A phylogenomic resolution for the taxonomy of Aegean green lizards

Lacerta pamphylica and Lacerta trilineata are two currently recognized green lizard species with a historically problematic taxonomy. In cases of tangled phylogenies, next-generation sequencing and double-digest restriction-site-associated DNA protocols can provide a wealth of genomic data and resolve difficult taxonomic issues. Here, we generated genome-wide SNPs and mitochondrial sequences, and applied molecular species delimitation approaches to provide a stable taxonomy for the Aegean green lizards. Mitochondrial gene trees, genetic cluster delimitation and population structure analyses converged into recognizing the populations of (a) L. pamphylica, (b) east Aegean islands, Anatolia and Thrace (diplochondrodes lineage), (c) central Aegean islands (citrovittata), and (d) remaining Balkan populations and islands (trilineata), as separate clusters. Phylogenomic analyses revealed a split into two major clades, east and west of the Aegean Barrier, unambiguously showing a sister–clade relationship between pamphylica and diplochondrodes, rendering L. trilineata paraphyletic. Species delimitation models were tested in a Bayesian framework using the genomic SNPs: lumping all populations into a single ‘species’ had the lowest likelihood but the current taxonomy was also outperformed by all other models. All lines of evidence support the Pamphylian green lizard as a valid species; thus, east Aegean L. trilineata should also be considered a distinct species under the name Lacerta diplochondrodes. Finally, evidence from the mitochondrial and nuclear genomes is overwhelmingly in favour of recognizing the morphologically distinct Cycladian green lizards as a distinct species. We propose their elevation to full species under the name Lacerta citrovittata. All remaining insular and continental populations of the Balkan Peninsula represent the species L. trilineata.     

A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA

Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTag^TM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1^st and 2^nd immunization; however, this difference was not statistically significant after 3^rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.     

Widespread Changes in White Matter Microstructure after a Day of Waking and Sleep Deprivation

BackgroundElucidating the neurobiological effects of sleep and waking remains an important goal of the neurosciences. Recently, animal studies indicated that sleep is important for cell membrane and myelin maintenance in the brain and that these structures are particularly susceptible to insufficient sleep. Here, we tested the hypothesis that a day of waking and sleep deprivation would be associated with changes in diffusion tensor imaging (DTI) indices of white matter microstructure sensitive to axonal membrane and myelin alterations.MethodsTwenty-one healthy adult males underwent DTI in the morning [7:30AM; time point (TP)1], after 14 hours of waking (TP2), and then after another 9 hours of waking (TP3). Whole brain voxel-wise analysis was performed with tract based spatial statistics.ResultsA day of waking was associated with widespread increases in white matter fractional anisotropy, which were mainly driven by radial diffusivity reductions, and sleep deprivation was associated with widespread fractional anisotropy decreases, which were mainly explained by reductions in axial diffusivity. In addition, larger decreases in axial diffusivity after sleep deprivation were associated with greater sleepiness. All DTI changes remained significant after adjusting for hydration measures.ConclusionsThis is the first DTI study of sleep deprivation in humans. Although previous studies have observed localized changes in DTI indices of cerebral microstructure over the course of a few hours, further studies are needed to confirm widespread DTI changes within hours of waking and to clarify whether such changes in white matter microstructure serve as neurobiological substrates of sleepiness.     

Prenatal Exposure to Cadmium,Placental Permeability and Birth Outcomes in Coastal Populations of South Africa

Background

The impact of prenatal exposure to cadmium (Cd) on birth outcomes is an area of concern. This study aimed to assess an impact of prenatal Cd exposure on birth outcomes in distinct coastal populations of South Africa.

Methods

Cadmium was measured in maternal blood (CdB) (n = 641), cord blood and in maternal urine (n = 317). This investigation assessed the associations between CdB (non-transformed) and birth outcomes across the 25^th, 50th, and 75th percentile for birth weight, birth length and head circumference, to test for a linear trend. Associations between natural log-transformed maternal CdB, size at birth and other factors were further evaluated using linear mixed-effects modelling with random intercepts.

Results

The average gestational age in the total sample was 38 weeks; 47% of neonates were female, average birth weight was 3065 g and 11% were of low birth weight (< 2500 g). The geometric mean (GM) of the maternal CdB level was 0.25 μg/L (n = 641; 95% CI, 0.23–0.27). The cord blood Cd level was 0.27 μg/L (n = 317; 95% CI, 0.26–0.29) and urine (creatinine-corrected) Cd level was 0.27 μg/L (n = 318; 95% CI, 0.24–0.29). The CdB cord:maternal ratio in the sub-cohort was 1, suggesting that the placenta offers no protective mechanism to the foetus. An inverse association was found between CdB and the lower birth weight percentile in female neonates only (β = - 0.13, p = 0.047). Mothers who reported eating vine vegetables daily had lower levels of CdB (β = - 0.55, p = 0.025). Maternal smoking was associated with an elevation in natural log-transformed CdB levels in both male and female cohorts.

Discussion

Significant inverse associations between prenatal Cd exposure and birth anthropometry were found in female neonates but not in male neonates, suggesting potential sex differences in the toxico-kinetics and toxico-dynamics of Cd.     

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.     

Calcium hypochlorite on mouse embryonic fibroblast cells (NIH3T3) in vitro cytotoxicity and genotoxicity: MTT and comet assay

Molecular Biology Reports - Antimicrobial irrigation solutions are widely used under clinical settings. Their effect on dental tissue is a subject of recent research, which aims for a safer...     

Phylogenetic relationships of the populations of Iranolacerta brandtii (de Filippi, 1863) (Squamata: Lacertidae) recently found in Eastern Anatolia,Turkey

The Persian Lizard, Iranolacerta brandtii, was until recently considered to be restricted to north-western Iran (Azerbaijan and Esfahan provinces). However, two recent studies have revealed the existence of populations in Eastern Anatolia, extending the range of this species for about 230?km westwards. The fragmented distribution of this species has been considered to be a consequence of the climatic oscillations during the Pleistocene and Holocene, which created events of alternating contact and isolation of populations in distinct glacial refugia. According to our obtained genealogy derived from three mitochondrial fragments (12S rRNA, 16S rRNA and cytb), the Turkish specimens cluster together but form an independent clade, sister to the individuals from Tabriz in Iran. The separation of these two clades is concurrent with the cladogenesis between the Esfahan and Ardabil clades, estimated to have taken place during the late Holocene.     

Comparative phylogeography of six herpetofauna species in Cyprus: late Miocene to Pleistocene colonization routes

The colonization patterns of oceanic islands are often interpreted through transmarine dispersal. However, in islands with intense human activities and unclear geological history, this inference may be inappropriate. Cyprus is such an island, whose geotectonic evolution has not been clarified yet to the desired level for biogeographical reconstructions, leaving the questions of ‘how the Cypriote biota arrived’ and ‘does the dispersal have the formative role in patterns of its diversification’ unanswered. Here, we address these issues through a reconstruction of the evolutionary history of six herptiles (Ablepharus budaki, Ophisops elegans, Acanthodactylus schreiberi, Telescopus fallax, Pelophylax cf. bedriagae, and Hyla savignyi) by means of mitochondrial DNA (cytochrome b and 16S rRNA), applying a Bayesian phylogenetic, biogeographical, and chronophylogenetic analyses. The phylogeographical analyses show that the colonization history of those species in Cyprus started in the late Miocene and extended into the Pliocene and Pleistocene, with geodispersal, transmarine dispersal, and human‐mediated dispersal having their share in shaping the diversification of Cypriote herptiles. The revealed patterns could be divided into three biogeographical categories: old colonizers that arrived in Cyprus during the late Miocene or early Pliocene either by a land bridge (geodispersal) which connected Cyprus with the mainland or by transmarine dispersal, younger colonizers that reached the island through transmarine dispersal from the Middle East, and new settlers that arrived through human‐induced (voluntary or not) introductions. This work advances our knowledge of the biogeography of Cyprus and highlights the need to consider both geo‐ and transmarine dispersal when dealing with islands whose associations do not have a straightforward interpretation. © 2013 The Linnean Society of London     

Molecular phylogeny and historical biogeography of the Anatolian lizard Apathya (Squamata,Lacertidae)

Apathya is a lacertid genus occurring mainly in south-east Turkey and its adjacent regions (part of Iran and Iraq). So far two morphological species have been attributed to the genus; A. cappadocica (with five subspecies, A. c. cappadocica, A. c. muhtari, A. c. schmidtlerorum, A. c. urmiana and A. c. wolteri) and A. yassujica. The first species occupies most of the genus’ distribution range, while A. yassujica is endemic of the Zagros Mountains. Here, we explored Apathya’s taxonomy and investigated the evolutionary history of the species by employing phylogenetic and phylogeographic approaches and using both mitochondrial (mtDNA) and nuclear markers. The phylogenetic relationships and the genetic distances retrieved, revealed that Apathya is a highly variable genus, which parallels its high morphological variation. Such levels of morphological and genetic differentiation often exceed those between species of other Lacertini genera that are already treated as full species, suggesting the necessity for a taxonomic revision of Apathya. The phylogeographical scenario emerging from the genetic data suggests that the present distribution of the genus was determined by a combination of dispersal and vicariance events between Anatolia and Southwest Asia dating back to the Miocene and continuing up to the Pleistocene. Key geological events for the understanding of the phylogeography of the genus are the movement of the Arabian plate that led to the configuration of Middle East (orogenesis of the mountain ranges of Turkey and Iran) and the formation of Anatolian Diagonal.