Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

Use of phytoplankton-derived dissolved organic carbon by different types of bacterioplankton

Phytoplankton and heterotrophic prokaryotes are major components of the microbial food web and interact continuously: heterotrophic prokaryotes utilize the dissolved organic carbon derived from phytoplankton exudation or cell lysis (DOCp), and mineralization by heterotrophic prokaryotes provides inorganic nutrients for phytoplankton. For this reason, these communities are expected to be closely linked, although the study of the interactions between them is still a major challenge. Recent studies have presented interactions between phytoplankton and heterotrophic prokaryotes based on coexistence or covariation throughout a time-series. However, a real quantification of the carbon flow within these networks (defined as the interaction strength, IS) has not been achieved yet. This is critical to understand the selectivity degree of bacteria responding to specific algal DOCp. Here we used microautoradiography to quantify the preferences of the major heterotrophic prokaryote phylogenetic groups on DOC derived from several representative phytoplankton species, and expressed these preferences as an IS value. The distribution of the ISs was not random but rather skewed towards weak interactions, in a similar way as the distributions described for stable complex non-microbial ecosystems, indicating that there are some cases of high specificity on the use of specific algal DOCp by some bacterial groups, but weak interactions are more common and may be relevant as well. The variety of IS patterns observed supports the view that the vast range of different resources (different types of organic molecules) available in the sea selects and maintains the high levels of diversity described for marine bacterioplankton.     

Mutations in neuromusculin,a gene encoding a cell adhesion molecule,cause nervous system defects

The Drosophila neuromusculin (nrm) gene encodes an immunoglobulin-like (Ig-like) cell adhesion molecule expressed in the precursors of the embryonic peripheral nervous system (PNS), in the midline precursors of the central nervous system (CNS), and in muscles. During the initial phases of CNS axonogenesis, nrm is expressed in cells involved in the development of commissures and longitudinal tracts. Mutations which alter expression of nrm mRNAs cause aberrant development of commissures and longitudinal axon pathways. Defects in the PNS and muscles of nrm mutants are also observed. In most nrm embryos, abnormal development can be detected in a subset of abdominal segments; however, in approximately 1 of 10 nrm embryos, the defects extend to all segments. Herein, we present evidence that nrm plays an important role in early morphogenesis, possibly by mediating or facilitating inductive cell contacts and movements.     

Does apple replant disease affect the soil patch selection behaviour and population growth of Collembolans?

Apple replant disease (ARD) is common to all major apple-growing regions in the world. It occurs when new apple trees are replanted on sites where previously the same or closely related crop species were grown. Biotic (fungi, bacteria and nematodes) and abiotic soil factors (poor soil structure, nutrition) contribute to the development and severity of ARD. However, the aetiology of ARD and effects on higher trophic levels are still unknown. In that sense, Collembola might play an important role, since they are one of the dominant mesofauna groups in many soils. They act as decomposer, fungivores and predators, representing different trophic levels in soil food webs. Therefore, any effect of ARD on the occurrence of Collembola could have ecological impacts on the soil quality and health. Here, we examined the colonization behaviour of two Collembolan species, Folsomia candida and Sinella curviseta, in choice tests and population growth tests using Apple Replant Diseased soil (ARD) and non-ARD soil samples from different field sites and standardized laboratory bioassays. Additionally, Collembola behaviour was quantified by continuous video observations to investigate short-term behavioural changes. Results showed that both Collembolan species significantly preferred colonization of the non-ARD soils compared with ARD soils, independent of the origin of the soil samples or specific disinfection treatments. Moreover, the detailed video analysis of the foraging behaviour indicates rapid colonization of soil samples and low dispersal rates. Most likely, volatile compounds and to a lesser extent feeding stimulants play a vital role for the colonization process for both Collembolan species. Finally, results showed negative effects of ARD on population growth of both Collembolan species already after an 8-week period, implying strong nutritional deficiencies in ARD affected soils. The hypothesis that ARD causing microorganisms directly affected orientation, colonization and population development of Collembola is discussed.     

Following the water? Landscape‐scale temporal changes in bat spatial distribution in relation to Mediterranean summer drought

Understanding how the spatial distribution of ecological resources shapes species’ diversity and abundance in human‐modified landscapes is a central theme in conservation biology. However, studies often disregard that such patterns may vary over time, thereby potentially missing critical environmental constraints to species persistence. This may be particularly important in highly mobile species such as bats, which are able to track temporal variations in spatial resource distribution. Here we test the hypothesis that bats in Mediterranean landscapes are strongly affected by the progressive reduction in water availability during the seasonal summer drought. We analyzed the effects of landscape composition and structure on bat diversity and activity, during pregnancy, lactation, and postlactation periods, and identified the most influential variables within and across periods. Water bodies showed the strongest positive effect on bats, followed by riparian habitats and areas with steeper (>30%) slopes. However, while during pregnancy, there were only small landscape effects, these increased during lactation and postlactation, highlighting a progressively stronger association with water habitats during the summer drought. The spatial projection of habitat models showed that the landscape distribution of bat diversity and activity hotspots changed markedly over time. During pregnancy, the spatial pattern of hotspot distribution was weakly defined, while during lactation and particularly postlactation, there was a concentration of hotspots along permanently flowing watercourses. Our study highlights that permanently flowing watercourses are critical for bat conservation in Mediterranean landscapes, calling for measures to counteract their ongoing degradation due in particular to climate change, water abstraction and damming. More generally, our study underlines the importance of considering the temporal dimension in habitat selection studies, without which there is the risk of overlooking the importance of habitats that are key for species persistence only at certain times of the year.     

An enzyme-coupled continuous spectrophotometric assay for glycogen synthases

The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.     

Evolutionary history of the European free-tailed bat,a tropical affinity species spanning across the Mediterranean Basin

The Mediterranean Basin is a global biodiversity hotspot, hosting a number of native species belonging to families that are found almost exclusively in tropical climates. Yet, whether or not these taxa were able to survive in the Mediterranean region during the Quaternary climatic oscillations remains unknown. Focusing on the European free-tailed bat (Tadarida teniotis), we aimed to (a) identify potential ancient populations and glacial refugia; (b) determine the post-glacial colonization routes across the Mediterranean; and (c) evaluate current population structure and demography. Mitochondrial and nuclear markers were used to understand T. teniotis evolutionary and demographic history. We show that T. teniotis is likely restricted to the Western Palearctic, with mitochondrial phylogeny suggesting a split between an Anatolian/Middle East clade and a European clade. Nuclear data pointed to three genetic populations, one of which is an isolated and highly differentiated group in the Canary Islands, another distributed across Iberia, Morocco, and France, and a third stretching from Italy to the east, with admixture following a pattern of isolation by distance. Evolutionary and demographic reconstruction supports a pre-Last Glacial Maximum (LGM) colonization of Italy and the Anatolian/Middle East, while the remaining populations were colonized from Italy after the Younger Dryas. We also found support for demographic expansion following the Iberian colonization. The results show that during the LGM T. teniotis persisted in Mediterranean refugia and has subsequently expanded to its current circum-Mediterranean range. Our findings raise questions regarding the physiological and ecological traits that enabled species with tropical affinities to survive in colder climates.     

Adrenergic Stimulation of Cyclic GMP Formation Requires NO-Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α₁-adrenergic receptors. The effects of N -monomethyl- l -arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α₁-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l -arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.     

The effects of l-DOPA on root growth, lignification and enzyme activity in soybean seedlings

In the present study, we investigated the effects of l-DOPA (l-3,4-dihydroxyphenylalanine), an allelochemical exuded from the velvetbean (Mucuna pruriens L DC. var. utilis), on the growth and cell viability of soybean (Glycine max L. Merrill) roots. We analyzed the effects of l-DOPA on phenylalanine ammonia lyase (PAL), cinnamyl-alcohol dehydrogenase (CAD) and cell wall-bound peroxidase (POD) activities as well as its effects on phenylalanine, tyrosine and lignin contents in the roots. 3-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), with or without 0.5?mM l-DOPA, in a growth chamber at 25?°C for 6, 12, 18 or 24?h with a day/night regime of 1:1, and a photon flux density of 280???mol?m^?2 s^?1. In general, the length, fresh weight and dry weight of the roots decreased followed by a significant loss of cell viability. Phenylalanine, tyrosine and lignin contents as well as PAL, CAD and cell wall-bound POD activities increased after l-DOPA treatment. These results reinforce the susceptibility of soybean to l-DOPA, which increases the enzyme activity in the phenylpropanoid pathway and, therefore, provides precursors for the polymerization of lignin. In brief, these findings suggest that the inhibition of soybean root growth induced by exogenously applied l-DOPA may be due to excessive production of lignin in the cell wall.     

Infrared thermal imaging associated with pain in laboratory animals

The science of animal welfare has evolved over the years, and recent scientific advances have enhanced our comprehension of the neurological, physiological, and ethological mechanisms of diverse animal species. Currently, the study of the affective states (emotions) of nonhuman animals is attracting great scientific interest focused primarily on negative experiences such as pain, fear, and suffering, which animals experience in different stages of their lives or during scientific research. Studies underway today seek to establish methods of evaluation that can accurately measure pain and then develop effective treatments for it, because the techniques available up to now are not sufficiently precise. One innovative technology that has recently been incorporated into veterinary medicine for the specific purpose of studying pain in animals is called infrared thermography (IRT), a technique that works by detecting and measuring levels of thermal radiation at different points on the body’s surface with high sensitivity. Changes in IRT images are associated mainly with blood perfusion, which is modulated by the mechanisms of vasodilatation and vasoconstriction. IRT is an efficient, noninvasive method for evaluating and controlling pain, two critical aspects of animal welfare in biomedical research. The aim of the present review is to compile and analyze studies of infrared thermographic changes associated with pain in laboratory research involving animals.