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Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease worldwide, causing severe ecological damage and economic losses to forestry. Two forms of this nematode have been reported, i.e., with strong and weak virulence, commonly referred as virulent and avirulent strains. However, the pathogenicity-related genes of B. xylophilus are not sufficiently characterized. In this study, to find pathogenesis related genes we re-sequenced and compared genomes of two virulent and two avirulent populations. We identified genes affected by genomic variation, and functional annotation of those genes indicated that some of them might play potential roles in pathogenesis. The performed analysis showed that both avirulent populations differed from the virulent ones by 1576 genes with high impact variants. Demonstration of genetic differences between virulent and avirulent strains will provide effective methods to distinguish these two nematode virulence forms at the molecular level. The reported results provide basic information that can facilitate development of a better diagnosis for B. xylophilus isolates/strains which present different levels of virulence and better understanding of the molecular mechanism involved in the development of the PWD. Electronic supplementary material The online version of this article (10.1007/s00438-020-01739-w) contains supplementary material, which is available to authorized users.

Arion vulgaris Moquin-Tandon, 1855 is regarded as one of the 100 most invasive species in Europe. The native distribution range of this species is uncertain, but for many years, the Iberian Peninsula has been considered as the area of origin. However, recent studies indicate that A. vulgaris probably originated from France. We have investigated the genetic structure of 33 European populations (Poland, Norway, Germany, France, Denmark, Switzerland) of this slug, based on two molecular markers, mitochondrial cytochrome c oxidase subunit I (COI, mtDNA) and nuclear zinc finger (ZF, nDNA). Our investigation included published data from two previous studies, giving a total of 95 populations of A. vulgaris from 26 countries. This comprehensive dataset shows comparable haplotype diversity in Central, North and Western Europe, and significantly lower haplotype diversity in the East. All haplotypes observed in the East can be found in the other regions, and haplotype diversity is highest in the Central and Western region. Moreover, there is strong isolation by distance in Central and Western Europe, and only very little in the East. Furthermore, the number of unique haplotypes was highest in France. This pattern strongly suggests that A. vulgaris has originated from a region spanning from France to Western Germany; hence, the slug is probably alien/invasive in other parts of Europe, where it occurs. Our results indicate the necessity to cover as much of the distribution range of a species as possible before making conclusive assumptions about its origin and alien status.

The quarantine nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease in Asia and Europe. Differentiation between B. xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. In this paper we described the results of the optimized multiplex real-time PCR protocol that utilizes two universal primers and three specific TaqMan probes to identify and differentiate the three Bursaphelenchus species, simultaneously. Species-specific real-time PCR reactions gave expected products for B. xylophilus, B. mucronatus and B. fraudulentus templates. The optimized primer combination together with designed species-specific probes has produced reliable results in multiplex PCR assays with eleven geographically distant isolates of B. xylophilus, eleven of B. mucronatus, seven of B. fraudulentus, and no cross-reactions were observed between tested samples and with other species (i.e., B. piniperdae, B. pinophilus, B. populi and Parasitaphelenchus papillatus). All species-specific real-time PCR reactions performed on DNA extracted from a single nematode individual consistently gave specific amplicons, confirming the reproducibility of the method. Moreover, the conducted study revealed that 30 fg was the lowest DNA input still detected in multiplex real-time PCR analysis. The described approach is simple, time-saving, and reliable in comparison to conventional PCR, and it can be used for simultaneous identification of the above three species within the xylophilus group.

Niniejszy artykuł poświęcono analizie związków frazeologicznych wykorzystanych przez Linę Kostenko w poetyckim tomie Триста поезій. Opisano fenomen osobowości językowej, wskazując na fakt, iż Lina Kostenko bez wątpienia należy do jednej z najbardziej jaskrawych osobowości językowych w literaturze ukraińskiej. Wspomniane frazeologizmy poddano analizie, uwzględniając ich podział na wyrażenia słownikowe, autorskie oraz aforyzmy. Zaproponowano właściwe tendencje, a także określono perspektywę badań.

Summary Bursaphelenchus michalskii sp. n. is described from the bark of the European white elm, Ulmus laevis. All propagative stages of the nematode were found in larval galleries of the large elm bark beetle, Scolytus scolytus, and in overlapping gallery systems of this species and the small European elm bark beetle, S. multistriatus. Dauer juveniles of the new nematode are transmitted to new breeding trees under elytra of adult S. scolytus. Bursaphelenchus michalskii sp. n. is characterised by the female body length of 953 (838-1108) μm and male body length of 893 (811-971) μm, very slender body (a = 53.9 (46.1-58.5) and 60.9 (52.2-72.0) in female and male, respectively), lateral fields with three incisures (two bands), excretory pore usually located anterior to the median bulb, lack of vulval flap, long post-uterine sac, relatively small spicules 12.3 (10.8-13.3) μm long with no cucullus and with distinct, somewhat thorn-like, dorsally bent or reflexed condylus and a conical or digitate rostrum, and the arrangement of the seven male caudal papillae (i.e., a single precloacal ventromedian papilla (P1), one pair of adcloacal ventrosublateral papillae (P2) at or just anterior to cloacal slit, one ventrosublateral, postcloacal pair (P3) located at ca 60% of the tail length, posterior to cloacal slit, and one pair (P4) of ventrosublateral papillae located near the base of the bursa). The newly described species shares most of the key morphological characters with members of the eremus-group (sensu Braasch et al., 2009). However, B. michalskii sp. n. is unique amongst Bursaphelenchus species by a combination of female tail and spicule shape, excretory pore position, and other morphometric characters. These findings were confirmed by DNA sequencing and phylogenetic analysis of the 18S and 28S rDNA regions and by the unique molecular profile of the ITS region (ITS-RFLP).

Summary The first spontaneous morphological/anatomical and behavioural mutation Bxy-rol(tom3) has been identified and characterised in the pine wood nematode, Bursaphelenchus xylophilus. The mutation is recessive to its wild-type counterpart. The mutant phenotype designated as Roller (Rol) is distinctive for the helical twist of the whole body around its longitudinal axis. The twist of the nematode body wall cuticle is followed by a similar twist of hypodermis, muscles and other organs and cuticular structures. These morphological and anatomical changes are responsible for the altered pattern of the nematode movement. Besides these changes, the Bxy-rol(tom3) mutation does not visibly affect any other essential character or function of B. xylophilus. Both wild-type and Rol nematodes present similar morphometrics, life cycle length, and reproductive potential. The Rol mutant easily cross-bred with individuals from any other tested strains/isolates of B. xylophilus and produced viable intra-specific hybrid offspring in vitro, on B. cinerea/potato dextrose agar plates. The mutants also seem to be well adapted to the nematode’s natural environment in pine wood. They dispersed inside pine seedlings, successfully mated, produced stable and fully viable offspring in this environment, and were as pathogenic to the host tree as the wild-type B. xylophilus. Potential of the characterised mutation as a marker for direct comparative study on strain/species reproductive compatibility, competition for space, food and vector insect, and for intra- and inter-specific hybridisation of B. xylophilus and B. mucronatus, is discussed.

Isoquinoline alkaloids (papaverine, noscapine, berberine, emetine, and quinine) were determined in medicinal plants and herbs used in Traditional Chinese Medicine and Ayurveda by liquid chromatogra...

Hydrophobic polymeric (0.45 and 0.20 μm polytetrafluoroethylene (PTFE) and 0.10 μm polypropylene (PP)) membranes were efficiently applied for cactus pear juice and sugars solutions concentration using Osmotic Membrane Distillation (OMD) process. It is reported that the PTFE membranes gave higher permeate fluxes and higher final juice concentrations. Furthermore, the filtration of raw juice prior to OMD process, the extension of the process time and the increase of processing temperature resulted in the increase of the final juice concentration. Using 0.45 μm PTFE membrane, the final concentration of the filtered juice (FJ) was 16.4 °Brix ± 0.1 (at 20 °C, after 5 h) and 23.4 °Brix ± 0.2 (at 35 °C, after 18 h). The juice quality was assessed by determining the juice viscosity, the total phenolic content, total flavonoids content, antioxidant activity and phenolic acids content. It was revealed that the physicochemical properties were preserved in the concentrated juice.

Differentiation between Bursaphelenchus xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. We report the results of a multiplex PCR that utilises four primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex PCR yielded DNA fragments of 767, 305 and 132 bp, for B. xylophilus, B. mucronatus and B. fraudulentus, respectively. This primer combination has produced reliable results in multiplex PCR assays with a number of different populations of the listed species, and no cross-reactions were observed with other Bursaphelenchus species. The described approach is simple, reliable and cheaper than other molecular methods presently used for simultaneous identification of the above three species within the xylophilus group.

Mycotoxins and selected hazardous alkaloids in the medicinal plants (Panax ginseng, Angelica sinensis, and Withania somnifera) and dietary supplements were determined. Purine alkaloids were found in majority of samples; however, isoquinoline alkaloids were less abundant than indole. The predominant alkaloids appear to be caffeine (purine group), harman (indole group) and berberine (isoquinoline). Examined medicinal plants and dietary supplements were contaminated by mycotoxins (especially ochratoxin A 1.72–5.83 µg kg−1), and many species of mold (e.g. Cladosporium, Eurotium, Aspergillus, Rhizopus, Penicillium). MTT cytotoxicity tests revealed that plant and supplements extracts exhibited medium or high cytotoxicity (only Dong quai—low). Moreover, antioxidant activity, total phenolics content and selected phytochemicals were analyzed by spectrophotometric and chromatographic methods. Quercetin and rutin were predominant flavonols (1.94-9.51 and 2.20–7.28 mg 100 g−1, respectively). Analysis of phenolic acids revealed—gallic acid, as the most abundant, except Panax ginseng, where ferulic acid was prevailing. The results were analyzed by chemometric methods (cluster analysis, ANOVA).

Anti-nutritional components such as: oxalates, thiocyanates and phytic acid were determined in 20 plants samples originated from Traditional Chinese Medicine and Ayurveda (e.g., Andrographis paniculata, Angelica sinensis, Emblica officinalis, Withania somnifera, Polygonum multiflorum, Bacopa monnieri, Pueraria lobata, Cola accuminata, Curcuma longa, Ocimum sanctum, Mucuna pruriens, Garcinia cambogia, Panax ginseng, Bupleurum sinensis, Salviae miltiorrhizae, Schisandra sinensis, Terminalia arjuna). Extraction and analysis procedures for these anti-nutritional components determination were modified and optimized. Water-soluble oxalates, thiocyanates and phytic acid were simultaneously determined by new procedure with ion chromatography method, resulting in analyzed samples: 0.38–145.39 mg 100 g−1 dry mass (d.m.) for oxalates, 0.94–34.34 mg 100 g−1 d.m. for thiocyanates, whereas 0.42–16.13 mg 100 g−1 d.m. for phytic acid. The highest of the total content of anti-nutritional components was observed for the O. sanctum, but the lowest for C. longa. Moreover, oxalates and phytic acid were determined by titration, whereas thiocyanates by spectrophotometric method.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD). This nematode is considered to be an indigenous to North America and was introduced to Japan in the late 19th century. Subsequently, it has spread throughout Japan and in many other countries, China, Taiwan, and South Korea. In 1999, B. xylophilus was discovered in Portugal, and in 2008 in Spain. So far the studies have revealed that the pathogenicity of B. xylophilus varies between different isolates. The conducted study compared the pathogenicity of five isolates of B. xylophilus, originating from different parts of Japan, to 3-year-old Pinus sylvestris, and their ability to reproduce in the seedlings. The results revealed diverse virulence of B. xylophilus resulting in plant mortality. Three isolates S10, Ka4, and T4 caused 100% mortality of plants within three months while at the same time, the other two isolates, C14-5 and OKD-1 did not cause any disease symptoms on plants. After seven months, some dieback occurred on two seedlings, but similar symptoms were also found on the control plant. Moreover, a significant positive correlation was found between nematode virulence and the number of nematodes reproducing on pine seedlings.

Selected plants used in Traditional Chinese Medicine (Garcinia cambogia, Emblica officinalis, Angelica sinensis, Schisandra sinensis, Scutellaria baicalensis, Salvia miltiorrhiza, and Ocimum sanctum), European herbs (Echinacea purpurea, Cichorium intybus, and Vitis vinifera), and supplements derived from these products were analyzed for their macroelements (K, Na, Ca, and Mg) and microelements (Zn, Cu, Cr, Se, Mn, Mo, and Fe) concentrations by wavelength-dispersion x-ray fluorescence (WD-XRF), scanning electron microscopy–energy dispersive x-ray spectrometry (SEM–EDX), ion chromatography (IC), and inductively coupled plasma–mass spectrometry (ICP–MS). In addition, relevant aspects of heavy metal toxicity are discussed.

The potentially toxic metals content was determined in selected plants, used in Traditional Chinese Medicine (Angelica sinensis, Bacopa monnieri, Bupleurum sinensis, Curcuma longa, Cola accuminata, Emblica officinalis, Garcinia cambogia, Mucuna pruriens, Ocimum sanctum, Panax ginseng, Pueraria lobata, Salvia miltiorrhiza, Schisandra sinensis, Scutellaria baicalensis, Siraitia grosvenorii, Terminalia arjuna and Terminalia chebula), and some European herbs (Echinacea purpurea, Hypericum perforatum, Vitis vinifera). Samples were mineralized in a closed microwave system using HNO3 and the concentrations of Cd, Pb, Al, As, Ba, Ni and Sb were determined by ICP-MS method. Some relevant aspects of potential toxicity of metallic elements and their compounds were also discussed. Results of metal content analysis in dietary supplements available on Polish market, containing studied plants, are presented as well. The results were analyzed by principal component analysis (PCA) and cluster analysis.

Bursaphelenchus fagisp. n. is described from the bark of European beech,Fagus silvatica. All propagative stages of the nematode are numerous in larval galleries of the beech bark beetle,Taphrorychus bicolor, while dauer juveniles aggregate in Malpighian tubules of adult beetles. The new species is characterised by the body length of 871 (763-1110) μm in female and 852 (718-992) μm in male, very slender body (a = 56.8 (50.4-67.1) and 64.5 (56.5-73.8) in female and male, respectively), and spicules 15.2 (14.0-17.0) μm long. The extended anterior vulval lip in female, lateral fields with four incisures, and number (7) and arrangement of male caudal papillae may indicate thatB. fagisp. n. is closely related to thexylophilusgroup. It differs from this group by the relatively small, claw-like spicules with narrow capitulum, indistinct condylus in almost continuous line with dorsal lamina, and lack of distinct cucullus at the spicule tip. The close relation ofB. fagisp. n. with thexylophilusgroup has been confirmed by DNA sequencing and phylogenetic analysis of the 28S rDNA region.Bursaphelenchus fagisp. n. most closely resemblesB. tokyoensisandB. idius, but can be separated by differences in the arrangement of the male caudal papillae, a unique feature for each species. The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). The mass presence of dauer juveniles ofB. fagisp. n. in Malpighian tubules of adultT. bicolorleads to degradation of the tubule cellular epithelium and local expansion of its basement membrane. These changes clearly indicate direct pathogenicity of the nematode to its vector insect. In laboratory rearing,B. fagisp. n. can grow and reproduce onBotryotinia fuckelianacultures.

The procedure involving water and water-methanol extraction, RP-HPLC-C18 column chromatography with PDA detection was developed for determination of cinnamic acid and benzoic acid derivatives in grapevine’s dietary supplements (LV, RW, VIN, VIC, and DK) available on the Polish market. Phenolic acids were analysed before and after acidic and basic hydrolysis and identified against standards. Totalamount of studied phenolic acids determined by HPLC-PDA was compared with total polyphenols content (TPC) by Folin-Ciocalteu method. The average content of studied phenolic acids (70.54±0.21; 122.95±0.49; 87.67±0.10; 132.21±0.24; 266.78 ±0.39, and 18.16±0.09 mg/100 g d.m. (dry mass) for LV, RW, VIN, VIC, DK, and WW, respectively) were higher than the TPC (1489.91±0.39, 1648.19±0.14, 1574.38±0.33, 1643.64±0.12, 1984.75±0.97, and 715.55±0.36 mg/100 g d.m. for LV, RW, VIN, VIC, DK, and WW, respectively). The new developed method was validated for specifi city, repeatability, and accuracy and can be suitable for routi...

The capillary isotachophoretic method was optimized and used for histidine determination in food samples. The optimum conditions for histidine separation and determination were found on the experimental conditions such as: selectivity, separation speed, pH, concentration of the leading and terminating electrolytes, and electroosmotic flow additives. The optimum electrolytes composition [leading electrolyte: 7 mM NH4OH + 15 mM 2-(N-morpholino)ethanesulfonic acid + 1% hydroxyethylcellulose; pH = 6.10 and terminating electrolyte: 15 mM aminocaproic acid +5 mM acetic acid +40% methanol; pH = 5.10] and conditions of analysis were adopted for histidine determination in food samples (meat and fish products). The proposed electrolyte system was characterized by linearity (10–100 and 100–430 mg · L−1 with R2 = 0.9976 and 0.9991), accuracy (99.5% and 98%), intra-assay of the relative step height (1.40% for standard and 3.20% for food samples analysis), inter-assay of the relative step height (3.65% and 6.30%) and s...

Bursaphelenchus masseyi sp. n. is described from trunks of unhealthy trembling aspen, Populus tremuloides, affected by sudden aspen decline (SAD) in Colorado, USA. All propagative stages of the nematode were present in larval galleries of a bark beetle, Trypophloeus populi. The dauer juveniles occupy the haemocoel of older larvae, pupae and adult beetles. The characteristic morphology of the male spicules with small but distinct cucullus, extended anterior vulval lip in female, lateral fields with four incisures, and number and arrangement of male caudal papillae, indicate that B. masseyi sp. n. is closely related to the xylophilus group. This relation has been confirmed by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The new species is characterised by the body length of 958 (765-1203) μm in female and 874 (691-1122) μm in male, moderately slender body (a = 38.2 (33.5-41.1) and 38.0 (33.1-44.1) in female and male, respectively), and spicules 29.2 (26.2-34.7) μm long with a small cucullus (1.3-1.5 μm in diam.) at their tips. Bursaphelenchus masseyi sp. n. can be separated from other species in the xylophilus group by the morphology of spicules which have a short capitulum and unique rostrum that is pointed somewhat anteriorly, relatively thick vulval flap, which is straight, parallel to the body long axis and bent towards the body wall at its distal end, and other morphological and morphometric characters. The new species most closely resembles B. trypophloei, but differs by the morphology of spicules (short but distinct condylus vs condylus in a continuous line with dorsal lamina, and smaller cucullus). The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). Diallelic cross-breeding in vitro revealed also that B. masseyi sp. n. and B. trypophloei are reproductively incompatible. The new species showed poor ability to develop and reproduce on Botrytis cinerea cultures, although it grew vigorously on laboratory cultures of Polish and Colorado isolates of Cytospora chrysosperma, the fungus naturally associated with galleries of the nematode vector, T. populi.

Mycotoxins and selected hazardous alkaloids in the medicinal plants (Panax ginseng, Angelica sinensis, and Withania somnifera) and dietary supplements were determined. Purine alkaloids were found in majority of samples; however, isoquinoline alkaloids were less abundant than indole. The predominant alkaloids appear to be caffeine (purine group), harman (indole group) and berberine (isoquinoline). Examined medicinal plants and dietary supplements were contaminated by mycotoxins (especially ochratoxin A 1.72–5.83 µg kg−1), and many species of mold (e.g. Cladosporium, Eurotium, Aspergillus, Rhizopus, Penicillium). MTT cytotoxicity tests revealed that plant and supplements extracts exhibited medium or high cytotoxicity (only Dong quai—low). Moreover, antioxidant activity, total phenolics content and selected phytochemicals were analyzed by spectrophotometric and chromatographic methods. Quercetin and rutin were predominant flavonols (1.94-9.51 and 2.20–7.28 mg 100 g−1, respectively). Analysis of phenolic acids revealed—gallic acid, as the most abundant, except Panax ginseng, where ferulic acid was prevailing. The results were analyzed by chemometric methods (cluster analysis, ANOVA).

Volatile organic compounds (VOCs) released from or taken up by Streptococcus pneumoniae and Haemophilus influenzae cultures were analysed by means of GC-MS after adsorption of headspace samples on multi-bed sorption tubes. Sampling was performed at different time points during cultivation of bacteria to follow the dynamics of VOC metabolism. VOCs were identified not only by spectral library match but also based on retention times of native standards. As many as 34 volatile metabolites were released from S. pneumoniae and 28 from H. influenzae, comprising alcohols, aldehydes, esters, hydrocarbons, ketones and sulfur-containing compounds. For both species, acetic acid, acetaldehyde, methyl methacrylate, 2,3-butanedione and methanethiol were found in strongly elevated concentrations and 1-butanol and butanal in moderately elevated concentrations. In addition, characteristic volatile biomarkers were detected for both bacterial species and exclusively for S. pneumoniae, also catabolism of aldehydes (3-methylbutanal and hexanal) was found. The results obtained provide important input into the knowledge about volatile bacterial biomarkers, which may become particularly important for detection of pathogens in upper airways by breath-gas analysis in the future.

Phenolic acids and derivatives of quercetin in Kudzu root were determined by high-performance liquid chromatography with diode array detector. The calibration curves were constructed using phenolic compounds standards (the coefficient of determination (R2) was 0.9990–0.9997 for phenolic acids and 0.9989–0.9994 for flavonols. respectively). Ethanol was the most efficient solvent in extracting flavonols and phenolic acids from dried Kudzu roots at the optimal extraction time 60 min. Due to acidic hydrolysis the content of free phenolic acids was increasing. The predominant phenolic acid in Kudzu root determined by HPLC-PDA method with hydrolysis digestion is p-hydrozybenzoic acid (39.57 ± 1.44 mg/g d.m.), next one was: p-coumaric acid (6.20 ± 0.11 mg/g d.m.), followed by ferulic acid (11.52 ± 0.21 mg/g d.m.).On the other hand, the highest content of flavonols was found for routine (6.13 ± 0.11 mg/g d.m.). The contents of the other flavonols in kudzu root are between 0.73 – 2.52 mg/g d.m. for Rh and Q.

Nematology , 2010, Vol. 12(1), 157-160 Short communication Species-specific polymerase chain reaction primers for simple detection of Bursaphelenchus fraudulentus (Nematoda: Parasitaphelenchidae) Anna F I L I P I A K 1 , ∗ , Agata J AKUBOW SKA 1 , 2 and Marek T OMALAK 1 Bursaphelenchus fraudulentus Ruhm, 1956 (Nema- toda: Parasitaphelenchidae) is a tree-inhabiting species frequently found in Europe (Ruhm, 1956; Schauer-Blume & Sturhan, 1989; Tomalak, 2004; Carletti et al ., 2005) and Asia (Braasch et al ., 2001). It is primarily found in dy- ing or dead deciduous trees (Ruhm, 1956; Schauer-Blume & Sturhan, 1989), although occasionally reported from conifers (Braasch et al ., 2001). Within the genus the spe- cific morphological characters (i.e. , vulval flap, shape of spicules, position of caudal papillae and presence of four incisures in the lateral fields) place B. fraudulentus into a distinct xylophilus -group which comprises eight other, morphologically similar, species, including the quarantine pest B. xylophilus (Gu et al ., 2008). Reliable methods of taxonomic identification of these species are therefore of particular interest to plant quarantine services. Several molecular techniques have been developed and used for identification of Bursaphelenchus spp. (Wang et al. , 1999; Matsunaga & Togashi, 2004; Burgermeister et al ., 2005; Castagnone et al. , 2005; Leal et al ., 2005, 2007) with ITS-RLFP analysis the most widely used in research and quarantine practice. An alternative method, based on a simple PCR amplification with primers specific for B. xylophilus and B. mucronatus (Matsunaga & Togashi, 2004), enables rapid and precise species identification, even from a single nematode (Filipiak et al ., 2007). Both native European species from the xylophilus - group, i.e. , B. mucronatus and B. fraudulentus , are rela- tively common and harmless to trees. They can be mor- phologically difficult to distinguish from each other and from the quarantine pest B. xylophilus. The provision of PCR primers specific for the DNA of B. fraudulentus , in addition to those already existing for B. xylophilus and B. 1 Department of Biological Pest Control and Quarantine, Institute of Plant Protection, Wladyslawa Wegorka 20, Poznan, 60-318, Poland 2 Department of Genetics, University of Valencia, Dr. Moliner 50, 46-100, Burjassot, Spain ∗ Corresponding author, e-mail: A.Filipiak@ior.poznan.pl Received: 22 October 2008; revised: 26 June 2009 Accepted for publication: 29 June 2009 Keywords: Bursaphelenchus xylophilus -group, diagnostics, molecular. mucronatus (Matsunaga & Togashi, 2004), enhances the utility of this method. The main objectives of our research were to sequence the ITS regions of the B. fraudulentus genome and to design specific primers for PCR amplifica- tion. Eight isolates of B. fraudulentus , two of B. mucrona- tus and one of B. xylophilus were examined. The Aus- trian (Osterreich), Russian (DE10w), German (Helmstedt and H26), and Hungarian (Ungarn) isolates of B. fraudu- lentus and the isolate of B. xylophilus (China) were ob- tained from the nematode collection of the Federal Re- search Centre for Cultivated Plants (formerly Federal Bi- ological Research Centre for Agriculture and Forestry – BBA), Braunschweig, Germany (Dr T. Schroder). All the remaining nematode populations were isolated from in- fested trees in Poland. The populations of B. fraudulentus originated from Poznan (PL-01 and PL-05) and Kornik (PL-04) and those of B. mucronatus from Nowy Tomysl (NTo-01) and Slawa Slaska (SlS-01). Their taxonomic identification was originally based on morphological and morphometric characteristics and subsequently confirmed by ITS-RLFP analysis according to the protocol described by Burgermeister et al. (2005). Prior to examination, all isolates were reared on Botrytis cinerea /malt agar (4.5%) at 25 ◦ C for ca 2 weeks. Propagated nematodes were col- lected by the Baermann funnel method and stored in 10 μ l H 2 O at − 20 ◦ C until use. Extraction of DNA was performed according to the method described by Iwahori et al. (1998), with mi- nor modifications to the composition of the lysis buffer: 100 mM Tris, pH 8.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, 1% β -mercaptoethanol, 100 μ g ml − 1 proteinase K per 100 μ l buffer. Before incubation, the mixture was © Koninklijke Brill NV, Leiden, 2010 DOI:10.1163/138855409X12469543767319 Also available online - www.brill.nl/nemy 157

Bursaphelenchus populi sp. n. is described from dying and dead aspen trees, Populus tremula, in Poland. The nematode was found in trees infested with a long-horn beetle, Saperda perforata, and is vectored in the insect haemocoel. The characteristic morphology of male spicules, extended anterior vulval lip, lateral fields with four incisures, and number and arrangement of male caudal papillae, place B. populi sp. n. in the xylophilus group. Bursaphelenchus populi sp. n. can be separated from all other species in that group by the distinctive vulval flap, which is always bent with its distal half sunken in a conspicuous, sharp depression posterior to the vulva, and other morphological and morphometric characters, i.e., female tail shape, excretory pore position, spicule length (32.1 (25.7-37.0) μm (as measured along arc) and shape, and a relatively long (i.e., L,= 1020 (909-1111) μm in females), and L = 850 (756-1055) μm in males), and slender body (a = 45.4 (40.2-52.4) and 42.0 (36.1-49.3) in female and male, respectively). The status of the new species is confirmed by the unique molecular profile of the ITS region (ITS-RFLP). DNA sequencing and phylogenetic analysis of 28S rDNA region placed the new species close to B. fraudulentus in the xylophilus group. In vitro cross-breeding of the new nematode with B. xylophilus, B. mucronatus, B. fraudulentus and B. doui revealed full reproductive incompatibility between these species. In laboratory experiments on 2-year-old seedlings B. populi sp. n. was specific to aspen. It did not develop on pine or oak and reproduced only in weakened or dead aspen seedlings. No pathogenicity to the tree host or vector insect was observed. In Botrytis cinerea PDA cultures, body dimensions were significantly altered in both sexes and female tail morphology differed when compared to individuals extracted from aspen wood.

A few extraction methods for the reverse-phase high performance liquid chromatography (RP-HPLC) determination of alkylphenolic compounds in fish tissues were examined. The best results of the isolation of 4-nonylphenol (4-n-NP) and 4-tert-octylphenol (4-t-OP) from Rainbow trout were obtained using soxhlet extraction; the recoveries reached 64.64 ± 7.26% and 65.26 ± 5.29%, respectively. The subsequent solid phase extraction (SPE) cleanup process of fish extract was optimized using four different sorbents and the highest recoveries of the analytes were obtained for aminopropyl silica cartridges. Their biological concentration factors (BCF) expressing the ratio between the concentration in Rainbow trout and in water were determined for two temperatures, 15 and 20°C.

The real-time PCR-HRM analysis was developed for the detection and discrimination of the quarantine nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. A set of primers was designed to target the ITS region of rDNA. The results have demonstrated that this analysis is a valuable tool for differentiation of these both species.

Konwencja Narodów Zjednoczonych o umowach międzynarodowej sprzedaży towarów (zwana dalej CISG) przyjęta została w ponad 60 państwach, w tym w krajach wiodących w handlu zagranicznym. Dziś już, po ponad 20|latach od chwili jej sporządzenia, nie ma wątpliwości, że stanowi pierwszą udaną próbę unifikacji prawa w zakresie umowy sprzedaży w skali światowej. Z tego względu szczególnego znaczenia nabiera wymóg jednolitości stosowania i wykładni przepisów CISG (art. 7 zd. 1 CISG). W literaturze przedmiotu podkreśla się, że CISG jest autonomicznym aktem prawnym i powinna być interpretowana w sposób odrębny od prawa krajowego. Zasada autonomicznej wykładni konwencji wymaga z kolei, aby strony umów i sąd stosujący CISG uwzględniały m.in. interpretację CISG przyjmowaną w innych państwach. Niezbędne jest zatem rozpowszechnianie orzecznictwa wydawanego w materii regulowanej przez CISG. Cel ten osiągnąć można, analizując rozstrzygnięcia podejmowane przez sądy innych państw konwencyjnych. Przedmiotem niniejszego opracowania jest analiza przepisów CISG dotyczących ryzyka przypadkowej utraty lub uszkodzenia towaru (art. 66-70 CISG) na przykładzie orzeczenia sądu niemieckiego (Oberlandesgerichtshof Schleswig-Holstein) z dnia 29 października 2002 r., w którym poruszone zostały dwa zasadnicze zagadnienia dotyczące: I) rozdziału wspomnianego ryzyka w umowach sprzedaży z zastrzeżeniem warunku; 2) zakresu swobody stron umowy w ustalaniu reguł przejścia ryzyka. Omawiane orzeczenie daje asumpt do podjęcia głębszej analizy wpływu zastrzeżenia warunku i jego ziszczenia (nieziszczenia) się na rozkład ryzyka przypadkowej utraty lub uszkodzenia towaru, zwłaszcza że CISG nie rozstrzyga w sposób wyraźny powyższych kwestii, a w literaturze przedmiotu przedstawiane są w tym zakresie zróżnicowane stanowiska.

Antioxidant activity, total phenolics content and selected phytochemicals (alkaloids and andrographolides) were determined in Andrographis paniculata and in dietary supplements containing this plant. Antioxidant activity was measured by FRAP, CUPRAC and DPPH procedures and ranged from 503.36 to 6164.09μmol TE/100g d.m. depending on methods, part of plant and kind of dietary supplement. The total phenolics (175.13-1723.79mg GAE/100g) and andrographolides content (19.44-85.13mg/g) in the studied samples were correlated with antioxidant activities determined by CUPRAC, FRAP and DPPH (r>0.95, p

Existing methods for the early detection of infections in mechanically ventilated (MV) patients at intensive care units (ICUs) are unsatisfactory. Here we present an exploratory study assessing the feasibility of breath VOC analyses for the non-invasive detection of pathogens in the lower respiratory tract of ventilated patients. An open uncontrolled clinical pilot study was performed by enrolling 28 mechanically ventilated (MV) patients with severe intracranial disease, being at risk for the development of or already with confirmed ventilation-associated pneumonia (VAP). The recently developed sampling technique enabled the collection of breath gas with a maximized contribution of alveolar air directly from the respiratory circuit under continuous capnography control, adsorptive preconcentration and final analysis by means of gas chromatography-mass spectrometry (GC-MS).VAP was confirmed in 22/28 preselected patients (78%). The most common microorganisms were Staphylococcus aureus (5/22 VAP patients), Escherichia coli (5/22 VAP patients) and Candida spp. (5/22 VAP patients). 12/32 metabolites released by S. aureus in our previous in vitro studies were also detected in the end-tidal air of VAP patients infected with this pathogen. A similar overlap was seen in Candida albicans infections (8/29 VOCs). Moreover, the concentration profile of selected compounds correlated with the course of the infection.This prospective pilot study provides proof of the concept that the appearance and the concentration profile of pathogen-derived metabolites (elucidated from in vitro experiments) in the breath of ventilated patients during clinically confirmed VAP correlates with the presence of a particular pathogen.

Summary High Resolution Melting Analysis (PCR-HRM – Polymerase Chain Reaction-High Resolution Melting) is a recently developed technique for fast, high-throughput post-PCR analysis of genetic mutations or variance in sequences of nucleic acid. It enables researchers to rapidly detect and categorize genetic mutations, identify new genetic variants without sequencing, or determine the genetic variation in a population prior to sequencing. PCR-HRM technique was also used to detect and distinguish Bursaphelenchus xylophilus from morphologically and genetically similar nematode species B. mucronatus . In the research reported here PCR-HRM was conducted on the rDNA of B. xylophilus (China) and Polish isolates of B. mucronatus (Mdz-01, Rad-03 and Wro-01), with the use of primers specifically designed from the ITS-1 region of B. mucronatus . The conducted study revealed that PCR-HRM is an effective essay which allows one to distinguish of B. xylophilus from B. mucronatus . This technique is very sensitive and enables detection of differences even in single nucleotides. PCR-HRM technique is also much cheaper when compared to commonly used PCR-RFLP.

Breath analysis for the purpose of non-invasive diagnosis of lung cancer has yielded numerous candidate compounds with still questionable clinical relevance. To arrive at suitable volatile organic compounds our approach combined the analysis of different sources: isolated tumor samples compared to healthy lung tissues, and exhaled breath from lung cancer patients and healthy controls. Candidate compounds were further compared to substances previously identified in the comparison of transformed and normal lung epithelial cell lines. For human studies, a breath sampling device was developed enabling automated and CO2-controlled collection of the end-tidal air. All samples were first preconcentrated on multibed sorption tubes and analyzed with gas chromatography mass spectrometry (GC-MS). Significantly (p < 0.05) higher concentrations in all three types of cancer samples studied were observed for ethanol and n-octane. Additional metabolites (inter alia 2-methylpentane, n-hexane) significantly released by lung cancer cells were observed at higher levels in cancer lung tissues and breath samples (compared to respective healthy controls) with statistical significance (p < 0.05) only in breath samples. The results obtained confirmed the cancer-related origin of volatile metabolites, e.g. ethanol and octane that were both detected at significantly (p < 0.05) elevated concentrations in all three kinds of cancer samples studied. This work is an important step towards identification of volatile breath markers of lung cancer through the demonstration of cancer-related origin of certain volatile metabolites.

The antioxidant activity (AA), total phenolic content (TPC) and total flavonoids content (TFC) in Dong quai (DQ, Angelica sinensis) raw materials and dietary supplements (DS) containing this plant were determined using the CUPRAC, FRAP and fluorescence methods. The antioxidant activity for DQ aqueous extracts revealed by CUPRAC was (1330.45 ± 1.30) μmol Trolox equivalent (TE) per 100 g of dry mass (DM), whereas the antioxidant activity as determined by FRAP was (1813.9 ± 2.0) μmol of TE per 100 g of DM. Lower values were noted for the fluorescence method than for CUPRAC and FRAP (ranging from (35.96 ± 0.3) to (304.6 ± 1.4) μmol of TE per 100 g of DM). The highest TPC values were determined for an aqueous extract of DQ ((3330.3 ± 2.3) μmol of TE per 100 g of DM), while TFC for ethanolic extracts of DQ was ((146.50 ± 0.5) mg of quercetin equivalent (QE) per 100 g of DM). Cinnamic acid, isomers of benzoic acid and derivatives of quercetin were analysed by HPLC-PDA. The ferulic acid concentration in an ethanolic extract of DQ was (21.83 ± 0.07) mg per 100 g of DM. Of the flavonols detected, rutin exhibited the highest concentration in ethanolic extract of DQ ((3.32 ± 0.13) mg of QE per 100 g of DM). Other phytochemicals (alkaloids, saponins, flavonoids, anthraquinones, tannins, steroids, etc.) were identified by phytoscreening colour reaction. The results were analysed by principal component analysis (PCA), cluster analysis and one-way ANOVA tests.

The effect of amino acid peroxides, relatively stable products of irradiation of amino acid solutions, on erythrocyte components was studied. Interaction of proline, lysine, valine, and leucine peroxides (100–300 μM) with erythrocyte membranes brought about a decrease of membrane protein -SH group content and of activities of (Na + , K + )-ATPase and Ca 2+ -ATPase, and induced aggregation of membrane proteins, due mainly to the formation of interpeptide disulfides. Interaction of amino acid peroxides with hemoglobin brought about hemoglobin oxidation to methemoglobin. The effects of amino acid peroxides are similar to those of t -butyl hydroperoxide. These results indicate that peroxides of amino acid and proteins, which can also be formed under physiological conditions, may be mediators of the cellular action of reactive oxygen species.

Summary The real-time PCR (polymerase chain reaction) based on TaqMan chemistry was used to detect and distinguish Bursaphelenchus xylophilus from other wood-inhabiting nematode species of the xylophilus group. In the study reported here DNA (deoxyribonucleic acid) samples of B. xylophilus and two other most common in Europe and both morphologically and genetically similar nematode species, i.e. B. fraudulentus and B. mucronatus, were examined in detail. The study confirmed a high efficiency of the reaction primers and TaqMan probe. These components allowed detection of B. xylophilus in such small target DNA samples as 3 pg. In contrast, in none of the examined concentrations of DNA samples derived from B. mucronatus and B. fraudulentus any signal was produced. These results showed high sensitivity and specificity of the real-time PCR assay and its potential for rapid and accurate molecular identification of B. xylophilus, even in samples contaminated with DNA of other, most closely related nematode species. Therefore, we conclude that this technique could be of a great value to plant quarantine inspection.

Non-invasive disease monitoring on the basis of volatile breath markers is a very attractive but challenging task. Several hundreds of compounds have been detected in exhaled air using modern analytical techniques (e.g. proton-transfer reaction mass spectrometry, gas chromatography-mass spectrometry) and have even been linked to various diseases. However,the biochemical background for most of compounds detected in breath samples has not been elucidated; therefore, the obtained results should be interpreted with care to avoid false correlations. The major aim of this study was to assess the effects of smoking on the composition of exhaled breath. Additionally, the potential origin of breath volatile organic compounds (VOCs) is discussed focusing on diet, environmental exposure and biological pathways based on other's studies. Profiles of VOCs detected in exhaled breath and inspired air samples of 115 subjects with addition of urine headspace derived from 50 volunteers are presented. Samples were analyzed with GC-MS after preconcentration on multibed sorption tubes in case of breath samples and solid phase micro-extraction (SPME) in the case of urine samples. Altogether 266 compounds were found in exhaled breath of at least 10% of the volunteers. From these, 162 compounds were identified by spectral library match and retention time (based on reference standards). It is shown that the composition of exhaled breath is considerably influenced by exposure to pollution and indoor-air contaminants and particularly by smoking. More than 80 organic compounds were found to be significantly related to smoking, the largest group comprising unsaturated hydrocarbons (29 dienes, 27 alkenes and 3 alkynes). On the basis of the presented results, we suggest that for the future understanding of breath data it will be necessary to carefully investigate the potential biological origin of volatiles, e.g., by means of analysis of tissues, isolated cell lines or other body fluids. In particular, VOCs linked to smoking habit or being the results of human exposure should be considered with care for clinical diagnosis since small changes in their concentration profiles(typically in the ppt(v)–ppb(v) range) revealing that the outbreak of certain disease might be hampered by already high background.

Background The routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs). Results Headspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole). Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml]. Conclusions The results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.

The approach for breath-VOCs' collection and preconcentration by applying needle traps was developed and optimized. The alveolar air was collected from only a few exhalations under visual control of expired CO(2) into a large gas-tight glass syringe and then warmed up to 45 °C for a short time to avoid condensation. Subsequently, a specially constructed sampling device equipped with Bronkhorst® electronic flow controllers was used for automated adsorption. This sampling device allows time-saving collection of expired/inspired air in parallel onto three different needle traps as well as improvement of sensitivity and reproducibility of NT-GC-MS analysis by collection of relatively large (up to 150 ml) volume of exhaled breath. It was shown that the collection of alveolar air derived from only a few exhalations into a large syringe followed by automated adsorption on needle traps yields better results than manual sorption by up/down cycles with a 1 ml syringe, mostly due to avoided condensation and electronically controlled stable sample flow rate. The optimal profile and composition of needle traps consists of 2 cm Carbopack X and 1 cm Carboxen 1000, allowing highly efficient VOCs' enrichment, while injection by a fast expansive flow technique requires no modifications in instrumentation and fully automated GC-MS analysis can be performed with a commercially available autosampler. This optimized analytical procedure considerably facilitates the collection and enrichment of alveolar air, and is therefore suitable for application at the bedside of critically ill patients in an intensive care unit. Due to its simplicity it can replace the time-consuming sampling of sufficient breath volume by numerous up/down cycles with a 1 ml syringe.

Phenolic acids and derivatives of quercetin in Indian (amla) and European gooseberry were determined by high-performance liquid chromatography with diode array detector. The calibration curves were constructed using phenolic compounds standards (the coefficient of determination (R 2) was 0.9990–0.9997 for phenolic acids and 0.9989–0.9994 for flavonols, respectively). The lowest detection limit was 0.28 mg L−1 and 0.35 mg L−1 for hyperoside and gallic acid, respectively, whereas the highest was 1.80 mg L−1 and 7.98 mg L−1 for quercetin and chlorogenic acid, respectively. The quantification limits calculated were 0.85–24.04 mg L−1 for hyperoside and chlorogenic acid, respectively. The predominant phenolic acid in amla and gooseberry is gallic acid: (5.37 ± 0.04) mg per 100 g of dry mass (d.m.) and (3.21 ± 0.03) mg per 100 g of d.m., respectively. The next one was caffeic acid, 0.65–1.22 mg per 100 g of d.m., followed by p-coumaric acid, 0.84–1.17 mg per 100 g of d.m. Out of the flavonols, rutin is predominant: (3.11 ± 0.13) mg per 100 g of d.m. and (2.12 ± 0.03) mg per 100 g of d.m., respectively. Anti-oxidant activity was also determined.

A capillary isotachophoretic method (cITP) using three electrolyte systems for determination of promethazine, thioridazine and chlorpromazine hydrochlorides in pharmaceutical preparations was demonstrated. Proposed systems were characterised by linearity range 5-100 mg/L with R2 in all cases were higher than 0.999. The elaborated method was tested on pharmaceutical preparations. The recovery values were from 96.5% to 101.3%. The proposed isotachophoretic method is simple with acceptable precision and accuracy and can be suitable for routine analysis of studied biological active compounds.

Hypericum perforatum is a medicinal plant which has been known in traditional medicine as an antiinflammatory and healing agent. Nowadays the use of Hypericum extracts is concerned mainly with antidepressive applications. In the present work, HPLC – RP- C18 column chromatography with photodiode array detection was applied for the determination of the derivatives of cinnamic and benzoic acid (e.g., caffeic, chlorogenic, ferulic, sinapic, gallic acids) (Fig.1.) and flavonols - quercetine derivatives (quercetine, rhamnetine, quercitrin, mirycetine, keampferol and rutin) in Hypericum Perforatum. Phenolic compounds were extracted from the sample matrix with ethanol and ethanolwater mixture in different ratios solvent (3:7; 8:2; v/v) at 30°C and 60°C in water-bath shaker and by ultrasonic extraction and then analyzed before and after acid and basic hydrolysis. The total amount of studied flavonols and phenolic acids were compared with the total flavonoids content (TFC) and with total polyphenols content (TPC). UV-Vis spectrometry was used to investigate methods for qualitative and quantitative determination of these compounds.

The aim of this study was to confirm the existence of volatile organic compounds (VOC) specifically released or consumed by the lung cancer cell line A549, which could be used in future screens as biomarkers for the early detection of lung cancer. For comparison, primary human bronchial epithelial cells (HBEpC) and human fibroblasts (hFB) were included. VOCs were detected in the headspace of cell cultures or medium controls following adsorption on solid sorbents, thermodesorption, and analysis by gas chromatography mass spectrometry. Using this approach, we identified VOCs that behaved similarly in normal and transformed cells. Thus, concentrations of 2-pentanone and 2,4-dimethyl-1-heptene were found to increase in the headspace of A549, HBEpC, and hFB cell cultures. In addition, the ethers methyl tert-butyl ether and ethyl tert-butyl ether could be detected at elevated levels in the case of A549 cells and one of the untransformed cell lines. However, especially branched hydrocarbons and alcohols were seen increased more frequently in untransformed than A549 cells. A big variety of predominantly aldehydes and the ester n-butyl acetate were found at decreased concentrations in the headspace of all cell lines tested compared with medium controls. Again, more different aldehydes were found to be decreased in hFB and HBEpC cells compared with A549 cells and 2-butenal was metabolized exclusively by both control cell lines. These data suggest that certain groups of VOCs may be preferentially associated with the transformed phenotype. Cancer Epidemiol Biomarkers Prev; 19(1); 182–95