Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.     

Real-time diagnosis of chemically induced hepatocellular carcinoma using a novel mass spectrometry-based technique

Real-time analyses of hepatocellular carcinoma were performed in living mice to assess the applicability of probe electrospray ionization–mass spectrometry (PESI–MS) in medical diagnosis. The number of peaks and the abundance of ions corresponding to triacylglycerols (TAGs) were higher in cancerous tissues than in noncancerous tissues. Multiple sequential scans of the specimens were performed along a predetermined line extending over the noncancerous region to detect the boundary of the cancerous region. Our system successfully discriminated the noncancerous and cancerous tissues based on the intensities of the TAG ions. These results highlight the potential application of PESI–MS for clinical diagnosis in cancer.     

Refinement and application of a conductiometric standpipe technique for measuring interstitial flow velocity in salmonid spawning gravels

A refinement to the conductiometric standpipe method for determining interstitial flow velocities is described. Three modifications to the original calibration are presented: (i) development of calibration curves for gravels of varying permeability; (ii) statistical validation of a practicable field run time; and (iii) integration of zero velocity flow data to the calibration procedure. These modifications are shown to improve the conductiometric probe’s ability to delineate interstitial flow velocities considered critical to salmonid incubation success. Field deployment of the probe highlighted its practical application for determining interstitial flow velocities in salmonid spawning gravels.     

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.)     

A novel encapsulation technique for the production of artificial seeds

A novel technique for the encapsulation of plant material in calcium alginate hollow beads was tested. The technique involves suspending plant material (i.e. plant cells, tissues, organs, shoot tips, somatic embryos) in a solution containing carboxymethylcellulose and calcium chloride and then dripping it into a stirred sodium alginate solution. In initial experiments with Daucus carota (carrot), it was found that after 14 days of cultivation, 100 % of seeds encapsulated in calcium alginate hollow beads would germinate in the liquid core and that 13% would burst the capsules. Embryogenic calli developed inside hollow beads and formed somatic embryos while calli in conventional calcium alginate beads became detached from the beads early in development, and no somatic embryogenesis occurred. With Solanum tuberosum (potato), development of calli was observed in 50% of hollow beads. Eighty-one percent of shoot tips encapsulated in hollow beads sprouted and grew out of the capsules. Received: 28 October 1999 / Revision received: 11 February 2000 / Accepted: 22 February 2000     

A novel technique for the enumeration of bacteriophage from water

Abstract A novel method has been developed for concentrating and enumerating bacteriophages in water. Host bacterial cells are added to water samples to adsorb phage and then the infected bacteria are collected by centrifugation at 4000 rev./min for 30 min. The pellets are resuspended in small volumes and assayed for phages using the agar-overlay method. This procedure was more successful than three established techniques in recovering Bacillus phages from seeded samples of tap water. It also gave efficient recovery of phages from samples of river, lake and sea-water.     

A technique and apparatus for placement of test substances into the intestines of mosquito larvae

A method for injection of substances into the intestinal lumen via the anal pore is described for fourth instar larvae of Aedes aegypti. Both oral and anal injections were possible with fourth instar larvae of Toxorhynchites amboinensis.

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Résumé On a pu introduire à partir du pore anal des échantillons liquides d'environ 1/3 l dans la lumière intestinale de larves du 4e stade d'Aedes aegypti. Un colorant injecté s'est réparti dans l'intestin postérieur et l'intestin moyen.11 l de solution ont pu être injectés oralement ou analement dans des larves de 4e stade de Toxorhynchites amboinensis. Les mortalités moyennes ont été de 20% (0 à 30%) pour des larves de A. eegypti et de 4% (0 à 10%) pour les larves de T. amboinensis. Nous avons évalué l'interêt de cette technique pour l'étude de l'action directe de toxines et d'autres substances sur les cellules intestinales. Nous avons souligné les possiblités de prélèvement local et de mesures physiologiques (pH, potentiel électrique transépithélial).

     

Use of dual-beam acoustic technique for detecting young whitefish, Coregonus sp., juveniles: first experiments in an enclosure

We tested the possibility of using a high-frequency, BioSonics, dual-beam, acoustic system with an upwards-looking transducer to detect very young whitefish, Coregonus sp., juveniles. These first experiments were conducted in an enclosure with 100 fish (20–30 mm total length).
Observations over 24-h periods showed wide variations of the fish vertical distribution (> 5 m) and two typical aggregation patterns in near-surface waters at dawn and dusk. No direct relationship was found between light intensity and distribution of the fish during the day.
The mean target strength of young whitefish juveniles was found to be – 56 dB when fish were staying near the surface and – 63 dB as they were moving up- or downwards.     

Use of a perfusion technique for measurements of respiratory activity in cultured cells

Summary A method for measuring respiratory activity in anchorage-dependent cultured cells has been developed. This method is based on a technique that permits the perfusion of standard plastic culture dishes with attached cells. Basal respiratory activities were studied in two continuous cell lines of neural origin, neuroblastoma C1300 clone 41A₃ and glioma 138MG. As compared to traditional measurements on detached cells, a fourfold increase in value was obtained. Investigations on membrane permeability suggested that the observed difference could be attributed to alterations in cell membrane integrity. Pretreatment with dibutyryl cyclic AMP, known to induce a morphological and biochemical differentiation in C1300 and 138MG cells, caused in both cell lines an enhanced respiration. This work was supported by grants from the National Swedish Board for Technical Development (grant 82-5025).     

A new species of Legeriomyces and other Harpellales reported for the first time in larval insects from Hungary

Trichomycetes from the guts of aquatic insects have been surveyed for the first time in Hungary. Legeriomyces hungaricus sp. nov. is described along with two new geographic records, L. ramosus and Stachylina grandispora, all of them Harpellales (Kickxellomycotina). The new species is distinguished from others by having smaller zygospores and a characteristic bell-shaped (campanulate) holdfast, unknown so far in other species of Legeriomyces. An unidentified species of Paramoebidium was also observed along with L. ramosus in nymphs of Baetis rhodani (Ephemeroptera). This preliminary report adds to the existing data on gut fungi and we hope will encourage further research on these cryptic endosymbionts in Hungary and its vicinity.     

A novel technique for the study of bacterial cell mechanical properties

A micromanipulation method is described for measuring the bursting forces of bacteria and relating them to cell size. At a compression speed of 6.2 m s^–1, bursting forces of three samples of rapidly growing Staphylococcus epidermis from a batch culture varied from 3 to 34 N with an average value of 13.8 N (standard error 0.8 N). Escherichia coli grown in continuous culture at a specific growth rate of 0.5 h^–1 had bursting forces varying from 1 to 9 N with an average value of 3.6 N (standard error 0.4 N). In squeeze-hold experiments, force relaxation was observed, which was attributed to water loss from the cells, or viscoelasticity, or both. At high compression speed, such as 6.2 m s^–1, this relaxation could be neglected. Micromanipulation strength measurements might be used in studies of cell mechanical disruption and of the dependence of cell strength on cell physiology.     

Development and testing of a novel microcantilever technique for measuring the cohesive strength of intact biofilms

Cohesive strength is an important parameter for understanding and modeling the mechanics of biomass detachment from bacterial biofilms. It is challenging to measure the mechanical properties of biofilms, however, because biofilms may desiccate when removed from liquid medium and they are inherently fragile. Poppele and Hozalski (Poppele and Hozalski, 2003, J Microb Methods 55:607–615) presented a microcantilever method for measuring the tensile strength of detached biofilm fragments while submersed in liquid medium. Here we present a modification of the microcantilever method to quantify the strength of intact bacterial biofilms. Initial testing was performed on Pseudomonas aeruginosa biofilms and on Staphylococcus epidermidis biofilms grown in rotating disk reactors. The cohesive strength values were highly variable (i.e., coefficients of variation ranging from 71% to 143%) and ranged from 59 to 18,900 Pa for the P. aeruginosa biofilms and from 61 to 5,840 Pa for the S. epidermidis biofilms. The biofilms also appeared to be isotropic as strength did not vary with angle of testing relative to the direction of applied shear. Strength testing using both the intact and fragment methods was performed on five samples of P. aeruginosa biofilms, and the strength populations were not from the same distribution in three cases. Equivalent diameters for the fragments detached from biofilms during strength testing ranged from 5 to 500 µm, which is within the range of size of biofilm fragments observed in the effluents of lab‐scale and full‐scale bioreactors. The microcantilever is a simple yet powerful tool for measuring the cohesive strength of intact biofilms at a relevant scale. Biotechnol. Bioeng. 2010;105: 924–934. © 2009 Wiley Periodicals, Inc.     

Gonad maturation and spawning times of Siganus sutor off the Keny a coast: evidence for definite spawning seasons in atropical fish

The gonad maturation cycle of Siganus sutor (Valenciennes, 1835) (Osteichthyes-Siganidae) is described for both males and females using macroscopic criteria for the testes and both macroscopic and microscopic ones for staging the ovaries. Siganus suror has two major spawning seasons: one in January/February and the other in May/June. The presence of these seasons is established by (a) the temporal variations in the condition factor and in the relative weight of the gonads, (b) the progression of peaks of maturity stages with seasonal occurrence of spent fish in the samples, and (c) the seasonal appearance ofjuveniles. This is a significant result for a tropical marine fish.     

Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae

Background and Aims The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the ‘viscid sepals’ of Specklinia endotrachys were visited by a ‘small fly’. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species.Methods Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography–mass spectometry using those same pheromones as standards.Key Results Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species.Conclusions Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.     

Use of the fluorescent antibody technique for the evaluation of Arthroderma uncinatum in soil

The fact that Arthroderma uncinatum can grow through soil has been demonstrated by the fluorescent antibody technique which appears to be a useful tool in investigating the ecology of keratin-decomposing fungi in the soil.