4). On some occasions the monkeys would have numerous ‘eye-closed’ periods of short duration or only a few eye-closed epochs of extended RGFP966 duration. It was notable that the vast majority of the Type 1 neurons described here had regular firing patterns during sleep, as illustrated for a typical single neuron in Fig. 8. The same property was described by Rolls et al. (2003) for single neurons in the subgenual cingulate cortex BA25 during periods of eye-closure. However, of note is that a few

Type 1 cells showed minor variations in the fine temporal patterning of neuronal firing during some ‘eyes-closed’ epochs, with some exhibiting ‘burst-like’ responses. The quantitative areal distribution of cell Types 1, 2 and 3 neurons in mPFC are given in Table 2 (see also Fig. 1C–E). Finally, it was not possible to ascertain unequivocally whether the neurons being studied electrophysiologically were excitatory projection pyramidal cells or local circuit inhibitory neurones. However, the likelihood is that most of the recorded cells were pyramidal projection neurons

as the spike durations were typically greater than 1.2 ms, which is highly characteristic Palbociclib mw of cortical pyramids (Rolls et al., 2003). The principal results of this study indicate that there are two populations of neurons throughout the monkey mPFC that significantly altered their firing rates when the subjects ‘closed’ or ‘opened’ their eyes. Type 1 cells (8.4% of all cells recorded) significantly increased their firing rate when the monkey became drowsy or closed its eyes, whilst Type 2 cells (1.8%) significantly decreased

their firing rate on eye-closure. Together these electrophysiological cell types represent a modest population (10.2%) of all the mPFC neurons screened in this study. Histological reconstructions confirmed that the cells studied electrophysiologically were in BAs 9, 10, 13 m,14c, 24b (dorsal anterior cingulate cortex) and 32 (pregenual cingulate cortex in primates), why with many of the recorded cells being located in the deep layers of the cortex (see Fig. 1C–E). A previous paper from our laboratory reported that neurons in BA25 (subgenual cingulate cortex) of the macaque mPFC also significantly increased their firing rates when monkeys went to sleep (Rolls et al., 2003). Of note is that comparable to the neurons reported here, the cells studied by Rolls et al. (2003) did not respond to gustatory, olfactory and most visual stimuli. Rolls and colleagues also presented evidence of four neurons in the orbitofrontal cortex (BA13) responding in a similar manner. The present study thus confirms and extends to further areas of mPFC the observations of the earlier companion paper. Taken together these two studies indicate that there are distributed populations of neurons throughout the mPFC of monkeys that selectively respond to being either ‘asleep’ or ‘awake’.

CRB of lactobacilli was growth-temperature-dependent and significantly higher (P < 0.05) for strains grown at 37 °C than Metformin at 30 °C (data not shown). CRB of L. crispatus 12005, L. rhamnosus GG, L. paracasei F8, L. plantarum F44, L. paracasei F19, and E. coli MC4 100 increased at a low pH and at a high ionic strength except for L. paracasei F8 at pH 3 and 4 (Fig. 2c). CRB of all five strains and E. coli MC4 100 was reduced significantly in the presence of 100 μg mL−1 of cholesterol

(Fig. 2). CRB was more than 95% for E. coli MC4 100 at high ionic strength combined with a low pH (3–4) and was completely inhibited in the presence of cholesterol at pH 3 and reduced to 10% at pH 8.0 (Fig. 2a–f). Pretreatment with proteolytic enzymes significantly reduced CRB of all five lactobacilli strains, including the S-layer-producing strain L. crispatus 12005 (Table 2). CRB by L. plantarum F44, L. paracasei F8, L. crispatus 12005 and L. paracasei F19 cells was significantly enhanced when these strains were grown in MRS with 0.5% TA (P < 0.05) Cetuximab molecular weight or 5% PB (P < 0.05) compared with cells grown in the MRS broth. The CRB of the L. paracasei F8 and L. paracasei F19 strains was significantly

enhanced with 0.25% mucin (P < 0.05), unlike L. rhamnosus 18243, which was unaltered when grown in MRS with bile or mucin. The SAT values of the less hydrophobic strains L. rhamnosus 18243, L. plantarum F44 and L. paracasei F19 dropped from 3.2 to 0.02 M, that is the cells were more hydrophobic when grown in MRS with 0.25% mucin, 0.5% TA or 5% PB, indicating an enhanced CSH in gut-simulated conditions (Fig. 3a–c). Three non-AA strains, L. rhamnosus 18243, L. plantarum F44 and L. paracasei F19, expressing

high CSH showed a dense biofilm formation when grown in MRS broth with either 5% PB or 0.5% TA compared Selleckchem Erastin with cells grown in MRS broth alone or in this broth with 0.25% mucin (Fig. 4a–c). The AA strains L. paracasei F8 and L. crispatus 12005 formed biofilm in MRS broth alone and MRS broth with 0.25% mucin (Fig. 4). Growth in MRS with 0.5% TA or 5% PB significantly reduced biofilm formation of the two AA strains (Fig. 4d and e). However, in the presence of 0.5% TA, the CRB ability of L. crispatus 12005 was significantly increased, and therefore the biofilm formation was increased (P < 0.05). Biofilm-forming lactobacilli strains, except L. paracasei F8, bound significantly (P < 0.05) more CR when cells were grown in MRS with 0.5% TA. Biofilm formation of five lactobacilli strains was studied after 24 and 72 h of growth (Fig. 5a). The AA strain L. crispatus 12005 showed higher biofilm formation with 0.5% TA and 5% PB stained with CR after 24 and 72 h of growth. The other four strains bound CR significantly more after 24 h in MRS with 0.5% TA compared with 72 h, and the CRB was similar for biofilm-forming cells after 24 and 72 h growth in MRS with 5% PB.

e. they occurred within the range of therapeutic doses), and 65% were classified as intermediate reactions. The susceptibility factors associated most frequently with ADRs were comorbidities (i.e. the presence of diseases that were considered as risk factors to developing an ADR; 36%), age (26%) and exogenous factors MK-2206 (i.e. the presence of drug interactions that were involved in the occurrence of ADRs; 17%). Fifty per cent of the ADRs could have been prevented. Conclusions  ADRs are very frequent in hospitalized patients and a significant

proportion of them is preventable. The DoTS classification allowed complete evaluation of the types of ADR encountered. We are currently carrying out a much larger prospective study. “
“The treatment of childhood cancer with chemotherapy, radiotherapy or surgery predisposes the child to a number of potential ‘late effects’, often complex and inter-related which may adversely influence growth, bone development and body composition or almost any endocrine gland function, depending on the treatment modality involved. As increasing time from cancer treatment is one of the risk factors for the development of endocrine dysfunction, effective long-term follow-up arrangements are necessary

for these patients to monitor for the development of such problems. Uncertainties remain about how services such as these should be organized and delivered in the longer term. “
“The prelims comprise: Half-Title ERK inhibitor Page Title Page Copyright Page Table of Contents Preface Numbers, conversions and tables “
“Hypocalcaemia and rickets may present relatively frequently in childhood.

A careful clinical and biochemical assessment of these cases should ensure that the relatively common causes are distinguished from rarer subtypes and treated appropriately. By contrast, hypercalcaemia is relatively rare and often resolves without PRKACG therapy. Osteoporosis is associated with substantial morbidity, but bisphosphanate therapy is proving a highly effective therapy. “
“Hypoglycaemia may be due to reduced glucose availability or increased glucose consumption, and requires urgent investigation and management to avoid neurological damage and unnecessary diagnostic tests later. The majority of causes present in neonatal life and are transient in nature. However, severe and persistent hypoglycaemia may be a consequence of significant endocrine dysfunction or inborn errors of metabolism, and should be managed in conjunction with a specialist centre from an early stage. “
“All change in history, all advance, comes from nonconformity. If there had been no troublemakers, no dissenters, we should still be living in caves.’ (AJP Taylor.) In the November/December 2010 issue of Practical Diabetes International, MEJ Lean’s personal comment reported on ‘How not to die from diabetes in a mountain hut’.

Supplementary searches to find additional published and unpublished studies were conducted in the Food and Drug Administration check details registry (accessed 11 October 2009) and the Clinical Trials registry (clinicaltrials.gov; accessed 11 October 2009). References of the articles included in our systematic review were also manually reviewed. Two reviewers independently reviewed all titles and abstracts for eligibility in an unblinded and standardized manner; all disagreements were resolved by a third reviewer. A data

extraction form was created, pilot-tested on four eligible studies, and then refined accordingly. Data collected were study design, trial participant characteristics, inclusion/exclusion criteria, study intervention including dosages and duration of follow-up, and primary/secondary outcome measurements. Data from all of the included studies Roscovitine ic50 were then abstracted and summarized independently by two reviewers using the data extraction form. Discrepancies were resolved by repeated review and discussion between reviewers. Data in the clinical trials registry were compared with those of the published journal article when possible. We assessed the methodological quality of all articles included in our systematic review using the Risk of Bias Tool

recommended by the Cochrane Handbook for Systematic Reviews of Interventions [12]. The data gathered from the risk of bias assessment are presented in our systematic review to identify studies that were compromised in terms of methodological quality, but these data were not utilized in our calculations of summary effect. Each study was graded as having a low risk, high risk, or unclear risk of bias in accordance with the Cochrane Collaboration Tool. The summary of the differences in treatment effects between GH axis treatments and placebo is given in terms of weighted mean differences (WMDs). No qualitative measures are

included in the treatment effects reported. For nine of our ten included articles, no calculations or estimates were needed to replace data missing from the published reports. We were unable to reach Waters et al. to request additional unpublished data, and thus we estimated values of LBM based on graphs provided in their study. We used a standardized formula to calculate the standard deviations from Edoxaban confidence intervals [12]. Summary treatment effects were calculated with the Cochrane Collaboration Review Manager Version 5 (revman 5) using the random effects model. This model provides a conservative estimation and takes into account variance between studies in terms of quality and sample size. A test of heterogeneity was applied to the included studies to evaluate the magnitude of differences between the studies for the overall treatment effect of GH axis drugs vs. placebo, as well as for subgroup analyses for each GH axis drug vs. placebo.

Supplementary searches to find additional published and unpublished studies were conducted in the Food and Drug Administration Ku-0059436 concentration registry (accessed 11 October 2009) and the Clinical Trials registry (clinicaltrials.gov; accessed 11 October 2009). References of the articles included in our systematic review were also manually reviewed. Two reviewers independently reviewed all titles and abstracts for eligibility in an unblinded and standardized manner; all disagreements were resolved by a third reviewer. A data

extraction form was created, pilot-tested on four eligible studies, and then refined accordingly. Data collected were study design, trial participant characteristics, inclusion/exclusion criteria, study intervention including dosages and duration of follow-up, and primary/secondary outcome measurements. Data from all of the included studies SB203580 clinical trial were then abstracted and summarized independently by two reviewers using the data extraction form. Discrepancies were resolved by repeated review and discussion between reviewers. Data in the clinical trials registry were compared with those of the published journal article when possible. We assessed the methodological quality of all articles included in our systematic review using the Risk of Bias Tool

recommended by the Cochrane Handbook for Systematic Reviews of Interventions [12]. The data gathered from the risk of bias assessment are presented in our systematic review to identify studies that were compromised in terms of methodological quality, but these data were not utilized in our calculations of summary effect. Each study was graded as having a low risk, high risk, or unclear risk of bias in accordance with the Cochrane Collaboration Tool. The summary of the differences in treatment effects between GH axis treatments and placebo is given in terms of weighted mean differences (WMDs). No qualitative measures are

included in the treatment effects reported. For nine of our ten included articles, no calculations or estimates were needed to replace data missing from the published reports. We were unable to reach Waters et al. to request additional unpublished data, and thus we estimated values of LBM based on graphs provided in their study. We used a standardized formula to calculate the standard deviations from Miconazole confidence intervals [12]. Summary treatment effects were calculated with the Cochrane Collaboration Review Manager Version 5 (revman 5) using the random effects model. This model provides a conservative estimation and takes into account variance between studies in terms of quality and sample size. A test of heterogeneity was applied to the included studies to evaluate the magnitude of differences between the studies for the overall treatment effect of GH axis drugs vs. placebo, as well as for subgroup analyses for each GH axis drug vs. placebo.

, 1999) and the role of these receptors in the cardiovascular system has been studied in detail (Knaus et al., 2007a,b). Briefly, adra2a/2c-ko mice display elevated plasma concentrations of catecholamines, increased blood pressure and cardiac hypertrophy in adulthood (Hein et al., 1999; Knaus et al., 2007a,b). The developmental consequences of constitutive deletions of adra2a, adra2c and adra2a/2c in the central nervous system are not striking and

the brains of these animals appear to be grossly normal. Quantification of the distribution of GAD65-GFP+ interneurons in adra2a-ko or adra2c-ko mice did not reveal any significant changes in the distribution of cortical interneurons at P21, suggesting compensatory regulatory mechanisms following constitutive developmental deletion of either of these receptors. Interestingly a significant increase in the percentage of GAD65-GFP+ cells in upper cortical layers II/III were detected in the somatosensory Galunisertib Alectinib cortex of adra2a/2c-ko mice, indicating that combined deletion of adra2a and adra2c receptors significantly modifies the distribution of cortical interneurons in vivo. The intracellular mechanism mediating the effects of adra2 stimulation on interneuron migration is likely to involve different transduction pathways. Adra2 are G-protein-coupled receptors negatively coupled to adenylate

cyclase, and modifications in the levels of cAMP could thus constitute a downstream effector of adra2 stimulation. Cyclic AMP is a key molecule regulating growth cone dynamics (Song & Poo, 2001), and experimental manipulation of the ratio of cAMP to cGMP determines the responsiveness of axonal growth cones to guidance cues (Nishiyama et al., 2003). In the embryonic brain cAMP is critical for proper axonal pathfinding of olfactory sensory neurons (Chesler et al., 2007). In migrating

neurons, alteration in the levels of cAMP decreases the migratory speed of cerebellar granule cells (Cuzon et al., 2008) and modulates the effects of serotonin on migrating cortical interneurons (Riccio et al., 2009). Interestingly, there is a functional pathway linking adra2a, cAMP and hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN channels; Wang et al., 2007). HCN channels have been shown to regulate axonal targeting of olfactory sensory many neurons during development (Mobley et al., 2010) and thus represent an attractive downstream developmental target of cAMP that could regulate interneuron migration. Calcium could also be another downstream effector mediating the effects of adra2 activation on migrating interneurons. In other cellular systems, it has been shown that adra2a stimulation regulates intracellular calcium levels through the modulation of voltage-gated N-type calcium channels and that this process occurs independently of cAMP modulation (Lipscombe et al., 1989; Ikeda, 1996).

, 2004). The previously designed approach to produce and resuscitate NC cells was applied to study M. smegmatis

strain with inactivated hlp gene. Our experiments revealed that M. smegmatisΔhlp strain and its derivatives developed in the modified Hartman-de-Bont medium (without K+) at similar growth rates compared with the wild-type strain (data not shown). At the stationary phase, the Δhlp strain entered the NC state (0 CFU) later than the Wt-pMind strain with Talazoparib concentration the empty plasmid (90–96 vs. 68–70 h) (Fig. 1a) or wild type (not shown). Complemented strain Δhlp∷hlp harboring the hlp gene on the plasmid entered the NC state only 2 h later than the Wt-pMind strain (data not shown). Next, NC cells of Wt-pMind and Δhlp Sirolimus strains

were tested for their ability to resuscitate in the presence of recombinant M. luteus RpfSm protein, which appeared to be more active and stable during storage compared with full-length M. luteus Rpf (unpublished data). Contrary to Wt-pMind, NC cells of the strain Δhlp failed to resuscitate in the liquid medium with RpfSm (Fig. 2). NC cells of the complemented strain Δhlp∷hlp were partially resuscitated by RpfSm (Fig. 2). Therefore, the lack of Hlp resulted in transition of mycobacterial cells to an irreversibly NC (or likely, moribund) state under chosen conditions (K+ depletion) but not to a resuscitatable dormancy. Taken together, these data led to the conclusion that Hlp does not affect the onset of the transition to nonculturability but Carnitine dehydrogenase seems to be essential for cell viability at a later stage of dormancy. It was noteworthy that the strain Δhlp∷rpf, lacking the hlp gene and harboring the plasmid-carried rpf gene, substantially differed from the Δhlp in culturability when cultivated in the modified Hartman-de-Bont medium. In particular, Δhlp∷rpf cells maintained the ability to produce colonies even during a prolonged (180 h) stationary phase, as opposed to Δhlp and Δhlp-AGH strains (Fig. 1b). However, insertion of the rpf gene to

wild-type M. smegmatis (Wt-AGR) caused the development of NC state of cells incubated in the same medium and conditions (Fig. 1b), as reported previously (Shleeva et al., 2004). To ensure that the maintenance of plateability in long-stored Δhlp∷rpf cultures was indeed due to Rpf production, we studied the Δhlp∷rpf mut strain (Table 1) with the rpf gene disrupted by site-directed mutagenesis (Mukamolova et al., 2006). Our experiments demonstrated that mutations in the rpf gene restored the ability to adopt the NC state under the given cultivation conditions (Fig. 1b). Thus, the combined action of a downshift of Hlp and an upshift of Rpf greatly prolonged the ability of M. smegmatis to endure starvation in a completely culturable state, revealing opposite effects of two proteins on the formation of ‘nonculturability’. Earlier we found that M.

, 1998). The genome size of the bacteriophages (φVh1, φVh2, φVh3, and φVh4) based on PFGE was minimal (0.8–3.2 kb) and was estimated to be 85, 58, 64, and 107 kb, respectively, by PFGE. The genome size of the members of the family Siphoviridae is reported to range from 14.5 kb in Lactococcus prophage bIL311 to 134.4 kb in Bacillus phage SPBc2 (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=10699). The genome size of the VHS1 Siphoviridae phage of V. harveyi described earlier was approximately 80 kb (Pasharawipas et al., 2005), and six of selleck products them described by Shivu and others had genome sizes ranging from 44 to 94 kb as determined by REA

(Shivu et al., 2007). The phylogenetic analysis showed that the four bacteriophages were distinct from one another as revealed by cluster analysis. The clustering pattern based on both REA and PFGE showed distinct genetic nature of φVh3. A marine phage capable of specifically transducing the tryptophan region was described almost three and a half decades back (Keynan et al., 1974). In the present study, all the four bacteriophages were capable of transducing the plasmid DNA between V. harveyi with a transduction frequency ranging from 4.1 × 10−7 to 2 × 10−9 PFU−1. A similar efficiency was reported with indigenous marine phage host isolates in an earlier report (Jiang & Paul, 1998). It has been demonstrated that the vibriophages in the coastal

environment transfer genes from O1 El Tor strain to ERK inhibitor non-O1/O139 through transduction, suggesting the process as one of the mechanisms of pathogenicity evolution among environmental Y-27632 in vivo V. cholerae

(Choi et al., 2010). Possibilities of genetic interaction among the bacteriophage genomes and chromosomal and plasmid-borne DNA of vibrios such as Vibrio parahaemolyticus strains and of genetic transmission among strains through filamentous phages have been suggested (Chang et al., 1998). The use of a wide variety of antibiotics in aquaculture has resulted in the emergence of antibiotic-resistant bacteria in aquaculture environments (Cabello, 2006). The abundant occurrence of bacteria along with their bacteriophages in seawater and aquatic sediments is known to facilitate such a transfer (Fuhrman, 1999). In conclusion, results from this study provide description of three bacteriophages of the family Siphoviridae and one of the family Podoviridae. Literature search shows that the latter group of bacteriophages has not been reported from the shrimp aquaculture ecosystem so far. The significance of the present study is that these bacteriophages were able to bring about generalized transduction and can transfer genetic elements such as antibiotic resistance or pathogenicity traits among V. harveyi and possibly in other vibrio species in the brackishwater aquaculture ecosystem. Authors are thankful to the Indian Council of Agricultural Research, New Delhi for the financial assistance [F.No.

Demographic, lifestyle and laboratory data were prospectively collected on each patient with HIV infection. The anti-HEV IgG seroprevalence in patients with HIV infection was compared with that in controls and demographic risk factors for HEV exposure were explored using logistic regression models. There was no difference in anti-HEV

IgG seroprevalence between the HIV-infected patients and controls. The only risk factor predictive of anti-HEV seropositivity was the consumption of raw/undercooked pork; sexual risk factors were unrelated. No patient with HIV infection had evidence of chronic coinfection with HEV Anti-HEV seroprevalence is similar in controls and patients with HIV infection. Risk factor analysis suggests that HEV is unlikely to be transmitted sexually. Chronic coinfection with HEV was absent, indicating that Galunisertib chronic HEV/HIV coinfection is not a common problem in this cohort. Hepatitis E virus (HEV) is endemic in many parts of the developing world and globally it is the

commonest cause of acute viral hepatitis. In developing countries, hepatitis E usually results in a self-limiting hepatitis, except in pregnant women in whom the mortality is approximately 20% [1]. Autochthonous (locally acquired) HEV infection is an emerging health issue in developed countries [1] and is thought in many cases to be a porcine zoonosis. In developed countries, acute HEV infection mainly affects the middle-aged and elderly and is more common in male individuals [2–7]. Recently, chronic HEV infection Ixazomib with rapidly progressive cirrhosis has been demonstrated in immunosuppressed transplant recipients [8], and in individuals with haematological malignancies [9]. In 2009, chronic HEV coinfection was documented in ALOX15 the UK [10] and France [11] in two HIV-infected patients, in association with established cirrhosis.

However, little is currently known about the extent or outcomes of HEV and HIV coinfection. The aim of this study was to document the incidence of chronic HEV coinfection in an unselected group of patients with HIV infection and to determine the anti-HEV seroprevalence in patients with HIV infection and compare it with that of a control population. Consecutive, unselected patients with documented HIV infection were approached to participate in the study between July 2009 and May 2010. The patients were attending the Departments of HIV Medicine at two teaching hospitals in southwest England (Royal Cornwall Hospital, Truro, and Southmead Hospital, Bristol, UK). After the patients had provided informed consent, a serum sample was taken and frozen at −70 °C, prior to being tested for HEV by reverse transcriptase-polymerase chain reaction (RT-PCR) and anti-HEV immunoglobulin G (IgG) and IgM immunoassays. Samples were also tested for hepatitis A virus (HAV) RNA by RT-PCR. Demographic, lifestyle and laboratory data were prospectively collected on each patient.

We then immersed them in

an intermediate solvent (propylene oxide; Nisshin EM) for 10 min and in a mixture (1 : 1 v/v) of propylene oxide and Spurr’s resin (Spurr, 1969; Polysciences, Warrington, PA) for 6 h at room temperature. We then placed the samples in pure Spurr’s resin at 4 °C for 3 days. The specimens were then embedded in resupinated gelatin capsules (Nisshin EM) and polymerized at 70 °C for 24 h. To observe the cellular reactions at contact sites between hyphae, we cut the blocks parallel to the contact regions with a microtome blade (Feather Safety Razor, Gifu, Japan) under the stereomicroscope. The blocks were then cut with a Porter-Blum MT-1 ultramicrotome (Ivan Sorvall, Norwalk, CT) and a diamond knife (Diatome, Bienne, Switzerland). We prepared ultrathin sections (c. 80 nm thick). Copper grids (Thin Bar grid, Gilder, Grantham, UK) were coated with 2% collodion in isoamyl Ivacaftor order acetate (Nisshin EM) 30 min before use. Sections were stained

with 4% aqueous uranyl acetate for 10 min and then with modified Sato’s lead solution (Sato, 1968) at room temperature for 10 min. Every staining step was followed by washing with distilled water. The stained sections were observed with an electron microscope (H7100, Hitachi, Ibaraki, Japan) at an accelerating voltage of 75 kV. When we www.selleckchem.com/products/BIBW2992.html noticed that some cell structures had collapsed at the hyphal contact zones, we rated the parts of the cell contents that had collapsed in each interaction zone. Proportions of the mafosfamide collapsed cell components were calculated using observations of 50 hyphal contact zones. Mycelia

were grown in 1/10-strength oatmeal liquid medium (2.6 g L−1 oatmeal, 5 g L−1 sucrose) for 1 week. The mycelial mass was cut into small pieces using a homogenizer (Nihon Seiki Kaisha Ltd, Tokyo) at 10 000 r.p.m. for 5 s. The homogenized mycelia were mixed in compatible and incompatible combinations and spread on cellulose membranes laid on oatmeal agar plates. Cellulose membranes with attached mycelia were stripped from the plates after 5 and 8 days inoculation, ground to a fine power in liquid nitrogen, and then dissolved in DNA isolation buffer (10 mM Tris-HCl pH 7.5, 100 mM LiCl, 100 mM EDTA, 0.5% w/v SDS). After incubation of the solutions at 60 °C for 30 min, we precipitated the mycelial debris by centrifugation at 10 000 g for 10 min at 4 °C. The total nucleic acids were extracted with an equal volume of phenol : chloroform : isoamyl alcohol (PCI; 25 : 24 : 1 v/v) and precipitated with an equal volume of isopropanol by centrifugation at 10 000 g at 4 °C for 15 min. Total nucleic acids were resuspended in 100 μL TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and were electrophoresed on 1.0% agarose gel in TAE buffer (40 mM Tris-acetate pH 8.0, 1 mM EDTA) to confirm the quality of the total nucleic acids.