Vascular architecture in shoots of early divergent vascular plants, Lycopodium clavatum and Lycopodium annotinum

Lycopodium represents a phylogenetically distinct clade of basal vascular plants with anatomical characters that have no parallel in other lineages. Thus, knowledge of lycopod structure and development may reveal important information about the common ancestors of all vascular plants. Here we report the unique architecture of the conducting system in Lycopodium annotinum and Lycopodium clavatum. Based on multiple series of anatomical sections, we reconstructed spatial relationships between microphylls and the stelar system. Analysis revealed that protoxylem ribs (PXR) were vertical, regardless of type of phyllotaxis, and their numbers were variable. Microphyll traces (MTr) were randomly distributed between ribs, resulting in the absence of defined sympodia and varied lengths of MTr. Dichotomous branching contributed to additional features, for example occurrence of mesarch protoxylem, affecting stele structure and PXR numbers. Our data showed limited interrelationships between lycopod vasculature and microphyll phyllotaxis. This may suggest that both systems developed independently, then evolved together to form the integrated supply system. Thus vasculature in extant lycophytes may be less functionally efficient than in seed plants, where consistent leaf-trace lengths guarantee predictable energy utilization during ontogeny. Differences may result from the phylogenetically different origin of microphylls, and the level of vascular complexity.     

The molecular basis of urokinase inhibition: from the nonempirical analysis of intermolecular interactions to the prediction of binding affinity

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a crucial role in angiogenesis process. In addition to its physiological role in healthy organisms, angiogenesis is extremely important in cancer growth and metastasis, resulting in numerous attempts to understand its control and to develop new approaches to anticancer therapy. The α-aminoalkylphosphonate diphenyl esters are well known as highly efficient serine protease inhibitors. However, their mode of binding has not been verified experimentally in details. For a group of average and potent phosphonic inhibitors of urokinase, flexible docking calculations were performed to gain an insight into the active site interactions responsible for observed enzyme inhibition. The docking results are consistent with the previously suggested mode of inhibitors binding. Subsequently, rigorous ab initio study of binding energy was carried out, followed by its decomposition according to the variation–perturbation procedure to reveal stabilization energy constituents with clear physical meaning. Availability of the experimental inhibitory activities and comparison with theoretical binding energy allows for the validation of theoretical models of inhibition, as well as estimation of the possible potential for binding affinity prediction. Since the docking results accompanied by molecular mechanics optimization suggested that several crucial active site contacts were too short, the optimal distances corresponding to the minimum ab initio interaction energy were also evaluated. Despite the deficiencies of force field-optimized enzyme-inhibitor structures, satisfactory agreement with experimental inhibitory activity was obtained for the electrostatic interaction energy, suggesting its possible application in the binding affinity prediction. Figure The comparison of an arrangement of inhibitors within uPA active. Amino acid residues form S1 pocket that binds the variable part of inhibitor molecules     

Cryptic diversity of Italian bats and the role of the Apennine refugium in the phylogeography of the western Palaearctic

The Mediterranean Basin is typified by a high degree of species rarity and endemicity that reflects its position, geomorphology, and history. Although the composition and cryptic variation of the bat faunas from the Iberian and Balkan Peninsulas are relatively well studied, data from the Apennine Peninsula are still incomplete. This is a significant shortfall, given the presumed refugial role of this region in the context of Europe's Pleistocene phylogeography. It was thus our aim to supplement the phylogeographical information from the region, generating mitochondrial sequences and reviewing published data, with a focus on the dispersal and diversification patterns characterizing taxa with different life strategies. Site‐specific lineages were ascertained, especially in the genera Myotis and Plecotus and amongst the pipistrelloid bats, representing speciose radiations. It was possible to observe disjunct ranges with patches isolated south of the Alps in several species, corresponding with evolution of elevated genetic distance. The genetic subdivision within the continuous Italian range into northern and southern lineages in several taxa indicated the possible past substructure of the refugium. Several shared lineages between the Apennine and Ibero‐Maghrebian regions were observed, indicating connectivity between the Adriatic and Atlantic?Mediterranean refuges, and raising questions as to which region these clades originated from and what was the direction of faunal exchange between them. In contrast to Europe's other two main refugia, the Apennine Peninsula is a smaller region with simpler phylogeographical patterns. Nevertheless, our results support the idea that the region generated novel lineages. Whereas diversification in sedentary bats may have been driven through the generation of in situ adaptations, specialization, and niche differentiation, the emergence of species with a tramp strategy could have entailed the utilization of faunal drift and the taxon cycle. © 2015 The Linnean Society of London     

Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

We report engineering Neurospora crassa to improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain of N. crassa (F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes, cre-1 and ace-1, on cellulase production were investigated. The F5 Δace-1 mutant showed no improvement over the wild type. The F5 Δcre-1 and F5 Δace-1 Δcre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1 Δcre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1 Δcre-1 ΔndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1 Δcre-1.     

Phylogeographical structure of the boreal‐montane orchid Malaxis monophyllos as a result of multi‐directional gene flow

We investigated the phylogeographical structure of the boreal‐montane orchid Malaxis monophyllos in its Eurasian geographical range. We analysed four sequences of plastid DNA (trnL, trnL–trnF, rps16 and accD‐psaI), resulting in 19 haplotypes and revealing a high level of intraspecific diversity (H_D = 0.702 and π = 0.196 × 10⁻²), but showing a lack of phylogeographical structure. This pattern might be caused by multiple phenomena and processes, e.g. broad‐fronted recolonization with accompanying multi‐directional gene flow between populations and expansion from at least two refugial areas. Despite the lack of phylogeographical structure, three centres of haplotype diversity were indicated in the European part of the range of M. monophyllos. According to these data, alpine and lowland glacial refugia located between the ice sheets in the European Alps and the Scandinavian glaciers seem most likely to be in Europe. Moreover, models of climatically suitable areas during the Last Glacial Maximum (LGM) confirmed the Alps as a possible refuge, and indicated an opportunity for the persistence of M. monophyllos populations in Beringia and parts of Siberia. Using two models [Model for Interdisciplinary Research on Climate (MIROC) and Community Climate System Model (CCSM)], we predicted a significant reduction in climatically suitable areas for M. monophyllos in the future (2080). Our study also demonstrated that the biological features of M. monophyllos, including breeding system and dispersal mode, seem to be crucial in understanding its phylogeographical pattern. Our results also highlighted the importance of anthropogenic habitats as reservoirs of genetic diversity and alternative habitats for this species in the context of declining natural populations. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 138–154.     

The rs340874 PROX1 type 2 diabetes mellitus risk variant is associated with visceral fat accumulation and alterations in postprandial glucose and lipid metabolism

Large-scale meta-analyses of genome-wide association studies have recently confirmed that the rs340874 single-nucleotide polymorphism in PROX1 gene is associated with fasting glycemia and type 2 diabetes mellitus; however, the mechanism of this link was not well established. The aim of our study was to evaluate the functional/phenotypic differences related to rs340874 PROX1 variants. The study group comprised 945 subjects of Polish origin (including 634 with BMI > 25) without previously known dysglycemia. We analyzed behavioral patterns (diet, physical activity), body fat distribution and glucose/fat metabolism after standardized meals and during the oral glucose tolerance test. We found that the carriers of the rs340874 PROX1 CC genotype had higher nonesterified fatty acids levels after high-fat meal (p = 0.035) and lower glucose oxidation (p = 0.014) after high-carbohydrate meal in comparison with subjects with other PROX1 genotypes. Moreover, in subjects with CC variant, we found higher accumulation of visceral fat (p < 0.02), but surprisingly lower daily food consumption (p < 0.001). We hypothesize that lipid metabolism alterations in subjects with the PROX1 CC genotype may be a primary cause of higher glucose levels after glucose load, since the fatty acids can inhibit insulin-stimulated glucose uptake by decreasing carbohydrate oxidation. Our observations suggest that the PROX1 variants have pleiotropic effect on disease pathways and it seem to be a very interesting goal of research on prevention of obesity and type 2 diabetes mellitus. The study may help to understand the mechanisms of visceral obesity and type 2 diabetes mellitus risk development.     

The utility of nanowater for ram semen cryopreservation

Nanowater (NW; water declusterized in the low-temperature plasma reactor) has specific physicochemical properties that could increase semen viability after freezing and hence fertility after artificial insemination (AI) procedures. The main goal of this study was to evaluate ram semen quality after freezing in the media containing NW. Ejaculates from 10 rams were divided into two equal parts, diluted in a commercially available semen extender (Triladyl®; MiniTüb GmbH, Tiefenbach, Germany) prepared with deionized water (DW) or NW, and then frozen in liquid nitrogen. Semen samples were examined for sperm motility and morphology using the sperm class analyzer system and light microscopy. Cryo-scanning electron microscopy (cryo-SEM) was employed to determine the size of extracellular water crystals in frozen semen samples. Survival time at room temperature, aspartate aminotransferase (AspAT) and alkaline phosphatase (ALP) concentrations post-thawing as well as conception/lambing rates after laparoscopic intrauterine AI of 120 ewes were also determined. There were no significant differences between DW and NW groups in sperm progressive motility (26.4 ± 12.2 and 30.8 ± 12.4%) or survival time (266.6 ± 61.3 and 270.9 ± 76.7 min) after thawing and no differences in the percentages of spermatozoa with various morphological defects before or after freezing. There were, however, differences (P < 0.05) in AspAT (DW: 187.1 ± 160.4 vs. NW: 152.7 ± 118.3 U/l) and ALP concentrations (DW: 2198.3 ± 1810.5 vs. NW: 1612.1 ± 1144.8 U/l) in semen samples post-thawing. Extracellular water crystals were larger (P < 0.05) in ejaculates frozen in NW-containing media. Ultrasonographic examinations on day 40 post-AI revealed higher (P < 0.05) conception rates in ewes inseminated with NW (78.3%) compared with DW semen (58.3%), and the percentages of ewes that carried lambs to term were 73.3% and 45.0% in NW and DW groups, respectively (P < 0.01). In summary, the use of a semen extender prepared with NW was associated with a substantial improvement in the fertilizing ability of frozen-thawed ram semen and lamb productivity of inseminated ewes.     

Design and synthesis of CK2 inhibitors

A series of new polybrominated benzimidazoles and benzotriazoles has been synthesized and their influence on the activity of protein kinase CK2 was evaluated. It was revealed that the most active inhibitors are those with methyl or ethyl substituent at benzene ring, namely 5,6,7-tribromo-4-methyl-1H-benzotriazole (38, IC(50) 0.51 μM) and 5,6,7-tribromo-4-ethyl-1H-benzotriazole (40, IC(50) 0.16 μM). The derivatives with large aromatic or heterocyclic substituents connected to benzimidazole or benzotriazole scaffold appeared to be less potent inhibitors.