The use of protein characteristics to assess the retrievability of ancient DNA from ancient bones

The ability to retrieve DNA from ancient specimens has been one of the greatest achievements of the past decade, and has opened a totally new field of research with applications in seemingly distant domains such as archeobotany, the molecular phylogeny of extinct genomes, human paleopathology and the genetic of ancient human populations. However, extraction of ancient DNA has often a very low rate of success, prompting researchers to develop screening methods for the selection of promising specimens. With this goal in mind, we studied the amino acid content of nine human bones of ancient origin. We demonstrate that a single HPLC chromatogram is indicative of the integrity of ancient bone proteins. Among five specimens containing amplifiable DNA, four exhibited a protein content similar to that of contemporary bone protein content. Three of the four specimens, from which we were unable to extract any amplifiable DNA, had an amino acid content strikingly different from that of contem-porary bone. A non-parametric statistical test, Kendall's tau, was used to show that protein content and PCR products, are probably correlated (at a 95% confidence level). In addition, the D/L Asp and D/L Glu racemization ratios obtained are indicative of the presence of ancient organic compounds. We propose that protein analysis should be systematically performed in studies where there are many samples in order to select the specimens that are most likely to contain retrievable ancient DNA.     

东北地区乳菇属的初步研究

本文报道了东北地区乳菇属(Lactarius)的29种,其中包括新种2个,长白乳菇(L.chanbainensis Y.Wang et Xie sp.nov.)温泉乳菇(L.wenquanensis Y.Wang et Xie sp.nov.);国内新记录种5个:茶绿乳菇[L.necator(Pers.ex Fr.)Farst.],条纹乳菇[L.oculatus(Peck)Burl.],变红乳菇[L.acris(Bolt.ex Fr.)Gray],复生乳菇(L.repraesentaneus Britz.)和点柄乳菇(L.maculatus Burl.)。新种有汉文和拉丁文描述以及形态、显微构造图;国内新记录种有汉文描述。     

基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。     

Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques

A well‐covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self‐primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four‐sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400–500 bp without bringing or inducing any sequence errors. About one‐third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well‐covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA‐labelled next‐generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality.     

Invasion from the cold past: extensive introgression of mountain hare (Lepus timidus) mitochondrial DNA into three other hare species in northern Iberia

Mitochondrial DNA introgression from Lepus timidus into Lepus granatensis and Lepus europaeus was recently reported in Iberia, although L. timidus presumably retreated from this region at the end of the last ice age. Here we assess the extent of this ancient mtDNA introgression by RFLP analysis of 695 specimens representing the three hare species present in Iberia. The introgressed L. timidus lineage was found in 23 of the 37 populations sampled. It is almost fixed in L. europaeus across its Iberian range in the Pyrenean foothills, and in L. granatensis, which occupies the rest of the peninsula, it is predominant in the north and gradually disappears further south. We also found it in Lepus castroviejoi, a species endemic to Cantabria. Multiple hybridizations and, potentially, a selective advantage for the L. timidus lineage can explain the remarkable taxonomic and geographical range of this mitochondrial introgression.     

中国横断山地区蓝藻型肺衣属地衣的三个新种

在形态和化学研究的基础上,结合分子生物学研究方法（ITS片段）,对中国横断山地区的830号与蓝细菌共生的肺衣属标本进行了分类学研究,共发现3个新种;其中,横断山肺衣L. hengduanensis的主要特征是具粉芽化裂芽,无脊肺衣L. irrugulosa的网脊不明显,以及宽叶肺衣L. latilobulata的裂片顶端宽且钝圆;3个种在ITS系统发育树上分别形成独立小枝。文中提供了中国肺衣属中与蓝细菌共生的物种检索表。     

The coralline genera Sporolithon and Heydrichia (Sporolithales,Rhodophyta) clarified by sequencing type material of their generitypes and other species

Interspecific systematics in the red algal order Sporolithales remains problematic. To re‐evaluate its species, DNA analyses were performed on historical type material and recently collected specimens assigned to the two genera Sporolithon and Heydrichia. Partial rbcL sequences from the lectotype specimens of Sporolithon ptychoides (the generitype species) and Sporolithon molle, both from El Tor, Egypt, are exact matches to field‐collected topotype specimens. Sporolithon crassum and Sporolithon erythraeum also have the same type locality; material of the former appears to no longer exist, and we were unable to PCR amplify DNA from the latter. A new species, Sporolithon eltorensis, is described from the same type locality. We have not found any morpho‐anatomical characters that distinguish these three species. No sequenced specimens reported as S. ptychoides from other parts of the world represent this species, and likely reports of S. ptychoides and S. molle based on morpho‐anatomy are incorrect. A partial rbcL sequence from the holotype of Sporolithon dimotum indicates it is not a synonym of S. ptychoides, and data from the holotype of S. episporum confirm its specific recognition. DNA sequences from topotype material of Heydrichia woelkerlingii, the generitype species, and isotype material of Heydrichia cerasina confirm that these are distinct species; the taxon reported to be H. woelkerlingii from New Zealand is likely an undescribed species. Type specimens of all other Sporolithon and Heydrichia species need to be sequenced to confirm that they are distinct species; morpho‐anatomical studies have proved inadequate for this task.     

DNA EXTRACTION METHODS FOR KELP (LAMINARIALES) TISSUE1

Newly adapted extraction methods for harvesting total cellular DNA from kelp (Laminariales; Phaeophyta) exploit the life-history stages of these heteromorphic algae. Earlier techniques, developed primarily for chloroplast DNA extraction, were time consuming and labor intensive and required large quantities of fresh sporophyte tissue. In contrast, the new methods expedite DNA extraction by employing dried sporophyte laminae or fresh gametophyte filaments and meiospores. The current methods require less tissue and procedural manipulation, reducing the time and labor involved in DNA extraction. Additionally, the current methods were successfully employed to extract DNA from herbarium specimens up to 22 years old. Total cellular DNA yields were 12.8 and 26.4 μg:g^?1 from dried laminae,: 1.9 and 11 μg from small volumes of concentrated gametophyte filaments (25 μL), and 43 to 54.5 μg from pellets of isolated meiospores (50 μL). Following purification on Sepharose columns, DNA from all three sources was sufficiently pure for molecular applications, including restriction endonuclease digestion, Southern blot-hybridizations, and amplification via the polymerase chain reaction.     

Use of laser scanning cytometry to study tumor microenvironment

The study of phenomena occurring in the tumor microenvironment is a challenging task because of technical difficulties, particularly when dealing with hypocellular specimens. Laser scanning cytometry (LSC) is a new laboratory technology that has been recently introduced to overcome the limitations of other traditional technologies. By combining the properties and the advantages of flow cytometry (FC) and immunohistochemistry (IHC), LSC allows the investigator to obtain objective information on DNA content, protein expression and cellular localization is combination with morphological features. It has been already shown that LSC results are reliable compared to more traditional technologies, and its implementation in the clinical routine is under way. Its use in oncology, which is rapidly expanding, spans from apoptosis analysis to DNA content quantitation and tumor cell phenotyping. Here we describe the technology underlying this novel fluorescence-based device, review its use in oncology by dissecting the phenomena occurring in the tumor microenvironment and propose its application for the immunological follow-up of malignant lesions undergoing immunotherapeutic manipulation.     

Range,population structure and morphological characterization of the small range endemic bush-cricket <Emphasis Type="Italic">Lluciapomaresius panteli</Emphasis> (Orthoptera: Tettigoniidae: Bradyporinae)

The bush-cricket genus Lluciapomaresius Barat, 2012 is endemic to the Iberian Peninsula. It includes at least seven species, most of which are narrow endemics restricted to single or few mountain ranges across the Peninsula. All of them are currently included under different categories in the IUCN red list. One of the species listed as data deficient is Pantel’s Saddle Bush-cricket L. panteli (Navàs 1899), an iconic species of the Montsant Natural Park. Up to now, it was thought to be restricted to few square kilometres in the Montsant range, in southern Catalonia (north-east Iberian Peninsula). The species was at some point suspected extinct, but it was rediscovered in 2000. More recently, a new species, L. nisae Olmo-Vidal 2017, was described based on specimens found in neighbouring Llaberia range, about 20 km south from Montsant. In this study, we present new data based on a systematic sampling of Lluciapomaresius across known and nearby locations in Montsant, Llaberia and surrounding ranges. The records and collected specimens allow us to provide a better delimitation of the actual distribution range and to conduct a detailed morphological study to shed light on the degree of differentiation of populations in different parts of the range, further supported by DNA sequence data of mitochondrial and nuclear genes. Our results show that L. nisae is a synonym of L. panteli as judged by the small genetic and morphological variability, better interpreted as the result of local differentiation of populations following post-glacial isolation. We further confirm the extremely small ranges and isolation of the Lluciapomaresius populations and propose to move the species towards the EN (endangered) category in the IUCN Red List. Additional research on the still unknown aspects of its biology and ecology is needed to implement reasoned and effective conservation measures that ensure its survival.     

Lepidochitona bullocki, a new species of chiton (Polyplacophora: Ischnochitonidae) from the Colombian Caribbean

The genus Lepidochitona is one of the most diverse in the Southern Caribbean. In order to describe this new species, twenty specimens of L. bullocki sp. nov. were collected by hand on the rocky coast, from 0.25 to 1.5m depth, and a shore distance of 1 to 5m, between the Northern section of Rodadero beach and Puerto Luz, in Santa Marta, Colombia. The specimens were small, with maximum observed size of 7.3mm. This is the fourth species of the genus Lepidochitona described for the Caribbean. The previously known species were L. liozonis (Dall & Simpson 1901), L. rosea (Kaas 1972) and L. rufoi (Garcia-Ríos 2010). L. bullocki differs from L. liozonis and L. rosea in having a rough tegmentum and color. It differs from L. rufoi in having longer and numerous hyaline spicules, bigger size and broader central radula tooth. Lepidochitona bullocki is the only Caribbean species of the genus frequently located on the illuminated side of rocks in the shallow sublitoral zone.     

Sequence repeats enlarge the internal transcribed spacer 1 region of the brown alga Colpomenia sinuosa (Scytosiphonaceae, Phaeophyceae)

Colpomenia sinuosa is an annual brown algal species that occurs in temperate to tropical waters of the world. In order to examine the genetic diversity among populations of the species and to discuss its current distribution, we analyzed the nuclear ribosomal DNA internal transcribed spacer (ITS) region from 18 specimens of C. sinuosa , and, for comparison, plastid rbc L from the same specimens. The C. sinuosa ITS region (2141–2534 base pairs) is approximately 2.2 times the length of most other brown algae. We found a long repeated sequence of approximately 190 base pairs in the first half of the ITS1 region; five repeats in Northern Hemisphere collections and three in those from the Southern Hemisphere, which result in ITS length variation. The unequal occurrence of tandem repeats of C. sinuosa corresponds to the geographical distribution of the species. The rbc L sequences from all the specimens of C. sinuosa , except the Canary Island samples, were identical, indicating that they indeed belong to the same species.     

A new species of Lepiota from China

In this report we describe the three species in Lepiota sect. Lepiota occurring in tropical China. Lepiota attenuata is a new species and is characterized by a pileus with brownish yellow squamules and radially sulcate striate margin, penguin-shaped spores that are distinctively narrowed toward the apex and inflated submoniliform or catenulate elements in the pileus covering. We compared the type specimens of L. metulispora and L. thrombophora with tropical Chinese specimens; both taxa occur in the study area. Phylogenetic relationships among the tropical Chinese species and other closely related species in the genus were inferred based on DNA sequences of the nuclear ribosomal genes (ITS, LSU and IGS) and the mitochondrial small ribosomal RNA gene (mtSSU).     

Leptodon Smith (Hedw.) Mohr in Ireland

Abstract

A new Irish locality is recorded for L. smithii, and the general features of the station are described. An account of the bryophyte succession on tree trunks in the area is given and the part played by L. smithii indicated. It is shown that gatherings from both Irish localities consisted entirely of male plants and the distribution and age of the antheridia on both specimens are recorded. It is suggested that rarity of sporophyte production in the British Isles is probably due to too great a spatial separation of male and female plants and not to the absence, or low production, of sex organs. It is also suggested that rarity of spores and impermanence of the habitat are the chief factors contributing to its rareness in climatically suitable areas of the British Isles. The known distribution of the plant in the British Isles is related to its European and world distribution, and it is shown that L. smithii may be expected in further Irish vice counties.

I have pleasure in acknowledging my thanks to Mr Wallace for the gift of a specimen of L. smithii from Knocklofty and for details of its habitat; to Mrs C. E. M. Shelswell-White for permission to enter and collect in the grounds of Bantry House, and to my colleague Dr J. G. Hawkes for confirming the identification of the two shrubs.     

Use of a dual priming oligonucleotide system to detect multiple sexually transmitted pathogens in clinical specimens

Aims:  To evaluate a new dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) assay for detection of six sexually transmitted pathogens, including Chlamydia trachomatis , Neisseria gonorrhoeae , Mycoplasma genitalium , Mycoplasma hominis , Ureaplasma urealyticum and Trichomonas vaginalis .
Methods and Results:  Using 130 clinical specimens, the results obtained by the multiplex PCR, previously established in-house PCR and COBAS Amplicor PCR assays were compared. The specimens frequently contained multiple pathogens (34/130 specimens). The multiplex PCR assay had an overall sensitivity of 96% and specificity of 100% compared to the in-house PCR assay at >20 μg ml⁻¹ of DNA concentrations in samples and there was no cross-reaction with nonpathogenic Neisseria species that cause the majority of false-positive results with the COBAS Amplicor PCR assay.
Conclusions:  The DPO-based multiplex PCR assay detected the six sexually transmitted pathogens in clinical specimens with a high sensitivity and specificity, although its sensitivity was dependent on the DNA content of the samples.
Significance and Impact of the Study:  It is the first report about the new DPO-based technique to detect multiple sexually transmitted pathogens in a single assay, which has considerable potential to diagnose the infections accurately and rapidly.     

Genetic polymorphism in Caulerpa taxifolia (Ulvophyceae) chloroplast DNA revealed by a PCR‐based assay of the invasive Mediterranean strain

Abstract An invasive, cold‐tolerant strain of the tropical green alga Caulerpa taxifolia was introduced recently in the Mediterranean Sea and along the Californian coast. We screened 50 aquarium and open‐sea C. taxifolia specimens for the presence/absence of an intron located in the rbcL gene of chloroplast DNA. We also reanalysed a total of 229 sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA, combining previously published sequences from different studies with 68 new sequences to complement rbcL data. The introduced Mediterranean strain was found to be characterized by the absence of the rbcL intron and by the occurrence of a particular monomorphic ITS type. A PCR assay based on rbcL gene was developed to detect new introductions of the invasive strain of C. taxifolia. This rapid and inexpensive test could be useful to assist environment managers in the preservation of coastal marine ecosystems.     

Recurrent introgression of mitochondrial DNA among hares (Lepus spp.) revealed by species-tree inference and coalescent simulations

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.     

Inhibition of Thymidine Kinase Activity and Deoxyribonucleic Acid Synthesis in L Cells Infected with the Meningopneumonitis Agent

The activities of enzymes related to deoxyribonucleic acid (DNA) synthesis were studied in uninfected L cells and in L cells infected with Chlamydia psittaci (strain meningopneumonitis). The meningopneumonitis agent multiplied normally but failed to induce the synthesis of thymidine kinase in LM (TK(-)) cells which contain no thymidine kinase in the uninfected state. It was concluded that this microorganism has no thymidine kinase of its own and that it does not depend on the functioning of the host enzyme for synthesizing its DNA. Exposure of clone 5b L cells to the meningopneumonitis agent was followed by a decline in their thymidine kinase activity to nearly zero levels, whereas the levels of uridine kinase and thymidylate synthetase remained unchanged. Inhibition of thymidine kinase activity in L cells occurred soon after infection and required new protein synthesis by the meningopneumonitis agent. This inhibition occurred before inhibition of host DNA synthesis, but it was not an essential prelude to the latter inhibition. On the basis of this and previous investigations and in light of present knowledge of the mammalian cell cycle, it was postulated that the meningopneumonitis agent inhibits macromolecular synthesis in L cells by preventing the initiation of a new cell cycle.     

A pressure cooking-based DNA extraction from archival formalin-fixed, paraffin-embedded tissue

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies.