Molecular,phytochemical and morphological characterization of the liverwort genus Radula in Portugal (mainland,Madeira, Azores)

Relationships of the eight species of the liverwort genus Radula occurring in Portugal (mainland, the Madeira and Azores archipelagos), including the Macaronesian endemics R. jonesii and R. wichurae, were evaluated based on molecular, phytochemical and morphological–anatomical data. Maximum parsimony and Bayesian analyses were performed with sequences from three plastid DNA markers (trnS _GGA-rps4 spacer, rps4 gene, trnL _UAA intron), volatile oil compounds, as well as qualitative morphological–anatomical characters. In addition, the molecular data were subjected to maximum likelihood analysis. The eight taxa, R. aquilegia, R. carringtonii, R. complanata, R. holtii, R. jonesii, R. lindenbergiana, R. nudicaulis and R. wichurae, can be clearly distinguished from each other, either by molecular data alone or by combination of characters from all three data sets. Radula aquilegia is monophyletic according to the molecular data, but shows considerable, yet undescribed intraspecific morphological and phytochemical variability. Recognition of R. complanata and R. lindenbergiana as separate species, previously based solely on the paroecious vs. dioecious sexual condition, is moderately supported by the molecular phylogenetic analyses and strongly supported by the phytochemical data. The Radula species, narrowly distributed in Macaronesia and Atlantic Europe, probably have two different origins. For Radula holtii and R. nudicaulis, connections with Radula species from the Neotropics are indicated. The other species, among them the two Macaronesian endemics, are closely related with the R. complanata/R. lindenbergiana complex, which is widely distributed in the northern hemisphere.     

Leptospira interrogans and Leptospira peptidoglycans induce the release of tumor necrosis factor a from human monocytes

Abstract Elevated plasma concentrations of the cytokine tumor necrosis factor α (TNFα) have been observed in patients affected by leptospirosis. In this study we found that a preparation of peptidoglycan of Leptospira interrogans , serovar copenhageni , was able to induce the release of TNFa from peripheral blood mononuclear cells. TNFa induction occurred in a dose dependent manner and was not affected by the endotoxin inhibitor polymixin B. This is the first report on induction of TNFa release by a peptidoglycan of spirochetes. Our findings are consistent with existing clinical data and provide a potential mechanism for TNFa production.     

Understanding differences between phylogenetic and pedigree-derived mtDNA mutation rate: a model using families from the Azores Islands (Portugal)

We analyzed the control region of the mitochondrial DNA (mtDNA)from maternally related individuals originating from the AzoresIslands (Portugal) in order to estimate the mutation rate ofmtDNA and to gain insights into the process by which a new mutationarises and segregates into heteroplasmy. Length and/or pointheteroplasmies were found at least in one individual of 72%of the studied families. Eleven new point substitutions werefound, all of them in heteroplasmy, from which five appear tobe somatic mutations and six can be considered germinal, evidencingthe high frequency of somatic mutations in mtDNA in healthyyoung individuals. Different values of the mutation rate accordingto different assumptions were estimated. When considering allthe germinal mutations, the value of the mutation rate obtainedis one of the highest reported so far in family studies. However,when corrected for gender (assuming that the mutations presentin men have the same evolutionary weight of somatic mutationsbecause they will inevitably be lost) and for the probabilityof intraindividual fixation, the value for the mutation rateobtained for HVRI and HVRII (0.2415 mutations/site/Myr) wasin the upper end of the values provided by phylogenetic estimations.These results indicate that the discrepancy, that has been reportedpreviously, between the human mtDNA mutation rates observedalong evolutionary timescales and the estimations obtained usingfamily pedigrees can be minimized when corrections for genderproportions in newborn individuals and for the probability ofintraindividual fixation are introduced. The analyses performedsupport the hypothesis that (1) in a constant, tight bottleneckgenetic drift alone can explain different patterns of heteroplasmysegregation and (2) in neutral conditions, the destiny of anew mutation is strictly related to the initial proportion ofthe new variant. Another important point arising from the dataobtained is that, even in the absence of a paternal contributionof mtDNA, recombination may occur between mtDNA molecules presentin an individual, which is only observable if it occurs betweenmtDNA types that differ at two or more positions.     

Characterization of Leptospira species isolated from soil collected in Japan

Leptospira were isolated from soil obtained from Hokkaido, the northernmost island, to Okinawa, the southernmost island, of Japan using sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5‐ fluorouracil. Fifty of 132 soil samples (37.9%) were culture‐positive. On the basis of 16S‐rDNA sequences, 12 of the isolated Leptospira were classified into a pathogenic species clade that is closely associated with L. alstonii and L. kmetyi. Nine isolates were classified as intermediate species and were found to be similar to L. licerasiae. Twenty‐seven isolates were classified as non‐pathogenic species, of which 23 were found to be related to L. wolbachii. Non‐pathogenic Leptospira are commonly distributed in environmental soil.     

问号钩体粘附侵袭相关基因特征分析

使用NCBI ,Swissprot/TrEMBL ,ProDom ,Pfam ,Tmpred ,SignalP ,ClustW等网络资源和软件 ,根据问号钩体黄疸出血型赖株粘附侵袭相关基因诠释结果 ,对mce ,invA ,mviN和atsE四个粘附侵袭相关基因编码蛋白的结构域、跨膜区域和信号肽等进行了详细分析 ,并使用Bioedit,Mega2软件进行氨基酸多重序列比较并绘制系统发生树。结果显示 ,mce和mviN为穿膜蛋白 ,invA和atsE为菌体内蛋白质 ;许多对哺乳动物和对植物致病的微生物具有mce ,invA ,mviN和atsE四个粘附侵袭相关基因 ,其表达的蛋白质在感染宿主过程中起重要作用 ,钩体的粘附侵袭相关蛋白与它们在一级结构上有较高相似性。据生物信息学结果推测 ,问号钩体黄疸出血型赖株粘附侵袭相关基因和钩体致病性间有密切关系 ,其编码蛋白在致病过程中可能起重要作用     

Genetic differences among the LPS biosynthetic loci of serovars of Leptospira interrogans and Leptospira borgpetersenii

The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar. Two groups of loci were delineated based on DNA hybridization and sequence analysis. Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis. Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci. These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37). Serovars Australis, Pomona and Autumnalis did not contain orf37. Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13. In the Group 2 loci, comparison of the genetic layout at the 5' end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity.     

Comparison of Leptospira species isolated from environmental water and soil in Japan

Leptospira was isolated from environmental water in central Japan using selective medium comprising five antibiotics, namely sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5‐fluorouracil. Of 100 water samples 57 (57%) were culture‐positive and 50 pure cultures were isolated. Of the 50 cultures isolated from water 48 were classified into a saprophytic clade on the basis of 16S ribosomal RNA gene sequences. However, it was previously reported that isolates from soil in Japan belonged to pathogenic, intermediate, and saprophytic clades, the current findings suggest less diversity of Leptospira species in environmental water than that in soil in Japan.     

Testing hierarchical levels of population sub-structuring: the Azores islands (Portugal) as a case study

The Azores archipelago (Portugal) is formed by nine islands whose relative positions define them as three geographical groups: Eastern (S. Miguel and Sta. Maria), Central (Terceira, Faial, Pico, Graciosa and S. Jorge) and Western (Flores and Corvo). Using the father's surname of 187,398 individuals living on the nine Azorean Islands, a population analysis based on inter-island relationship and hierarchical organization was conducted. The relation between islands was investigated using summary statistics, analysis of molecular variance (AMOVA) as well as graphical methods. When the values of heteronymy were contrasted with values of gene diversity based on haplogroup frequencies of the Y chromosome, it was possible to verify that Graciosa and Sta. Maria appeared to have the least diverse populations, and that Flores, despite its smaller population size and geographical isolation, has considerably higher levels of diversity. As for inter-island relationships, the difficulty of directly interpreting summary statistics values was evidenced. The AMOVA revealed that only 0.77% of the variation in surnames can be attributed to among-island variation, a value that, although small, can be considered significant. Application of Malécot's model revealed that geographic distance has an important impact in the genetic structure of the archipelago. Monmonier's maximum-difference algorithm demonstrated that the most isolated island of the archipelago appears to be Graciosa, followed by the islands of the Western group and by Sta. Maria. After integrating values of summary statistics with results from AMOVA and graphical methods, a more accurate genetic profile of the Azores, highly supported by genetic data, has emerged.     

Molecular and phenotypic characterization of Leptospira johnsonii sp. nov., Leptospira ellinghausenii sp. nov. and Leptospira ryugenii sp. nov. isolated from soil and water in Japan

In a previous study, 50 of 132 soil samples collected throughout Japan were found to be Leptospira‐positive. In the present study, three strains identified in the collected specimens, three, E8, E18 and YH101, were found to be divergent from previously described Leptospira species according to 16S ribosomal RNA gene sequence analysis. These three strains have a helical shape similar to that of typical Leptospira and were not re‐isolated from experimental mice inoculated with the cultured strains. Upon 16S ribosomal RNA gene sequence analysis, E8 was found to belong to the intermediate Leptospira species clade and E18 and YH101 to belong to the saprophytic Leptospira species clade. Based on analyses of genome‐to‐genome distances and average nucleotide identity in silico using whole genome sequences and DNA–DNA hybridization in vitro, these isolates were found to be distinct from previously described Leptospira species. Therefore, these three isolates represent novel species of the genus Leptospira for which the names Leptospira johnsonii sp. nov., (type strain E8 ^T, = JCM 32515 ^T = CIP111620 ^T), Leptospira ellinghausenii sp. nov., (type strain E18 ^T, = JCM 32516 ^T = CIP111618 ^T) and Leptospira ryugenii sp. nov., (type strain YH101 ^T, = JCM 32518 ^T = CIP111617 ^T) are proposed.     

Conocephalum salebrosum Szweykowski,Buczkowska & Odrzykoski (Marchantiopsida): modelling the occurrence of a hygrophytic species new to the bryoflora of Portugal,Madeira and Azores

Abstract

Conocephalum salebrosum Szweykowski, Buczkowska & Odrzykoski, a hygrophytic species, is newly reported for several localities in continental Portugal as well as in Madeira and Azores Islands. Information from studied specimens, and a map showing the actual distribution of this species and its closely related species, C. conicum (L.) Dumort., are provided as the basis for a discussion on the chorology and ecological ranges of both species. Additionally, a model of the potential distribution for both species in continental territory is developed through the Maximum Entropy Modelling technique, followed by an explanation of an hypothesis on the preferences of each species of Conocephalum known in this territory.     

Effect of osmolarity and viscosity on the motility of pathogenic and saprophytic Leptospira

The motility of bacteria is an important factor in their infectivity. In this study, the motility of Leptospira, a member of the spirochete family that causes a zoonotic disease known as leptospirosis, was analyzed in different viscous or osmotic conditions. Motility assays revealed that both pathogenic and saprophytic strains increase their swimming speeds with increasing viscosity. However, only pathogenic Leptospira interrogans maintained vigorous motility near physiological osmotic conditions. This suggests that active motility in physiological conditions is advantageous when Leptospira enters hosts and when it migrates toward target tissues.     

问号钩端螺旋体基因组DNA芯片的研制

目的 研制并初步评估问号钩端螺旋体(简称钩体)赖型赖株的基因组DNA芯片。方法 利用Primegens引物设计软件筛选出问号钩体赖型赖株全基因组中的特异性基因进行引物设计。对成功设计出相应引物的3 290个基因用聚合酶链反应方法进行扩增,以纯化后的产物点样制备芯片。并用双色荧光杂交策略对芯片质量进行了初步平估。结果 共获得3 290个基因产物用于点样。参考株自身杂交实验结果表明:该芯片有较高的点一致性、信噪比和较低的假阳性率。结论 成功制备了包含问号钩体赖型赖株3 290个目的基因的基因组DNA芯片,并可用于基于该芯片的问号钩体比较基因组学的研究。     

A New Species of Goniopholis from the Upper Jurassic of Portugal

The new goniopholidid crocodile Goniopholis baryglyphaeus sp. nov. from the Kimmeridgian of Guimarota/Leiria, Portugal is based on the oldest known relatively complete skeleton of Goniopholis from Europe and consists of a nearly complete skull together with a mandible and various postcranial remains. G. baryglyphaeus shows characters that hitherto have only been described for Goniopholis simus : a caudal expansion of the prefrontal that excludes the frontal from the medial margin of the orbit; and the loss of contact between the paroccipital process and the lateral surface of the squamosal and the quadrate, which exposes the cranioquadrate canal laterally. This new species can be distinguished from other European Goniopholis species by an articular facet placed ventral to the retroarticular process, and directed slightly medioventrally; a caudal margin of the mandible forming almost a right angle at its caudoventral corner; an almost square rostrolateral corner of the cranial table; a straight transverse suture between the parietal and the frontal with a median rostral process; a rostrally tapering wedge-like lacrimal, and a regular heavy skull-sculpturing. The new find of Goniopholis in Portugal shows that this genus was distributed in a larger area, and was present much earlier, than hitherto reported in south-western Europe.     

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.     

钩端螺旋体赖型017鞭毛钩相关蛋白K基因的克隆及序列分析

在以抑制消减杂交比较强毒株赖型钩端螺旋体017株和无毒株双曲钩体Patoc　I株　的基因组差异时,获得了一系列仅存在于强毒株而无毒株缺如的差异片段.选取差异片段AF325810设计特异性引物,以赖型钩端螺旋体017株基因组DNA为模板,进行巢式PCR扩增,PCR纯化产物T载体克隆,选取阳性克隆测序,进一步进行生物信息学分析,以获得强毒株赖型钩端螺旋体017株特有的毒力相关基因,DOT BLOT显示其在钩端螺旋体各株间有不同分布PCR扩增得到了产物为2kb大小的DNA片段,序列分析结果显示得到了问号钩端螺旋体赖型017株的鞭毛钩相关蛋白K基因的上游序列,为进一步探索钩端螺旋体的致病机制奠定了基础.     

Population structure of seabass in Portugal: evidence from allozymes

Starch-gel electrophoresis was used to assess the level and distribution of genetic variation in juvenile European seabass sampled from five different nursery grounds along the coast of Portugal between November 1992 and February 1994. Thirty-eight loci were examined, of which six ( AAT-3*, ADA*, GPI-1*, GPI-2*, G3PDH-2*, SOD* ) were polymorphic at the 99% level and were used in population surveys. Low but statistically significant multilocus F _ST values suggest that population structuring exists along the Portuguese coast. There is some restriction in gene flow between the population at Faro and all other sites to the north.     

Characterization of cheW genes of Leptospira interrogans and their effects in Escherichia coli

The motility and chemotaxis systems are critical for the virulence of leptospires. In this study, the phylogenetic profiles method was used to predict the interaction of chemotaxis proteins. It was shown that CheW1 links to CheA1, CheY, CheB and CheW2, CheW3 links to CheA2, MCP （LA2426）, CheB3 and CheD1; and CheW2 links only to CheW1. The similarity analysis demonstrated that CheW2 of Leptospira interrogans strain Lai had poor homology with Chew of Escherichia coli in the region of residues 30-50. In order to verify the function of these proteins, the putative cheW genes were cloned into pQE31 vector and expressed in wild-type E. coli strain RP437 or chew defective strain RP4606. The swarming results indicated that CheW1 and CheW3 could restore swarming of RP4606 while CheW2 could not. Overexpression of CheW1 and CheW3 in RP437 inhibited the swarming of RP437, whereas the inhibitory effect of CheW2 was much lower. Therefore, we presumed that CheW1 and CheW3 might have the function of CheW while CheW2 does not. The existence of multiple copies of chemotaxis homologue genes suggested that L. interrogans strain Lai might have a more complex chemosensory pathway.     

Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA

Bacterial specific primers were used to amplify 23S rRNA genes from a representative strain from each of the 23 serogroups of the pathogenic Leptospira interrogans and 8 strains from 6 serogroups of the non-pathogenic Leptospira biflexa. Only regions of extreme variability, which had been identified on the basis of homology-based search of all the 23S rRNA sequences available in GenBank database, were sequenced from the amplified products. PCR primers that had the potential to distinguish L. interrogans from L. biflexa species were designed from the derived sequences and a sensitive PCR protocol developed. The PCR method enabled the differentiation of the 59 strains of the 23 serogroups of L. interrogans from the 8 strains of 6 serogroups of L. biflexa. Further investigation by 16S rDNA sequencing of two strains of L. interrogans, which gave unexpected PCR results, provided evidence that they had been misclassified and hence we propose to reassign them to L. biflexa.     

Peopling of the Azores Islands (Portugal): data from the Y chromosome

Nine Atlantic islands with approximately five and a half centuries of demographic history constitute the Portuguese archipelago of the Azores. Despite the recent peopling history of these islands, written records regarding the specific origin and relative proportions of the first settlers are scarce and incomplete. To gain insights into the history of the peopling of the Azores and to evaluate to what extent population imports described in historical sources left their marks on the genetic constitution of the present-day populations, we analyzed 11 Y-chromosome biallelic markers in a sample of 145 unrelated individuals of Azorean ancestry. The main results of this study indicate that the genetic profile of the Azorean male population shows high affinities with that of mainland Portugal, in accordance with the general knowledge, derived from historical sources, that the Portuguese were the major contributors to the Azorean founding population. Nevertheless, genetic traces of settlers from other origins also mentioned in historical records can still be found in the present-day population. Thus typically sub-Saharan male lineages were detected in the archipelago, in contrast to what has been described for mainland Portugal. Furthermore, compared to what has been described for the mainland Portugal population, our data support a stronger influence of people of Jewish origin, as detected by an increased frequency of lineages belonging to haplogroup J.