Applications of gene sequence polymorphisms in evolutionary genetic studies of Atlantic salmon (Salmo salar) and other teleost fish species

Evolutionary genetics incorporates traditional population genetics and studies of the origins of genetic variation by mutation and recombination, and the molecular evolution of genomes. Among the primary forces that have potential to affect the genetic variation within and among populations, including those that may lead to adaptation and speciation, are genetic drift, gene flow, mutations and natural selection. The main challenges in knowing the genetic basis of evolutionary changes is to distinguish the adaptive selection forces that cause existent DNA sequence variants and also to identify the nucleotide differences responsible for the observed phenotypic variation. To understand the effects of various forces, interpretation of gene sequence variation has been the principal basis of many evolutionary genetic studies. The main aim of this thesis was to assess different forms of teleost gene sequence polymorphisms in evolutionary genetic studies of Atlantic salmon (Salmo salar) and other species. Firstly, the level of Darwinian adaptive evolution affected coding regions of the growth hormone (GH) gene during the teleost evolution was investigated based on the sequence data existing in public databases. Secondly, a target gene approach was used to identify within population variation in the growth hormone 1 (GH1) gene in salmon. Then, a new strategy for single nucleotide polymorphisms (SNPs) discovery in salmonid fishes was introduced, and, finally, the usefulness of a limited number of SNP markers as molecular tools in several applications of population genetics in Atlantic salmon was assessed. This thesis showed that the gene sequences in databases can be utilized to perform comparative studies of molecular evolution, and some putative evidence of the existence of Darwinian selection during the teleost GH evolution was presented. In addition, existent sequence data was exploited to investigate GH1 gene variation within Atlantic salmon populations throughout its range. Purifying selection is suggested to be the predominant evolutionary force controlling the genetic variation of this gene in salmon, and some support for gene flow between continents was also observed. The novel approach to SNP discovery in species with duplicated genome fragments introduced here proved to be an effective method, and this may have several applications in evolutionary genetics with different species - e.g. when developing gene-targeted markers to investigate quantitative genetic variation. The thesis also demonstrated that only a few SNPs performed highly similar signals in some of the population genetic analyses when compared with the microsatellite markers. This may have useful applications when estimating genetic diversity in genes having a potential role in ecological and conservation issues, or when using hard biological samples in genetic studies as SNPs can be applied with relatively highly degraded DNA.

Oral health and menopause

Hormone therapy (HT) is widely used to relieve climacteric symptoms in order to increase the well-being of the women. The benefits as well as side-effects of HT are well documented. The principal menopausal oral symptoms are dry mouth (DM) and sensation of painful mouth (PM) due to various causes. Profile studies have indicated that HT users are more health-conscious than non-users. The hypothesis of the present study was that there are differences in oral health between woman using HT and those not using HT. A questionnaire study of 3173 women of menopausal age (50-58 years old) was done to investigate the prevalence of self-assessed sensations of PM and DM. Of those women participating in the questionnaire study, a random sample of 400 (200 using, 200 not using HT) was examined clinically in a 2-year follow-up study. Oral status was recorded according to WHO methods using DMFT and CPITN indices. The saliva flows were measured, salivary total protein, albumin and immunoglobulin concentrations and selected periodontal micro-organisms were analysed, and panoramic tomography of the jaws was taken. The patients filled in a structured questionnaire on their systemic health, medication and health habits. According to our questionnaire study there was no significant difference in the occurrence of self- assessed PM or DM between the HT users and non-users. According to logistic regression analyses, climacteric complaints significantly correlated with the occurrence of PM (p=0.000) and DM (p=0.000) irrespective of the use of HT, indicating that PM and DM are associated with climacteric symptoms in general. There was no difference between the groups in DMFT index values at follow up. The number of filled teeth (FT) showed a significant (p<0.05) increase in the HT group at follow-up. Periodontitis was diagnosed in 79% of HT users at baseline and in 71% at the follow-up. The values for non-HT users were 80% vs. 76%, respectively (Ns.). The mean numbers of ≥ 6 mm deep periodontal pockets were 0.9 ± 1.7 at baseline vs. 1.1 ± 2.1 two years later in the HT group, and 1.0 ± 1.7 vs. 1.2 ± 1.9, respectively, in the non-HT group. In a large Finnish national health survey, the prevalence of peridontitis of women of this age group was lower, but the prevalence of severe periodontitis seemed to be higher than in our study. Salivary albumin, IgG and IgM concentrations decreased in the HT group during the 2-year follow up (p<0.05), possibly indicating an improvement in epithelial integrity. No difference was found in any other salivary parameters or in the prevalence of the periodontal bacteria between or within the groups. In conclusion, the present findings showed that 50 to 58 year old women living in Helsinki have fairly good oral and dental health. The occurrence of PM and DM seemed to be associated with climacteric symptoms in general, and the use of HT did not affect the oral symptoms studied.

Oral health, smoking and adolescence

The present cross-sectional study examined the effect of smoking on oral health in a birth cohort of 15 to 16-year-old Finnish adolescents. The hypothesis was that oral health parameters were poorer among smoking than non-smoking subjects and that a tobacco intervention program could be effective among the adolescents. The study was conducted in the Kotka Health Center, Kotka, Finland. Altogether 501 out of 545 subjects (15- to 16-year-old boys [n = 258] and girls [n = 243]) were clinically examined in 2004 and 2005. The sample frame was a birth cohort of all subjects in 1989 and 1990, living in Kotka. A structured questionnaire was also filled in by the participants to record their general health and health habits, such as smoking, tooth brushing, and medication used. The participants were classified into nonsmokers, current smokers, and former smokers. Subgingival pooled plaque samples were taken and stimulated salivary samples were also collected. The subjects were asked from which of seven professional groups (doctors, school nurses, dental nurses, general nurses, dentists, teachers and media professionals) they would prefer to receive information about tobacco. The two most popular groups they picked up were dentists and school nurses. Current smokers (n=127) were then randomly assigned into three groups: the dentist group (n =44), the school-nurse group (n =42), and the control group (n =39). The intervention was based on a national recommendation of evidence based guidelines by The Finnish Medical Society Duodecim ( 5A counseling system). Two months after the intervention, a second questionnaire was sent to the smokers in the intervention groups. Smoking cessation, smoking quantity per week, and self-rated addiction for smoking (SRA) were recorded. The results were analyzed using the R-statistical program. The results showed that 15% of the subjects had periodontitis. Smokers (25%) had more periodontitis than non-smokers (66%) (p < 0.001). Smoking boys (24%) also had more caries lesions than non-smokers (69%) (p < 0.001), and they brushed their teeth less frequently than non-smokers. Smoking significantly impaired periodontal health of the subjects, even when the confounding effects of plaque and tooth brushing were adjusted. Smoking pack-years, intensified the effects of smoking. Periodontal bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia and Treponema denticola were more frequently detected among the smokers than non-smokers, especially among smoking girls. Smoking significantly decreased the values of both the salivary periodontal biomarkers MMP-8 (p=0.04) and PMN elastase (p=0.02) in boys. The effect was strengthened by pack years of smoking (MMP-8 p=0.04; elastase p0.01). Of those who participated in the intervention, 19 % quit smoking. The key factors associated with smoking cessation were best friend`s influence, nicotine dependence and diurnal type. When the best friend was not a smoker, the risk ratio (RR) of quit smoking after the intervention was 7.0 (Cl 95% 4.6 10.7). Of the diurnal types, the morning people seemed to be more likely to quit (RR 2.2 [Cl 95% 1.4 3.6]). Nicotine dependence also elicited an opposite effect: those who scored between 3 and 5 dependence scores were less likely to quit. In conclusion, smoking appears to be a major etiological risk factor for oral health. However, the early signs of periodontal disease were mild in the subjects studied. Based on the opinions of the adolescent s, dental professionals may have a key position in their smoking cessation. The harmful effects of smoking on oral health could be used in counselling. Best friend`s influence, nicotine dependence and diurnal type, all factors associated with smoking cessation, should be taken more carefully into account in the prevention programs for adolescents.

Oral health and kidney disease with emphasis on diabetic nephropathy

Chronic kidney disease (CKD) is a worldwide health problem, with adverse outcomes of cardiovascular disease and premature death. The ageing of populations along with the growing prevalence of chronic diseases such as diabetes and hypertension is leading to worldwide increase in the number of CKD patients. It has become evident that inflammation plays an important role in the pathogenesis of atherosclerosis complications. CKD patients also have an increased risk of atherosclerosis complications (including myocardial infarction, sudden death to cardiac arrhythmia, cerebrovascular accidents, and peripheral vascular disease). In line with this, oral and dental problems can be an important source of systemic inflammation. A decline in oral health may potentially act as an early marker of systemic disease progression. This series of studies examined oral health of CKD patients from predialysis, to dialysis and kidney transplantation in a 10-year follow-up study and in a cross-sectional study of predialysis CKD patients. Patients had clinical and radiographic oral and dental examination, resting and stimulated saliva flow rates were measured, whilst the biochemical and microbiological composition of saliva was analyzed. Lifestyle and oral symptoms were recorded using a questionnaire, and blood parameters were collected from the hospital records. The hypothesis was that the oral health status, symptoms, sensations, salivary flow rates and salivary composition vary in different renal failure stages and depend on the etiology of the kidney disease. No statistically significant difference were seen in the longitudinal study in the clinical parameters. However, some saliva parameters after renal transplantation were significantly improved compared to levels at the predialysis stage. The urea concentration of saliva was high in all stages. The salivary and plasma urea concentrations followed a similar trend, showing the lowest values in kidney transplant patients. Levels of immunoglobulin (Ig) A, G and M all decreased significantly after kidney transplantation. Increased concentrations of IgA, IgG and IgM may reflect disintegration of the oral epithelium and are usually markers of poor general oral condition. In the cross-sectional investigation of predialysis CKD patients we compared oral health findings of diabetic nephropathy patients to those with other kidney disease than diabetes. The results showed eg. more dental caries and lower stimulated salivary flow rates in the diabetic patients. HbA1C values of the diabetic patients were significantly higher than those in the other kidney disease group. A statistically significant difference was observed in the number of drugs used daily in the diabetic nephropathy group than in the other kidney disease group. In the logistic regression analyses, age was the principal explanatory factor for high salivary total protein concentration, and for low unstimulated salivary flow. Poor dental health, severity of periodontal disease seemed to be an explanatory factor for high salivary albumin concentrations. Salivary urea levels were significantly linked with diabetic nephropathy and with serum urea concentrations. Contrary to our expectation, however, diabetic nephropathy did not seem to affect periodontal health more severely than the other kidney diseases. Although diabetes is known to associate with xerostomia and other oral symptoms, it did not seem to increase the prevalence of oral discomfort. In summary, this series of studies has provided new information regarding the oral health of CKD patients. As expected, the commencement of renal disease reflects in oral symptoms and signs. Diabetic nephropathy, in particular, appears to impart a requirement for special attention in the oral health care of patients suffering from this disease.

Análise do processo sinérgico da deglutição em pacientes portadores de bronquiectasia atendidos no Hospital Universitário Pedro Ernesto

A respiração e a deglutição são vitais para o homem. Enquanto a primeira diz respeito a um ato primitivo da vida, a hematose, a segunda trata da manutenção da vida, oferecendo a energia necessária, nutrindo e hidratando, perpassando pelo prazer alimentar, ato tão importante na sociedade contemporânea. A relação funcional entre essas funções ainda não foi totalmente elucidada, porém é crescente o interesse e o número de estudos sobre esta temática. Considerando que a deglutição eficiente tem como pressuposto a capacidade de proteger via aérea inferior, a alteração de deglutição primária ou secundária a um dano pulmonar pode trazer repercussões severas para a integridade do sistema respiratório. O objetivo desse estudo é analisar o processo sinérgico da deglutição em portadores de bronquiectasia, a fim de verificar se há alteração na fisiologia da deglutição e caracterizá-la, assim como, identificar se há correspondência entre alteração da função pulmonar e alteração da deglutição.Para tal foram selecionados randomicamente 30 pacientes na faixa etária de 18 a 65 anos, atendidos no ambulatório de bronquiectasia do HUPE. Destes, 26 indivíduos responderam a um questionário dirigido sobre hábitos alimentares e possíveis dificuldades de alimentação; foram submetidos à avaliação clínica da deglutição; 22 fizeram espirometria no setor de prova de função pulmonar no HUPE e 17 avaliação videofluoroscópica da deglutição. Dos 26 indivíduos estudados 10 eram homens e 16 mulheres, com média de idade de 46,3 anos. Na avaliação clínica da deglutição observaram-se alterações estruturais e funcionais em todos os indivíduos estudados. As principais alterações estruturais detectadas referem-se e a dinâmica laríngea; enquanto na avaliação funcional detectou-se alterações referentes à ejeção oral, dinâmica hiolaríngea, trânsito faríngeo e presença de deglutições múltiplas. Com relação à espirometria 06 indivíduos apresentaram distúrbio obstrutivo leve; 04 distúrbio obstrutivo moderado e 09 distúrbio obstrutivo acentuado. A videofluoroscopia da deglutição corroborou os achados da avaliação clínica da deglutição e evidenciou episódios de penetração e aspiração laríngea Pode-se concluir que: (1) a avaliação clínica da deglutição associada à avaliação videofluoroscópica são métodos eficientes para a análise do processo sinérgico da deglutição; (2) identificou-se alteração do processo sinérgico da deglutição, nos indivíduos avaliados; (3) a ausculta cervical isoladamente, não demonstrou ser um método eficiente para predizer aspiração e/ou penetração laringotraqueal; (4) houve correspondência entre os resultados da avaliação clínica funcional da deglutição e videofluoroscópica, exceto quanto a presença de penetração e/ou aspiração e quanto a capacidade de avaliar a ejeção oral; (5) não foi possível identificar se há correspondência entre alteração da função pulmonar e processo sinérgico da deglutição.

Genetic variation between outer-coastal and fjord populations of Pacific herring (Clupea pallasii) in the eastern Gulf of Alaska

Pacific herring (Clupea pallasii) from the Gulf of Alaska were screened for temporal and spatial genetic variation with 15 microsatellite loci. Thirteen collections were examined in this study: 11 from Southeast Alaska and 2 from Prince William Sound, Alaska. Although FST values were low, a neighbor-joining tree based on genetic distance, homogeneity, and FST values revealed that collectively, the Berners Bay and Lynn Canal (interior) collections were genetically distinct from Sitka Sound and Prince of Wales Island (outer-coastal) collections. Temporal genetic variation within regions (among three years of Berners Bay spawners and between the two Sitka Sound spawners) was zero, whereas 0.05% was attributable to genetic variation between Berners Bay and Sitka Sound. This divergence may be attributable to environmental differences between interior archipelago waters and outer-coast habitats, such as differences in temperature and salinity. Early spring collections of nonspawning Lynn Canal herring were nearly genetically identical to collections of spawning herring in Berners Bay two months later—an indication that Berners Bay spawners over-winter in Lynn Canal. Southeast Alaskan herring (collectively) were significantly different from those in Prince William Sound. This study illustrates that adequate sample size is needed to detect variation in pelagic fish species with a large effective population size, and microsatellite markers may be useful in detecting low-level genetic divergence in Pacific herring in the Gulf of Alaska.

Genetic enhancement and conservation of aquatic biodiversity in Africa

There is a pressing need to enhance fish production in Africa through improved farm management and the use of improved fish breeds and/or alien species in aquaculture while at the same time conserve the aquatic genetic diversity. This paper presents the outcome of the Expert Consultation on Biosafety and Environmental Impact of Genetic Enhancement and Introduction of Improved Tilapia Strains/Alien Species in Africa held in Nairobi, Kenya on 20-23 February 2002. The main topics discussed were status of aquaculture in Africa and the role of genetic enhancement; potential benefits and risks involved in introduction of genetically improved strains and/or alien species with specific reference to tilapias; existing policies and legislation for the conservation of biodiversity, their strengths and weaknesses; capacity for undertaking genetic enhancement research and implementation of policies for the conservation of aquatic biodiversity.

The genetic structure and phylogenetics of pikeperch (Sander lucioperca) Anzali and Amirkolaye wetlands and perch (Perca fluviatilis) populations in Aras Dam and south-west of the Caspian Sea

The genetic structure of pikeperch (Sander lucioperca) and perch (Perca fluviatilis) populations was studied using microsatellite technique. A total of 207 specimens of adult pikeperch were collected from Aras dam (57 specimens), Anzali wetland (50 specimens), Talesh (50 specimens) and Chaboksar (50 specimens) coasts. Also a total of 158 specimens of adult perch were collected from Anzali (Abkenar (50 specimens)and Hendekhale(48 specimens)) and Amirkolaye(60 specimens) wetlands. About 2 g of each specimen's dorsal fin was removed, stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using ammonium-acetate method. The quality and quantity of DNA was assessed using 1% agarose gel electrophoresis. Polymerase Chain Reaction (PCR) was conducted on the target DNA using 15 pairs of microsatellite primers. PCR products were electrophoresed on poly acryl amide gels (6%) that were stained that were stained using silver nitrate. DNA bands were analyzed with BioCapt software. Allele count and frequency, genetic diversity, expected and observed heterozygosity , allele number and the effective allele number, genetic similarity and genetic distance, Fst, Rst, Hardy Weinberg Equilibrium based on X2 and Analysis of Molecular Variance (AMOVA) at 10% confidence level was calculated using the Gene Alex software. Dendogram for genetic distances and identities were calculated using TFPGA program for any level of hierarchy. The results for P. fluviatilis showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 4.1±1.1 and mean observed and expected heterozygosity was 0.56±0.12 and 0.58±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.095) with Nm=2.37 was observed between Hendekhale and Amirkolaye and the lowest Fst (0.004) with Nm=59.31 was observed between Abkenar and Hendekhale. According to AMOVA Significant difference (P<0.05) was observed between recorded Rst in the studied regions in Anzali and Amirkolaye lagoons. In another words there are two distinct populations of this species in Anzali and Amirkolaye lagoons. The highest genetic distance (0.181) and lowest genetic resemblance (0.834) were observed between specimens from Hendekhale and Amirkolaye and the lowest genetic distance (0.099) and highest genetic 176 resemblance (0.981) were observed between specimens from Abkenar and Hendekhale. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Anzali and Amirkolaye wetlands have the same ancestor. On the other hand there is no noticeable genetic distance between the specimens of these two regions. Also the results for S. lucioperca showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 3.0±0.6 and mean observed and expected heterozygosity was 0.52±0.21 and 0.50±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.093) with Nm=2.43 was observed between Aras dam and Anzali wetland and the lowest Fst (0.022) with Nm=11.27 was observed between Talesh and Chaboksar coasts. Significant differences (P<0.05) were observed between recorded Rst in the studied regions exept for Talesh and Chaboksar Coasts. In another words there are three distinct populations of this species in Caspian sea, Anzali wetland and Aras dam. Highest genetic distance (0.110) and lowest genetic resemblance (0.896) were observed between specimens from Aras dam and Anzali wetland and the lowest genetic distance (0.034) and highest genetic resemblance (0.966) were observed between specimens from Talesh and Chaboksar coasts. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Talesh and Chaboksar coasts have the lowest genetic distance. On the other hand the main population of this species belongs to Anzali wetland. Phylogenetic relationship of these two species was inferred using mitochondrial cytochrome b gene sequencing. For this purpose 2 specimens of P. fluviatilis from Anzali wetland, 2 specimens of S. lucioperca from Aras dam and 2 specimens of S. lucioperca from Anzali wetland were sequenced and submitted in Gene Bank. These sequences were aligned with Clustal W. The phylogenic relationships were assessed with Mega 4. The results of evolutionary history studies of these species using Neighbor-Joining and Maximum Parsimony methods showed that the evolutionary origin of pikeperch in Aras Dam and Anzali wetland is common. On the other hand these two species had common ancestor in about 4 million years ago. Also different sequences of any region specimens are supposed as different haplotypes. 177 As a conclusion the results of this study showed that microsatellite and mtDNA sequencing methods respectively are effective in genetic structure and phylogenic studies of P. fluviatilis and S. lucioperca.

Genetic structure analysis of natural Sargassum muticum (Fucales, Phaeophyta) populations using RAPD and ISSR markers

Sargassum muticum is important in maintaining the structure and function of littoral ecosystems, and is used in aquaculture and alginate production, however, little is known about its population genetic attributes. In this study, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four populations of S. muticum and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China. The selected 24 RAPD primers and 19 ISSR primers amplified 164 loci and 122 loci, respectively. Estimates of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate level of genetic variations within each S. muticum population, and a high level of genetic differentiations were determined with pairwise unbiased genetic distance (D) and fixation index (F-ST ) among the populations. The Mantel test showed that two types of matrices of D and F-ST were highly correlated whether from RAPD (r = 0.9706, P = 0.009) or ISSR data (r = 0.9161, P = 0.009). Analysis of molecular variance (AMOVA) was conducted to apportion the variations among and within the S. muticum populations. It indicated that variations among populations were higher than those within populations, being 55.82% verse 44.18% by RAPD and 55.21% verse 44.79% by ISSR, respectively. Furthermore, the Mantel test suggested that genetic differentiations among populations were related to the geographical distances (r > 0.6), namely, conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. On the whole, the high genetic structuring among the four S. muticum populations along the distant locations was clearly indicated in RAPD and ISSR analyses (r > 0.9, P < 0.05) in our study.

Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers

Genetic variation of four populations of Sargassum thunbergii (Mert.) O. Kuntze and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China was studied with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. A total of 28 RAPD primers and 19 ISSR primers were amplified, showing 174 loci and 125 loci, respectively. Calculation of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate levels of genetic variations within each S. thunbergii population. High genetic differentiations were determined with pairwise Nei's unbiased genetic distance (D) and fixation index (F-ST) between the populations. The Mantel test showed that two types of matrices of D and FST were highly correlated, whether from RAPD or ISSR data, r=0.9310 (P = 0.008) and 0.9313 (P=0.009) respectively. Analysis of molecular variance (AMOVA) was used to apportion the variations between and within the S. thunbergii populations. It indicated that the variations among populations were higher than those within populations, being 57.57% versus 42.43% by RAPD and 59.52% versus 40.08% by ISSR, respectively. Furthermore, the Mantel test suggested that the genetic differentiations between the four populations were related to the geographical distances (r > 0.5), i.e., they conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. As a whole, the high genetic structuring between the four S. thunbergii populations along distant locations was clearly indicated in the RAPD and ISSR analyses (r > 0.8) in our study.

Plastid microsatellites for the study of genetic variability in the widespread Cephalanthera longifolia, C. damasonium and C. rubra (Neottieae, Orchidaceae), and cross-amplification in other Cephalanthera species

Plastid microsatellite loci developed for Cephalanthera longifolia were used to examine the level of genetic variation within and between populations of the three widespread Cephalanthera species (C. damasonium, C. longifolia and C. rubra). The most detailed sampling was in C. longifolia (42 localities from Ireland to China; 147 individuals). Eight haplotypes were detected. One was detected in the vast majority of individuals and occurred from Ireland to Iran. Three others were only found in Europe (Ireland to Italy, England to Italy and Austria to Croatia). Two were only found in the Middle East and two only in Asia. In C. damasonium, 21 individuals from 10 populations (England to Turkey) were sampled. Only one haplotype was detected. In C. rubra, 34 individuals from eight populations (England to Turkey) were sampled. Although it was not possible to amplify all loci for all samples of this species, nine haplotypes were detected. Short alleles for the trnS-trnG region found in two populations of C. rubra were characterized by sequencing and were caused by deletions of 26 and 30 base pairs. At this level of sampling, it appears that C. rubra shows the greatest genetic variability. Cephalanthera longifolia, C. rubra and C. damasonium have previously been characterized as outbreeding, outbreeding with facultative vegetative reproduction and inbreeding, respectively. Patterns of genetic variation here are discussed in the light of these reproductive system differences. The primers used in these three species of Cephalanthera were also demonstrated to amplify these loci in another five species (C. austiniae, C. calcarata, C. epipactoides, C. falcata and C. yunnanensis). Although it is sometimes treated as a synonym of C. damasonium, the single sample of C. yunnanensis from China had a markedly different haplotype from that found in C. damasonium. All three loci were successfully amplified in two achlorophyllous, myco-heterotrophic species, C. austinae and C. calcarata. © 2010 The Linnean Society of London.

An introduction to China's rail transport Part 2: Urban rail transit systems, highway transport and the reform of China's railways

In this paper, an introduction is provided to some of the components of China's transport system. The authors include the urban rail transit systems, the highway transport systems and its competition for China's railways and the reform of China's railway industry. This is the second of two papers on the situation of rail transport in China.

BELFAST nonagenarians; Nature or Nurture? Immunological, cardiovascular and genetic factors

Nonagenarians are the fastest growing sector of populations across Western European and the developed world. They are some of the oldest members of our societies and survivors of their generation and may help us understand how to age not only longer, but better.The Belfast Longevity Group enlisted the help of 500 community-living, mobile, mentally competent, 'elite' nonagenarians, as part of an ongoing study of ageing. We assessed some immunological, cardiovascular, nutritional and genetic factors and some aspects of their interaction in this group of 'oldest old'.Here we present some of the evidence related to genetic and nutritional factors which seem to be important for good quality ageing in nonagenarians from the Belfast Elderly Longitudinal Free-living Ageing STudy (BELFAST).

Cannabinoid Receptor influences Chromatin Remodelling in Mouse Spermatids by affecting Content of Transition Protein 2 mRNA and Histone Replacement

Marijuana smokers and animals treated with ?9-tetrahydrocannabinol, THC, the principal component of marijuana, show alterations of sperm morphology suggesting a role for cannabinoids in sperm differentiation and/or maturation. Since the cannabinoid receptor 1 (CNR1) activation appears to play a pivotal role in spermiogenesis, the developmental stage where DNA is remodeled, we hypothesized that CNR1 receptors might also influence chromatin quality in sperm. We used Cnr1 null mutant (Cnr1-/-) mice to study the possible role of endocannabinoids on sperm chromatin during spermiogenesis. We demonstrated that CNR1 activation regulated chromatin remodeling of spermatids by either increasing Tnp2 levels or enhancing histone displacement. Comparative analysis of WT, Cnr1+/- and Cnr1-/- animals suggested the possible occurrence of haploinsufficiency for Tnp2 turnover control by CNR1, while histone displacement was disrupted to a lesser extent. Further, flow cytometry analysis demonstrated that the genetic loss of Cnr1 decreased sperm chromatin quality and was associated with sperm DNA fragmentation. This damage increased during epididymal transit, from caput to cauda. Collectively, our results show that the expression/activity of CNR1 controls the physiological alterations of DNA structure during spermiogenic maturation and epididymal transit. Given the deleterious effects of sperm DNA damage on male fertility, we suggest that the reproductive function of marijuana users may also be impaired by deregulation of the endogenous endocannabinoid system.