Monofiletičnost porodice Proteidae (Amphibia: Caudata) testirana filogenetskom analizom sekvenci mitohondrijalne 12s rDNA

A phylogenetic analysis of partial 12S rRNA mitochondrial gene sequences from representatives of salamandroid families provided moderate support for the monophyly of the family Proteidae, comprising the North American genus Necturus and the European blind cave salamander Proteus.Filogenetska analiza djelomičnih mitohondrijskih genskih sekvenci 12S rRNA predstavnika salamandroidnih porodica umjereno podržava monofiletičnost porodice Proteidae, obuhvaćajući sjevernoamerički rod Necturus i europski rod špiljske čovječje ribice Proteus

Gradijentna HPLC analiza raloksifen hidroklorida i primjena u kontroli kvalitete

A rapid, sensitive and selective method for the determination of raloxifene hydrochloride (RLX) in pure drug and in tablets was developed using gradient high performance liquid chromatography (HPLC). The devised method involved separation of RLX on a reversed phase Hypersil ODS column and determination with UV detection at 284 nm. The standard curve was linear (R = 0.999) over the concentration range of 50-600 μg mL1 with a detection limit of 0.04 μg mL1 and a quantification limit of 0.16 μg mL1. Intra-day and inter-day precision and accuracy of the method were established according to the current ICH guidelines. Intra-day RSD values at three QC levels (250, 450 and 550 μg mL1) were 0.20.5% based on the peak area. The intra-day relative error (er) was between 0.2 and 0.5%. The developed method was successfully applied to the determination of RLX in tablets and the results were statistically compared with those obtained by a literature method. Accuracy, evaluated by means of the spike recovery method, was excellent with percent recovery in the range 97.7103.2 with precision in the range 1.62.2%. No interference was observed from the conformulated substances. The method was economical in terms of the time taken and the amount of solvent used.Koristeći gradijentnu tekućinsku kromatografiju visoke učinkovitosti razvijena je brza, osjetljiva i selektivna metoda za određivanje raloksifen hidroklorida (RLX), čiste supstancije i u tabletama. U radu je primijenjena reverzno-fazna kolona Hypersil ODS te UV detekcija pri 284 nm. Standardna krivulja bila je linearna (R = 0,999) u koncentracijskom području 50600 μg mL1. Granica detekcije bila je 0,04 μg mL1 a granica određivanja 0,16 μg mL1. Repetabilnost, intermedijalna preciznost i ispravnost ispitivane su prema važećim ICH uputama. Mjerenjem površine ispod pika na tri koncentracijske razine (250, 450 i 550 μg mL1) procijenjena je repetabilnost na 0,20,5%. Relativna pogreška procijenjena unutar jednog dana (er) bila je između 0,2 i 0,5%. Razvijena metoda uspješno je primijenjena za određivanje RLX u tabletama. Rezultati su statistički uspoređeni s rezultatima dobivenim prema ranije objavljenoj metodi. Analitički povrat bio je u rasponu 97,7103,2 uz preciznost od 1,6 do 2,2%. Nije primijećena interferencija pomoćnih tvari. Metoda je ekonomična s obzirom na utrošeno vrijeme i količine upotrebljenog otapala

Extensive sampling sheds light on species-level diversity in Palearctic Placobdella (Annelida: Clitellata: Glossiphoniiformes)

The bloodfeeding leech genus Placobdella is dominated by North American diversity, with only a single nominal species known from Central America and one from the Palearctic region. This is likely due to considerable underestimation of Palearctic biodiversity, but investigations into potential hidden diversity are lacking. To shed light on this, the present study introduces new data for specimens initially identified as Placobdella costata from Ukraine (close to the type locality), Italy, Germany, Latvia, Montenegro, Bulgaria, Slovenia, Turkey, Azerbaijan, Tunisia, and Algeria, and uses both nuclear (Internal Transcribed Spacer [ITS] region) and mitochondrial (cytochrome c oxidase subunit I [COI]) sequence data in phylogenetic and DNA barcoding frameworks, in order to better understand species-level diversity. Seven independent lineages are present in the trees, five of which show adequate separation at the COI locus to suggest their unique species-level status (COI distances between these clades range from 4.86 to 8.10%). However, the ITS data suggest that speciation is recent or incipient in these clades, and that not enough time has passed for clear separation at this locus. We discuss the evolutionary and taxonomic implications of our findings and speculate on dispersal events that may have contributed to shaping this pattern of geographic distribution

Fundamental research questions in subterranean biology

Five decades ago, a landmark paper inSciencetitledThe Cave Environmentheralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field.Peer reviewe

Islands beneath islands: phylogeography of a groundwater amphipod crustacean in the Balearic archipelago

<p>Abstract</p> <p>Background</p> <p>Metacrangonyctidae (Amphipoda, Crustacea) is an enigmatic continental subterranean water family of marine origin (thalassoid). One of the species in the genus, <it>Metacrangonyx longipes</it>, is endemic to the Balearic islands of Mallorca and Menorca (W Mediterranean). It has been suggested that the origin and distribution of thalassoid crustaceans could be explained by one of two alternative hypotheses: (1) active colonization of inland freshwater aquifers by a marine ancestor, followed by an adaptative shift; or (2) passive colonization by stranding of ancestral marine populations in coastal aquifers during marine regressions. A comparison of phylogenies, phylogeographic patterns and age estimations of clades should discriminate in favour of one of these two proposals.</p> <p>Results</p> <p>Phylogenetic relationships within <it>M. longipes </it>based on three mitochondrial DNA (mtDNA) and one nuclear marker revealed five genetically divergent and geographically structured clades. Analyses of cytochrome oxidase subunit 1 (<it>cox1</it>) mtDNA data showed the occurrence of a high geographic population subdivision in both islands, with current gene flow occurring exclusively between sites located in close proximity. Molecular-clock estimations dated the origin of <it>M. longipes </it>previous to about 6 Ma, whereas major cladogenetic events within the species took place between 4.2 and 2.0 Ma.</p> <p>Conclusions</p> <p><it>M. longipes </it>displayed a surprisingly old and highly fragmented population structure, with major episodes of cladogenesis within the species roughly correlating with some of the major marine transgression-regression episodes that affected the region during the last 6 Ma. Eustatic changes (vicariant events) -not active range expansion of marine littoral ancestors colonizing desalinated habitats-explain the phylogeographic pattern observed in <it>M. longipes</it>.</p

Potential efficacy of mitochondrial genes for animal DNA barcoding: a case study using eutherian mammals

<p>Abstract</p> <p>Background</p> <p>A well-informed choice of genetic locus is central to the efficacy of DNA barcoding. Current DNA barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase 1 gene (<it>CO1</it>) to diagnose and delimit species. However, there is no compelling <it>a priori </it>reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal <it>CO1 </it>barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. Four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation.</p> <p>Results</p> <p>Based on 1,179 mitochondrial genomes of eutherians, we found that the universal <it>CO1 </it>barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate (> 90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. Our results also demonstrated that any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels.</p> <p>Conclusions</p> <p>We suggest that the <it>CO1 </it>barcoding region, the universal DNA barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. Nevertheless, DNA barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups.</p

Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells

It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected

Structural and micro-anatomical changes in vertebrae associated with idiopathic-type spinal curvature in the curveback guppy model

Background: The curveback lineage of guppy is characterized by heritable idiopathic-type spinal curvature thatdevelops during growth. Prior work has revealed several important developmental similarities to the human idiopathicscoliosis (IS) syndrome. In this study we investigate structural and histological aspects of the vertebrae that areassociated with spinal curvature in the curveback guppy and test for sexual dimorphism that might explain a femalebias for severe curve magnitudes in the population.Methods: Vertebrae were studied from whole-mount skeletal specimens of curved and non-curved adult males andfemales. A series of ratios were used to characterize structural aspects of each vertebra. A three-way analysis of variancetested for effects of sex, curvature, vertebral position along the spine, and all 2-way interactions (i.e., sex and curvature,sex and vertebra position, and vertebra position and curvature). Histological analyses were used to characterize microarchitecturalchanges in affected vertebrae and the intervertebral region.Results: In curveback, vertebrae that are associated with curvature demonstrate asymmetric shape distortion,migration of the intervertebral ligament, and vertebral thickening on the concave side of curvature. There is sexualdimorphism among curved individuals such that for several vertebrae, females have more slender vertebrae than domales. Also, in the region of the spine where lordosis typically occurs, curved and non-curved females have a reducedwidth at the middle of their vertebrae, relative to males.Conclusions: Based on similarities to human spinal curvatures and to animals with induced curves, the concaveconvexbiases described in the guppy suggest that there is a mechanical component to curve pathogenesis incurveback. Because idiopathic-type curvature in curveback is primarily a sagittal deformity, it is structurally more similarto Scheuermann kyphosis than IS. Anatomical differences between teleosts and humans make direct biomechanicalcomparisons difficult. However, study of basic biological systems involved in idiopathic-type spinal curvature incurveback may provide insight into the relationship between a predisposing aetiology, growth, and biomechanics.Further work is needed to clarify whether observed sex differences in vertebral characteristics are related to the femalebias for severe curves that is observed in the population

Altered sensory-weighting mechanisms is observed in adolescents with idiopathic scoliosis

BACKGROUND: Scoliosis is the most common type of spinal deformity. In North American children, adolescent idiopathic scoliosis (AIS) makes up about 90% of all cases of scoliosis. While its prevalence is about 2% to 3% in children aged between 10 to 16 years, girls are more at risk than boys for severe progression with a ratio of 3.6 to 1. The aim of the present study was to test the hypothesis that idiopathic scoliosis interferes with the mechanisms responsible for sensory-reweighting during balance control. METHODS: Eight scoliosis patients (seven female and one male; mean age: 16.4 years) and nine healthy adolescents (average age 16.5 years) participated in the experiment. Visual and ankle proprioceptive information was perturbed (eyes closed and/or tendon vibration) suddenly and then returned to normal (eyes open and/or no tendon vibration). An AMTI force platform was used to compute centre of pressure root mean squared velocity and sway density curve. RESULTS: For the control condition (eyes open and no tendon vibration), adolescent idiopathic scoliosis patients had a greater centre of pressure root mean squared velocity (variability) than control participants. Reintegration of ankle proprioception, when vision was either available or removed, led to an increased centre of pressure velocity variability for the adolescent idiopathic scoliosis patients whereas the control participants reduced their centre of pressure velocity variability. Moreover, in the absence of vision, adolescent idiopathic scoliosis exhibited an increased centre of pressure velocity variability when ankle proprioception was returned to normal (i.e. tendon vibration stopped). The analysis of the sway density plot suggests that adolescent idiopathic scoliosis patients, during sensory reintegration, do not scale appropriately their balance control commands. CONCLUSION: Altogether, the present results demonstrate that idiopathic scoliosis adolescents have difficulty in reweighting sensory inputs following a brief period of sensory deprivation