, 2000, 2004, 2008; Starkey et al., 2007; Yli-Mattila et al., 2009; Sarver et al., 2011). All F. graminearum sensu stricto strains (lineage 7) can produce click here sexual progeny (ascospores) without contact with a sexual partner, which is known to be important for initiating the disease cycle (Trail et al., 2002). However, this self-fertility varies among the other members of the Fg complex. For

example, Fusarium asiaticum (lineage 6), which is widely distributed in Asia, exhibited a lower self-fertility than the highly fertile F. graminearum strains (Lee et al., 2012). The sexual ability of the Fg complex is controlled by master regulators called mating-type (MAT) loci (Debuchy & Turgeon, 2006). Unlike their heterothallic relatives, the Fg complex strains carry two MAT loci (MAT1-1 and MAT1-2) in a single nucleus for controlling sexual development, but the structural organization of individual MAT genes is similar to those in Sordariomycetes fungi (e.g. Neurospora crassa, Podospora

anserina, and Sordaria macrospora; Yun et al., 2000; Debuchy & Turgeon, 2006). Three (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and one (MAT1-2-1) transcripts are located at both loci, among which the deduced product of MAT1-1-1 carries a DNA-binding motif called the alpha box, BIBW2992 supplier those of MAT1-1-3 and MAT1-2-1 contain an HMG box domain, and that of MAT1-1-2 includes a newly proposed DNA-binding PHP domain (Yun et al., 2000; Debuchy & Turgeon, 2006). An additional transcript, MAT1-2-3, has been proposed as a new MAT gene at the MAT1-2 locus in the heterothallic Fusarium verticillioides and F. graminearum (Martin et al., 2011). However, it contains no known DNA-binding motifs and its role(s) in sexual development are unknown. To date, gene deletion analyses have confirmed that both MAT loci are essential for sexual development in F. graminearum Selleckchem Depsipeptide (Lee et al., 2003; Desjardins et al., 2004) but the functional requirement for the individual MAT genes, except MAT1-2-1, has not been intensively demonstrated.

Recently, the transgenic strains deleted for MAT1-1-1 and MAT1-1-3, respectively, have become available (Son et al., 2011). Despite the importance of MAT loci in sexual development, transcriptional expression or regulation of MAT genes has remained largely unknown in filamentous fungi. Only a few reports are available (Leubner-Metzger et al., 1997; Czaja et al., 2011), and only the expression pattern of MAT1-1-2 is available from microarray analysis in F. graminearum (Hallen et al., 2007). The functions of each MAT gene in a self-fertile S. macrospora have been determined; Smt A-1 and Smt A-3, which are comparable to MAT1-1-1 and MAT1-1-3, respectively, are dispensable for fruiting body formation (Klix et al., 2010).

The finding of a larger amplitude of the N1 component over the right as compared with the left hemisphere sites and of a more widespread group difference in the N1 peak amplitude over the right hemisphere in our Staurosporine in vitro study is noteworthy. Although lateralization effects in ERP results should be interpreted with caution, our results do agree with reports of greater right hemisphere involvement in the processing of spectral information and of timbre in particular (e.g. Belin et al., 2000; Zatorre & Belin, 2001; von Kriegstein

et al., 2003). While the N1 enhancement in musicians was present to all sound types, the relationship between its peak amplitude and measures of musical proficiency was limited to the NAT condition. More specifically, individuals who rated their own musical ability more highly had a larger N1 peak amplitude to both music

and voice deviants. Additionally, individuals with higher MAP scores had higher N1 peak amplitude to music deviants. A similar but weaker relationship was also present between MAP find more scores and N1 to voice deviants. A relationship between N1 and either the age at onset of training or the duration of training was not significant. In part this may be due to the fact that we tested amateur musicians, who on average started their training later than what would be typical for professional musicians. Overall, however, reports of correlation between either the age at the onset of musical training or the duration HAS1 of such training and the enhancement of early ERP responses are not consistent (e.g. Pantev et al., 1998; Shahin et al., 2003; Musacchia et al., 2007). Our evaluation of timbre encoding in musicians and non-musicians has its limitations. Our main task probed the ability of the two groups of participants to resist distraction

and did not measure overt timbre perception. Therefore, whether enhanced N1 peak amplitude to complex sounds in musicians actually translates into better timbre identification and/or discrimination requires future studies. Related to the above point is the fact that the design of our study required that we use only a small set of sounds to represent vocal and musical timbres. In contrast, studies of the FTPV component used a large range of vocal and non-vocal sounds. Future studies that use a larger set of timbre examples and focus on the FTPV component may help determine whether musicians’ neural encoding of voices as a perceptual category (compared with voices’ acoustic properties as in the current study) is superior to that in non-musicians. In summary, musicians showed an enhanced N1 ERP component not only to musical and vocal sounds but also to never before heard spectrally-rotated sounds.

Participants self-reported improved confidence levels aligned to different situations, including the ability to adjust insulin accordingly and achieve target blood glucose (4–7mmol/L). Scores increased significantly in a number of scenarios, e.g. confidence to do a sporting activity 8(6.5–8.2) to 9(8.0–9.0), confidence to adjust insulin for the activity 5(3.8–6.2) to 8(6.9–8.3), and confidence that glucose levels will be in target after insulin adjustment 4(3.4–5.6) IDH inhibitor to 8(6.6–8.0). Attendance at a structured group education programme can significantly increase individuals’ self-efficacy to manage type 1 diabetes. Copyright © 2012 John Wiley & Sons. “
“We report

the case of a six-year-old male paediatric patient diagnosed with type 1 diabetes following an emergency department admission for treatment of an asthma attack. The patient’s elevated blood glucose level of 22.9mmol/L was consistent with current guidelines for the diagnosis of diabetes. However, he remained on the same dose of insulin

for three years (despite steady, normal growth). During this time he had no problems with blood glucose control and no hypoglycaemic attacks. The diagnosis of diabetes was, therefore, questioned. The values for steroid induced hyperglycaemia were markedly higher in this patient than anecdotal values. This prompted the suspicion that the steroid induced hyperglycaemia had been further exacerbated Epigenetic inhibitor cell line by salbutamol administration in the setting of an acute stress response. The findings of this case illustrate selleck kinase inhibitor the importance of understanding drug induced hyperglycaemia in the presence of intercurrent illness. Copyright © 2010 John Wiley & Sons. “
“Approximately half a million people die in the United Kingdom each year, of whom more than three-quarters are aged 75 years and over. Calculations based on the prevalence of diabetes indicate that 6–9% of those dying will have diabetes. In ethnic minority groups the prevalence will be significantly higher and

in all groups the majority will have type 2 diabetes. Accurate data on the incidence of diabetes as a contributory factor to death are not available due to the vagaries of death certification. Excellent end of life care (EOLC) strategies such as the Liverpool Care Pathway for the Dying Patient are already in routine use. The aim of this position statement is to augment such EOLC tools with guidance specifically related to people with diabetes, their families and carers. Outside the scope of this statement are issues relating to the use of advance directives (although patient autonomy and choice are paramount at all times), preferred models of palliative care services to support patients with diabetes, and referral criteria for hospice care. Historically, the diabetes community has pioneered a patient-centred approach to care but the care of the dying patient with diabetes has been neglected and needs to be incorporated into our practice.

Participants self-reported improved confidence levels aligned to different situations, including the ability to adjust insulin accordingly and achieve target blood glucose (4–7mmol/L). Scores increased significantly in a number of scenarios, e.g. confidence to do a sporting activity 8(6.5–8.2) to 9(8.0–9.0), confidence to adjust insulin for the activity 5(3.8–6.2) to 8(6.9–8.3), and confidence that glucose levels will be in target after insulin adjustment 4(3.4–5.6) Selleck Torin 1 to 8(6.6–8.0). Attendance at a structured group education programme can significantly increase individuals’ self-efficacy to manage type 1 diabetes. Copyright © 2012 John Wiley & Sons. “
“We report

the case of a six-year-old male paediatric patient diagnosed with type 1 diabetes following an emergency department admission for treatment of an asthma attack. The patient’s elevated blood glucose level of 22.9mmol/L was consistent with current guidelines for the diagnosis of diabetes. However, he remained on the same dose of insulin

for three years (despite steady, normal growth). During this time he had no problems with blood glucose control and no hypoglycaemic attacks. The diagnosis of diabetes was, therefore, questioned. The values for steroid induced hyperglycaemia were markedly higher in this patient than anecdotal values. This prompted the suspicion that the steroid induced hyperglycaemia had been further exacerbated TGF-beta inhibitor by salbutamol administration in the setting of an acute stress response. The findings of this case illustrate Tyrosine-protein kinase BLK the importance of understanding drug induced hyperglycaemia in the presence of intercurrent illness. Copyright © 2010 John Wiley & Sons. “
“Approximately half a million people die in the United Kingdom each year, of whom more than three-quarters are aged 75 years and over. Calculations based on the prevalence of diabetes indicate that 6–9% of those dying will have diabetes. In ethnic minority groups the prevalence will be significantly higher and

in all groups the majority will have type 2 diabetes. Accurate data on the incidence of diabetes as a contributory factor to death are not available due to the vagaries of death certification. Excellent end of life care (EOLC) strategies such as the Liverpool Care Pathway for the Dying Patient are already in routine use. The aim of this position statement is to augment such EOLC tools with guidance specifically related to people with diabetes, their families and carers. Outside the scope of this statement are issues relating to the use of advance directives (although patient autonomy and choice are paramount at all times), preferred models of palliative care services to support patients with diabetes, and referral criteria for hospice care. Historically, the diabetes community has pioneered a patient-centred approach to care but the care of the dying patient with diabetes has been neglected and needs to be incorporated into our practice.

Barth, M. Battegay, E. Bernasconi, J. Böni, H.C. Bucher, P. Bürgisser, C. Burton-Jeangros, A. Calmy, M. Cavassini, M. Egger, L. Elzi, J. Fehr, M. Flepp, P. Francioli (President of the SHCS), H. Furrer (Chairman of the Clinical and Laboratory Committee), C.A. Fux, M. Gorgievski, H. Günthard (Chairman of the Scientific Board), B. Hasse, H.H. Hirsch, B. Hirschel, I. Hösli, C. Kahlert, L. Kaiser, O. Keiser, C. Kind, T. Klimkait, H. Kovari, B. Ledergerber, G. Martinetti, B. Martinez de Tejada, K. Metzner, N. Müller, D. Nadal, G. Pantaleo, A. Rauch, S. Regenass, M. Rickenbach (Head of Data Center), C. Rudin

(Chairman of the Mother & Child Substudy), P. Schmid, D. Schultze, selleck compound F. Schöni-Affolter, J. Schüpbach, R. Speck, P. Taffé, P. Tarr, A. Telenti, A. Trkola, P. Vernazza, V. von Wyl, R. Weber and Selleck 5-FU S. Yerly. Financial support: This study has been financed in the framework of the Swiss HIV Cohort Study, supported by the Swiss National Science Foundation. Further support was provided by an unrestricted educational grant from Gilead. The funding sources had no influence on study design, analysis or writing of the manuscript. “
“We prospectively investigated fever symptoms and maternal diagnosis

of malaria in pregnancy (MIP) in relation to child HIV infection among 2368 pregnant HIV-positive women and their infants, followed up from pregnancy until 6 weeks post-delivery in Tanzania. Doctors clinically diagnosed and treated MIP and fever symptoms during prenatal health care. Child HIV status was determined

via DNA polymerase chain reaction (PCR). Multivariable logistic regression models were used to estimate relative risks (RRs) and 95% confidence intervals (CIs) for HIV mother-to-child transmission (MTCT) by the 6th week of life. Mean gestational age at enrolment was 22.2 weeks. During follow-up, 16.6% of mothers had at least one MIP diagnosis, 15.9% reported fever symptoms and 8.7% had both fever and MIP diagnosis. Eleven per cent of HIV-exposed infants were HIV-positive by 6 weeks. The RR of HIV MTCT was statistically similar for infants whose mothers were ever vs. never clinically diagnosed with MIP (RR 1.24; 95% CI 0.94–1.64), were diagnosed with one vs. no clinical MIP episodes (RR 1.07; 95% Ribociclib concentration CI 0.77–1.48) and had ever vs. never reported fever symptoms (RR 1.04; 95% CI 0.78–1.38) in pregnancy. However, the HIV MTCT risk increased by 29% (95% CI 4–58%) per MIP episode. Infants of women with at least two vs. no MIP diagnoses were 2.1 times more likely to be HIV infected by 6 weeks old (95% CI 1.31–3.45). Clinical MIP diagnosis, but not fevers, in HIV-positive pregnant women was associated with an elevated risk of early HIV MTCT, suggesting that malaria prevention and treatment in pregnant HIV-positive women may enhance the effectiveness of HIV prevention in MTCT programmes in this setting. Future studies using a laboratory-confirmed diagnosis of malaria are needed to confirm this association.

, 2003). Sequences of the PCR products were analyzed by blast search, and the most closely related species were determined. DNA or protein sequences were aligned with clustalw algorithm implemented in bioedit software using the default parameters. The aligned and trimmed sequence regions were used Compound C as the input files to infer phylogenetic trees based on neighbor joining of genetic distance with bootstrapping in molecular evolutionary genetics analysis (mega) software version 4.0.2. The accession numbers for the partial sequences of

BE74 16S rRNA gene and phzD genes are HM588007 and HM588008. The primers used for amplifying the ∼340-bp phzD fragment were as follows: PhzD254-282F, AAC AGC GCG GYC TSC TCA AGG ACT TCT GG and PhzD571-592R, SSG CRC AGC GCT CGG CGG CGT A. Mycelia of BE74 were collected from one agar plate or 1 mL liquid culture for RNA isolation using an RNA isolation kit (Ribopure-Bacteria, Ambion). Total RNAs were treated with DNase for half an hour and extracted using the standard phenol–chloroform method. Reverse transcription (RT) was performed with ∼200 ng RNA, SuperScript II reverse transcriptase (Invitrogen) and random hexamers. Two microliters of the RT reaction were subjected to a PCR reaction with the

primers PLX4032 purchase designed for an ∼162-nt fragment within the phzD gene of BE74: NocPhzD_F1, AAC AGC GCG GCC TCC TCA AGG ACT TCT GG and NocPhzD_R2, TTG GTG AGC AGG AGG TCC TCA CCG TCG. The annealing

temperature was 64 °C and there were 30 PCR cycles. Initially, a small number OSBPL9 of adult worker honeybees (N=6) were collected in September 2008 from hives at six locations (separated by 3–20 miles) in southeastern Ohio. After the processing and selective isolation of actinomycetes with the AIA, the purified actinomycete colonies were analyzed using morphology (colors of aerial and substrate mycelia, pigments and starch lysis zone, etc.) and sequences of the amplified partial 16S rRNA gene. The results confirmed the presence of actinomycetes, mainly a diverse group of Streptomyces, in the guts of honeybees. Three to eight different Streptomyces species could be identified, with six bees from each of five locations. Bees of the remaining one location did not yield any actinomycete-like colonies on the AIA, but did produce a large number of nonactinomycete colonies. DNA typing showed that these nonactinomycete isolates were related to at least five Bacillus species (identity of the 16S rRNA gene >97%). They were Bacillus cereus, Bacillus gibsonii, Bacillus pumilus, Bacillus firmus and Bacillus marisflavi.

Interestingly, those who had disclosed their status to close friends were more likely to report difficulty taking ART than those who had not disclosed their status to close friends. Of the attitudes evaluated (outlined in Fig. 1), not believing in the benefits of ART, concern about the effectiveness of ART in the future, reporting that tablets were an unwanted reminder of HIV infection, negative body image/changes, a negative impact of HIV/AIDS on sex and relationships and a Protein Tyrosine Kinase inhibitor high degree of confidence that unprotected sex was not a risky behaviour were associated with increased likelihood of reporting difficulty taking ART at

a level of α=0.05. A positive health attitude and/or the adoption of positive strategies to manage one’s health was associated with a reduced likelihood of reporting difficulty taking ART. Deeming safe sex to be nonessential because of treatment effects also met the criterion for inclusion in multivariable analysis. The level Silmitasertib nmr of support from a range of sources (HIV-positive

friends, close friends, parents, family in general, a counsellor and the respondent’s doctor) was the only socioeconomic factor associated with reported difficulty taking ART at a level of α=0.05. Education level, urbanicity and additional support variables (support from partner/spouse and PLWH groups) also met the criterion for entry into multivariable analyses (see Fig. 1 for the full list of socioeconomic factors investigated). Of the treatment-related variables assessed (see Fig. 1), dosing frequency, the type of regimen taken, the

length of time on ART, and experiencing physical adverse events in the last 12 months or health service discrimination in the last 2 years were associated with reported difficulty taking ART at a level of α=0.05. No additional variables met the criterion for inclusion in the multivariable analyses. Of the disease-related factors assessed (outlined in Fig. 1), diagnosis of an ADI was associated with reported difficulty taking ART at a level of α=0.05. The respondent’s most recent CD4 cell count also met the criterion for inclusion in multivariable analyses. Variables that had shown a significant association in bivariate analyses at the level of α=0.2 were included in multivariable Nutlin-3 analysis. Initially, we set up logistic regression models of clusters of variables that were expected to exhibit a high degree of collinearity (step 1 models). At step 1, we created four models: (i) a substance use model, (ii) an other personal factors and attitudes model, (iii) a socioeconomic factor model, and (iv) a treatment-related and disease-related factor model. Variables that remained significantly associated with reported difficulty taking ART at step 1 at the level of α=0.1 were included in the step 2 logistic regression model. The following variables maintained an independent association with reported difficulty taking ART at the level of α=0.

In early December 2008, the patient with other friends ingested undercooked wild boar meat slices. A few days later, during Christmas and the New Year holidays, they consumed homemade dry meat made with pork from the wild boar. Two weeks later, the patient buy Z-VAD-FMK developed fever, generalized pain, abdominal distension, lack of appetite, and diarrhea. These symptoms continued for 10 days, then the fever ceased. Almost 30 days after ingestion of the infected meat, the patient developed generalized muscle pain, a nonitchy rash on his back, periorbital edema, and abdominal distension. Five days after returning to Switzerland,

he presented to our outpatient clinic. On examination, he was in relatively good health with an erythematous rash on the back, diffused pain, and tenderness of the muscles. Laboratory tests revealed eosinophilia (3,200/mL) and increased muscle enzyme (CK). Anti-Trichinella IgG (titer 113 U/mL, normal values <1 U/mL) were detected by a proprietary ELISA at the Institute of Parasitology of the University of Bern. Another sample collected 4 weeks later showed an increase of antibodies

to 170 U/mL, confirming the diagnosis of trichinellosis. The patient was treated with albendazole 400 mg b.i.d. for 14 days in combination Nutlin-3a order with prednisone 50 mg/d with a favorable outcome. Fourteen days after the beginning of the therapy, the patient was symptom free. In Bosnia, two persons who consumed pork from the same wild boar showed a similar symptomatology and trichinellosis was confirmed by serology (Bosnian Health Care System, personal communication). In Switzerland, Trichinella sp. infection has

not been documented in domestic pigs and wild boars in the last 50 years.3 A few cases of infection with Trichinella britovi have been reported among foxes and lynxes from the south of the country.4 Until 1976, human trichinellosis has been rarely documented in Switzerland.5 Lupinc describes a 34-year-old male of Bosnian origin who visited the Emergency Department of Zurich University Hospital on January 14, 2003. He complained of fever and generalized muscular pain. Laboratory tests revealed eosinophilia and an increase of liver enzymes. Trichinellosis was diagnosed by serology. Two others members of his family (a 31-year-old woman and a 12-year-old girl) developed generalized muscle pain, fever and eosinophilia. Trichinellosis PAK5 was also confirmed in these cases. All these patients were treated with albendazole with a favorable outcome. The epidemiological research showed a cluster of cases that included other hospitalized patients with a similar symptomatology, who were treated in a health service of Bosnia. All infected persons had eaten smoked pork during holidays in Bosnia. No information is available on the species of the etiological agent; however, since T britovi and Trichinella spiralis are endemic in the region, they may have been the species involved in these cases.

In early December 2008, the patient with other friends ingested undercooked wild boar meat slices. A few days later, during Christmas and the New Year holidays, they consumed homemade dry meat made with pork from the wild boar. Two weeks later, the patient MS-275 in vivo developed fever, generalized pain, abdominal distension, lack of appetite, and diarrhea. These symptoms continued for 10 days, then the fever ceased. Almost 30 days after ingestion of the infected meat, the patient developed generalized muscle pain, a nonitchy rash on his back, periorbital edema, and abdominal distension. Five days after returning to Switzerland,

he presented to our outpatient clinic. On examination, he was in relatively good health with an erythematous rash on the back, diffused pain, and tenderness of the muscles. Laboratory tests revealed eosinophilia (3,200/mL) and increased muscle enzyme (CK). Anti-Trichinella IgG (titer 113 U/mL, normal values <1 U/mL) were detected by a proprietary ELISA at the Institute of Parasitology of the University of Bern. Another sample collected 4 weeks later showed an increase of antibodies

to 170 U/mL, confirming the diagnosis of trichinellosis. The patient was treated with albendazole 400 mg b.i.d. for 14 days in combination Panobinostat nmr with prednisone 50 mg/d with a favorable outcome. Fourteen days after the beginning of the therapy, the patient was symptom free. In Bosnia, two persons who consumed pork from the same wild boar showed a similar symptomatology and trichinellosis was confirmed by serology (Bosnian Health Care System, personal communication). In Switzerland, Trichinella sp. infection has

not been documented in domestic pigs and wild boars in the last 50 years.3 A few cases of infection with Trichinella britovi have been reported among foxes and lynxes from the south of the country.4 Until 1976, human trichinellosis has been rarely documented in Switzerland.5 Lupinc describes a 34-year-old male of Bosnian origin who visited the Emergency Department of Zurich University Hospital on January 14, 2003. He complained of fever and generalized muscular pain. Laboratory tests revealed eosinophilia and an increase of liver enzymes. Trichinellosis was diagnosed by serology. Two others members of his family (a 31-year-old woman and a 12-year-old girl) developed generalized muscle pain, fever and eosinophilia. Trichinellosis Carbohydrate was also confirmed in these cases. All these patients were treated with albendazole with a favorable outcome. The epidemiological research showed a cluster of cases that included other hospitalized patients with a similar symptomatology, who were treated in a health service of Bosnia. All infected persons had eaten smoked pork during holidays in Bosnia. No information is available on the species of the etiological agent; however, since T britovi and Trichinella spiralis are endemic in the region, they may have been the species involved in these cases.

digitatum have been limited. Mitochondria are generally accepted as having a common origin and play an important role in phylogenetic studies. Many programmes,

such as the Fungal Mitochondrial Genome Project (Paquin et al., 1997), have significantly increased the data on fungal mitochondria and more than 80 complete fungal mitochondrial genomes are available in NCBI (www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=4751&opt=organelle). Information acquired from mitochondria, e.g. gene content and arrangement, exon–intron structure, as well as molecular phylogeny based on single or concatenated mitochondrial protein sequences, has largely increased our knowledge of Penicillium and its closely related Aspergillus species (Woo et al., TSA HDAC 2003; Juhasz et al., 2004, 2008). However, phytopathogenic Penicillium species have not been well described.

To reveal the mechanisms of molecular plant–pathogen interactions, whole genome sequencing of P. digitatum has been initiated in our laboratory. Here we reveal the mitochondrial genome and use comparative analysis to further confirm the species’ evolutionary degree, and to explore polymophism in Penicillium mitochondrial genomes. Penicillium digitatum strain Pd01 was isolated from green mould diseased citrus fruit collected in Zhejiang province, China, in 2000. It was maintained on potato GDC-0980 solubility dmso dextrose agar medium at 4 °C. The mycelium of Pd01 was cultured in potato dextrose broth in a rotary shaker at 150 g at 25 °C for 4 days. Fresh harvested Pd01 mycelia (100 mg) were homogenized in a mortar precooled with liquid nitrogen.

The subsequent powder was transferred to a 2-mL Eppendorf tube, then incubated at 65 °C for 1 h after adding 0.8 mL Y-27632 2HCl CTAB buffer (1% CTAB, 1 M NaCl 100 mM Tris, 20 mM EDTA; 1% polyvinyl polypyrolidone and 1% β-mercaptoethanol). Thereafter, 0.8 mL chloroform/isoamyl alcohol (24 : 1, v/v) was added to the tube. After being vortexed for 10 min, the mixture was centrifuged at 14 000 g for 10 min. The aqueous phase was transferred to a new tube, and extracted with a mixture of equal volume of phenol/chloroform for 1 min, and centrifuged at 4000 g for 10 min. The extraction was repeated twice. The supernatant was transferred to a new tube containing isopropanol (two-thirds the volume of supernatant), then gently mixed at room temperature for 10 min, and centrifuged at 14 000 g for 10 min. After pouring off and being dried in air, the obtained pellet was suspended in 0.3 mL TE buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0), then stored at −20 °C for 1 h after adding 0.2 mL 5 M NaCl and 1 mL frozen ethanol. The obtained DNA was precipitated by centrifugation at 14 000 g for 15 min and washed twice with 75% ethanol, then re-suspended in 50 μL TE buffer. The DNA was qualified and quantified by agar electrophoresis and spectrophotometrics, as described by Sambrook & Russell (2001).