Applications of MALDI-TOF-MS in clinical microbiology laboratory

For twenty years, mass spectrometry (MS) has emerged as a particularly powerful tool for analysis and characterization of proteins in research. It is only recently that this technology, especially MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization Time-Of-Flight) has entered the field of routine microbiology. This method has proven to be reliable and safe for the identification of bacteria, yeasts, filamentous fungi and dermatophytes. MALDI-TOF-MS is a rapid, precise and cost-effective method for identification, compared to conventional phenotypic techniques or molecular biology. Its ability to analyse whole microorganisms with few sample preparation has greatly reduced the time to identification (1-2 min). Furthermore, this technology can be used to identify bacteria directly from clinical samples as blood culture bottles or urines. Future applications will be developed in order to provide direct information concerning virulence or resistance protein markers.     

Cardenolides of Leptadenia madagascariensis from the Madagascar dry forest

Investigation of the endemic Madagascar plant Leptadenia madagascariensis Decne. (Apocynaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the four new cardenolides 1-4. The structure elucidations of these compounds were based on analyzes of their 1D and 2D NMR spectra and mass spectrometric data. The cardenolides were strongly antiproliferative to the A2780 ovarian cancer cell line, with IC₅₀ values of 0.18, 0.21, 0.17, and 0.29 ??M line, and to the H460 human lung cancer cell line, with IC₅₀ values of 0.16, 0.68, 0.37, and 0.48 ??M, respectively.     

Conspecifics as informers and competitors: an experimental study in foraging bumble-bees

Conspecifics are usually considered competitors negatively affecting food intake rates. However, their presence can also inform about resource quality by providing inadvertent social information. Few studies have investigated whether foragers perceive conspecifics as informers or competitors. Here, we experimentally tested whether variation in the density of demonstrators ('none', 'low' and 'high'), whose location indicated flower profitability, affected decision-making of bumble-bees Bombus terrestris. Bumble-bees foraged on either 'simple' (two colours) or 'complex' (four colours) artificial floral communities. We found that conspecifics at low density may be used as sources of information in first flower choices, whereas they appeared as competitors over the whole foraging sequence. Low conspecific densities improved foragers' first-visit success rate in the simple environment, and decreased time to first landing, especially in the complex environment. High conspecific densities did not affect these behavioural parameters, but reduced flower constancy in both floral communities, which may alter the efficiency of pollinating visits. These results suggest that the balance of the costs and benefits of conspecific presence varies with foraging experience, floral community and density. Spatio-temporal scales could thus be an important determinant of social information use. This behavioural flexibility should allow bumble-bees to better exploit their environment.     

Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells

Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions.     

New Paleocene Sepiid Coleoids (Cephalopoda) from Egypt: Evolutionary Significance and Origin of the Sepiid ‘Rostrum’

New coleoid cephalopods, assignable to the order Sepiida, are recorded from the Selandian/Thanetian boundary interval (Middle to Upper Paleocene transition, c. 59.2 Ma) along the southeastern margin (Toshka Lakes) of the Western Desert in Egypt. The two genera recognised, Aegyptosaepia n. gen. and ?Anomalosaepia Weaver and Ciampaglio, are placed in the families Belosaepiidae and ?Anomalosaepiidae, respectively. They constitute the oldest record to date of sepiids with a ‘rostrum-like’ prong. In addition, a third, generically and specifically indeterminate coleoid is represented by a single rostrum-like find. The taxonomic assignment of the material is based on apical parts (as preserved), i.e., guard, apical prong (or ‘rostrum-like’ structure), phragmocone and (remains of) protoconch, plus shell mineralogy. We here confirm the shell of early sepiids to have been bimineralic, i.e., composed of both calcite and aragonite. Aegyptosaepia lugeri n. gen., n. sp. reveals some similarities to later species of Belosaepia, in particular the possession of a distinct prong. General features of the phragmocone and protoconch of the new form are similar to both Belocurta (Middle Danian [Lower Paleocene]) and Belosaepia (Eocene). However, breviconic coiling and the presence of a longer ventral conotheca indicate closer ties with late Maastrichtian–Middle Danian Ceratisepia. In this respect, Aegyptosaepia n. gen. constitutes a link between Ceratisepia and the Eocene Belosaepia. The occurrence of the new genus near the Selandian/Thanetian boundary suggests an earlier origin of belosaepiids, during the early to Middle Paleocene. These earliest known belosaepiids may have originated in the Tethyan Realm. From northeast Africa, they subsequently spread to western India, the Arabian Plate and, probably via the Mediterranean region, to Europe and North America.     

Honeybee Colony Disorder in Crop Areas: The Role of Pesticides and Viruses

As in many other locations in the world, honeybee colony losses and disorders have increased in Belgium. Some of the symptoms observed rest unspecific and their causes remain unknown. The present study aims to determine the role of both pesticide exposure and virus load on the appraisal of unexplained honeybee colony disorders in field conditions. From July 2011 to May 2012, 330 colonies were monitored. Honeybees, wax, beebread and honey samples were collected. Morbidity and mortality information provided by beekeepers, colony clinical visits and availability of analytical matrix were used to form 2 groups: healthy colonies and colonies with disorders (n = 29, n = 25, respectively). Disorders included: (1) dead colonies or colonies in which part of the colony appeared dead, or had disappeared; (2) weak colonies; (3) queen loss; (4) problems linked to brood and not related to any known disease. Five common viruses and 99 pesticides (41 fungicides, 39 insecticides and synergist, 14 herbicides, 5 acaricides and metabolites) were quantified in the samples.The main symptoms observed in the group with disorders are linked to brood and queens. The viruses most frequently found are Black Queen Cell Virus, Sac Brood Virus, Deformed Wing Virus. No significant difference in virus load was observed between the two groups. Three acaricides, 5 insecticides and 13 fungicides were detected in the analysed samples. A significant correlation was found between the presence of fungicide residues and honeybee colony disorders. A significant positive link could also be established between the observation of disorder and the abundance of crop surface around the beehive. According to our results, the role of fungicides as a potential stressor for honeybee colonies should be further studied, either by their direct and/or indirect impacts on bees and bee colonies.     

The Urinary Cytokine/Chemokine Signature of Renal Hyperfiltration in Adolescents with Type 1 Diabetes

Objective

Urinary cytokine/chemokine levels are elevated in adults with type 1 diabetes (T1D) exhibiting renal hyperfiltration. Whether this observation extends to adolescents with T1D remains unknown. Our first objective was to determine the relationship between hyperfiltration and urinary cytokines/chemokines in normotensive, normoalbuminuric adolescents with T1D using GFR_cystatin. Our second aim was to determine the relationship between urine and plasma levels of inflammatory biomarkers, to clarify the origin of these factors.

Methods

Urine and serum cytokines/chemokines (Luminex platform) and GFR_cystatin were measured in normofiltering (n = 111, T1D-N, GFR<135 ml/min/1.73 m²) and hyperfiltering (n = 31, T1D-H, GFR≥135 ml/min/1.73 m²) adolescents with T1D (ages 10–16), and in age and sex matched healthy control subjects (HC, n = 59).

Results

We noted significant step-wise increases in urinary cytokine/chemokine excretion according to filtration status with highest levels in T1D-H, with parallel trends in serum analyte concentrations. After adjusting for serum glucose at the time of sampling, differences in urinary cytokine excretion were not statistically significant. Only serum IL-2 significantly differed between HC and T1D (p = 0.0076).

Conclusions

Hyperfiltration is associated with increased urinary cytokine/chemokine excretion in T1D adolescents, and parallel trends in serum cytokine concentration. The GFR-associated trends in cytokine excretion may be driven by the effects of ambient hyperglycemia. The relationship between hyperfiltration, glycemia, and variations in serum and urine cytokine expression and their impact on future renal and systemic vascular complications requires further study.     

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.