Whale sharks are a declining species for which little biological data is available. While these animals are protected in many parts of their range, they are fished legally and illegally in some countries. Baseline biological and ecological data are needed to allow the formulation of an effective conservation plan for whale sharks. It is not known, for example, whether the whale shark is represented by a single worldwide panmictic population or by numerous, reproductively isolated populations. Genetic analysis of population structure is one essential component of the baseline data required for whale shark conservation.o = 0.66 and He = 0.69. All but one of the eight loci meet the expectations of Hardy-Weinberg equilibrium. Analysis of these loci in whale sharks representing three major portions of their range, the Pacific (P), Caribbean (C), and Indian (I) Oceans, determined that there is little population differentiation between animals sampled in different geographic regions, indicating historical gene flow between populations. FST values for inter-ocean comparisons were low , and only C×I approached statistical significance (p = 0.0495).We have identified 8 polymorphic microsatellites in the whale shark and used these markers to assess genetic variation and population structure in a panel of whale sharks covering a broad geographic region. This is the first record of microsatellite loci in the whale shark, which displayed an average of 9 alleles per locus and mean HWe have shown only low levels of genetic differentiation between geographically distinct whale shark populations. Existing satellite tracking data have revealed both regional and long-range migration of whale sharks throughout their range, which supports the finding of gene flow between populations. Whale sharks traverse geographic and political boundaries during their life history and interbreed with animals from distant populations; conservation efforts must therefore target international protection for this species. Rhincodon typus , is the largest shark and the largest fish The whale shark, Whale sharks have been the target of widespread active fisheries in the past, and while they are currently protected in many waters, open fisheries remain in several countries. Whale sharks are listed as Vulnerable in the IUCN Red List of Threatened Species, and in 2002 the species was placed on CITES Appendix II. A slow growth rate and late time to sexual maturity make animals such as the whale shark particularly slow to recover from overfishing or habitat disruption, and current evidence indicates that whale sharks are declining in number. Aerial surveys, mark-recapture and photo identification have been used to track the abundance over time of whale sharks in various regions. While not all studies agree, most data suggest that whale shark aggregations have fewer sharks of smaller average size in recent years The use of microsatellites as a tool to understand the population genetics of a species has revolutionized the field of conservation biology A total of 68 whale shark samples were collected from 11 different sites: Veraval, India (8); Utila, Bay Islands, Honduras (6); Galapagos Islands, Ecuador (5); Dania Beach Florida, USA (1); Mossel Bay, South Africa (1); Mahe, Seychelles (3); Cocos Island, Costa Rica (1); Ningaloo Reef, Western Australia (5); Gulf of Tadjoura, Djibouti (15); Bahia de La Paz, Baja California, Mexico (5); and Male, Maldives (18) . These sHaeIII and RsaI, blunt-ended with Klenow and T4 DNA polymerase, dephosphorylated with calf intestinal phosphatase and purified using QIAquick (Qiagen). The forSNX (5′-CTAAGGCCTTGCTAGCAGAAGC-3′) and revSNX (5′-pGCTTCTGCTAGCAAGGCCTTAGAAAA-3′) linkers were annealed, then ligated to the blunt-ended DNA using T4 DNA ligase at room temperature overnight. The linkered DNA was purified using QIAquick and hybridized with 30-mer CA and GA biotinylated oligonucleotides in 12× SSC, 0.1% SDS hybridization buffer at 65 degrees for 2 hours. Repeat-containing DNA fragments were isolated using streptavidin coated magnetic beads  according to the manufacturer's protocol. The beads were washed once wash at room temperature in 2× SSC, 0.1% SDS and once at 50 degrees with 1× SSC, 0.1% SDS. Purified DNA was eluted in 10 mM Tris at 95 degrees, and amplified by PCR using the SNX primer. The amplified DNA was cloned using the TopoTA kit (Invitrogen), and individual clones were sequenced and analyzed for the presence of repeats  Following extraction, DNA samples were quantified using a nanodrop spectrophotometer, and 30 ng DNA per locus was amplified by PCR using primers against unique sequences flanking each microsatellite. Amplification employed 6-Fam labeled forward primers, and reverse primers tailed with the sequence Although a total of 68 whale shark samples were analyzed, some samples did not yield sufficient quantities of DNA to analyze all loci, and some DNAs did not yield useful data for all loci. Different numbers of samples are therefore reported for the different loci. In all cases, where fewer than 68 animals were used for analysis, this is indicated in the methodology. In individual tests for genetic differentiation (Structure and PCA), the single South African animal was treated individually. In analyses where animals were grouped into populations, this animal was included with the Indian Ocean group. Input files for various software were constructed using the program Create when possible A Bayesian approach using genotype data for individual animals was performed to detect any population structure across the entire data set using the program STRUCTURE 2.2 s) was produced using the program POPULATIONS 1.2.30 Traditional tests for population differentiation were performed by calculation of F-statistics using FSTAT and Microsatellite Analyzer (MSA) 4.05 e) was performed using ARLEQUIN 2.0 θH), was calculated from the expected homozygosity (HomE). Assuming that the population is in mutation-drift equilibrium, Theta(θH) is θH = (1−HomE)/HomE, where HomE = 1−HE, and HE is expected heterozygosity. To evaluate the utility of microsatellite genotypes as individual genetic tags, we estimated the probability of identity for individual loci and over all eight loci using Cervus 3.0.3 Calculation of effective population size (Na) per locus ranged from 3 for Rtyp8, to 34 for Rtyp6, with a mean of 9. The Rtyp6 locus was unusual in its degree of polymorphism, and Na excluding Rtyp6 was 3–8, with an average of 5.4. The range for observed heterozygosity (Ho) was 0.45–0.85, and for expected heterozygosity (He) 0.40–1.00. The loci were analyzed with the program MicroChecker, and with the exception of Rtyp6 all conformed to Hardy-Weinberg equilibrium, with no evidence for scoring error, null alleles or large allele dropout Whale sharks are the only genus within their family, Rhincodontidae, and microsatellites had not previously been described from this species. Microsatellites have been isolated from members of the sister families Stegostomatidae and Ginglymostomatidae, but microsatellite sequences are highly unlikely to be conserved across families S), a measure of genetic diversity that describes the number of alleles per locus independent of sample size, could not be calculated individually for several populations due to small sample size. This value was therefore examined after the animals had been pooled into ocean-specific populations, as described below. Values for RS ranged from 2.0 to 8.7 . Allelic richness . FST values for P×I and P×C were not statistically significant, while the value for C×I approached statistical significance (p = 0.0495). The Caribbean animals may therefore be more differentiated from Indian Ocean sharks than they are from Pacific, or than Pacific and Indian are from each other. Those animals for which sex is known were analyzed separately as male and female populations using FSTAT; this analysis did not detect any statistically significant genetic difference between males and females (data not shown).In this study, small sample numbers precluded analyzing individual sampling locations as populations of whale sharks. Since the Bayesian analysis programs did not discern inherent structure among the individual whale sharks in our data set, the animals were grouped for further analysis into three ocean-specific populations—Pacific , consisting of samples from La Paz, Galapagos and Cocos Island; Caribbean , consisting of samples from Utila and Florida; and Indian , consisting of samples from Djibouti, Seychelles, Maldives, Veraval and Ningaloo . The sinS), which describes the similarity between two groups of individuals based on allele frequencies using the Infinite Alleles Model, was calculated using the program POPULATIONS 1.2.30. Pairwise values for DS were P×C = 0.248, C×I = 0.169 and P×I = 0.078. These data indicate a lack of strong genetic differentiation between any of the analyzed whale shark populations.Nei's standard genetic distance (De) reflects the number of breeding adults and the potential for inbreeding and genetic drift in the species. Effective population size was estimated based on the θH value, which is θH = 4Neμ, where μ is the mutation rate. We used a mutation rate of 1×10−3 mutants/generation/locus The effective population size , while FST values for the Caribbean population were somewhat larger when compared to both the Pacific and Indian animals . Only the C×I population approached statistical significance for genetic differentiation (p = 0.0495). It is likely this value would be more highly significant if additional Caribbean animals were available for analysis. FST for C×I remains low, however, indicating subtle differentiation between Caribbean and Indian whale sharks. Despite this finding, the data show that there has been significant gene flow between geographically disparate populations. The individual-based analyses (STRUCTURE and PCA) also indicate no clear genetic clusters of whale sharks based on sampling location  were found to consist of a single continuous population displaying isolation by distance throughout the North Atlantic, with genetically distinct Iberian and Black Sea populations only Xiphias gladius) mitochondrial DNA showed that fish from the Pacific, Mediterranean, Northern Atlantic and Southern Atlantic comprise four genetically distinct populations Balaenoptera brydei) have a lifestyle seemingly similar to that of whale sharks, as they inhabit tropical and warm temperate waters, and have been shown to undertake equatorial migrations. Nonetheless, microsatellites and mitochondrial DNA analysis was used to show that these whales comprise distinct oceanic populations in the Northern Pacific, Southwestern Pacific, Southeastern Pacific and Indian oceans, with low levels of gene flow Thunnus thynnus) has yielded conflicting results for population structure between the Mediterranean and northwest Atlantic ST values were statistically significant between three of the four populations, the values were quite small, ranging from 0.005 to 0.034 Sphyrna lewini) showed significant genetic divergence between Atlantic and Indo-Pacific populations, and identified a cryptic species within the northwest Atlantic High rates of gene flow are typical in large, vagile marine species, which have few barriers to migration between populations. For some species, this migratory activity supports a single panmictic population that shows little genetic variability. For example, harbour porpoises . These data indicate significant gene flow between Indian and Pacific Ocean populations, with reduced levels of interaction with Atlantic animals, a result that parallels the data from our microsatellite analysis. It is possible that the somewhat greater degree of Caribbean/Atlantic population differentiation seen using mitochondrial DNA markers, in comparison to the microsatellites described here, indicates some level of female site fidelity. This hypothesis must be qualified, however, given the small number of animals analyzed in both studies.Two previous studies have employed mitochondrial DNA sequence analysis to compare whale shark populations. Analysis of the mitochondrial control region was used to study the relatedness of 41 whale sharks in the Gulf of California ST values of 0.81 between South Africa and Australia, and of 0.89 between South Africa and New Zealand ST = 0.063–0.067) of nuclear markers was found, particularly between Belize and the other nurseries. Genetic differentiation was far greater using analysis of mitochondrial DNA, however, providing evidence for female philopatry. Any demonstration of female philopatry in whale sharks must await the sampling and analysis of larger numbers of these animals.Female philopatry is found in numerous shark species. White sharks are highly migratory, for example, yet genetic analysis has shown strong population structure. Mitochondrial DNA analysis of sharks from South Africa found evidence for population differentiation in comparison to sharks from Australia/New Zealand, with FSatellite tracking studies of whale sharks have demonstrated both short and long range migratory movements, which support the gene flow inferred with microsatellite markers. Sharks tagged near Taiwan, at Gladden Spit off the coast of Belize, and at Ningaloo reef in Western Australia, all recorded short range movements within their ocean basins Ecological data also support frequent movements of whale sharks between populations. Our sample set displays a striking absence of females, with 2.8 males for every 1 female, reflecting the observation that most feeding aggregations are composed largely of immature males. For example, most sharks observed at Ningaloo Reef, at Gladden Spit, and off the coast of Djibouti are juvenile males −9. As has been demonstrated in other sharks, seals and rays, the ability to genetically identify any individual within a population can discern breeding structure, paternity and sibship While early estimates proposed that the world population of whale sharks might number no more than a few thousand, identification of larger aggregations of animals in previously unknown locations has revised that estimate upwards. Data from tagging and mark-recapture studies has shown that surprisingly few animals are resighted at most locations. Of 72 whale sharks tagged at Gladden Spit, only 17 individuals were resighted over 5 years The work presented here describes the first identification of microsatellite loci in the whale shark, and the use of these loci to analyze population structure across a panel of whale shark DNAs from three different ocean basins. This much-needed first look at whale shark population structure using nuclear markers showed little genetic differentiation between geographic populations. Rather, the data confirm a history of gene flow between populations, supporting migration and interbreeding between these seemingly disparate groups. Such data are supported by satellite tracking studies that show frequent mid-range and periodic long-range migrations. Though this level of gene flow is sufficient to genetically normalize populations, it is unlikely to be sufficient to reestablish depleted populations. As whale sharks cross geographic and political boundaries in their movements, international protection should be sought to ensure the continued survival of this species. In addition, it should be kept in mind that genetic methods of population study reflect only the history of the species. They cannot detect more recent changes in behavior that may be caused by overfishing, habitat disruption, tourism, or other anthropogenic activities currently impacting whale shark populations.


In all, 45 (50%) patients suffered one or more WHO grade 3/4 toxicity, grade 3 in 93% cases. Patients received more than 90% of the planned chemoradiation schedule. The median overall survival was 26  months, 51%  and 26%  surviving 2 and 5 years, respectively. Advanced stage, particularly T4 disease, was associated with a worse prognosis. Patients considered not suitable for surgery for reasons other than their disease, mainly co-morbidity, had a significantly better outcome, median survival 40  months, 2- and 5-year survivals 67%  and 32% , respectively (P<0.001). This schedule is a feasible, tolerable and effective treatment for patients with oesophageal cancer considered unsuitable for surgery.We performed a retrospective study of 90 consecutive cases with inoperable carcinoma of the oesophagus treated with definitive chemoradiation at a single cancer centre between 1995 and 2002. For the last 4 years, 73 patients have received therapy according to an agreed protocol. This outpatient-based regimen involves four cycles of chemotherapy, cycles 3 and 4 given concurrently with 50 Gy external beam radiotherapy (XRT) delivered in 25 fractions over 5 weeks. Cisplatin 60 mg m Carcinoma of the oesophagus is becoming more common in Europe and North America. In the UK, there are 7000 new cases per year, the eighth most common malignancy, and in some regions it has become more common than gastric cancer . DespiteIn the UK, if the disease appears resectable and patients are sufficiently fit, surgery remains the mainstay of therapy. However, despite better staging and improving perioperative care, the outcome from this treatment remains poor, with 30–45% patients surviving 2 years after radical resection . The proRadical radiotherapy (RT), or chemoradiation (CRT), has lead to long-term survival in some reported series, though it is not absolutely clear how these patients were selected for nonsurgical therapy . Patients were deemed not suitable for surgery because of factors relating to their disease, usually infiltration of tumour into other mediastinal organs, or co-morbidity, making surgery unacceptably hazardous. This study assesses the outcome for this group of patients.This is a retrospective study of 90 consecutive patients treated between 1 March 1995 and 1 October 2002, who received definitive CRT at the Velindre Cancer Centre. One patient was diagnosed and treated in 1995 and three in 1997, and, based on encouraging early results at our centre and elsewhere, a further 86 patients were treated between 1998 and 2002. All patients were discussed at multidisciplinary meetings attended by one or more specialist gastro-enterologists, radiologists, pathologists, surgeons, oncologists, palliative care and upper GI nurses. Patients were initially seen by the surgical team and counselled regarding the risks and likely success of an oesophagectomy. The reasons for patients not receiving surgery are shown in All patients were confirmed histologically to have oesophageal carcinoma. Patients had disease starting at or below 17 cm ab oral, and included patients with Siewert types I and II gastro-oesophageal junctional disease  to assess locoregional disease, except for six, of whom five had malignant stricturing which precluded EUS evaluation.−1 or where there was a 25% rise in the serum creatinine after cisplatin treatment. Patients being considered for surgery underwent echocardiograms or MUGA scans, lung function testing with spirometry and arterial blood gas analysis.Physiological assessment included routine haematological and biochemical assays. Renal function was assessed further by calculating the glomerular filtration rate (GFR) using the EDTA clearance method or using the Cockroft formula, when an EDTA test was carried out if the predicted GFR was less than 60 ml min−2  as a day case on Day 1 of each of the three weekly cycles. Carboplatin (AUC=5) is given instead of cisplatin if the GFR is less than 40 ml min−1 or if significant neuro-/nephrotoxicity occurs during treatment. During the neoadjuvant phase of treatment the 5FU is given as a continuous infusion of 300 mg m−2 day−1, reduced to 225 mg m−2 day−1 during the RT, via a central venous catheter. Patients are given prophylactic warfarin 1 mg day−1.Of the 90 patients, 73 received the current treatment protocol for definitive CRT. This consists of four cycles of cisplatin and 5-fluorouracil (5FU), cycles three and four being given concurrently with 5 weeks of RT. The cisplatin is given at 60 mg mThe RT dose is 50 Gy in 25 fractions given over 5 weeks in two treatment phases. Phase 1 is given with an anterior–posterior parallel pair field arrangement giving 26–30 Gy, according to the normal tissue tolerance of the spinal cord, heart and lungs. The remainder is given using an anterior and two posterior oblique, three-field plan. The dose was prescribed to the ICRU 50 reference point, usually being the mid-plane of opposed fields and central axis intersection point of multiple fields.The conformal planned target volume (PTV) is the EUS-defined gross tumour volume (GTV), with a 2–3 cm margin in all directions. Both phases are planned early during the neoadjuvant phase of therapy using a 3D Helax planning system. The GTV is drawn directly onto the axial planning images using distances of the defined primary and nodal disease ab oral and to the tracheal bifurcation, taken as a reference point, derived from the EUS. The fields are checked with a barium study in the simulator prior to treatment. Portal films are taken during the first week of each phase of treatment.Patients are reviewed by an oncologist or dedicated radiographer prior to the first day of the chemotherapy cycle, and weekly during the chemoradiation, noting treatment-related acute toxicities. The intent is to deliver this treatment in the outpatient department, though some patients require admission for ‘hotel’ needs. Enteral or parenteral feeding is not routinely used.Of the remaining 17 patients, 13 received the same regimen without the neoadjuvant phase of chemotherapy, one patient the above regimen omitting the 5FU, two received four cycles of epirubicin, cisplatin and infusional 5FU prior to chemoradiation and one patient received two cycles of mitomycin C, ifosfamide and cisplatin, concurrently with 50Gy radiation in 20 fractions.Following the completion of chemoradiation, a CT scan is performed to establish a disease ‘baseline’ after treatment, though comparison with scans prior to treatment are notoriously difficult to interpret. Patients no longer undergo EUS, as this invasive procedure does not accurately stage disease after CRT  – a database and patient activity system available in all Welsh hospitals, patient notes and the Welsh Cancer Information and Surveillance Unit registry. Data were analysed using SPlus 2000. The outcome was assessed using the Kaplan–Meier and log-rank methods, and contingency tables were analysed using Outcome data were available on all 90 patients who received definitive chemoradiation. The distribution of patient and disease characteristics is shown in There were similar numbers of adenocarcinomas and squamous cancers in this series. In six cases, due to poor differentiation of the tumour, it was not possible to classify the type. In all, 49 tumours (54%) were situated in the lower one-third of the oesophagus or crossed the gastro-oesophageal junction. The average length of the primary tumour was 6.9 cm (range 2–13 cm) and was ⩾8 cm in 22 cases (24%).The majority of patients received the chemoradiation schedule as prescribed, the commonest dose modifications being made to the chemotherapy treatment. A total of 37% of patients required a dose modification to the planned chemotherapy schedule. These were usually minor dose modifications or delays, most commonly resulting from 5FU-induced mucosal toxicity. Overall, the patients received 94 and 93% of the planned cisplatin and 5FU, respectively. Four patients were changed to carboplatin as a result of neuro-/nephrotoxicity. Only six (7%) patients received less than the prescribed dose of radiation or a delay of more than 1 week in their RT treatment.There were no deaths directly related to treatment, though five patients died during or within 30 days of completing treatment due to progressive disease. One patient had a suspected pulmonary embolism but a post mortem was not carried out. Significant toxicities are shown in In all, 17 patients required nonelective hospitalisation due to treatment complications, most commonly for rehydration as a result of dysphagia, electrolyte disturbance and mucosal toxicity. In addition, 18 patients (20%) required enteral feeding as a result of malignant dysphagia and five patients developed deep venous thrombosis, which required anticoagulation.During follow-up, nine (10%) patients developed nonmalignant strictures requiring dilatation and four patients developed tracheo-oesophageal fistulae, all of whom had a covered stent successfully placed in the oesophagus. Three patients had a persistent ulcerative oesophagitis and two a temporary radiation pneumonitis, which settled with appropriate treatment.As of 31 January 2003, 45 (50%) of patients were alive, 36 with no evidence of disease progression. One patient has been lost to follow-up. The median progression-free survival for the whole group was 18  months.P=0.024, Figure 2P=0.89; median survival times 15  and 26  months, respectively). In patients with T4 disease, the overall survival was significantly worse than in those with non-T4  months. For the whole series, the 2-, 3- and 5-year survivals were 51% , 45%  and 26% , respectively  months). In the latter group, the 2- and 5-year survivals were 67%  and 32% , respectively.In 38 patients where the extent of their disease determined that the chance of a complete resection (R0) was too low, the median survival was 11  months, whereas, in the 52 cases where patient-related factors precluded surgery, the survival was significantly better, (P>0.29). The only significant prognostic indicator was the presence of T4 disease, when compared with T1–T3 , P=0.01). The reason for not having surgery was excluded from the Cox model because this factor is partly derived from the stage of disease, and, as would be expected, the association between the two was highly significant . Of those cases deemed to be unsuitable for surgery because of their disease, 66% were T4.In a Cox regression model, none of the covariates age, sex, histological type, the presence of M1a disease (coeliac lymphadenopathy associated with a tumour of the lower third of the oesophagus) and nodal status were significantly associated with outcome  patients developed progressive disease during the period of study. A total of 17 patients (19%) had radiological or clinical evidence of disease progression at the end of or just after completion of treatment. In a further 21 cases (23%), disease recurrence was confined to locoregional sites, defined as lying within the planned RT target volume. In eight patients, the initial relapse was at a distant site and in three both local and distant disease progression occurred together.The 49 patients who relapsed were considered for second-line treatment, but in 23 (47%) best supportive care was deemed most appropriate. Of 26 patients who received active second-line palliative therapy, 22 (85%) underwent endoscopic therapy to palliate local disease, this being the placement of an oesophageal stent in 19 (73%) and laser therapy in three. For those patients who had distant metastases as a component of disease relapse, five (42%) underwent second-line chemotherapy. The median survival of patients after disease progression was 5.1  months.In this series, 90 patients with oesophageal cancer who received definitive chemoradiation, having been selected for nonsurgical therapy because of co-morbidity or locally advanced disease, had a median survival of 26 months, 51 and 26% being alive 2 and 5 years, respectively, after diagnosis.These results compare favourably with other published series evaluating the use of chemoradiation for oesophageal cancer  5FU was used. This has been shown to be superior to bolus treatments in other cancers  and optiIn a similar number of patients, How do these results compare to surgical series? There have recently been two large multicentred randomised controlled trials evaluating the role of chemotherapy prior to surgery. The UK MRC OE02 trial demonstrated a clear benefit for neoadjuvant treatment . The oveP<0.001, Figure 4There was a highly significant difference in outcome between those who did not receive surgery due to characteristics relating to the patients, for example, co-morbidity, performance status, patient choice, etc, compared with factors relating to their disease, for example, invasion of mediastinal organs, extensive lymphadenopathy, etc.,  patients did not show a significantly reduced survival rate than patients with stage III  disease may be that the latter patients had on average more bulky disease than the former. In this cohort, all those with Stage IVa disease had nonbulky coeliac nodal involvement with cancer in the lower on-third oesophagus (M1a disease). This extent of disease may have a more favourable outcome with or without therapeutic intervention than patients with stage III disease not receiving surgery. Cox regression showed that T4 disease had a greater impact on outcome than the presence of M1a, and in stage III cases the proportion of T4 tumours was higher (68%) than in stage IVa cases (32%).Although chemoradiation is considered to be superior in terms of local control and survival over RT alone, it is associated with increased toxicity (As well as the encouraging results with respect to overall survival, it is also particularly encouraging that, several weeks after completing this treatment, patients frequently feel very well and most notably their dysphagia has usually resolved. This, and other aspects of quality of life, is the subject of ongoing research by the South East Wales Oesophago-Gastric Unit.What proportion of patients may be suitable for this regimen? In 1995, an audit of all cases diagnosed with oesophagogastric cancer in Wales found the incidence of oesophageal cancer to be 11/100 000 (In the United States, results from the ‘Patterns of Care’ report reflect that chemoradiation is already a standard treatment option for patients with nonmetastatic disease (Undoubtedly, the way to improve outcomes is through well-conducted research. This disease site has been generally been poor in recruitment of patients into clinical studies, whereas chemoradiation trials in other cancer sites such as anal cancer demonstrate that high rates of accrual can be obtained . EvidencCapecitabine and oxaliplatin are being tested in advanced oesophagogastric cancer and would certainly provide a more convenient and hopefully more active systemic regimen. Irinotecan and taxanes have also shown high activity in Upper GI malignant disease, together with promising radiosensitising properties.These data show that definitive chemoradiation is feasible and tolerable for 50% of patients with oesophageal cancer, treated with potentially curative therapy. It supports other evidence that this modality is the treatment of choice in patients unsuitable for surgery on the basis of co-morbidity or locally advanced disease. It also suggests that it should be compared in a prospective study to a surgically based treatment strategy.


Background. Genetic polymorphisms of drug metabolizing enzymes involved in the detoxification pathways of carcinogenic substances may influence cancer risk. Methods. Case-control study that investigates the relationship between CYP1A1 Ile/Val, exon 4 mEH, and GSTM1 null genetic polymorphism and the risk of oral and pharyngeal cancer examining the interaction between these genes, tobacco, and alcohol. 92 incident cases and 130 consecutive hospital-based controls have been included. Results. No significant associations were found for any of the genotypes assessed. The estimated risk was slightly elevated in subjects with the wild type of the mEH gene and the null GSTM1 genotype. For exon 4 mEH heterozygous polymorphism, the risk was slightly lower for heavy smokers than for light smokers. The inverse association was observed for the GSTM1 null genotype. Conclusions. The results suggest that exon 4 mEH and GSTM1 null polymorphisms might influence oral and pharyngeal cancer. Oral and pharyngealcancers represent an important problem worldwide. The incidence and prevalencerates for these tumors are double in men than in women. Cancers of the oralcavity rank as the eighth most common cancer among men, being responsible for3% of the cancers diagnosed in this gender . MortaliTobacco and alcoholare the main risk factors for oral and pharyngeal cancers. In USA and Europe,they are responsible for 75–80% of thesetumors , 3. EpidThe reason why someindividuals develop cancer and others do not can be attributed, at least partly,to variations in genetic polymorphisms responsible for metabolizingcarcinogenic substances found in tobacco and alcohol. Although many genes havebeen associated with metabolism of these compounds, some with the highestrational of those beinginvolved are CYP1A1, mEH, and GSTM1.The CYP1A1 genebelongs to the CYP1 subfamily and encodes for the enzyme aryl hydrocarbonhydrolase, which is involved in the activation of many polycyclic aromatichydrocarbons and aromatic amines  and is pThe microsomal formof epoxide hydrolase is primarily associated with the metabolism of exogenousxenobiotic compounds. Its interest in oral and pharyngeal cancers comes fromthe fact that it has been detected in alltissues including the aerodigestive tract and catalyzes the hydrolosis of arene, alkene, and aliphatic epoxides frompolycyclic aromatic hydrocarbons and aromatic amines . EnzymatThe glutathione Stransferase (GST) comprises a family of phase II detoxifying enzymes thatcatalyze a great number of detoxification reactions that take place between thecytosolic glutathione and compounds containing an electrofilic centre . TheGSTIn the present case-controlstudy, we aimed to examine the relationship between the CYP1A1 Ile/Val, exon 4 mEH(139 Arg → His), and GSTM1 nullgenetic polymorphism and the risk of oral and pharyngeal cancers, investigatingalso the association with smoking, drinking, and the gene-gene interactions.The presenthospital-based case-control study was conducted at the Santiago de CompostelaUniversity Hospital Complex  between January 1996 and January 2000. Data was collected on a totalof 92 incident Caucasian male cases with histopathologically confirmeddiagnosis of primary oral or pharyngeal cancers. The study was restricted tonewly diagnosed patients over 20 years of age without a prior history ofcancer. For study purposes, tumors of the lip were excluded.A total of 130 consecutivecontrols were included from patients attending the Hospital ComplexPreoperative Unit for nonsmoking- and nonalcohol-related trivial surgery. Theinclusion criterion for controls was absence of prior history of cancer. Subjectsunder 20 years of age were excluded. The types of surgical procedures controls were scheduled to undergo mainlycomprised inguinal hernias, cataracts, and orthopedic surgery. Informed consentwas obtained from all study subjects prior to the interview and the extractionof total blood. Only 1 case and 6 controls refused to participate in the study. The study protocol was approved by the Galician Ethical Research Committee.All study subjectswere interviewed by a person purpose-trained to administer a structuredquestionnaire addressing various aspects of lifestyle, with special emphasis onsmoking habit, alcohol consumption, occupation, and other activities related to the development oforal and pharyngeal cancers. For cases, the interview was done as soon aspossible after cancer was detected and always within 15 days from diagnosis. The questionnaire used was previously applied to a group of individualsfulfilling the characteristics of the subjects going to be included and allquestions with difficult interpretation were duly amended.μg/mL. The genotypingassays were performed at the Molecular Medicine Unit of the Santiago deCompostela University Hospital Complex.After cases andcontrols were identified, whole blood samples of 6 mL were collected from eachsubject in heparin-containing tubes. The samples were stored at 4°C and centrifuged at 2800 rpmfor at least 10 minutes within the next 24 hours. The three independentfractions were isolated and stored at −84°C until analysis. DNA was extracted fromthe buffy coat of middle layer containing monocytes. The cells were washed withTE (10x) and centrifuged at 3000 rpm during 10 minutes several times until theywere cleaned. The pellet was then treated with lysis buffer and proteinase K in1% SDS previous to the extraction of DNA with phenol-chloroform and ethanolprecipitation similarly as previously described . The DNAμL containing 20 mM Tris-HCl (PH 8.4), 50 mM KCl, 3 mM MgCl2, 0.25 μMof each GSTM1 primer, 0.25 μM of each β-globin primer , 1 U Taq DNApolymerase (Promega), 2 μMeach dNTPs, and 600–900 ng of DNA. The GSTM1 primers were 5′-GAACTCCCTGAAAAGCTAAGC and 5′-GTTGGGCTCAAATATACGGTGG. A negative control was included in allbatches. The PCR conditions were 94° for 3 minutes, followed by 35 cyclesof 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 45 seconds, and afinal extension of 72°C for 7 minutes.Genotyping for GSTM1was carried out in the whole sample using a modified PCR method describedpreviously . Reactio139 arg), a 357-bp fragment containing thepolymorphic site was amplified pyrene-7,8-diol epoxide, the activated form ofbenzo[a]pyrene  but the Our study suggeststhat the effect of the GSTM1 null polymorphism is more noticeable among lightdrinkers, although this association was nonsignificant. This relationship wasmaintained when we took out the nondrinkers from the analysis (data not shown). This differential effect could be explained by the fact that the GSTM1isoenzyme, together with the alcohol dehydrogenase, is involved in theoxidation of ethanol to acetaldehyde . Even thEven though theCYP1A1 mutation has been shown to increase microsomal activity for convertingprocarcinogens, including PAH aromatic amines, the results of various reportson smoking-related cancers are inconsistent , 66–70. This paper hasseveral limitations. The main one is the small sample size included. This is especiallyimportant when analyzing polymorphisms that have a very low frequency in thepopulation, such as CYP1A1 Ile/Val, but also limits the power to identifygene-environmental and gene-gene interactions in any polymorphismsinvestigated. Nevertheless, it has to be taken into account that for cancerswith a low incidence such as oral and pharyngeal ones, 92 cases can be arelatively good number. Another limitation could be the fact that our study washospital-based and this could result in selection bias. Even though it has beensuggested that studies with hospital controls can provide lower risk estimates,since diseases of controls could be associated with the polymorphisms understudy, previous meta- and pooled analysis that assessed these polymorphisms onhead and neck cancers found no differences for hospital-based studies inrelation to population-based studies , 66. TheThe present studyhas also some advantages. One of them is that the participation of cases andcontrols was very high. All the cases belonged to the same catchment area,which has a unique reference hospital, and were collected consecutively, makingthem representative of the cases in that area. The controls also belonged tothe same area and did not have any symptom or disease related to alcohol or tobaccoconsumption. Another advantage is the fact that the genes analysed here were ofphase I and phase II, which adds value to the results obtained. When studyingsusceptibility genes in many occasions, both types of genes are not studied andindividuals can have susceptibility in type I compensated with the activity of the other type suchas it was shown in Park et al. investigation .As a conclusion, itseems that even though none of the three genotypes analysed have a significantassociation with the risk of oral and pharyngeal cancer in this population, therisks of tobacco and alcohol consumption might be modified by GSTM1 null andexon 4 mEH polymorphisms. The small number of nonsmoking and nondrinking subjectsin our population limited our analysis, so we propose that further studies are carriedout to clarify this question. Even though we found no significant interactionbetween the GSTM1null genotype and the exon 4 mEH genotype, the GSTM1 absencedid show a slight rise in risk so this should also be investigated taking intoaccount other polymorphisms including the CYP1A1.


Rapid, easy, economical and accurate species identification of yeasts isolated from clinical samples remains an important challenge for routine microbiological laboratories, because susceptibility to antifungal agents, probability to develop resistance and ability to cause disease vary in different species. To overcome the drawbacks of the currently available techniques we have recently proposed an innovative approach to yeast species identification based on RAPD genotyping and termed McRAPD (Melting curve of RAPD). Here we have evaluated its performance on a broader spectrum of clinically relevant yeast species and also examined the potential of automated and semi-automated interpretation of McRAPD data for yeast species identification.A simple fully automated algorithm based on normalized melting data identified 80% of the isolates correctly. When this algorithm was supplemented by semi-automated matching of decisive peaks in first derivative plots, 87% of the isolates were identified correctly. However, a computer-aided visual matching of derivative plots showed the best performance with average 98.3% of the accurately identified isolates, almost matching the 99.4% performance of traditional RAPD fingerprinting.Since McRAPD technique omits gel electrophoresis and can be performed in a rapid, economical and convenient way, we believe that it can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories that are able to adopt this technique. It can also serve as a broad-range high-throughput technique for epidemiological surveillance. Over the past decades, patients have increasingly been colonized and infected with a variety of yeast species mainly due to immunocompromising conditions as well as due to increased use of invasive techniques and devices . TherWe have recently proposed an innovative technique termed McRAPD (Melting curve of Random Amplified Polymorphic DNA) which has the potential to provide rapid and accurate pathogenic yeast identification grown in pure culture in an easy and economical way . Here weS. cerevisiae and C. glabrata). Based on these results, the amount of 1 μl was evaluated as the best, because lower volumes did not always guarantee reliable amplification, whereas in higher volumes it cannot be excluded that too much PCR inhibitors may enter the reaction mixture. For comparison and reference, the commercial kit YeaStar Genomic DNA Kit  was used in parallel with 1 μl of crude colony lysates. Results of this comparison represented by melting curves and banding patterns are summarized in Figure C. glabrata, showed both markedly different banding patterns and melting curves, indicating that the performance of McRAPD with colony lysate was suboptimal in this case compared to the commercial kit. Our experience in routine experiments shows that the initial relative fluorescence intensity of a McRAPD sample after amplification should exceed the relative value of 15 at the standard 30% LED power as adjusted in melting protocol by user. When a sample does not meet this condition, repeating the assay including DNA extraction is strongly recommended for reliable results.To achieve rapid and economical performance of the McRAPD identification approach, we used the simplified DNA extraction technique described by Steffan et al. . HoweverIn addition, reproducibility of the simplified DNA extraction based on crude colony lysates was tested. DNA was extracted from 4 different yeast species, each represented by one strain, where 5 colonies were grown for different time periods in each strain and used for extraction. Sampling was performed in the interval between 12 and 24 h of colony growth, approximately every 3 h. Freshly prepared lysis buffer was always used for DNA extraction in each of the samples. The results clearly demonstrate that the time-point of colony sampling and different runs of the extraction procedure have little influence on the variability of McRAPD results , whereas those variably present longer RAPD products (> 900 bp) are reflected in the variable second portion of the domain , the result of McRAPD and RAPD identification cannot be considered as discrepant from the result of conventional phenotyping techniques. In the other cases of doubtful profiles (n = 12), McRAPD either suggested discrepant species identification result or did not suggest any identification. In such cases, the conventional phenotypic species identification was further verified using the ID 32C. Results of this verification are summarized in Table Clustering of McRAPD data was performed using the UPGMA algorithm performed with similarity coefficients obtained as described in Material and Methods . This is mainly because of different data processing in conventional RAPD versus McRAPD. In RAPD, differences in overall amplification efficiency result in differences in intensity of banding patterns. Therefore, it is strongly recommended not to include weak bands into comparison of RAPD fingerprints, because these can appear or disappear in different amplification runs. Also, the relative intensity of strong bands cannot be reliably taken into account for comparison. That is why we used the band-based Jaccard coefficient for processing of RAPD fingerprints, which takes the position of a band into account but neglects its intensity. In contrast, raw fluorescence measured during melting in the McRAPD procedure truly reflects the relative representation of individual RAPD products (bands in electrophoresis) in the sample. Inter-sample and inter-run differences in overall fluorescence of samples are subsequently proportionally equilibrated during numerical normalization of melting data. Then, relative representation of individual RAPD products is reflected in the slope of a normalized curve or in the height of a peak in a derivative curve and this is taken into account during further evaluation.To see whether the strain clustering patterns resulting from McRAPD and conventional RAPD are consistent, McRAPD genotypes were color-coded by ground tint colors in the dendrogram of RAPD fingerprints using different color saturation for different genotypes  in particular, should be the main competitors of McRAPD. Although MLST is more demanding concerning instrumentation, skills and labour, it has the advantage of unmatched interlaboratory reproducibility, enabling global epidemiology. However, it can hardly be expected that MLST can present an economically affordable alternative for routine identification and prospective epidemiological surveillance in near future. It can rather be expected that its use will be limited to retrospective epidemiological studies. Thus, McRAPD offers a promising choice for routine identification of pathogenic yeast species; in case of failure, it could be supplemented by other techniques, the best of which appears to be single-locus sequencing in our opinion.1. Crude colony lysates provide an economical, rapid and reliable alternative to elaborate DNA extraction techniques for the purposes of McRAPD when performed by skilled personnel.2. Our optimized McRAPD protocol shows excellent intralaboratory reproducibility and is able to delineate specific genotypes in some of the species studied.3. Computer-aided visual matching of first derivative plots shows best performance among the approaches tested for interpretation of mere numerical McRAPD data. Its performance almost matched the performance of traditional RAPD fingerprinting and was comparable to the performance of the ID32C commercial system.4. We believe that because of its advantages over conventional phenotypic identification approaches and competitive costs McRAPD can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories being able to adopt the technique. It can also serve as a broad-range high-throughput technique for crude epidemiological surveillance.Candida albicans (56.2%), C. glabrata (12.6%), C. krusei (8%), C. tropicalis (7.7%), Saccharomyces cerevisiae (3.1%), C. parapsilosis (2.5%), and C. lusitaniae (2%) were represented by at least 35 isolates each, whereas the less frequently isolated species C. guilliermondii (1.3%) and C. pelliculosa (1%) were represented by at least 15 isolates each. A few isolates of C. orthopsilosis and C. metapsilosis were also included into the study later, when described as cryptic species of C. parapsilosis [The 9 yeast species most frequently isolated from clinical samples in our settings, namely representing 94.3% of yeast species isolated from patient samples at our department, were included into the study. Among these, 7 more common species, i.e. psilosis . See alsAll of the isolates were identified using conventional phenotypic identification techniques, i.e. evaluation of micromorphology on rice agar and evaluation of biochemical properties using in-house prepared assimilation and fermentation tests  followed2, 2 mM dithiothreitol, 12 U of Zymolyase, all from Sigma-Aldrich, St. Louis, Missouri, USA). The mixture was incubated for 30 min at 37°C and centrifuged . The supernatant was transferred into a new tube, diluted with TE buffer to 300 μl and stored at -20°C until used. For comparison and reference, YeaStar Genomic DNA Kit  was also used for DNA extraction in selected strains following manufacturer's recommendations. Briefly, 1 ml of yeast submerged culture (approx. 1.5 × 107 cells) grown in YPG  in an Erlenmeyer flask shaken at 30°C was spun down and the pellet was subjected to enzyme lysis in 120 μl of YD Digestion Buffer (containing RNase A and Zymolyase) for 1 hour at 37°C. Then, 120 μl of YD Lysis Buffer and 250 μl of chloroform were added, mixed and spun down again. The aqueous supernatant was then loaded onto a fast spin-column, spun down, and the impurities were washed away using DNA Wash Buffer. Finally, DNA was eluted by 60 μl of water.Crude colony lysates described earlier as suitable for amplification were prepared by simple toothpick technique . Briefly2, 200 μM of each dNTP, 2.5 U of Taq polymerase Unis , 250 μg/ml BSA and LCGreen dye at 1× concentration . Either 1 μl of crude colony lysate or 1 μl of DNA extracted using the YeaStar Genomic DNA Kit was added into the reaction. Amplification was performed in a Rapid Cycler 2 apparatus  applying an empirically optimized protocol of initial denaturation at 95°C, 5 min, followed by 45 cycles of denaturation at 95°C for 5 s, annealing at 48°C for 10 s, and extension at 72°C for 40 s, with ramping 1°C/s, followed by final extension at 72°C for 5 min.PCR reaction was performed in a glass capillary in a total volume of 10 μl consisting of 0.5 μM primer ACGGGCCAGT , 10 mM TRAPD amplicons were subjected to melting analysis on a high-resolution melting instrument HR-1 . The samples were heated at ramping rate of 0.3°C/s with acquisition of fluorescence data ranging from 75 to 95°C. Results were analysed using the HR-1 melt analysis software. Relative fluorescence was first plotted versus temperature and fluorescence intensity values were normalized as recommended by the manufacturer. For this purpose, temperature ranges preceding and following the melting domain were optimized empirically to result in reproducible normalized melting curves in all of the yeast species examined. The optimized intervals for normalization were 75.5-77.5°C and 91.5-93.5°C, respectively. A simple procedure for comparison of normalized melting profiles was developed by us. Briefly, differences in McRAPD data of a pair of isolates were calculated by subtracting their normalized fluorescence values measured at each temperature point during melting analysis. Then, the sum of these subtracted values represented absolute numerical distance between the pair of isolates, i.e.:whereAD1,2 was absolute distance between isolates No. 1 and 2f1(t) was normalized fluorescence of isolate No. 1 measured at temperature tf2(t)was normalized fluorescence of isolate No. 2 measured at temperature tADmax. Relative distance values for the remaining pairs of isolates were calculated as a fraction of the highest absolute value, i.e.:After the absolute distance was established in all pairs (combinations) of isolates, the relative distance 1.0 was assigned to the highest absolute value obtained in the most dissimilar  pair of isolates, abbreviated as A matrix of relative distances was assembled for the isolates included into each comparison. Then, the matrix of relative distances was used to calculate tree data for a cladogram using the UPGMA method and Phylip software ,29. PhylFor additional analysis, plots of the first negative derivation of fluorescence depending on temperature were also prepared based on melting data normalized previously. To delineate the melting peaks better, smoothing of data was performed using the HR-1 analysis software as recommended by the manufacturer. In some cases this smoothing resulted in truncation of the left and/or right end of the derivative curve. This process was carefully observed to prevent any loss of potentially discriminatory peaks at both ends of the derivative curves. To prevent excessive simplification and loss of informative data, smoothing was performed only if it undoubtedly resulted in a distinct amelioration of peaks' discrimination.After melting analysis was performed, each sample was also subjected to gel electrophoresis in 2% agarose gel at 5 V/cm for 3 hours. The gels were stained by ethidium bromide added into them during preparation at the final concentration of 1 μg/ml and resulting banding patterns were photographed. Comparison of fingerprints was performed using GelCompar II software  applying the Jaccard coefficient at 1.5% positioning tolerance. Dendrograms were constructed using the UPGMA algorithm.JT performed most of the DNA extractions and McRAPD amplification, processed the acquired data, performed statistical analysis and drafted the paper. PP developed a software tool to facilitate comparison of normalized McRAPD data. LR participated in DNA extractions and McRAPD amplification. PH and DK performed conventional phenotypic identification of yeast species as well as ID 32C identification of selected strains and revised the paper critically. VR conceived and designed the study, developed the concept of automated processing of McRAPD data, participated in drafting the paper, revised it critically and gave final approval of the version to be published. All authors read and approved the final manuscript.Similarity coefficients. Listing of similarity coefficients obtained upon automated comparison of normalized melting curves within each species.Click here for fileDendrogram of RAPD fingerprints. Dendrogram based on RAPD fingerprints of all strains included in the study. Analysis of RAPD fingerprinting patterns always provided accurate identification except for 2 strains showing quite unique fingerprints (marked by arrows). For comparison of strain clustering between conventional RAPD and McRAPD, the strains of different species are color-coded by ground tint colors and their specific McRAPD genotypes are indicated by different saturation of colors. In case a strain was not assigned to a specific McRAPD genotype, it is not color-coded.Click here for fileAverage derivative curves. Plots of average McRAPD first negative derivative curves of species and genotypes included in the study.Click here for fileListing of clinical isolates and reference strains included in this study.Click here for file


Raisins are a significant source of dietary fiber and polyphenols, which may reduce cardiovascular disease (CVD) risk by affecting lipoprotein metabolism and inflammation. Walking represents a low intensity exercise intervention that may also reduce CVD risk. The purpose of this study was to determine the effects of consuming raisins, increasing steps walked, or a combination of these interventions on blood pressure, plasma lipids, glucose, insulin and inflammatory cytokines.Thirty-four men and postmenopausal women were matched for weight and gender and randomly assigned to consume 1 cup raisins/d (RAISIN), increase the amount of steps walked/d (WALK) or a combination of both interventions (RAISINS + WALK). The subjects completed a 2 wk run-in period, followed by a 6 wk intervention. Systolic blood pressure was reduced for all subjects (P = 0.008). Plasma total cholesterol was decreased by 9.4% for all subjects (P < 0.005), which was explained by a 13.7% reduction in plasma LDL cholesterol (LDL-C) (P < 0.001). Plasma triglycerides (TG) concentrations were decreased by 19.5% for WALK (P < 0.05 for group effect). Plasma TNF-α was decreased from 3.5 ng/L to 2.1 ng/L for RAISIN (P < 0.025 for time and group × time effect). All subjects had a reduction in plasma sICAM-1 (P < 0.01).This research shows that simple lifestyle modifications such as adding raisins to the diet or increasing steps walked have distinct beneficial effects on CVD risk. Cardiovascular disease (CVD) is the leading cause of death for both men and women in the United States . Risk faThe addition of raisins to the diet may decrease CVD risk, as they contain dietary fiber to lower LDL-C , as wellRegular exercise can decrease risk for CVD by decreasing TG and increasing HDL-C . ExercisThe purpose of this study was to determine if consumption of raisins, increasing steps walked, or a combination of these interventions affect risk for CVD by assessing resting blood pressure, plasma lipids, glucose, insulin and inflammatory cytokines.As indicated in Table Body weight and waist circumference were not changed as a result of the intervention Table . In contCarbohydrate intake during the intervention was significantly higher for RAISIN  and RAISIN + WALK  than WALK . In contrast, there was a significant group and group × time effect on plasma TNF-α concentrations  an increase in TG removal with elevated LPL, or 2) decreased TG secretion from the liver . ExercisA synthetic polyphenol, as well as resveratrol, stimulated AMPK in vitro; leading to a reduction in TG accumulation . The synThe reduction in blood pressure for RAISIN and RAISIN + WALK may have resulted from antioxidant effects of the raisin polyphenols. Scavenging of ROS inhibits NAD(P)H oxidase and subsequently decreasing superoxide production . This loThe lack of change in plasma glucose and insulin for RAISIN and RAISIN + WALK is expected given that plasma TG were unaffected by the interventions. A decrease in TG may lower insulin resistance by alleviating lipotoxicity . The incRaisins and walking had positive effects on markers of inflammation. It is expected that the inflammatory cytokines would be significantly decreased for RAISIN and RAISIN + WALK, as polyphenols may act to lower oxidative stress and inflammation ,19,20. GOral gavage of large doses of resveratrol significantly decreased TNF-α concentrations in rats . HoweverResearchers have indicated that regular aerobic exercise inhibits the production of TNF-α, potentially via an increase in IL-6 with each acute bout . IL-6 haThe decrease in sICAM-1 for RAISIN and RAISIN + WALK is supported by findings from previous research by Estruch et al. , where ~In conclusion, risk factors for CVD were affected significantly by consuming raisins or increasing steps walked/d. Blood pressure, plasma total cholesterol and LDL-C were significantly decreased by all interventions, while walking lowered plasma TG. All interventions significantly reduced plasma concentrations of sICAM-1, potentially preventing progression of atherosclerosis by decreasing adhesion of monocytes to the vascular endothelium. Raisins lowered the risk for inflammatory damage by decreasing TNF-α. The findings of this research show that simple lifestyle modifications such as adding raisins to the diet or increasing steps walked/d have distinct beneficial effects on risk factors for CVD.Kits for total cholesterol (TC) and triglycerides were from Roche Diagnostics ; glucose kits were from WACO .Raisins were provided by the California Table Grape Commission .2 were also excluded from the study. A total of 17 men and 17 postmenopausal women volunteered to participate in the study. Study protocols were approved by the Institutional Review Board.Men and postmenopausal women between the ages of 50–70 y were recruited from the University community for this study. Subjects provided informed consent and completed a medical history during recruitment. Exclusion criteria included taking blood-thinning medications, cigarette smoking, diabetes, cardiovascular disease, or renal disease. Subjects with a body mass index greater than 37 kg/mThe subjects in the study were matched according to sex and body mass, then randomly assigned to 1 of 3 groups: 1) a group that consumed 1 cup raisins/d (RAISIN), 2) a group that increased the amount of steps taken each day (WALK), or 3) a group that consumed 1 cup raisins/d and increased the amount of steps taken (RAISIN + WALK). The subjects completed a 2 wk washout period, followed by a 6 wk intervention.All subjects completed a 2 wk run-in period in order to standardize exercise and dietary habits. Subjects were asked to maintain there normal level of activity and abstain from polyphenol-rich foods, including grapes, berries, wine, chocolate, raisins, tea, vitamins, and any other supplements. These restrictions were continued during the 6 wk intervention in order to isolate the antioxidant effects of the raisins. The subjects that were asked to increase the amount of steps walked/d during the intervention were issued a pedometer. The subjects maintained their normal daily activity during the 2 wk period in order to obtain an estimate of the amount of steps/d. The subjects in RAISIN were asked to maintain their normal level of activity during the 6 wk intervention.The raisins consumed by the subjects in this study were provided by the researchers weekly with a checklist to determine compliance. A Registered Dietitian provided dietary instruction for substituting the raisins for other foods to ensure weight maintenance, and provided written materials to reinforce the information. The dietitian also counseled WALK subjects to ensure weight maintenance during the intervention. The subjects were encouraged to consume the raisins with other foods and space their intake throughout the day in order to make consumption easier. Subjects were also instructed on how to accurately complete a food frequency questionnaire and a detailed food record.WALK participants were instructed to increase their steps by walking an additional 10 min/d  every 2 wk in an attempt to increase their walking by approximately 1 km/d every 2 wk. Therefore, the subjects were walking an additional 10 min/d for the first 2 wk, 20 min/d for the second 2 wk, and 30 min/d for the last 2 wk . The subjects logged their steps daily, and noted any difficulties with the pedometer or unusual physical activity .Subjects completed a food frequency questionnaire after random assignment into groups to assess usual dietary intake. A physical activity questionnaire was also administered to estimate average activity levels. After the baseline information was collected, the subjects reported to the laboratory following an overnight fast (~12 h) at the beginning of the 2 wk washout period. At this point, pedometers were issued to subjects in the WALK and RAISIN + WALK groups, and subjects began the dietary restrictions. A phlebotomist collected 10 mL of blood from the antecubital vein into a tube containing EDTA to assess lipids for screening purposes.The subjects returned to the laboratory twice more at the start of the 6 wk intervention after the 2 wk washout period. Blood was drawn as before; 60 mL in the first visit, and 10 mL in the second visit (just for assessment of lipids). Body weight, height, waist circumference and resting blood pressure were also measured for each subject in the first visit. Plasma was separated by centrifugation at 2200 × g for 20 min at 4°C; and sodium azide (1 μL/mL), phenylmethyl-sulphonyl fluoride , and aprotinin (5 μL/mL) were added to the samples for preservation. Approximately 1 mL of plasma was aliquoted at both time points to assess plasma lipids. The remainder of the plasma was aliquoted into microcentrifuge tubes and stored at -80°C for later analysis. This procedure was completed again at the end of the 6 wk, as the subjects reported twice for blood collection in the same wk.Subjects completed a 5 d diet record, including 3 weekdays and two weekend days, during the study to assess normal dietary consumption. The days were not necessarily consecutive, but it was emphasized that the days should represent the subjects' typical intake. Subjects in the RAISIN and RAISIN + WALK groups received sheets to record raisin consumption; and subjects in the WALK and RAISIN + WALK groups received forms to record daily step totals. These forms were distributed weekly, and a researcher reviewed the data with each subject weekly. Raisins were also distributed weekly. Counseling was provided at this time to ensure that subjects adhered to dietary restrictions and increased their steps as appropriate.All dietary records were analyzed using the Nutrition Data System 5.0 . Total kcal intake, as well as carbohydrate, fat, protein, and dietary fiber were determined. Also, monounsaturated, polyunsaturated, and saturated fat was assessed, as well as dietary cholesterol and dietary fiber.Plasma lipids were determined by taking the average of 2 values obtained on separate days of the same wk before and after the intervention. This was done to account for day-to-day variability in these values. Plasma total cholesterol was measured in triplicate using enzymatic methods, with Roche Diagnostics standards and kits . Plasma Seated resting systolic and diastolic blood pressure were measured after 5 min of rest using a Welch Allyn Tycos blood pressure cuff.® technology on a Luminex ® IS 200 system with antibodies to these analytes from LINCO Research  [Plasma glucose was analyzed by an automated lactate/glucose analyzer . Plasma insulin and cytokines  were measured using xMAPles, MO) . Assays All statistical analyses were performed with SPSS 12.0 for Windows . One way ANOVA was used to determine difference in macronutrient intake for the groups. The least significant difference was used as a post-hoc test. Repeated measures ANOVA was utilized to determine changes in variables over time for the three groups. If a significant interactive effect was found, Tukey post hoc analyses were completed. Significance was set at P ≤ 0.05.Supported by a grant from The California Grape Commission to MLFMLF designed the study, with suggestions and help from MP and JV. MP, UV, SS, MTG, and RW participated in carrying out the study and data collection. MP, UV, SS, and RW completed the assays. MP and MLF drafted the manuscript. All authors read and made suggestions for the final manuscript.


Aim. Based on the previously established method, we developed a better and stable animal model of type 2 diabetes mellitus by high-fat diet combined with multiple low-dose STZ injections. Meanwhile, this new model was used to evaluate the antidiabetic effect of berberine. Method. Wistar male rats fed with regular chow for 4 weeks received vehicle (control groups), rats fed with high-fat diet for 4 weeks received different amounts of STZ once or twice by intraperitoneal injection (diabetic model groups), and diabetic rats were treated with berberine . Intraperitoneal glucose tolerance test and insulin tolerance test were carried out. Moreover, fasting blood glucose, fasting insulin, total cholesterol, and triglyceride were measured to evaluate the dynamic blood sugar and lipid metabolism. Result. The highest successful rate (100%) was observed in rats treated with a single injection of 45 mg/kg STZ, but the plasma insulin level of this particular group was significantly decreased, and ISI has no difference compared to control group. The successful rate of 30 mg/kg STZ twice injection group was significantly high (85%) and the rats in this group presented a typical characteristic of T2DM as insulin resistance, hyperglycemia, and blood lipid disorder. All these symptoms observed in the 30 mg/kg STZ twice injection group were recovered by the treatment of berberine. Conclusion. Together, these results indicated that high-fat diet combined with multiple low doses of STZ  proved to be a better way for developing a stable animal model of type 2 diabetes, and this new model may be suitable for pharmaceutical screening. Now there has been a tragic increase in diabetes across the world,paralleling the overweight and obesity epidemic. There are 95 percent of those people belonging to type 2 diabetes. Therefore, it is great urgency to find better treatments and novelprevention strategies for type 2 diabetes. To accomplish this goal, appropriate experimental models are considered as essential tools for understanding the molecularbasis, pathogenesis of the vascular and neural lesions, actions of therapeuticagents, and genetic orenvironmental influences that increase the risks of type 2 diabetes.Although there are numerous animal models  available for the study oftype 2 diabetes –4, thepβ celldeath through alkylation of DNA . To investigate differences in insulinsensitivity, we performed an ITT at different time points (Our data showed that 45 mg/kg STZ injection(DM4) had the highest successful rate and higher FBG level. Therefore, IPGTTand ITT were carried out in DM4 andcontrol groups to measure glucose tolerance and insulinsensitivity. As shown in e points . Insuline points . All theP < .01).P < .01]. Meanwhile at 8 weeks after STZ injection  had the higher successful rate and higher FBG level, IPGTT and ITT wereperformed further in this group at 4 weeks  and 8 wenjection , the are 4 weeks  and 8 we 4 weeks  STZ inje 4 weeks . However 4 weeks .This reP < .05).According to the data shown above, 30 mg/kg STZ twice injection developeda diabetic rat model with insulin resistance and hyperglycemia. Therefore, we measuredthe body weight and food intake between this group and the control group. As shown in From the data shown in the second section, 30 mg/kg STZ twice injectionmight develop a better type 2 diabetic rat model. Therefore, in the third sectionwe investigated the stability of this experimental type 2 diabetic model.As shown in The biochemical parameter and ISI at the end of study were presented in P < .05]. In the treatment group, the glucose concentrationdeclined rapidly during ITT. Meanwhile, berberine administration significantlydecreased FBG, TC, and TG levels compared to diabetic model group (P < .05),the fasting insulin was changed but not significantly, ISI was higher than the modelgroup, and the result indicated that berberine improved insulin sensitivity andabnormal blood lipid. The berberine treatmentdecreased the body weight slightly but with no significant difference compared with HFD-STZ group feeding rats develop insulinresistance –7. At thThe purpose of the present study was to develop an animal model of type 2diabetes that would imitate the natural history and the metaboliccharacteristics of the human syndrome and be responsive to the pharmaceutical treatment. On the other hand, the goal of the presentstudy was to develop an animal model which is neither inherited nor geneticallyobese, and which is easily accessible, fairly economical, and with high successfulrate. The results demonstrated that we had achieved our goals.The primary attempts of the present study were to identify the dose ofSTZ that was low enough to develop type 2 diabetes models in HFD rats withhigher successful rate and without much insulin deficiency. The different dosesof STZ  were studied. Injection of STZ after 4 weeks of high-fat diet caused frank hyperglycemia with 100% successrate, which is consistent with literature reports [β cells when they were given a singlehigh-dose injection of STZ ; interestingly, multiplelow-dose injections of STZ  could induce a gradual, autoimmunedestruction of β cells [β-cell. This might happen in decompensated phase of type 2 diabetes. High-fatdiet has been extensively used to develop insulin resistance [Our second study attempted to find if multiple low dosesof STZ could achieve our goal (high successful rate and performance of type 2 diabetes). Multiple low-dose STZ injection to induce diabetes and its complication model have been reported in many studies –14. Mult β cells . Wrightsistance . Therefoβ might be a cofactorof berberine in achieving its anti-inflammation and insulin-resistance-improvingeffects [α inliver [α isa positive regulator of PEPCK so an increase of HNF-4α would result inincreased gluconeogenesis. Hence, we administrated berberine asantidiabetic drug in the presentstudy, and our result also demonstratedthat berberine treatment could decrease the insulin resistance and improveimpaired glucose tolerance. Meanwhile, we also found that berberine could correctlipid metabolism disorders, which indicated that the treatment of berberinemight play an important role in diabetic complication. Altogether, the diabeticmodel we developed was suitable to investigate not only the pathogenesis but also the pharmaceutical selection.Meanwhile, the current study considered the incidence of diabetes in the group.This method will successfully produce type 2 diabetic rat models, as well asprovide the enough number of diabetic rats once which will make theinvestigation more convincing.Furthermore, the diabetic model we developed produces hyperglycemia around 14.5 mmol/L, whichis reasonable to be treated by therapeutic compound as practiced clinically. Therapeutically,it is difficult to reduce elevated blood glucose except for administration ofinsulin. Berberine, the major active constituent of Rhizoma coptidis, is used clinicallyin the treatment of diarrhea as an antimicrobial agent. Early as 1986, investigatorsfrom china have started to report the hypoglycemic effect of berberine. In 1999,Yuan reported that berberine exerted beneficial effect on the treatment ofdiabetes clinically; other investigators also proved its role in the treatmentof type 2 diabetes in clinic –18. The effects . The latinliver . HNF-4α Conclusively, our study demonstratesthat a combination of HFD and multiple low doses of STZ injection could beeffectively used to generate a rat model that mimics the natural history andmetabolic characteristics of type 2 diabetes in humans. It was also useful inevaluating the effect of therapeutic compounds on the treatment of type 2 diabetes.


Sarcopenia is a major public health problem in industrialized nations, placing an increasing burden on public healthcare systems because the loss of skeletal muscle mass and strength that characterizes this affection increases the dependence and the risk of injury caused by sudden falls in elderly people. Albeit exercise and caloric restriction improve sarcopenia-associated decline of the muscular performances, a more suitable and focused pharmacological treatment is still lacking.Ginkgo biloba extract used in chronic age-dependent neurological disorders, on the function of the soleus muscle in aged rats. EGb 761 induced a gain in muscular mass that was associated with an improvement of the muscular performances as assessed by biochemical and electrophysiological tests. DNA microarray analysis shows that these modifications are accompanied by the transcriptional reprogramming of genes related to myogenesis through the TGFβ signaling pathway and to energy production via fatty acids and glucose oxidation. EGb 761 restored a more juvenile gene expression pattern by regenerating the aged muscle and reversing the age-related metabolic shift from lipids to glucose utilization.In order to evaluate such a possible treatment, we investigated the effects of EGb 761, a Thus, EGb 761 may represent a novel treatment for sarcopenia both more manageable and less cumbersome than exercise and caloric restriction. Sarcopenia, a term coined by Rosenberg Ginkgo biloba extract, which has been shown to be an effective treatment in chronic age-dependent neurological disorders such as, for example, Alzheimer's disease soleus muscle isometric contractile force. These effects are associated, as shown by DNA microarray analysis, with the restoration of a more juvenile transcriptional activity of genes relevant for regeneration and energy production processes in the soleus of aged rats. This is the first time, to our knowledge, that EGb 761 has been shown to benefit the aged muscle in mammals suggesting that it may represent a novel treatment for sarcopenia.Muscle wasting and weakness associated with sarcopenia may benefit from drugs that target neurodegenerative diseases, since sarcopenia shares several features with different age-related neuromuscular disorders At the beginning of the experiment, 22 month-old rats were randomly separated in two groups and were weighted at regular intervals. The first group, Aged Control (AC), did not receive any treatment, while the second group, Aged Treated (AT), received EGb 761 in the drinking water. After 5 weeks of this regimen, the mean body weight of the AC group was increased by 2.2% while the rats in the AT group lost 4% (p = 0.02) of their initial body weight, indicating that EGb 761 counteracts the weight gain naturally occurring in aged laboratory rats .soleus muscle, a slow twitch muscle (type I fiber) with predominant oxidative metabolism. The animals were euthanized and the soleus muscles dissected and weighted. The soleus mean weight was increased by 6.5% in the AT compared to the AC group of rats. Moreover, the ratio of the soleus weight reported to the total body weight was increased by 13%  levels in AT rats (371±223 U/L), compared to AC rats (737±178 U/L). Interestingly, the CK value obtained in the AT rats was closely similar to the value observed in a group of young (Y) 4 month-old rats (370±53 U/L). Since CK serum levels act as a peripheral marker of muscle damage, these findings further suggest that EGb 761 has a protective effect against the damages that affect the aging muscle. In order to ascertain whether this protective effect was also associated with increased muscular performances, we compared the contractile force of the soleus muscles of AC and AT rats, either during a single intermittent isometric twitch or during tetanic contractions, evoked by direct muscle stimulation and through nerve stimulation  revealed that AT  muscles . Also, a muscles . Taken tsoleus muscle by DNA microarray analysis. The results already obtained point out to some precise phenomena based on regeneration or energy production amelioration for the improvement of muscular functions elicited by EGb 761. Therefore, the analysis of DNA microarray experiments investigating molecular events associated with aging and with EGb 761 treatment on global gene transcription was focused on genes implicated in these processes on a limited number of rats for each group.Thereafter, in order to gain insight on the molecular mechanisms underlying these anatomical, biochemical and physiological modifications elicited by EGb 761, we analyzed the global gene expression in the rat The DNA microarray analysis identified a total of 1518 genes, out of 9906, modulated either by aging or EGb 761 or both. Aging affected the expression of 1015 genes, 2/3 of them, for a total of 626, were down-regulated, while the remaining 389 were up-regulated, as assessed by comparing the old sarcopenic (AC) versus the young group (Y) of rats . EGb 761The microarray data was further investigated using hierarchical clustering analysis based on the overall similarity of the variations in the genes expression patterns. Permutation analysis was conducted on the three different groups of rats altogether. About 2/3 of the genes modulated by EGb 761 or Aging showed more than 30% of expression variability in each of the 3 groups. These genes were classified as non exploitable (NE) and were not used for the clustering analysis see  Fig. 3)Fig. 3). Ginkgo biloba extract on aging since these genes represent almost 60% (115 out of 198) of the genes regulated by EGb 761.Thus, according to the results obtained by clustering analysis, EGb 761 modified the expression of 30% (115 out of 378) of the genes affected by aging. This result stresses the impact of the soleus muscle, including those used for the microarray.In order to validate the DNA microarray results before further analysis, we analyzed the m-RNA expression levels of 13 selected genes having different levels of expression by real-time quantitative RT-PCR (qPCR) in the Acvr1, AdipoQ, Bambi, Col5A, Fasn, Fst and Gmfg, had a similar pattern of variation by DNA microarray and qPCR analysis both when comparing the young with the aged group of rats as well as when comparing the aged with the aged-treated group of rats. Among the remaining genes, Cd36, Id1 and Ucp3, displayed a similar pattern when comparing the young with the aged group of rats and Areg, Id3 and Tgf-β, when comparing the aged with the aged-treated group of rats  and transforming growth factor β2 (Tgfb2), which are ubiquitous factors that intervene also in the development and regeneration of the skeletal, nervous and connective tissues. Moreover, the EGb 761 treatment was also associated with the up-regulation by 50 to 70% of genes specifically implicated in skeletal muscle development such as Follistatin (Fst), Follistatin related protein (Fstl1), BMP and activin membrane-bound inhibitor (Bambi), α2-macroglobulin (A2m), Activin receptor type I (Acvr1), embryonic Myosin heavy chain (Myh3) and Ryanodine receptor 3 (Ryr3). Since several of these genes participates also in the TGFβ signaling pathway, which plays a relevant role in the muscle satellite cell proliferation and differentiation We classified all the genes belonging to clusters A and B according to their biological functions, using the Ingenuity pathway analysis software and the gene ontology database. The most relevant of these functions, according to the number of genes modified both by aging and EGb 761 treatment, are related to cell cycle, cell death, growth and proliferation, as well as metabolism . Among tFasn), responsible for fatty acid synthesis, the Acetyl-Coenzyme A acyltransferase2 (Acaa2), involved in cholesterol and lipoprotein synthesis and fatty acid elongation, the Hexokinase 1 (Hk1) and the Phosphofructokinase 1 (Pfk1), both implicated in glucose metabolism. The treatment induced as well the up-regulation of the transcripts for the Pyruvate dehydrogenase kinase 4 (Pdk4) and the Fatty acid translocase (Cd36) involved in lipid metabolism and transport, respectively, and the Uncoupling proteins 1 and 3 (Ucp1 and Ucp3), relevant for energy production . Moreover, according to our results, others transcripts such as Bambi, Fstl1 and A2m, all inhibitors of BMPs and Activin Fasn, Acaa2, Cd36, Hk1 and Pdk4, coding for key mitochondrial enzymes associated to lipid and glucose metabolism and involved in Krebs cycle, β2-oxidation, fatty acid transport and oxidative stress  gives another example of how EGb 761 may modulate transcripts that are relevant for muscle strength. Tnc, in the adult muscles, is an extracellular matrix (ECM) molecule that localizes in the tendon along with other components of the ECM such as the collagen I and III, playing an important role in the force transmission and tissue structure maintenance. The collagen constitutes 60–85% of the tendon dry weight and is predominantly type I (∼60%) arranged in tensile-resistant fibers, types III (≤10%), IV (∼2%) The two-fold increase of Tenascin C  that do not exist in other organisms and are extremely difficult to synthesize. Thus, the natural extract of such a living fossil, shaped by natural selection during several millions of years, may contain several unique molecules that are less disruptive than synthetic products and better suited for fitting with biological systems In conclusion, EGb 761 may represent a novel therapeutic intervention, endowed with a rejuvenation effect mediated by transcriptional reprogramming of a majority of genes implicated in aging, and with several advantages over CR and exercise to prevent sarcopenia in elderly people. Among these advantages there is a more easily implementation and therefore a better compliance with the treatment. Moreover, the effects of EGb 761 are mediated both by the restoration of the expression of genes that are pathologically down-regulated by aging and the boosting of the expression of genes that are physiologically up-regulated to counteract the effects of the aging process. This is another advantage over exercise and CR whose action is limited to slowing down the muscular ageing progression. It is also noteworthy that this effect may be unique to All animal procedures were performed under authorizations of the “Direction des Services Vétérinaires de Paris” of the “Prefecture de Police de Paris” and strictly followed the guidelines for the ethical treatment of the animals set forth by French law (decree N° 87-848 dated 11/19/1987 and modified by the decree N° 2001-464 dated 5/29/2001). The study was conducted with male Wistar rats on a group of young, 4-months old, rats (Young group = Y), and two groups of aged, 23-month old, rats. The aged rats either received (Aged Treated group = AT) or did not receive (Aged Control group = AC) EGb 761 treatment.Ginkgo Biloba extract (EGb 761), provided by IPSEN laboratories (France), was administered in the water at a dose of 75 mg/Kg body weight for two months. During the 60 days of the experiment, the animals were weighed each week and the volume of drinking solution was measured. The AC and AT groups were sacrificed at 23 months of age, young rats were sacrificed at 4 months.The Blood samples were taken from the hearts of anaesthetized rats, immediately before sacrifice. Activities of serum CK were determined using a Biomerieux kit (enzyline CK NAC optimized 10).Rats were anesthetized with Isoflurane  inhalation before being sacrificed by section of the carotid arteries.Soleus muscles with their attached nerves were carefully removed, mounted in a silicone-lined bath filled with Krebs-Ringer solution continuously perfused with 95% O2 and 5% CO2 throughout the experiment. The Krebs-Ringer solution was maintained at 22±0.5°C instead of 37°C in order to optimize the experimental conditions, since at a temperature of 37°C the risk of nerve conduction failure is greatly increased. One of soleus tendons was securely anchored onto the silicone-coated bath, the other tendon was attached to an FT03 isometric force transducer .via a suction microelectrode, with supramaximal current pulses of 0.15 ms duration, at frequencies indicated in the text, or by direct electrical stimulation. The resting tension was adjusted for each preparation investigated with a mobile micrometer stage  in order to obtain maximal contractile responses. Signals from the isometric transducer were amplified, collected, and digitized with the aid of a computer equipped with a DT2821 analogue to digital interface board , as previously reported /P/<0.05.Muscle twitches and tetanic contractions were evoked either by stimulating the motor nerve soleus muscles from each animal were excised, snap-frozen in liquid nitrogen and stored at −80°C.For microarrays and qPCR experiments the 260, and the quality was evaluated on nanochips with the Agilent 2100 Bioanalyzer .Total RNA was isolated from an aliquot of muscle thawed on ice; and extraction was performed by a silica gel-based purification method . For qPCR, total RNA was subjected to DNAse treatment (Qiagen) 30 min at room temperature. Total RNA yield was measured by the ODsoleus muscle was hybridized in duplicate . Thus 4 microarrays were utilized for each group of rats.Gene expression levels were measured by using rat oligonucleotide arrays as described previously After hybridization and washing, the slides were scanned using a Genepix 4000B scanner  and Genepix software. The scanned image files were analyzed using CodeLink expression software, version 4.0 .CodeLink software was used to normalize the raw hybridization signal. The threshold of detection was calculated using the normalized signal intensity of the 100 negative controls, spots with lower signal intensities were termed “absent”.ESTs were analyzed by BLAST research on the NCBI nucleotides databases. The clusters were generated using Pearson correlation. Statistical comparison and filtering were performed using Genespring software 7.0 (Agilent).P value of ≤0.05 in the Welch ANOVA parametric test, using a multiple test correction . Moreover, to avoid false positive genes, in particular for genes with small variation between groups, we removed from the two lists above genes with variability ≥30% inside each of the three groups , those genes were classified as non exploitable (NE). Thus, a gene was considered for the clustering analysis only if it met the above criteria in all pairwise comparisons and if the detected signal was above the background for at least one of the compared groups, thereby carrying a statistically significant absolute call ‘present’ or ‘marginal’ in all samples.A first selection of genes was performed by pair-wise comparisons between AT and AC rats (Egb761 effect) or Y and AT rats (Aging effect). Each sample from one group was compared with each sample from the other group. This permutation analysis comparing 3 different group allows for strengthening the relevance of the findings when a given differences is found across different groups. To identify significant changes, the following criteria were used: 1) p-value less than 0.05; 2) a fold change of 1.3 was used to define a set of significantly up- and down regulated genes; 3) the over 1.3 fold change should be reproducible across all comparisons. Therefore a given mRNA transcript was considered as differentially expressed in the comparison of any two samples if the difference gave a The qPCRs were performed as described previously Table S1(3.03 MB PDF)Click here for additional data file.Table S2(2.19 MB PDF)Click here for additional data file.Table S3(1.10 MB PDF)Click here for additional data file.Table S4(0.85 MB PDF)Click here for additional data file.Table S5(1.12 MB PDF)Click here for additional data file.Table S6(1.39 MB PDF)Click here for additional data file.