL. Since the advent of HAART, CMV infection may occasionally occur as part of immune reconstitution syndromes, but the overall incidence of CMV in individuals living with HIV has dramatically reduced [57]. 4.4.2.2 Presentation. CMV may affect all sections of the gut. Table 4.2 illustrates clinical presentation according to area affected. 4.4.2.3 Diagnosis. CMV viraemia, detected by polymerase chain reaction (PCR), may be positive in the absence of end-organ disease and several studies have shown this to be of negligible diagnostic use [58,59]. As indicated in Table 4.2, endoscopy may reveal classical CMV ulceration of the gut mucosa and biopsy with histopathological review may identify characteristic intranuclear and intracytoplasmic ‘owl’s eye’ inclusions [60]. The absence of ulceration makes a diagnosis of CMV colitis very unlikely [61].

2%) of participants self-identified as gay or homosexual. The cohort was highly educated, with more than half (51.9%) holding university or post-graduate qualifications, and 21.6% with tertiary diploma or technical and further education degrees. Nearly two-thirds of participants (913; 65.7%) were somewhat or very involved in the gay community in Sydney. By the end of the study in 2007, there were 53 HIV seroconversions identified, giving an overall HIV incidence of 0.78 per 100 PY [95% confidence interval

(CI) 0.59–1.02]. The total follow-up time was 5161 PY, and the median was 3.9 years per participant. Risk factor analysis was performed on 47 of the 53 HIV seroconverters who had sexual behaviour data available within 12 months of seroconversion. Risk factors associated with an HIV incidence of selleck chemicals more than 2 per 100 PY are summarized in Table 1. In order of incidence they included reports in the past 6 months of UAI with a known HIV-positive partner, any injecting drug use, receptive UAI with a casual partner, any anal STI, both oral erectile dysfunction medication and methamphetamine use, more than 50 casual partners, having an HIV-positive regular partner, any oral erectile dysfunction medication use and any psychedelic/hallucinogen use (Table 1). The remaining risk factors examined had an HIV incidence

of <2 per 100 PY. Circumcision status (HIV incidence in uncircumcised Ferrostatin-1 mouse participants of 1.04 per 100 PY; 95% CI 0.58–1.20) and the use of ‘any recreational drugs’ (0.83 per 100 PY; 95% CI 0.77–1.41) in the past 6 months were associated with an HIV incidence of approximately 1 per 100 PY. Daily alcohol consumption

(1.48 per 100 PY; 95% CI 0.74–2.96) and prior HBV infection (1.24 per 100 PY; 95% CI 0.71–2.19) were each associated with an HIV incidence of <2 per 100 PY. When demographic factors including age, education, income and occupation were examined individually, none was found to be related to an HIV incidence of ≥2 per 100 PY (data not shown). In total, there were nine risk factors identified with an HIV incidence of 2 per 100 PY or higher (Table 1). The stepwise procedure described above was used to rank these nine risk factors. Thirteen of the total 47 HIV seroconversions were among men who reported the highest risk behaviour of UAI with an HIV-positive Methane monooxygenase partner. The group of participants reporting UAI with an HIV-positive partner were excluded from the analysis and the incidence in the remaining eight high-risk groups was recalculated. Receptive UAI with casual partners was the next highest risk group identified (2.43 per 100 PY; 95% CI 1.38–4.28), accounting for 12 of the remaining 34 HIV seroconversions (Table 2). After exclusion of those men, reported use of both oral erectile dysfunction medication and methamphetamines had the highest HIV incidence (1.67 per 100 PY; 95% CI 0.84–3.34). The combined HIV incidence among men who reported at least one of these three risk factors (hereafter called the ‘high-incidence’ subgroup) was 2.

However, analysis of single (adra2a or adra2c) knockout animals revealed no alterations in interneuron distribution at the same age, suggesting the presence of compensatory regulatory mechanisms. Thus, for the first time, a specific role for adrenergic receptor activation has been postulated in interneuron migration and disposition. However, the intracellular mechanisms that Etoposide mediate this function remain to be elucidated. The study of Riccio and colleagues represents the first step in the effort to elucidate the role(s) of adrenergic receptors in cortical

neuron migration. Pyramidal neurons also express these receptors (Wang & Lidow, 1997), and it will be of interest to assess their role in the radial migration of this larger population of cortical cells. The results so far point to the notion that overstimulation of adrenergic receptors in the cortex by excessive levels of noradrenaline or by drugs may lead to alterations in

the formation of neuronal circuits and, consequently, of cortical function. “
“Taste stimuli increase extracellular dopamine (DA) in the nucleus accumbens (NAc) and in the medial prefrontal cortex (mPFC). This effect shows single-trial habituation in NAc shell but not in core or in mPFC. Morphine sensitization abolishes habituation of DA responsiveness in NAc shell but induces it in mPFC. These observations Meloxicam support the hypothesis of an inhibitory influence of mPFC DA on NAc DA. To test this hypothesis, we used in vivo microdialysis

Small molecule library to investigate the effect of mPFC 6-hydroxy-dopamine (6-OHDA) lesions on the NAc DA responsiveness to taste stimuli. 6-OHDA was infused bilaterally in the mPFC of rats implanted with guide cannulae. After 1 week, rats were implanted with an intraoral catheter, microdialysis probes were inserted into the guide cannulae, and dialysate DA was monitored in NAc shell/core after intraoral chocolate. 6-OHDA infusion reduced tissue DA in the mPFC by 75%. Tyrosine hydroxylase immunohistochemistry showed that lesions were confined to the mPFC. mPFC 6-OHDA lesion did not affect the NAc shell DA responsiveness to chocolate in naive rats but abolished habituation in rats pre-exposed to the taste. In the NAc core, mPFC lesion potentiated, delayed and prolonged the stimulatory DA response to taste but failed to affect DA in pre-exposed rats. Behavioural taste reactions and motor activity were not affected. The results indicate a top-down control of NAc DA by mPFC and a reciprocal relationship between DA transmission in these two areas. Moreover, habituation of DA responsiveness in the NAc shell is dependent upon an intact DA input to the mPFC. “
“Learning anatomy is similar to learning a language.

The restored phenotypes of the EN isolates are stable after several generations of growth in the absence of the stressors, suggesting the mechanism of stressor tolerance is an inherited consequence, rather than an adaptive consequence; therefore, next-generation DNA sequencing of the EN isolates genomes may be a viable strategy to identify potential candidate polymorphisms that are responsible for restoration of acid and detergent tolerance. Mutation of acpXL delays nodule development and interferes with proper bacteroid development in the host plant P. sativum cv. Early Alaska (Vedam Rapamycin clinical trial et al., 2003, 2004); however, it was unknown

whether other VLCFA mutations would have a similar effect. Pea plants were inoculated with the fabF2XL, fabF1XL mutant, and the number and size of nodules were monitored 10, 17, and 24 d.p.i. (Table 3). At 17 d.p.i., plants infected with the

fabF2XL, fabF1XL mutant had small, round, white nodules, while the wild-type plants had large, red, oblong nodules. By 24 d.p.i., the nodules from plants infected with the fabF2XL, fabF1XL mutant were indistinguishable from nodules of plants inoculated with wild type. In addition, plants inoculated with the mutant had a 1.75-fold increase in the number of nodules per plant (Table 3). Shoot dry weights were measured 24 d.p.i. and no differences were observed between peas inoculated with the wild-type and the R428 datasheet fabF2XL, fabF1XL mutant (Table 3). Complementation of the fabF2XL, fabF1XL mutation with the plasmid pCS115 restored the wild-type phenotypes for each time point tested (Table 3). We did not observe any differences in growth rate between the wild-type and mutant strains;

therefore, the delay in nodule development is probably not related to differences in generation time (data not shown). We also used nodulation assays with a ropB mutant to determine whether the ropB down-regulation observed in VLCFA mutants might contribute to the delayed nodulation phenotype. Mutation of ropB had no observable effect on nodule development in P. sativum, suggesting that the repression of ropB in the fabXL mutants for is probably not responsible for the delayed nodulation defect (Table 3). The TY sensitivity phenotype of the fabF2XL, fabF1XL mutant was also unrelated to altered ropB expression. These results indicate that the phenotypes of the fabXL mutants can be categorized as either ropB-dependent phenotypes, which include sensitivity to membrane stressors and ropB-independent phenotypes, which include delayed nodulation and sensitivity to the growth medium TY. The ropB gene is induced by peptide-containing media components (Foreman et al.

The PD0325901 mw presence of infection for a minimum of at least 14 years in our now dialysis-dependent patient prior to diagnosis of nephrotic syndrome is consistent with this observation. The association between P malariae infection and nephrotic syndrome remains controversial. In fact, there has been little reported in the literature about quartan malarial nephrotic syndrome since the 1970s, which some speculate may be because of improved nutritional status and availability of antimalarials in endemic locations, although the validity of the originally proposed theory of immune complex deposition as the cause of quartan malarial nephrotic syndrome is in question.11 Although children from Nigeria

and the Ivory Coast exhibited membranous glomerulopathy with focal and segmental glomerulosclerosis as in our patient, studies in Ugandan children with presumed quartan malarial nephrotic syndrome exhibited proliferative glomerulonephritis,11 a difference that is not explained by the immune complex theory. More recently, among 272 children in Nigeria with nephrotic syndrome prospectively followed over 12 years, only 38.7% had concomitantly detected HM781-36B chemical structure P malariae infection.12 Additionally, subsequent immunofluorescent examination of glomeruli from 76 cases of nephrotic syndrome in Nigeria detected P malariae antigens in only

25% of cases compared to hepatitis B in 24% of cases.13 In more recent reports, the prevalence of P malariae-associated nephrotic syndrome has declined in children and idiopathic nephrotic syndromes and those associated with sickle cell disease and HIV now occur more commonly.14,15 However, demonstrated difficulty in detecting sub-clinical P malariae through conventional means such as microscopic examination of the peripheral blood and antigen capture assays necessitates further studies with newer technologies like PCR to detect low-level parasitemia, as current infection rates among patients with

nephrotic syndrome may be underestimated. This case illustrates the importance of obtaining a detailed travel history, which should not be limited to recent travel. Increased ease of travel and consequent increased movement of people from areas where chronic infectious diseases are endemic to locations where such diseases are unknown and for which health care providers Cobimetinib nmr have limited or no experience necessitates increased emphasis on global health in medical education. Meeting the health care needs of world travelers will not only require better understanding of the clinical presentations for specific diseases but also the epidemiology and distribution of such diseases. Targeted laboratory screening of asymptomatic travelers for tropical disease has been shown to be of value in identifying clinically unapparent tropical infections in up to 25% of returning travelers when carried out by informed health care providers who obtain well-structured histories prior to testing.

[8] Another recent study from France suggests common patterns of involvement of arterial branches in patients with TAK.[9] As we discussed above, GCA is the other vasculitis

affecting large arteries. Recently, the similarities between TAK and GCA have been drawing attention.[10] Although both diseases clearly have a different etiology, there are many common pathological findings. GCA mainly affects older populations. Giant cells and granulomatous lesions can be found in patients with TAK and GCA. Maksimowicz-McKinnon et al. analyzed 69 and 75 patients with GCA and TAK, respectively, and found 73% of patients with GCA have lesions in large branches of the aorta.[11] The criteria for TAK by the Selleck ABC294640 American College of Rheumatology[12] are widely used. In Japan, the guideline provided by the Japanese Circulation Society[13] is also used for diagnosis. There are no studies to date comparing the diagnostic accuracy between the different criteria, but considering the difference between the items contained in each, using one criteria does not seem to result in a big difference in accuracy compared with using the other. It has been shown that many patients were diagnosed as having TAK more than several years

or as long as decades after they developed the disease.[6] A recent study reported that this discordance of time between development and diagnosis of TAK has become shorter and shorter.[14] This may reflect the development of Tacrolimus (FK506) imaging techniques and

prevailing information about this disease among physicians. Because occlusion or narrowing of arteries and branches of the aorta appear in advanced stages selleck of the disease, establishment of classification criteria, which could diagnose TAK in the early stage, is strongly desirable. Imaging of arteries is very useful in diagnosing TAK and for patient follow-up. Angiography is the gold standard to show narrowing or occlusion of the aorta or its main branches. Computed tomography (CT) angiography or magnetic resonance (MR) angiography are very useful tools to detect arterial lesions. Positron emission tomography (PET) is also useful to detect inflammation of arteries.[15] Atherosclerosis may display similar signals in PET so that special attention needs to be paid to aging and basic metabolic disease status to accurately evaluate the results of PET. Since establishment of classification criteria for early TAK is desired, PET could serve to detect active disease lesions before occlusion or narrowing of large branches of the aorta. Incorporation of MRI with enhancement or FDG-PET (PET with[18]F-fluorodeoxyglucose) would improve accuracy of early diagnosis.[15, 16] To date, no established biological markers specific to diagnose patients with TAK have been reported. Patients with TAK often present with increased inflammation markers, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR).

A microorgansim with arsenic replacing phosphate in such critical molecules as DNA and RNA appears to be equally science fiction. The findings start with the gradual adaptation of a new gammaproteobacterial click here strain to resistance to up to 40 mM added arsenate (not a surprise) and high intracellular arsenic bioaccumulation (unusual, but also reported previously). There is no difficulty in believing these

results. Growth curves show relatively good growth when 40 mM arsenate was added to medium that contained 3 μM phosphate (present as medium contamination). The cells look larger when grown in high arsenate than when grown in 1.5 mM phosphate, and the arsenate-rich cells have numerous vesicles (in cross-section transmission electron microscopy) that look much like polyhydroxybutyrate (likely) or polyphosphate (less likely) inclusions. There is no indication that the authors considered element-specific microprobing Akt inhibitor of those electron micrograph sections or whether such would be feasible, although Wolfe-Simon and colleagues have a figure with nanoSIMS (secondary ion mass spectroscopy) element-specific scanning of intact cells for 75As, 31P, and 12C content, and not cross sections with visible vesicles. A table shows

data that low P/high As-grown cells contain 20 × less P as a percent dry weight than high P/low As cells, and that the high As-grown intact cells contain a total of 7.3 As atoms for every P atom. The data are sufficient to calculate whether there was adequate P in the low P/high As cells for the needs of DNA, RNA, and phospholipids (as our casual calculations indicate is the case). However, Wolfe-Simon and colleagues did not make such a calculation from their data, although they calculated that a bacterial chromosome might need 7.5 × 106 P atoms. There is evidence that the cells bioaccumulate arsenic, but no need to

suggest that any arsenate is to be found in DNA or RNA diester linkages between sugar moieties. There is a figure showing gel electrophoresis pattern of total cellular DNA from high- and low-arsenic cells, with measurements indicating Alanine-glyoxylate transaminase that the ratio of As/C in the DNA from high-arsenic cells was 1 As per 105 C atoms, while DNA has a ratio closer to 1 P per 10 C atoms. The questionable conclusion of the paper appears as an established fact in the abstract (first paragraph of the report): ‘arsenate in macromolecules that normally contain phosphate, most notably nucleic acids and proteins.’ There are no data to support this claim, which is repeated. The data presented do not disprove the existence of arseno-ester bonds in cellular nucleic acids and proteins any more than they support such an interpretation. However, common sense and a little understanding of microbiology and biochemistry should have protected the authors from themselves. The Editor in Chief of Science is a biochemistry professor and author of the highly regarded basic text ‘The Molecular Biology of the Cell.

J Interferon Cytokine Res 2002; 22: 295–303. 112 Shepherd FA, Beaulieu R, Gelmon K et al. Prospective

randomized Fulvestrant trial of two dose levels of interferon alfa with zidovudine for the treatment of Kaposi’s sarcoma associated with human immunodeficiency virus infection: a Canadian HIV Clinical Trials Network study. J Clin Oncol 1998; 16: 1736–1742. 113 Kreuter A, Rasokat H, Klouche M et al. Liposomal pegylated doxorubicin versus low-dose recombinant interferon alfa-2a in the treatment of advanced classic Kaposi’s sarcoma; retrospective analysis of three German centers. Cancer Invest 2005; 23: 653–659. 114 Masood R, Cai J, Zheng T et al. Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS–Kaposi?sarcoma. Decitabine ic50 Proc Natl Acad Sci USA 1997; 94: 979–984. 115 Gavard J, Gutkind JS. VEGF controls endothelial-cell permeability by promoting the [beta]-arrestin-dependent endocytosis of VE-cadherin. Nat Cell Biol 2006; 8: 1223–1234. 116 Uldrick TS, Wyvill KM, Kumar P et al. Phase II study of bevacizumab in patients with HIV-associated Kaposi’s sarcoma receiving antiretroviral therapy. J Clin Oncol 2012; 30: 1476–1483. 117 Fife K, Howard MR, Gracie

F et al. Activity of thalidomide in AIDS-related Kaposi’s sarcoma and correlation with HHV8 titre. Int J STD AIDS 1998; 9: 751–755. 118 Little RF, Wyvill KM, Pluda JM et al. Activity of thalidomide in AIDS-related Kaposi’s sarcoma. J Clin Oncol 2000; 18: 2593–2602. 119 Koon HB, Honda K, Lee JY et al. Phase II AIDS Malignancy Consortium trial of imatinib in AIDS-associated Kaposi’s sarcoma (KS). J Acquir Immune Defic Syndr 2011; 56: 64–68. 120 Dezube BJ, Krown SE, Lee JY et al. Randomized phase II trial of matrix metalloproteinase inhibitor COL-3 in AIDS-related Kaposi’s sarcoma: an AIDS Malignancy Consortium Study. J Clin Oncol 2006; 24: 1389–1394. 121 Brinker BT, Krown SE, Lee JY et al. Phase 1/2 trial of BMS-275291 in patients with human immunodeficiency virus-related Kaposi sarcoma: a multicenter

trial of the AIDS Malignancy Consortium. Cancer 2008; 112: 1083–1088. 122 Little RF, Pluda JM, Wyvill KM et al. Activity of subcutaneous interleukin-12 in AIDS-related Kaposi Oxalosuccinic acid sarcoma. Blood 2006; 107: 4650–4657. 123 Lechowicz M, Dittmer DP, Lee JY et al. Molecular and clinical assessment in the treatment of AIDS Kaposi sarcoma with valproic Acid. Clin Infect Dis 2009; 49: 1946–1949. 124 Krown SE, Roy D, Lee JY et al. Rapamycin with antiretroviral therapy in AIDS-associated Kaposi sarcoma: an AIDS Malignancy Consortium study. J Acquir Immune Defic Syndr 2012; 59: 447–454. 125 Evans SR, Krown SE, Testa MA et al. Phase II evaluation of low-dose oral etoposide for the treatment of relapsed or progressive AIDS-related Kaposi’s sarcoma: an AIDS Clinical Trials Group clinical study. J Clin Oncol 2002; 20: 3236–3241. 126 Zhong DT, Shi CM, Chen Q et al.

After accepting electrons from NDH-2, menaquinol can be reoxidized via two alternative routes, ending with either a cytochrome aa3-type or a cytochrome bd-type terminal oxidase (Fig. 1, for a review, see Cox & Cook, 2007). In the energetically

more efficient route, menaquinol is oxidized by the cytochrome bc1 complex (consisting of subunits QcrA-C), which then transfers the electrons to the terminal cytochrome aa3-type oxidase (CtaC-F) (Matsoso et al., 2005). The cytochrome bc1 complex p38 MAPK phosphorylation and the cytochrome aa3 oxidase, thought to form a super complex in mycobacteria, are proton-translocating enzymes, assuring the high energetic yield of this route (Niebisch & Bott, 2003; Matsoso et al., 2005). Alternatively, menaquinol can be directly oxidized by a cytochrome bd-type terminal oxidase (CytA-B) (Kana et al., 2001). This reaction is not coupled to proton pumping; consequently, the cytochrome bd oxidase route is energetically Daporinad less efficient. However, cytochrome bd oxidase displays a higher affinity for oxygen and is thus used at low-oxygen tensions (Kana et al., 2001), whereas the cytochrome aa3-type enzyme is the predominant terminal electron acceptor during aerobic growth (Shi et al., 2005).

The energy of the proton motive force is subsequently utilized by ATP synthase for the synthesis of ATP. During dormancy, NDH-2 was found to be upregulated, whereas NDH-1 is strongly downregulated (Schnappinger et al., 2003; Shi et al., 2005). The cytochrome bc1 and cytochrome aa3 complexes are downregulated as well; however, the cytochrome bd-type oxidase is transiently upregulated, arguably to facilitate transition to the dormant state by contributing

to redox balance (Shi et al., 2005). The question of the predominant terminal electron acceptor in the dormant state is still open. It has been suggested that nitrate reductase (NarG-I) acts as an acceptor, and indeed, the enzymatic activity of nitrate Venetoclax reductase was found to be increased (Wayne & Hayes, 1998), and addition of nitrate increased the viability of dormant mycobacteria (Gengenbacher et al., 2010). Moreover, the nitrate transporter NarK2 is upregulated during dormancy (Schnappinger et al., 2003; Voskuil et al., 2003; Shi et al., 2005). The subunits of the ATP synthase complex were found to be downregulated using in vitro dormancy models as well as an in vivo mouse lung infection model (Shi et al., 2005; Koul et al., 2008). This considerable remodeling in dormant mycobacteria reflects reduced oxygen availability and decreased energy requirements in a state without growth. During dormancy, cellular ATP levels are ∼10-fold lower as compared with replicating bacilli (Starck et al., 2004; Koul et al., 2008; Rao et al., 2008; Gengenbacher et al., 2010). Nevertheless, dormant M. smegmatis are active in respiratory ATP synthesis and maintain an energized membrane (Koul et al., 2008). Furthermore, both replicating and dormant M.

Finally, the pellet was suspended with an equal volume of ice-cold 15% glycerol. Electroporation was conducted according to the protocol described in previous reports (Link et al., 1997b; Datsenko & Wanner, 2000). Fifty microliters of CP25e (5′-CCC ATT ATG CTT TGG CAG TTT ATT CTT GAC ATG TAG TGA GGG GGC TGG TAT AAT CAC ATA GTA CTG TTG GGT CTA GAT TAG GGT AAC TTT AAG GAG GTA TTC CTC-3′) and an equal volume of its complementary DNA (200 nM each) were mixed and incubated at 95 °C for 5 min, and then cooled to room temperature. Fifty microliters

of LacUV5 (5′-CTC ACT CAT TAG GCA CCC CAG GCT TTA CAC TTT ATG CTT CCG GCT CGT ATA ATG TGT GGA ATT GTG AGC GGA TAA CAA TTT CAC ACA GGA AAC AGC T-3′) and an equal volume of its complementary DNA (200 nM selleck products each) were mixed, and incubated at 70 °C for 10 min, and then cooled to room temperature. These were used as templates for PCR. PCR or cross-over PCR (Link et al., 1997b) was performed with AccuPrime Pfx DNA Polymerase, Opaganib Platinum Pfx DNA Polymerase (Invitrogen),

or Pyrobest DNA polymerase (Takara Bio Inc.). Primers and templates used in this study are listed in Table S2 (DNA sequences are listed in Table S3). Each PCR product specified by enzyme name in Table S2 was digested with the correspondent enzyme, dephosphorylated with alkaline phosphatase (Takara Bio Inc.), and then introduced into the donor vector with a DNA Ligation Kit Ver.2 (Takara Bio Inc.). The LacI promoter region of pKO3-lacI-35-10 (5′- TGG

CGC AAA ACC TTT CGC GGT ATG GCA TGA TAG CG -3′) was replaced with 5′- TGT TGA CAA ACC TTT CGC GGT ATG GTA TAA TAG CG -3′. Finally, we constructed the plasmids listed in Table S2. The DNA sequences of the constructed plasmids were confirmed by DNA sequencing analysis: the region amplified from genomic DNA was consistent with E. coli genomic DNA (GenBank accession no. NC_000913) or S. cerevisiae genomic DNA (768–995 base of GenBank accession no. X05731 for ubi4, and GenBank accession no. Z74170 for ubp1). Homologous recombination Dichloromethane dehalogenase with pKO3 or pKOV was performed according to the procedure reported previously with modifications (Link et al., 1997b). Briefly, derivatives of pKO3 or pKOV were transformed into the target strains. The strains were grown at 43 °C on LB agar plates containing CP (20 μg mL−1). Three colonies were picked and cultivated at 30 °C in 1 mL of LB broth, and 100 μL of cultured bacteria was further cultivated at 37 °C overnight on LB agar plates containing 10% (w/v) sucrose (Wako Pure Chemical Industries, Ltd). Finally, the strains grown only in LB containing sucrose but not in LB containing CP were selected as the strains containing neither pKO3 nor pKOV. Insertion of the target gene fragment into the appropriate sites in the chromosomal DNA was confirmed by PCR amplifying the region spanning both the target gene and the chromosomal site.