fil ion ucl ac uk/spm/) working on Matlab 2010b (MathWorks, Inc , working on Matlab 2010b (MathWorks, Inc.,, MA, USA). All functional volumes were subjected to standard preprocessing procedures (Friston, Ashburner, Kiebel, & Penny, 2007), including spatial realignment, co-registration with the anatomical scan, normalization [on the Montreal Neurological Institute (MNI)

space with 2 × 2 × 2 mm3 voxels] using the unified segmentation of the anatomical scan and smoothed with an isotropic 6 mm full width half-maximum (FWHM) Gaussian kernel. Time series from each voxel were high-pass filtered (1/128 Hz cutoff) and the preprocessed find protocol functional volumes were then submitted to fixed-effects analysis (i.e., first level analysis, FFX) using a block design, applying the general linear model to each voxel (Friston et al., 1995 and Worsley and Friston, 1995) and using an auto-regressive [AR(1)] function to account for temporal correlations between voxels across the whole brain. Afterwards, the data were submitted to second-level analysis (random effect analysis, RFX) in order to generalize the results for

the population. All conditions were modeled in a full factorial model (ANOVAs) 3 × 2 (F1: condition; F2: task). The coordinates derived from these analyses (cluster maxima) were converted from MNI coordinates to Talairach and Tournoux stereotaxic coordinates using the icbm2tal script ( Lancaster et al., 2007) in order to associate MAPK Inhibitor Library ic50 mafosfamide the results with an anatomical location ( Talairach & Tournoux, 1988). The WFU pickAtlas software ( Maldjian et al., 2004 and Maldjian et al.,

2003) was used to define anatomical locations based on the Talairach Daemon atlas database ( Lancaster et al., 2000) and the automatic anatomical labeling (AAL) tool ( Tzourio-Mazoyer et al., 2002). Anatomical labels were assigned according to the nearest gray matter position. All illustrations are based on this neurological convention. Statistical parametric maps (SPMs) were assessed to determine the brain activation associated with each experimental context (simple effects). Effects were recognized at p < .05 corrected for multiple comparisons at the voxel level (FWE). SPMs were also computed to compare brain activity across tasks in the active condition (dynamic vs static) as well as between AO + MI and AO in the dynamic task. Significant differences were recognized at p < .001, uncorrected at the voxel level but with an extended cluster threshold of 240 contiguous voxels (pcluster < .05; false discovery rate (FDR) corrected) for topological analysis ( Chumbley & Friston, 2009). In this manuscript, all locations are presented in MNI coordinates (x, y, z) and the Tables provide details of the local maxima for each cluster. In the first part of this study, the pattern of brain activation in each experimental task was studied with simple effect comparisons (contrast between task and resting state).

g De Fruyt et al , 2009, Hrebícková et al , 2002 and McCrae et a

g. De Fruyt et al., 2009, Hrebícková et al., 2002 and McCrae et al., 2005). This has led to them being empirically related to a cornucopia of concepts as well as used in mediation and moderation models of current behaviours, helping to define relationships and explain outcomes. In adolescence, personality PI3K inhibitor may even be a key mediator of individual differences in the course and treatment responses of youth with mental disorders that emerge at this period in development (Costello, Copeland, & Angold, 2011). However, on closer inspection, problems remain with personality measurement in adolescents. In comparison to adult research, studies with adolescents have found more cross loadings, and items that

do not load sufficiently on any factor. CDK inhibitor Additionally, the studies demonstrate that items from the Neuroticism and Conscientiousness scales perform better, whereas Extraversion, Agreeableness and Openness items have less reliability (e.g. Parker and Stumpf, 1998 and Sneed et al., 2002). The problems with factor replicability may be due to developmental changes that take place during this time; personality traits are still in flux throughout adolescence (McCrae et al., 2002) and the structure and coherence of the five factors vary at different ages (Soto, John, Gosling, & Potter, 2008). Therefore it is important to

determine if the precision of personality measurement can be maximised for use in behavioural and clinical studies in this age range. Item response theory (IRT) can be used to improve the measurement Buspirone HCl of adolescent personality. The application of IRT allows scale psychometric properties to be revealed with greater precision than other multivariate methodologies; analysing item level information can provide insights into measurement reliability and enables a thorough evaluation of the internal construct validity. IRT provides information by checking the validity of the items and delineating poor performing indicators. It does this by estimating each individual item’s discrimination on the latent trait (the

a parameter) and difficulty within a population (the b parameter) ( Embretson & Reise, 2000). An item’s discrimination reflects how the probability of endorsing an item changes as the level of the underlying trait increases. Thus, highly discriminating items more strongly represent the latent trait. The item’s difficulty corresponds to the likelihood of an individual endorsing it given their level of the latent trait. An item is considered easy if most people endorse it and the difficulty rises as the likelihood of endorsing it decreases. Therefore some items may be easy to endorse even at relatively low levels of the latent trait. IRT also provides estimates of each scale and item’s total information function through total and item information curves (TICs and IICs).

We confirm all patient/personal identifiers have

been rem

We confirm all patient/personal identifiers have

been removed or disguised so the patient/person(s) described are not identifiable and cannot be identified through the details of the story. The authors declare that there is no conflict of interest. JM had the idea for the study, led the data analyses and wrote the first draft of the report. AS undertook the BIBF 1120 mw interviews and participated in analysis of the resulting data. AQ assisted in conceptual work and data presentation. KN was the Principal Investigator for the CAMWEL trial and is the guarantor for this study. All authors participated in discussions about the design of this study, contributed to revisions of the report and approved the submission of the final report. The CAMWEL trial was funded by the Camden Primary Care Trust FDA-approved Drug Library (NHS Camden). JM is supported by a Wellcome Trust Research Career Development Fellowship in Basic Biomedical Science (WT086516MA). The sponsor and funder had no role in the decision to publish nor in the writing of this paper. “
“The devastating diagnosis of incurable cancer has a major effect on patients’ well-being [1],

and drastically alters patients’ perspective on the future [2]. Patients have to cope with a life limiting illness and many decisions are to be made [3], [4] and [5]. The impact of a bad news consultation is evident and patients often report strong emotions, such as anxiety [6] and [7] and depressive feelings [7] and [8]. However, emotional arousal might not be limited to self-reported psychological arousal. There is growing evidence that the body reacts to mental stress as well [9], [10], [11], [12], [13] and [14]. Stress, negative thoughts and emotions, as for example evoked by the diagnosis of incurable cancer, Ergoloid may activate the sympathetic nervous system (SNS) [15], [16], [17] and [18]. As a subsystem of the autonomic nervous system, the SNS controls visceral functions and operates mostly unconsciously. Activation of the SNS leads to the so-called fight-flight response, which increases physiological arousal and prepares the body for action

[18] and [19]. Physiological arousal is an important underlying component in emotional experiences [15] and [16] and is expected to influence memory of provided information [18]. Indeed, patients’ recall of medical information is problematic: on average patients forget about 40 to 80% of the provided information [5], [20], [21], [22] and [23]. Previous research reported that only 49 to 83% of newly diagnosed cancer patients were able to recall provided information about the proposed treatment correctly [21]. In older cancer patients, recall is even worse; only 21.9% of recommendations nurses made in a consultation about chemotherapy were remembered [5]. The emotional arousal, evoked by the bad news, might be responsible for the poor information recall during medical consultations [5].

The pig model of paraoxon poisoning used here exhibited reproduci

The pig model of paraoxon poisoning used here exhibited reproducible prolonged respiratory distress and delayed mortality, with signs and symptoms characteristic of organophosphate poisoning [21]. The most important finding in the present study was the dramatic effect of Cuirass technique in reducing the paraoxon-induced mortality (Figure 2). This Cuirass technique was found to be superior to bag-valve mask ventilation, a common

ventilation procedure, expected to be used Sunitinib following both single exposure and on-scene mass casualty event. Earlier studies have demonstrated that respiratory failure was the predominant cause of death in nerve agent poisoning and that significant cardiovascular depression occurred only after cessation of respiration [24] and [25]. This emphasizes the importance of respiratory support over cardiovascular support during early stages following OP poisoning. Biphasic Cuirass Ventilation has been reported as an easily-adopted and rapidly-applied method suitable for use by non-medical personnel, Obeticholic Acid purchase even while wearing protective gear [20]. In addition,

Ben-Abraham et al. [19] have indicated that physicians wearing full personal protective gear applied the cuirass and instituted ventilation faster than performing endotracheal intubation followed by positive pressure ventilation. Unfortunately, as we have shown here for the first time, the bag-valve mask ventilation did not sufficiently improve the impact of OP exposure unless continuously implemented. While animals survived during ventilation, shortly after its termination the animals died and mortality rates resembled that of the non-ventilated Control group. In contrast, ventilation with the cuirass for the same period of time prevented 24 h mortality and the animals recovered better and Vasopressin Receptor faster with no deterioration

following cessation of ventilation. An additional advantage of the Cuirass relates to airway management. In pre-hospital ventilation, a jaw thrust into the BVM is required to avoid the tongue occluding the airway, assuming the supine position of the casualty. This adds to the difficulties of using BVM in the pre-hospital setting of a chemical event. When using the cuirass there is no need for a jaw thrust, as the use of a guedel is enough. In our study there was no need for that since the animals were in a prone position. In recent years several studies described a successful use of supraglottic airways and intubation in the pre-hospital setting [26], [27], [28] and [29]. Endotracheal intubation is still regarded as the golden standard, and supraglottic airways are regarded a bridge until definite airway control is achieved [30]. When looking at the success rates, supraglottic airways are easier to manage, including in a chemical event [26], [27], [28], [29] and [30].

Poród noworodka z podejrzeniem obustronnej agenezji nerek powinie

Poród noworodka z podejrzeniem obustronnej agenezji nerek powinien być zaplanowany w ośrodku referencyjnym,

zapewniającym możliwość prowadzenia intensywnej terapii noworodka, diagnostyki obrazowej z wykorzystaniem różnych technik obrazowania oraz leczenia nerkozastępczego u noworodka. Brak miąższu obu nerek w prenatalnym badaniu USG w połączeniu z małowodziem nasuwa podejrzenie obustronnej agenezji Ipilimumab purchase nerek – wady skutkującej głębokimi zaburzeniami rozwoju płodu. Bezpośrednią konsekwencją braku czynnego miąższu nerkowego jest brak wytwarzania moczu płodowego, a w efekcie znaczny deficyt płynu owodniowego, czyli małowodzie [5, 6]. Całość zaburzeń powstających w wyniku braku czynnego miąższu nerkowego jest określana mianem zespołu Potter. Zespół Potter jest uznawany za zaburzenie letalne. Jeśli ciąża kończy się urodzeniem żywego dziecka, bezpośrednią przyczyną zgonu noworodka jest niewydolność oddechowa na tle hipoplazji płuc i niewydolność nerek [5, 6]. Przy braku miąższu jednej nerki stwierdzonym w badaniu prenatalnym i prawidłowej ilości płynu owodniowego nie jest konieczne poszerzanie diagnostyki ani rozważanie interwencji terapeutycznej w okresie prenatalnym Brak miąższu jednej nerki w badaniu prenatalnym nasuwa podejrzenie jej agenezji. Oznacza to całkowity brak zawiązka nerki, któremu towarzyszy brak

moczowodu i brak części trójkąta pęcherza moczowego [7]. Jednostronna agenezja nerki w około 30% przypadków współistnieje z innymi anomaliami rozwojowymi [8]. Brak miąższu jednej Akt inhibitor nerki w badaniu prenatalnym przy prawidłowej strukturze nerki drugiej sugeruje wadę wiążącą się

z niskim ryzykiem zaburzonego rozwoju płodu oraz zwykle dobrym odległym rokowaniem [7, 8]. Przy prawidłowej ilości płynu owodniowego nie jest konieczne poszerzanie diagnostyki ani rozważanie interwencji terapeutycznej w okresie prenatalnym [7]. Należy jednak zwrócić szczególną uwagę na ewentualne współistnienie innych anomalii rozwojowych (zwłaszcza układu krążenia) [8]. Postępowanie w przypadku podejrzenia agenezji jednej nerki zaprezentowano na rycinie 2. Dysplazja wielotorbielowata. Pierwsze badanie ultrasonograficzne dziecka z podejrzeniem dysplazji torbielowatej obu nerek powinno odbyć Cell Penetrating Peptide się w ciągu 24–48 godzin po porodzie. Dysplazja wielotorbielowata (DWN) jest najczęściej występującą formą dysplazji nerek. Częstość DWN waha się od 1:3640 do 1:4300 żywych urodzeń [9, 10]. Może występować rodzinnie, jednak w większości przypadków pojawia się sporadycznie. Dotyczy zwykle jednej nerki. W przypadku zmian dotyczących obu nerek rokowanie jest zwykle niepomyślne, a zgon występuje najczęściej w okresie okołoporodowym. Noworodki z zachowaną częściowo funkcją nerek wymagają zwykle dializoterapii w 1. roku życia.

, 1991 and Tallal et al , 1994) Although the correspondence betw

, 1991 and Tallal et al., 1994). Although the correspondence between the two sets of studies is impressive the pattern of abnormalities in SLI also differs from that seen in the KE family

in several ways. In the current study, grey matter in the posterior temporal cortex in SLI is significantly RAD001 order decreased relative to controls, whereas it was increased in affected KE family members. Similarly, the putamen was found to have increased grey matter in affected KE family members, whereas we found no structural differences in the putamen in SLI. Finally, the caudate nucleus was found to be significantly reduced in volume in affected KE family members relative to their unaffected relatives and functionally overactive in a PET study of word repetition (Watkins et al., 1999). In our SLI study and the functional MRI study of the KE family, the caudate nucleus was not reliably activated by the task used and no group differences in function were detected. Also, the unaffected siblings in our study had significantly less grey matter in the caudate nucleus bilaterally relative to the typically developing controls

and did not differ significantly from their siblings with SLI. The latter suggests that reduced caudate nucleus volume can be considered a heritable risk factor for SLI but requires additional deficits Androgen Receptor Antagonist to affect language development. Alternatively, the siblings in our study have some protective factors, plasticity or compensatory mechanisms available to them that are unavailable to their affected

siblings. The increased grey 3-mercaptopyruvate sulfurtransferase matter of the left central opercular cortex in the unaffected siblings relative to the SLI and control groups might reflect such compensatory mechanisms. The similarities between the functional and structural abnormalities in this group of people with SLI and the affected members of the KE family are likely a reflection of the similarities in their behavioural deficits. Both groups have impairments in nonword repetition and oromotor function. Whether any of the individuals with SLI that we studied also have a mutation in FOXP2 is unknown, but is unlikely, given the rarity of such mutations in individuals with SLI ( Newbury & Monaco, 2010). In a larger population of SLI, however, allelic variation in a downstream target gene of FOXP2, CNTNAP2 was found to correlate with performance on nonword repetition ( Vernes et al., 2008), so investigations of this gene in our participants are warranted. Previous developmental studies measuring grey matter volume and cortical thickness have revealed gradual linear and nonlinear reductions in these measures that continue into the second decade (e.g., Giedd et al., 1999, Giorgio et al., 2010 and Gogtay et al., 2004).

For analysis, responses were collapsed into ‘Strongly Agree or Ag

For analysis, responses were collapsed into ‘Strongly Agree or Agree’, ‘Neutral’ and ‘Strongly Disagree or Disagree’. Participants were asked to respond to one item on confidence: How confident do you feel about discussing obesity with clients? (1 = very confident, 2 = confident, 3 = somewhat unsure, and 4 = completely unsure), and one item on training needs: Do you feel that you need

more training on how to discuss obesity with clients? (1 = yes, more training is essential, 2 = yes, more training would Navitoclax mw be useful, 3 = no, the training I have received is adequate, 4 = no, the training I have received is excessive). For analysis, responses were collapsed into ‘Very confident or confident’ and ‘Less confident or unconfident’, and ‘Yes, more training is useful or essential’ and ‘No, more training is not required’, respectively. In the final section, participants were asked record their educational degree, year of study, gender, age, weight, and height. Participants were not asked any information regarding their ethnic background AZD2281 in vivo as previous research involving trainee HCPs studying at The University of Nottingham

demonstrated little variance with the majority being Caucasian [50]. This study received approval from the Nottingham University Medical School Ethics Committee. All responses were anonymous. Participants were considered to have consented to taking part in the study if they completed and returned a questionnaire. By way of a small token of appreciation, participants were offered the opportunity to enter a prize-draw

to win one of three £50 book vouchers. Data Adenosine triphosphate entry was conducted by three members of the research team. A randomly selected 10% sample of each members’ data was checked by an independent researcher for accuracy of entry and revealed an error rate of <1%; below the threshold considered to have any significant effect on the data analysis [51]. Prior to analysis, the data set was screened for missing values, normality and univariate outliers [52]. Categorical demographic data were analyzed for differences between student groups using Chi-squared tests. As continuous demographic data were non-Gaussian, analyses relating to student group effects employed Kruskal–Wallis nonparametric analysis of variance tests followed up with post hoc Mann–Whitney U-tests. As the distribution of scores of the 11 preferred terms approximated to normal, a one-way repeated measures ANOVA was conducted to compare scores. A post hoc analysis was performed using Tukey’s studentized range test to identify statistically significant difference between pairs of terms. A one-way between-groups MANOVA was also conducted to investigate sex differences and differences between the courses that students were registered on. Once again, post hoc analysis was performed using Tukey’s studentized range test to identify statistically significant difference between pairs of terms.

There were three replicates for each temperature treatment Popul

There were three replicates for each temperature treatment. Population identity was maintained at all times through the separation of populations into floating mesh tubs within the same tank so that northern barramundi (N) could be distinguished from southern barramundi (S) at cool (N22 from S22), control (N28 from S28) and hot (N36 from S36) temperatures. During growth trials fish were reared for a period of approximately 3.5 months (106 days), Akt inhibitor while at all times water chemistry, dissolved oxygen (> 5 mg/ml), pH, and temperature (experimental conditions ± 1 °C) were rigorously maintained. After this time, fish were humanely

sacrificed in accordance with Animal Ethics Permit A1249 and their weight was recorded as a measure of growth over the rearing experiment. White muscle tissue was chosen for gene expression analysis due to its growth responsiveness and high metabolic rate and was immediately dissected from each fish and snap frozen in liquid nitrogen. Total RNA was extracted by homogenizing frozen muscle tissue in Ultraspec solution (Biotecx; using

a PRO200 homogenizer (PRO scientific Inc.; RNA was precipitated in a solution containing 0.5 vol of RNA precipitation solution (1.2 M sodium chloride, 0.8 M disodium citrate) (Sambrook and Russel, 2001) and 0.5 vol of isopropyl alcohol. RNA quality and quantity were verified using a Nanodrop spectrophotometer (Nanodrop technology; via examination

MG-132 datasheet of absorbance ratios at OD 260/280 (range 1.98–2.06) and OD 260/230 (range 1.96–2.07) and by the visual inspection of the 18S Meloxicam and 28S ribosomal bands (and possible DNA contaminants) on a 1.5% agarose gel. After Nanodrop quantification, four RNA pools were constructed by combining individual fish RNA samples representing northern barramundi reared at 22 °C and 36 °C, and cool-adapted barramundi reared at 22 °C and 36 °C. Each sample pool consisted of 5 μg of total RNA from a total of eight separate individuals so that any potential variation between individual fish could be captured. Each RNA pool was then treated with a Turbo DNA-free kit (Ambion; http://www/ as a precaution to eliminate trace DNA contamination before being sent for further processing including sample quality and quantity verification on an Agilent RNA Bioanalyzer chip directly prior to sequencing on an Illumina Genome Analyzer IIx (Macrogen Inc.; Four mRNA-seq libraries were constructed representing pooled samples from northern and southern populations of barramundi reared at 22 °C and 36 °C incorporating unique bar-coding for each pool library. Illumina transcriptome mRNA pair-end sequencing (101 bp reads) was performed using standard protocols and reagents according to the manufacturer’s recommendations (Illumina Inc.;

In addition, although the original Report used and recommended th

In addition, although the original Report used and recommended the symbols NAD and NADH2 for the oxidized and reduced forms respectively of the coenzyme, they also suggested NAD+ and NADH respectively as alternatives. This latter system has the advantage that it allows the plain symbol NAD to refer to the two forms collectively, but it has the disadvantage that it assigns a+superscript to what

is in reality an anion. In practice the system with NAD+ and NADH has become overwhelmingly the most used, and when it became adopted in Enzyme Nomenclature there was a feeling that the equation looked unbalanced with unequal charges on the left and right-hand sides. In what Alberty in particular

considered as a misguided move, this was then “corrected” by including protons in equations. A suggested way to avoid Obeticholic Acid molecular weight the problem (Alberty and Cornish-Bowden, 1993), in which the two forms of coenzyme were to be written as NADox and NADred has received no significant adoption in the literature. Ipilimumab research buy As the entry for acetate kinase considered above is one of the simpler examples, with no comments or specificity information (with the implication that the enzyme catalyses that one reaction only) it is useful to examine a more typical entry: EC Accepted name: hexokinase Reaction: ATP+d-hexose=ADP+d-hexose 6-phosphate Other name(s): hexokinase type IV, glucokinase; oxyclozanide hexokinase d; hexokinase type IV; hexokinase (phosphorylating); ATP-dependent hexokinase; glucose ATP phosphotransferase Comments: d-Glucose, d-mannose, d-fructose, sorbitol and d-glucosamine

can act as acceptors; ITP and dATP can act as donors. The liver isoenzyme has sometimes been called glucokinase. Systematic name: ATP:d-hexose 6-phosphotransferase Links to other databases: BRENDA, EXPASY, GTD, IUBMB, KEGG, METACYC, PDB, UM-BBD, CAS registry number: 9001-51-8 References: 1. Bailey, K. andWebb, E.C. Purification of yeast hexokinase and its reaction with ββ′-dichlorodiethyl sulphide. Biochem. J.42 (1948) 60–68. [PMID: 16748250]. 2. Berger, L., Slein, M.W., Colowick, S.P. and Cori, C.F. Isolation of hexokinase from baker׳s yeast. J. Gen. Physiol.29 (1946) 379–391. 3. Kunitz, M. and McDonald, M.R. Crystalline hexokinase (heterophosphatase). Method of isolation and properties. J. Gen. Physiol.29 (1946) 393–412. 4. Pollard-Knight, D. and Cornish-Bowden, A. Mechanism of liver glucokinase. Mol. Cell. Biochem. 44 (1982) 71–80. [PMID: 7048063]. 5. Ureta, T., Radojković, J., Lagos, R., Guixé, V. and Núñez, L. Phylogenetic and ontogenetic studies of glucose phosphorylating isozymes of vertebrates. Arch. Biol. Med. Exp.12 (1979) 587–604. [PMID: 233226]. 6. Cárdenas, M.L., Rabajille, E. and Niemeyer, H. Fructose: A good substrate for rat-liver ‘glucokinase’ (hexokinase d). Biochem. J. 222 (1984) 363–370.

In this context, computational

In this context, computational Selleckchem Epacadostat approaches for protein 3D modeling may assist in establishing genotype–phenotype and structure–function correlations, as well as predicting the structural and/or functional impact of each mutation. The goal of the present study was to identify the mutation(s) associated with odonto-HPP affecting monozygotic twin probands, establish a genotype–phenotype association, and use a 3D modeling approach to evaluate the impact of each mutation in the TNAP protein structure. Additionally, we evaluated the

expression of mutant protein and its subcellular localization in dental pulp cells from probands by Western blotting and immunocytochemistry. The probands were male monozygotic twins of Caucasian descent clinically diagnosed with odonto-HPP. Probands and their biological parents were examined in order

to identify potential mutations in the ALPL gene. The family was provided with study information and consented to participate (IRB #065/2005). The clinical diagnosis of odonto-HPP in the probands (by physical and dental examinations, radiographs, and blood chemistry assays) and subsequent Hydroxychloroquine datasheet management of dental symptoms have been reported previously [18] and [19]. Briefly, probands (patients A and B), at the age of two, were brought to the Piracicaba Dental School, University of Campinas, Brazil for dental evaluation. Parents reported premature exfoliation of the anterior primary teeth, with signs of partial root resorption. Physical examination and radiographs (long bones, joints, and skull) showed age-appropriate growth and development. Routine laboratory testing revealed low serum ALP activity for both probands (patient A: 62 U/L, patient B: 63 U/L; normal range for children 151–471 U/L), while serum phosphate and calcium levels remained within normal limits [18], [19] and [20]. Genomic DNA of probands and their parents

was isolated from peripheral blood leukocytes using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) Demeclocycline following the manufacturer’s instructions. Primer sequences were designed to amplify all TNAP coding exons (2–12), as previously reported [21], allowing analysis of the whole coding sequence, including intron–exon borders. Polymerase chain reaction (PCR) was performed in a final volume of 50 μL with 100 ng of DNA, 30 μM forward and reverse primers, 0.2 mM dNTP mix (Invitrogen™, Life Technologies, Carlsbad, CA, USA Life Technologies, Gaithersburg, MD, USA), 0.75 U Gold Tap® Flexi DNA polymerase (Promega), and 1–3 mM MgCl2. Cycle conditions and annealing temperature were optimized for each primer pair.