Surprisingly, commercial sex workers and clients selleckchem of commercial sex workers were not less likely to have their source tested than the rest of the study population. The difference between heterosexual and homosexual subjects could not be explained by differences in frequency of anonymous contacts, as one

might have expected. However, it is possible that the definition of anonymous contacts did not encompass the same realities in the two groups, as many anonymous MSM contacts occurred in bathhouses with truly untraceable contacts. Testing the source also allowed us to detect 11 undiagnosed HIV infections. The HIV prevalence of the source population of unknown HIV status was therefore 3.7%, a proportion 10 times higher than that reported in the general population in Switzerland [27]. When source subjects that were reported to be HIV MEK inhibitor positive were included, the prevalence increased to 24%,

which is consistent with other reports [13,17]. Sixty-two per cent of those for whom information was available were not treated and 69% had a detectable viral load. These data underscore the risk of undiagnosed and untreated HIV infection in the population of source subjects and therefore support the prescription of nPEP in cases of exposure to persons of unknown HIV status belonging to high-risk groups. However, in this study, a significant proportion (58%) of subjects reporting heterosexual contact with an anonymous or a casual partner were prescribed nPEP, although the source was not reported to belong to any risk group for HIV infection. Although this practice is not endorsed by our national guidelines, antiretroviral prophylaxis was provided in these cases because the source

was reported to have multiple sexual partners and believed to be at risk for HIV infection. We observed two seroconversions. Neither was linked to nPEP failure, as infection occurred after ongoing risk behaviour. The fact that one of the two patients was not offered prophylaxis at the time of consultation does not call into question Pregnenolone our policy to withhold nPEP when the source is tested negative. Indeed, fourth-generation tests have recently been shown in percutaneous occupational exposures to detect p24 antigen during acute HIV infection when antibodies are still undetectable [28]. The absence of nPEP failure, however, cannot be considered proof of its efficacy as the sample size was too small to allow assessment of such a rare phenomenon. A major limitation of our study was the high drop-out rate throughout the follow-up period. Overall, 16% of patients for whom nPEP was initiated never came back for assessment of regimen completion and drug toxicity and 49% of all participants never had a second HIV test at 3 months.

However, clinicians must decide whether the attributed benefits are clinically significant, considering the costs and potential risks of GH axis treatments. A limitation of this study is the small number of studies available of each GH axis drug class. Disorders of body fat metabolism and associated metabolic buy MK0683 alterations are common in patients infected with HIV [1]. While the definition is not standardized, a diagnosis of HIV-associated lipodystrophy describes metabolic derangements including insulin resistance and changes in lipid metabolism, which result in lipoatrophy (peripheral adipose wasting) and lipohypertrophy (visceral adipose accumulation)

[1]. The pathogenesis of HIV-associated lipodystrophy is multifactorial and includes genetic predisposition, effects of antiretroviral agents, HIV infection itself, and other host factors [2]. Highly active antiretroviral therapy (HAART) is comprised of potent antiretroviral agents, which have dramatically improved clinical outcomes in patients with HIV infection. Unfortunately, HAART is often associated with the

onset, or exacerbation, LDK378 cost of HIV-associated lipodystrophy [1]. The prevalence of HIV-associated lipodystrophy is 4% in untreated patients and 13–40% in patients on HAART [3]. The associated fat redistribution syndrome can lead to negative social, psychological and medical consequences

[4]. Cosmetic changes in body shape may result in decreased compliance with HAART, which can result in increased viral replication and associated morbidities and mortality [4]. The metabolic derangements in HIV-associated lipodystrophy are difficult to reverse. A number of treatments for this condition have been explored, including metformin, thiazolidinediones (TZDs), testosterone and growth hormone (GH) axis drugs. Metformin has been shown to reduce visceral adipose tissue (VAT) mass but accelerates peripheral adipose tissue loss. TZDs and testosterone are not effective in reducing triclocarban VAT. However, some studies have shown that GH axis drugs can both decrease VAT and help to maintain or improve peripheral adipose tissue mass [5]. Although the underlying mechanism for the development of HIV-associated lipodystrophy and related disorders such as metabolic syndrome is not fully understood, evidence suggests that neurohormonal dysregulation plays a role in causing these debilitating conditions [6–8]. GH is a polypeptide hormone secreted episodically by the adenohypophysis that affects protein, carbohydrate and lipid metabolism. There is also evidence that it plays a role in skeletal and visceral growth. Specifically, GH affects the metabolism of fats by causing cells to switch from using carbohydrates for fuel to burning fats for energy.

Thus, it is suspected that augmenting the GH axis in patients with HIV-associated lipodystrophy results in improved utilization of fat stores and subsequent redistribution of adipose tissue [9]. GH axis drugs investigated for the treatment of HIV-associated lipodystrophy include recombinant growth hormone (GH), growth FK506 cost hormone releasing hormone (GHRH), tesamorelin, also known as growth hormone releasing factor (GHRF), and insulin-like growth factor-1 (IGF-1). There are some concerns with this class of drug. GH, the most studied GH axis drug, costs approximately $52 per milligram, and is estimated to cost approximately US$10 000–US$30 000

per year of treatment [10]. Significant treatment-associated side effects of these drugs include arthralgias, myalgias, peripheral oedema,

insulin resistance and diabetes [11]. Considering the expense and side effects associated with these drugs, it is important to evaluate the evidence regarding the efficacies of these treatments to allow the patient and health care provider to make informed decisions. In the present systematic review, we evaluate randomized controlled trials comparing the effects of GH axis treatments with those of placebo in changing VAT, subcutaneous adipose tissue (SAT) and lean body mass (LBM) in patients with GW-572016 in vitro HIV-associated lipodystrophy. A detailed protocol was written prior to conducting the review. The protocol and documentation of all changes made after construction of the protocol are available upon request. Inclusion criteria were as follows (all were required to be met): (1) the study design must be a randomized controlled trial; (2) study participants were adult patients with HIV-associated lipodystrophy;

(3) the intervention was a GH axis drug (GH, GHRH, tesamorelin or IGF-1); (4) the comparison group was treated with placebo; and (5) the study included one of the primary outcomes. There were no exclusion criteria. Our primary outcomes of interest included changes in VAT mass, SAT mass or LBM. The secondary these outcomes included changes in extremity fat, levels of fasting plasma glucose, high-density lipoprotein (HDL) cholesterol and triglycerides, and waist circumference. Potential harms of treatment were also evaluated. The following databases were searched for studies: OVID MEDLINE (1996 to present; accessed 6 June 2010), The Cochrane Library [Cochrane Central Register of Controlled Trials and Cochrane Database of Randomized Controlled Trials (CENTRAL); accessed 11 October 2009], Web of Science (accessed 11 October 2009), Summons (accessed 13 October 2009), Google Scholar (accessed 11 October 2009) and PubMed (accessed 5 June 2010). Search terms included: HIV, AIDS, growth hormone, Serostim, GH releasing hormone, tesamorelin, IGF-1, HIV-associated lipodystrophy, adipose tissue and body composition. A comprehensive list of all search terms is available upon request.

A traditional defining characteristic of members of the Shewanella genus is the inability to use glucose as a substrate for growth. Shewanella spp. isolates, however, have the common ability to use a diverse array of substrates, allowing them to survive in a range of environments (Hau & Gralnick, 2007; Fredrickson et al.,

2008). Members of the Shewanella genus show great flexibility with regard to alteration of growth strategy and metabolism based on the availability of different carbon sources (Tang et al., 2009). In keeping with this flexibility, the traditional view of inability to use glucose has changed as many Shewanella spp. isolates have since been found to use glucose (Bowman et al., 1997; Nogi et al., 1998; Leonardo et al., 1999; Brettar et al., 2002; Gao et al., 2006; Zhao et al., 2006; Xiao et al., 2007; Rodionov et al., 2010). To this end, Shewanella oneidensis selleckchem MR-1, isolated from sediment in Lake Oneida, NY, has not been yet observed to use glucose as a fermentation substrate, under short-term growth experiments without initial glucose exposure in a rich growth medium (Myers & Nealson, 1988; Venkateswaran et al., 1999; Rodionov et al., 2010). In microbial fuel cells (MFCs) with complex

growth media, however, S. oneidensis Tamoxifen in vitro has been found to generate current upon extended glucose addition (Biffinger et al., 2008, 2009). This response to glucose addition was slow, suggesting that S. oneidensis may be able to use glucose, given ample time to induce the appropriate genetic mechanisms, for a mutator population to proliferate (Chao & Cox, 1983; Giraud et al., 2001a, b) and/or

develop a ‘growth advantage in stationary phase’ (GASP) mutant (Finkel & Kolter, 1999). Mutator bacteria contain mutations that inactivate mutation-avoidance genes yielding higher spontaneous mutation rates, which in turn yield an increased evolutionary pace (Chao & Cox, 1983; Mao et al., 1997; Giraud et al., 2001a, b). GASP refers to the genetic alterations (not physiological adaptations) that occur in cells incubated in long-term batch cultures that confer a competitive advantage to these cells over younger ‘naïve’ cultures (Finkel, 2006). Given that the S. oneidensis genome suggests the ability to use glucose as a Liothyronine Sodium fermentation substrate via the Entner–Doudoroff pathway, the current study seeks to show whether S. oneidensis can indeed utilize glucose as a sole carbon source given an initial glucose exposure, as suggested by previous MFC studies (Biffinger et al., 2008, 2009). Shewanella oneidensis MR-1 (Venkateswaran et al., 1999), obtained from the American Type Culture Collection (ATCC#700550) and stored at −80 °C, was grown up in Luria–Bertani (LB) broth for 24 h, shaking (100 r.p.m.) at 25 °C. From the LB culture, S. oneidensis MR-1 was serially passed every 24 h for 96 h into LB broth amended with 10 mM glucose.

More recently, C. vulgaris NJ-7, a strain isolated from the Antarctic, was used to investigate the adaptation of eukaryotic microbes to permanently cold environments (Hu et al., 2008; Li et al., 2009). The strain NJ-7 possesses the same 18S rRNA gene sequence as that of UTEX259, a strain isolated

from the temperate region, but shows a significantly intensified freezing tolerance (5- to  1000-fold higher viability) than the temperate strain. Comparative studies of the two C. vulgaris strains provide opportunities to understand how intra-species learn more evolution is undertaken in eukaryotic microbes to adapt to the Antarctic or other extreme environments. HIC6 is a group-3 late embryogenesis abundant (LEA) protein found in C. vulgaris. Together with HIC12, it was first identified by 2D-HPLC and SDS-PAGE to be hardening (cold treatment)-induced in the strain C-27 (Honjoh et al., 1995). Its cDNA was also identified by differential screening of a cDNA library (Joh et al., 1995) or suppression subtractive hybridization (Machida et al., 2008). LEA Rapamycin mouse proteins were initially found at the late stage of embryogenesis in cotton (Galau et al., 1986) and were subsequently found in algae (such as C. vulgaris), cyanobacteria (Close & Lammers, 1993), nematodes (Browne

et al., 2002) and fungi (Abba’ et al., 2006). The proteins can be divided into different groups on the basis of similarities in amino acid sequences (Colmenero-Flores et al., 1997; Cuming, 2005; Battaglia et al., 2008). Like many other LEA proteins, HIC6 remained soluble under boiling conditions and showed in vitro cryoprotective activities on lactate dehydrogenase (LDH) (Honjoh et al., 2000). Overexpression of HIC6 in plant or yeast could enhance their freezing tolerance (Honjoh et al., 1999, 2001), and

in the transgenic plant, HIC6 was localized to mitochondria (Honjoh et al., 2001). In strains NJ-7 and UTEX259, the encoding gene hiC6 was also induced upon exposure to cold, and the expression was intensified in strain NJ-7 in comparison with UTEX259 (Li et al., 2009). These results suggest that the enhanced expression of hiC6 is probably involved in the development of freezing tolerance in C. vulgaris. The intensified expression of hiC6 in NJ-7 could be due to gene duplication, increased transcription or post-transcriptional regulation. Obatoclax Mesylate (GX15-070) In our previous study, only one hiC6 gene was identified in each of the two Chlorella strains, NJ-7 and UTEX259 (Li et al., 2009). In the present study, however, sequencing of that chromosomal region revealed that multiple hiC6 genes are organized in tandem in both strains. The tandem-arrayed genes encode different HIC6 isoforms and are differentially expressed. Chlorella vulgaris strains were grown in BG11 (Stanier et al., 1971) in the light of 50 μE m−2 s−1 at 20 °C with aeration. Cells grown at 20 °C were cooled to 4 °C in a water bath and transferred to a 4 °C refrigerator with aeration and illumination (50 μE m−2 s−1) for different periods of time.

1b and c). The noncovalent inhibitors including benzamidine, leupeptin and

nafamostat mesylate also showed weak inhibition of HsaD (Fig. 1b) compared to PMSF and DCI. MGL like HsaD catalyses the turnover of highly hydrophobic substrates: as such the inhibitors LY294002 mw that have been identified tend to be insoluble (e.g. pristimerin and NAM). Although pristimerin is the most active noncovalent inhibitor tested (35% inhibition at 50 μM – Fig. 2a), further investigation was hampered by its poor aqueous solubility under conditions that are required for HsaD to remain active. NAM and JZL184 are covalent inhibitors: JZL184 like DCI and PMSF modifies the catalytic serine of MGL (Long et al., 2009), while NAM modifies a cysteine in the active site of MGL (Saario et al., 2005). Consistent http://www.selleckchem.com/products/Staurosporine.html with the lack of a cysteine residue in the active site of HsaD, NAM does not significantly inhibit HsaD (Fig. 2a). JZL 184 proved a better inhibitor (Fig. 2a) but was difficult to work with due to its hydrophobic nature and hence poor solubility. A series of specific acetylcholinesterase inhibitors were tested for inhibition of HsaD (Fig. 2b). These included eserine, edrophonium, tacrine, neostigmine, pyridostigmine and trichlorfon. After incubation with HsaD, trichlorfon inhibited poorly. Eserine and neostigmine show better inhibition, but still not as strong as was observed with DCI (c. 30% inhibition at 1 mM). The other

acetylcholinesterase inhibitors did not significantly inhibit HsaD. Two mechanisms have been proposed for the hydrolysis of substrates by MCP hydrolases. The first is based on the mechanism known to occur in serine proteases and proceeds via an acyl enzyme and tetrahedral intermediate (Ruzzini et al., 2012). The second requires a keto-enol tautomerization resulting in a gem-diol intermediate (Horsman et al., 2007). Recent mutagenesis experiments combined with structural studies resulted in trapping of the acyl enzyme intermediate of HOPDA hydrolysis, by another member of the C-C bond hydrolase family, BphD (Ruzzini et al., 2012) strongly supporting the first mechanism. Inhibition by PMSF and

DCI is also consistent with this mechanism as PMSF and DCI act as tetrahedral and acyl enzyme intermediate analogues, respectively, when they modify the active Oxalosuccinic acid site serine. The most successful inhibitors were those that covalently modify HsaD (e.g. DCI). The primary issue with DCI and other covalent inhibitors tends to be their broad specificity profile making them poor starting points for inhibitor design. To help understand the specificity observed among the covalent inhibitors, the structure of HsaD modified with PMSF was solved (Fig. 3). Although density was observed for the sulphonate group covalently linked to Ser114, there was insufficient density to accommodate the phenylmethyl group of PMSF. A lack of electron density for PMSF in the structure with HsaD might suggest that PMSF acts reversibly.

1b and c). The noncovalent inhibitors including benzamidine, leupeptin and

nafamostat mesylate also showed weak inhibition of HsaD (Fig. 1b) compared to PMSF and DCI. MGL like HsaD catalyses the turnover of highly hydrophobic substrates: as such the inhibitors find protocol that have been identified tend to be insoluble (e.g. pristimerin and NAM). Although pristimerin is the most active noncovalent inhibitor tested (35% inhibition at 50 μM – Fig. 2a), further investigation was hampered by its poor aqueous solubility under conditions that are required for HsaD to remain active. NAM and JZL184 are covalent inhibitors: JZL184 like DCI and PMSF modifies the catalytic serine of MGL (Long et al., 2009), while NAM modifies a cysteine in the active site of MGL (Saario et al., 2005). Consistent Selleck LY2157299 with the lack of a cysteine residue in the active site of HsaD, NAM does not significantly inhibit HsaD (Fig. 2a). JZL 184 proved a better inhibitor (Fig. 2a) but was difficult to work with due to its hydrophobic nature and hence poor solubility. A series of specific acetylcholinesterase inhibitors were tested for inhibition of HsaD (Fig. 2b). These included eserine, edrophonium, tacrine, neostigmine, pyridostigmine and trichlorfon. After incubation with HsaD, trichlorfon inhibited poorly. Eserine and neostigmine show better inhibition, but still not as strong as was observed with DCI (c. 30% inhibition at 1 mM). The other

acetylcholinesterase inhibitors did not significantly inhibit HsaD. Two mechanisms have been proposed for the hydrolysis of substrates by MCP hydrolases. The first is based on the mechanism known to occur in serine proteases and proceeds via an acyl enzyme and tetrahedral intermediate (Ruzzini et al., 2012). The second requires a keto-enol tautomerization resulting in a gem-diol intermediate (Horsman et al., 2007). Recent mutagenesis experiments combined with structural studies resulted in trapping of the acyl enzyme intermediate of HOPDA hydrolysis, by another member of the C-C bond hydrolase family, BphD (Ruzzini et al., 2012) strongly supporting the first mechanism. Inhibition by PMSF and

DCI is also consistent with this mechanism as PMSF and DCI act as tetrahedral and acyl enzyme intermediate analogues, respectively, when they modify the active Resminostat site serine. The most successful inhibitors were those that covalently modify HsaD (e.g. DCI). The primary issue with DCI and other covalent inhibitors tends to be their broad specificity profile making them poor starting points for inhibitor design. To help understand the specificity observed among the covalent inhibitors, the structure of HsaD modified with PMSF was solved (Fig. 3). Although density was observed for the sulphonate group covalently linked to Ser114, there was insufficient density to accommodate the phenylmethyl group of PMSF. A lack of electron density for PMSF in the structure with HsaD might suggest that PMSF acts reversibly.

Short-chain fatty acids (SCFAs) were determined by gas chromatography (GC-14B; Shimadzu, Kyoto, Japan). Succinate and d-/l-lactate were measured by commercial assay kits (Megazyme, Wicklow, Ireland). To monitor the growth of each bacterial strain in culture, copy number of 16S rRNA gene was quantified by real-time PCR. Repeated bead beating plus column method

(Yu & Morrison, PS-341 in vitro 2004) was employed for DNA extraction and purification from 1 mL of inocula and cultures at 48 and 96 h. PCR targeting the 16S rRNA gene was performed with a LightCycler 480 system (Roche Applied Science, Mannheim, Germany) and a KAPA SYBR FAST qPCR kit (KAPA Biosystems, Woburn, MA). Primer sets specific to each bacterial strain were used as follows: U2_Fw (5′-CTAGGTGTAGGGGGTATC-3′) and U2_Rv (5′-GCTGCCCTCTGTCGTTG-3′) for strain R-25 (Koike et al., 2010), 193f (5′-GGTATGGGATGAGCTTGC-3′) and 654r (5′-GCCTGCCCCTGAACTATC-3′) for F. succinogenes S85 (Tajima et al., 2001) and Sele.rumi_Fw (5′-TGCTAATACCGAATGTTG-3′) and Sele.rumi_Rv (5′-TCCTGCACTCAAGAAAGA-3′) for S. ruminantium S137 (Tajima et al., 2001). All other quantification procedures, including the standard plasmids, PCR conditions, and calculations, were according to Koike et al. (2007, 2010). To measure the fibrolytic activity in culture, fibrolytic find more enzyme assays were carried out for extracellular and intracellular fractions.

Culture supernatant and bacterial cells from strains R-25 and F. succinogenes S85 monocultures and their coculture were separated by centrifugation (16 000 g, 4 °C, 10 min). The supernatant was placed in dialysis tubing (12 000- to 14 000-Da cut-off, Seamless Cellulose Tubing, Sanko-junyaku, Tokyo,

Japan) in potassium phosphate buffer (50 mM, pH 6.8) overnight. The dialyzed fraction was condensed with polyethylene glycol (MW 20,000) and used in extracellular enzyme assays. Cell-free extract was obtained by ultrasonic disruption of the cell pellet (10 × 1 min on ice, 20 kHz, 25 watts) using a VC-70 O-methylated flavonoid Ultrasonic Processor (Sonics and Materials, Newton, CT) followed by centrifugation (16 000 g, 4 °C, 20 min) and was used in intracellular enzyme assay. The carboxymethylcellulase (CMCase) and xylanase activities were determined by monitoring the increase in reducing sugar formation from the substrates using dinitrosalicylic acid reagents, as described by Cotta (1988). Carboxymethylcellulose and oat spelt xylan were dissolved in 50 mM potassium phosphate buffer (pH 6.8) at 1% (w/v) and used as the substrates. The protein concentration was determined using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) with bovine gamma globulin as a standard. Enzyme activity was expressed as specific activity (formation of 1 nmol of sugar min−1 mg of protein−1) or total activity mL−1 culture (formation of 1 nmol of sugar min−1 mL−1 of original culture).

Mass spectra were acquired by a Finnigan™ LCQ™ DECA ion trap instrument. An ionization device was used for sample analyses (sheath gas: 80 mL min−1, auxiliary gas: 20 mL min−1, spray voltage: 5 kV, capillary temperature: 300 °C, capillary voltage: 46 kV, and tube lens: −60 kV). The Xcalibur 2.0

SR2 software (copyright Thermo Electron Corporation 1998–2006) was used. Morphological and cultural studies of the most productive isolate containing the ts gene, SBU-16, including conidial morphology, the mechanism PI3K Inhibitor high throughput screening of conidia production, and growth characteristics on PDA, potato-carrot agar (PCA), and on the firm base of an alfalfa stem were carried out according to Simmons (2001). The isolate of SBU-16 was grown on the media in a culture chamber under check details a 10-h photoperiod provided by 56 W cool-white fluorescent lamps (Philips Master, Holand) at 22 °C. Anamorph and telomorph populations were examined at 4–5 days and 2–6 weeks, respectively. The size and morphology of 100 mature conidia and 50 conidiophores

in lactic acid were recorded by light microscopy at 100× magnification and photographed. A total of 25 isolates separated from the inner bark of T. baccata were screened for the presence of the ts gene. Based on the conserved region of the ts gene, the specific primers were designed and synthesised for the amplification of the core DNA fragment of ts from 25 isolated endophytic fungi. Following PCR amplification, a 334-bp product was obtained. Of 25 isolates, 4 (SBU-16, SBU-17, SBU-69 and SBU-71) showed PCR positive for the conserved sequence of the ts gene (Fig. 1). Taxol and 10-DAB III were extracted from culture filtrates and mycelia of the four ts PCR positive fungi and then analyzed selleck products by HPLC-DAD. Under the same analysis conditions, the samples containing chemical reference substances of 10-DAB III and taxol were also compared with fungal extracts (Fig. 2). Further convincing evidence for the identity of 10-DAB III and taxol was obtained by high-performance

liquid chromatography-mass spectrometry (LC-MS). Characteristically, standard 10-DAB III and taxol yielded both an [M + H]+ peak at a molecular weight of 854 and an [M + Na]+ peak at a molecular weight of 876, respectively (see Fig. 3a and b). By comparison, fungal taxol also produced peaks, [M + H]+ at m/z 854 and [M + Na]+ at m/z 876. The peaks corresponding to taxol exhibited mass-to-charge (m/z) ratios corresponding to the molecular ions (M + H)+ of standard taxol (at 854), confirming the presence of taxol in the fungal extracts. It was evident that taxol was much more complex because its molecular weight (from high-resolution mass spectrometry) was 854, which corresponds to a molecular formula of C47H51NO14 as reported earlier (McClure & Schram, 1992). The results of the quantification analysis among the four ts PCR positive isolates showed that SBU-16, which was isolated for the first time in our laboratory, produces taxol (6.9 ± 0.2 μg L−1) and its intermediate compound, 10-DAB III (2.

Mass spectra were acquired by a Finnigan™ LCQ™ DECA ion trap instrument. An ionization device was used for sample analyses (sheath gas: 80 mL min−1, auxiliary gas: 20 mL min−1, spray voltage: 5 kV, capillary temperature: 300 °C, capillary voltage: 46 kV, and tube lens: −60 kV). The Xcalibur 2.0

SR2 software (copyright Thermo Electron Corporation 1998–2006) was used. Morphological and cultural studies of the most productive isolate containing the ts gene, SBU-16, including conidial morphology, the mechanism Pexidartinib clinical trial of conidia production, and growth characteristics on PDA, potato-carrot agar (PCA), and on the firm base of an alfalfa stem were carried out according to Simmons (2001). The isolate of SBU-16 was grown on the media in a culture chamber under CB-839 concentration a 10-h photoperiod provided by 56 W cool-white fluorescent lamps (Philips Master, Holand) at 22 °C. Anamorph and telomorph populations were examined at 4–5 days and 2–6 weeks, respectively. The size and morphology of 100 mature conidia and 50 conidiophores

in lactic acid were recorded by light microscopy at 100× magnification and photographed. A total of 25 isolates separated from the inner bark of T. baccata were screened for the presence of the ts gene. Based on the conserved region of the ts gene, the specific primers were designed and synthesised for the amplification of the core DNA fragment of ts from 25 isolated endophytic fungi. Following PCR amplification, a 334-bp product was obtained. Of 25 isolates, 4 (SBU-16, SBU-17, SBU-69 and SBU-71) showed PCR positive for the conserved sequence of the ts gene (Fig. 1). Taxol and 10-DAB III were extracted from culture filtrates and mycelia of the four ts PCR positive fungi and then analyzed ADAMTS5 by HPLC-DAD. Under the same analysis conditions, the samples containing chemical reference substances of 10-DAB III and taxol were also compared with fungal extracts (Fig. 2). Further convincing evidence for the identity of 10-DAB III and taxol was obtained by high-performance

liquid chromatography-mass spectrometry (LC-MS). Characteristically, standard 10-DAB III and taxol yielded both an [M + H]+ peak at a molecular weight of 854 and an [M + Na]+ peak at a molecular weight of 876, respectively (see Fig. 3a and b). By comparison, fungal taxol also produced peaks, [M + H]+ at m/z 854 and [M + Na]+ at m/z 876. The peaks corresponding to taxol exhibited mass-to-charge (m/z) ratios corresponding to the molecular ions (M + H)+ of standard taxol (at 854), confirming the presence of taxol in the fungal extracts. It was evident that taxol was much more complex because its molecular weight (from high-resolution mass spectrometry) was 854, which corresponds to a molecular formula of C47H51NO14 as reported earlier (McClure & Schram, 1992). The results of the quantification analysis among the four ts PCR positive isolates showed that SBU-16, which was isolated for the first time in our laboratory, produces taxol (6.9 ± 0.2 μg L−1) and its intermediate compound, 10-DAB III (2.