Molecular phylogeny of RNA polymerase II gene reveals the relationships of tetraploid species with St genome (Triticeae: Poaceae)

To evaluate phylogeny of tetraploid with St genome, phylogenetic analyses of RNA polymerase II (RPB2), a member of the nuclear gene family encoding the second largest subunit, were performed. Our results showed that: (1) Roegneria magnicaespes and Roegneria alashanica are related to Pseudoroegneria. (2) Roegneria elytrigioides has StStStSt genomes and should therefore be classified as Pseudoroegneria elytrigioides. (3) Pseudoroegneria tauri and Pseudoroegneria deweyi which have StStPP genomes should be transferred to Douglasdeweya and be renamed as Douglasdeweya wangii and Douglasdeweya deweyi, respectively. (4) Pseudoroegneria geniculata ssp. scythica is related to Pseudoroegneria and Lophyrum, and hence should be identified as a species of Trichopyrum. (5) Pseudoroegneria libanotica might be a parental donor for Elytrigia caespitosa rather than Elytrigia caespitosa ssp. nodosa. It is unreasonable to recognize El. caespitosa ssp. nodosa as a subspecies of El. caespitosa. (6) Interspecific and intergeneric variations are detected in St genome of these tetraploid species.     

Phytoaccumulation of cadmium through Azolla from aqueous solution

Azolla, which is an aquatic fern, has proved to be effective in the uptake and accumulation of metals from polluted waters. Azolla spp., namely A. microphylla cv. MH3 and A. caroliniana Willd, were chosen as model plants so that Cd(II) could be accumulated from aqueous solution. An increase in uptake time and the concentration of Cd(II) in aqueous solution resulted in more Cd(II) accumulation in both species. Modified Michaelis-Menten equation was employed to describe the concentration-dependent kinetics of Cd(II) uptake through the roots of A. microphylla cv. MH3, and the values of K_m and V_max were found to be 0.23 mg/L and 16.49 μg/(g.f.wt.h), respectively. Cd(II) uptake by A. microphylla cv. MH3 occurs partly through Ca(II) channels and has the potential to be mediated by Zn(II) transporters.     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes

Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours.     

Pericarp structure and phylogeny of tribe Mentheae (Lamiaceae)

Pericarp structure is investigated in 143 specimens belonging to 59 out of ca. 65 genera and 134 out of ca. 2,000 species of Lamiaceae subfamily Nepetoideae tribe Mentheae. Some of the data presented by earlier authors are believed to be incorrect. The variation in pericarp structure has been found to be strongly correlated to the variations in DNA and gross morphological characters. The groups of two-staminate genera (Salvia, etc.) and four-staminate genera of Salviinae (Lepechinia and Chaunostoma) can be distinguished from each other and from other labiates by differences in pericarp structure. The other two subtribes, Menthinae and Nepetinae, differ considerably, but their variations strongly overlap. The differences in pericarp structure also suggest that Menthinae can be divided into three main monophyletic groups based on other data: (1) the main bulk of the tribe, (2) a group of Prunella and Cleonia and perhaps also Horminum, and (3) the characteristic genus Lycopus. Pericarp data disagree with a suggestion that the genus Melissa should be included in the subtribe Salviinae. Modifications of the subtribal classification of Mentheae are discussed.     

Phytoalexin production by Ononis species

Following exposure to short wavelength (254 nm) ultra-violet light, the detached leaflets of Passaea (Ononis) ornithopodioides and 31 species and subspecies of Ononis have been found to accumulate substantial quantities of the isoflavonoid (pterocarpan) phytoalexin, medicarpin. Apart from P. (Ononis) ornithopodioides, O. cristata, O. fruticosa, O. pubescens and O. rotundifolia, leaf tissues of all the species investigated similarly contained small amounts of the related fungitoxic pterocarpan, maackiain. Isoflavan phytoalexins common in genera such as Medicago and Trifolium (tribe Trifolieae) were absent from both Ononis and Passaea. The phytoalexin data suggest that Passaea should probably be combined with Ononis and, in conjunction with information on constitutive isoflavonoids, that Ononis itself should be assigned to the Trifolieae rather than to the distinct tribe Ononideae. Chemical evidence for and against an especially close taxonomic association between Ononis and Cicer (tribe Cicereae) is also briefly discussed.     

Mortality of Pratylenchus penetrans by Volatile Fatty Acids from Liquid Hog Manure

As part of our research program assessing the use of liquid hog manure (LHM) to control root-lesion nematodes, Pratylenchus penetrans, a series of acute toxicity tests was conducted to: (i) examine if non-ionized forms of volatile fatty acids (VFA) are responsible for the mortality of P. penetrans exposed to LHM under acidic conditions, (ii) determine if Caenorhabditis elegans can be a surrogate for P. penetrans in screening tests by comparing their sensitivities to VFA, (iii) characterize the nematicidal effect of individual VFA in LHM to P. penetrans, and (iv) determine whether individual VFA in LHM interact in their toxicity to P. penetrans. LHM was significantly (P ≤ 0.05) more toxic to P. penetrans than a mixture of its main VFA components at concentrations of 5% and 10% (vol. VFA or LHM /vol. in buffer). Pratylenchus penetrans was more sensitive to acetic acid than C. elegans, whereas the sensitivity of both nematode species to n-caproic acid was similar. Individual VFA vary in their lethality to P. penetrans. n-valeric acid was the most toxic (LC₉₅= 6.8 mM), while isobutyric acid was the least toxic (LC₉₅ = 45.7 mM). Individual VFA did not interact in their toxicity to P. penetrans, and their effects were considered additive. VFA account for the majority of the lethal effect of LHM to P. penetrans under acidic conditions. Caenorhabditis elegans cannot be used as a surrogate to P. penetrans in toxicity studies using VFA. The efficacy of LHM to control P. penetrans can be evaluated by assessing its VFA content prior to application, and this evaluation is facilitated by the fact that the interaction of individual VFA appears to be simply additive.     

The genus Juniperus in Mexico and Guatemala: Numerical and chemosystematic analysis

The variation leaf constituents (mostly terpenoids) was analyzed from each of the taxa of Juniperus in Mexico and Guatemala by numerical taxonomic methods. These results were compared with those of a previous study utilizing morphological characters. In general, the two agree on the major groups. Differences between more closely related species were more apparent with the chemical data, whereas more distantly related taxa sometimes appeared to be more closely related chemically due to the presence of a single component such as α-pinene in high concentration in each of the oils. Four large groups are apparent; the deppeanan; flaccidan; monticolan; and the oneseeded (pinchotii) complex. Some taxa (J. durangensis, J. standleyi, J.jaliscana) are still of uncertain affinities. This study confirms the morphological data indicating that J. patoniana Martínez should be reduced to a variety of J. deppeana (J. deppeana var. patoniana (Martínez) Zanoni, comb. et stat. nov). No samplestypical of J. monosperma were found in Mexico and J. monosperma var. gracilis (sensu Martinez) was found to not be closely allied with J. monosperma from the USA but has some uncertain affinities with species of the one-seeded complex. These relationships need to be examined in more detail. J. blancoi appears to be closely related to J. scopulorum. This information on the junipers of Mexico and Guatemala should prove invaluable to future studies on the evolution of the Juniperus in North America.     

Genes implicated in Caenorhabditis elegans and human health regulate stress resistance and physical abilities in aged Caenorhabditis elegans

Recently, nine Caenorhabditis elegans genes, grouped into two pathways/clusters, were found to be implicated in healthspan in C. elegans and their homologues in humans, based on literature curation, WormBase data mining and bioinformatics analyses. Here, we further validated these genes experimentally in C. elegans. We downregulated the nine genes via RNA interference (RNAi), and their effects on physical function (locomotion in a swim assay) and on physiological function (survival after heat stress) were analysed in aged nematodes. Swim performance was negatively affected by the downregulation of acox-1.1, pept-1, pak-2, gsk-3 and C25G6.3 in worms with advanced age (twelfth day of adulthood) and heat stress resistance was decreased by RNAi targeting of acox-1.1, daf-22, cat-4, pig-1, pak-2, gsk-3 and C25G6.3 in moderately (seventh day of adulthood) or advanced aged nematodes. Only one gene, sad-1, could not be linked to a health-related function in C. elegans with the bioassays we selected. Thus, most of the healthspan genes could be re-confirmed by health measurements in old worms.     

What happens to the mycorrhizal communities of native and exotic seedlings when Pseudotsuga menziesii invades Nothofagaceae forests in Patagonia,Argentina?

Pseudotsuga menziesii is one of the most widely planted conifers in the Patagonian Andes of Argentina, having invading characteristics that are broadly reported. We studied the mycorrhizal status of seedlings along six Nothofagaceae + P. menziesii invasion matrices to investigate their role in the invasive process, according to these hypothesis: a) The abundance and richness of EM will be higher in seedlings grown in their own soil; b) In the presence of native EM inoculum, the invasive plant will be associated with generalist mycorrhizae (EM and/or AM), c) AM associations will be more abundant in P. menziesii seedlings grown in Interface or native forest soils, d) Mycorrhizal community differences between treatments will alter host fitness (growth and nutritional parameters). Seedlings from Nothofagus dombeyi, N. antarctica, Lophozonia alpina, L. obliqua and Pseudotsuga menziesii were set up in a soil-bioassay that included soils from non-invaded Nothofagaceae forests, pure P. menziesii plantations, and the interface between both. Pseudotsuga menziesii seedlings showed a decreasing, although never null, ectomycorrhizal (EM) colonization pattern from plantations to non-invaded forests, mainly with exotic EM species. Hebeloma mesophaeum and Wilcoxina sp. 1, two EM species with cosmopolitan distribution, were found to be shared by both tree species. Hebeloma hiemale and Wilcoxina sp. 1, common mycorrhizal partners of P. menziesii in Patagonia although not registered from Nothofagaceae forest, were found to be associated with N. antarctica, being the first report for both fungal species. Pseudotsuga menziesii seedlings showed the ability to form different arbuscular mycorrhiza (AM) colonization types (Paris-, Arum-, Both- and Intermediate-types) depending on the treatments, with significantly higher presence of Intermediate-type in the Interface treatment, where colonization was low. The shared EM species and the presence of different AM colonization types imply enhanced possibilities for invasive P. menziesii seedlings establishment and development. Seedling features and EM colonization rates evidenced that P. menziesii invasion could produce maladaptation (defined as a relative decline in host fitness due to altered mycorrhizal communities from native settings) of mycorrhizal communities, seriously injuring native ecosystem.     

A Tea Hydroperoxide Lyase Gene,CsiHPL1, Regulates Tomato Defense Response Against Prodenia Litura (Fabricius) and Alternaria Alternata f. sp. Lycopersici by Modulating Green Leaf Volatiles (GLVs) Release and Jasmonic Acid (JA) Gene Expression

Hydroperoxide lyases (HPLs) play important roles in modulating plant defense by regulating the release of green leaf volatiles (GLVs) and the jasmonic acid (JA) pathway. CsiHPL1—a chloroplast-localized tea gene that encodes HPL—was previously cloned and predicted to be a regulator of plant defense responses. CsiHPL1 was expressed constitutively in transgenic tomato (Solanum lycopersicum) plants to define its function in plant defense. CsiHPL1 overexpression caused tomato to release more constitutive and wound-induced GLVs [including (Z)-hexenal and (Z)-3-hexen-1-ol]. CsiHPL1 transgenic lines also exhibited lower levels of resistance to the larva of the tomato-chewing herbivore Prodenia litura (Fabricius) but enhanced resistance to the necrotrophic fungus Alternaria alternata f. sp. lycopersici (AAL). Furthermore, transgenic lines exhibited decreased expression levels of JA-related genes (SlAOS and SlPI-II) induced by P. litura and AAL infection. We thus concluded that constitutive expression of CsiHPL1 can regulate tomato resistance to P. litura and AAL by modulating GLV release and JA gene expression. Application of these results will be helpful in controlling plant defenses against herbivore attack and fungal disease.     

The trans-generational phase accumulation in the desert locust: Morphometric changes and extra molting

To understand the underlying trans-generational phase accumulation, a classical morphometric characteristic, the F/C ratio (F, hind femur length; C, maximum head width), of adult desert locusts (Schistocerca gregaria) was monitored over eight consecutive generations. Adult F/C ratios, which are larger in solitarious locusts than in gregarious ones, were negatively correlated to the darkness of body color at hatching. Two successive generations were required for a complete shift from the gregarious (crowd-reared) to the solitarious (isolated-reared) phase and vice versa in the laboratory. That is (1) female adults needed to be exposed to crowded (or isolated) conditions so that their hatchlings would become large (or small) and dark (or green) in color, and (2) the hatchlings then needed to be exposed to crowded (or isolated) conditions for their entire nymphal stage. Solitarious locusts exhibited extra molting that influenced the F/C ratio in the adult stage, but did not exert significant influences on the trans-generational changes in this trait because the incidence was low. The incidence of extra molting was negatively correlated with nymphal survival rates. The morphometric trans-generational changes may be explained without assuming any accumulating internal factor.     

Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran

Leishmania major is the causative agent and Phlebotomus papatasi is the main vector of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran and elsewhere. Nested PCR protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene) in female P. papatasi. In the current investigation, L. major was found in Natanz, Isfahan province in centre of Iran, in a focus of rural ZCL. Ten (1.8%) out of 549 female P. papatasi was found to be infected with L. major based on the PCR detection and sequencing of parasite ITS-rDNA.Nine (1.8%) out of 498 female P. papatasi infected with L. major came from animal shelters, inside houses and yards. And one (1.9%) out of 51 female P. papatasi infected with L. major came from gerbil borrows. Infection rates were higher for females containing red blood meals, large eggs (semi-mature and mature) than for those without either blood meals or eggs. From the 10 infections detected three different haplotypes of L. major were identified. Two haplotypes were found to be novel. The other haplotypes of L. major was found to be identical to that of isolates of L. major from Iran and in elsewhere using GenBank data. Comparisons of infection rates between habitats will be inaccurate when the proportions of blood-fed and gravid flies differ among sandfly samples.     

Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum

The use of lacZ from Thermoanaerobacterium thermosulfurigenes (encoding β-galactosidase) and lucB from Photinus pyralis (encoding luciferase) as reporter genes in Clostridium acetobutylicum was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of lacZ revealed a sequence error in the original database entry, which resulted in β-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The lacZ reporter data showed that bdhA (encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by sol (encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and bdhB (encoding butanol dehydrogenase B) expression. adc (encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between adc and bdhB (higher) and bdhA and the sol operon (lower). The lucB reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data). lucB reporter measurements confirmed the expression pattern of the sol and ptb-buk (encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the lacZ reporter and showed that the expression level from the ptb promoter is 59-fold higher than that from the sol operon promoter.     

Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle

The aim of this study was to investigate the prevalence and transferability of resistance in tetracycline-resistant Escherichia coli isolates recovered from beef cattle in South Korea. A total of 155 E. coli isolates were collected from feces in South Korea, and 146 were confirmed to be resistant to tetracycline. The tetracycline resistance gene tet(A) (46.5%) was the most prevalent, followed by tet(B) (45.1%) and tet(C) (5.8%). Strains carrying tet(A) plus tet(B) and tet(B) plus tet(C) were detected in two isolates each. In terms of phylogenetic grouping, 101 (65.2%) isolates were classified as phylogenetic group B1, followed in decreasing order by D (17.4%), A (14.2%), and B2 (3.2%). Ninety-one (62.3%) isolates were determined to be multidrug resistant by the disk diffusion method. MIC testing using the principal tetracyclines, namely, tetracycline, chlortetracycline, oxytetracycline, doxycycline, and minocycline, revealed that isolates carrying tet(B) had higher MIC values than isolates carrying tet(A). Conjugation assays showed that 121 (82.9%) isolates could transfer a tetracycline resistance gene to a recipient via the IncFIB replicon (65.1%). This study suggests that the high prevalence of tetracycline-resistant E. coli isolates in beef cattle is due to the transferability of tetracycline resistance genes between E. coli populations which have survived the selective pressure caused by the use of antimicrobial agents.     

Molecular Identification of Sibling Species of Sclerodermus (Hymenoptera: Bethylidae) That Parasitize Buprestid and Cerambycid Beetles by Using Partial Sequences of Mitochondrial DNA Cytochrome Oxidase Subunit 1 and 28S Ribosomal RNA Gene

The species belonging to Sclerodermus (Hymenoptera: Bethylidae) are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI) and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1–5). A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances) between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5) averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ) tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1–4) clustered together and only Sclerodermus sp. (No. 5) clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5) might be a new species of Sclerodermus.     

Novel amide alkaloids from the roots of Piper guineense

Two novel amide alkaloids, wisanine and wisanidine, have been isolated from the petroleum-extract of the roots of Piper guineense, and found to be N-piperidyl-5 (2-methoxy-4,5-methylenedioxyphenyl)-trans-2-trans-4-pentadienamide and N-pyrrolidyl-5-(2-methoxy-4, 5-methylenedioxyphenyl)-trans-2-trans-4-pentadienamide respectively. The structure of wisanidine has been confirmed by synthesis. N-Isobutyl)-trans-2-trans-4-eicosadienamide, recently reported to be present in the fruits of the plant as well as Piperine and Δ^α,β-dihydropiperine have also been found to be major constituents of the roots.     

Profundulus kreiseri,a new species of Profundulidae (Teleostei,Cyprinodontiformes) from northwestern?Honduras

A new species of Profundulus, Profundulus kreiseri (Cyprinodontiformes: Profundulidae), is described from the Chamelecón and Ulúa Rivers in the northwestern Honduran highlands. Based on a phylogenetic analysis using cytochrome b and the presence of synapomorphic characters (dark humeral spot, a scaled preorbital region and between 32-34 vertebrae), this new species is placed in the subgenus Profundulus, which also includes Profundulus (Profundulus) oaxacae, Profundulus (Profundulus) punctatus and Profundulus (Profundulus) guatemalensis. Profundulus kreiseri can be distinguished from other members of the subgenus Profundulus by having less than half of its caudal fin densely scaled. Profundulus kreiseri can further be differentiated from Profundulus (Profundulus) oaxacae and Profundulus (Profundulus) punctatus by the absence of rows of dark spots on its flanks. The new species can further be differentiated from Profundulus (Profundulus) guatemalensis by the presence of fewer caudal- and pectoral-fin rays. The new species is distinguished from congeners of the profundulid subgenus Tlaloc (viz., Profundulus (Tlaloc) hildebrandi, Profundulus (Tlaloc) labialis, Profundulus (Tlaloc) candalarius and Profundulus (Tlaloc) portillorum) by having a scaled preorbital region and a dark humeral spot. Profundulus kreiseri and Profundulus portillorum are the only two species of Profundulus that are endemic to the region south of the Motagua River drainage in southern Guatemala and northwestern Honduras.