The drivers of avian‐haemosporidian prevalence in tropical lowland forests of New Guinea in three dimensions

Haemosporidians are among the most common parasites of birds and often negatively impact host fitness. A multitude of biotic and abiotic factors influence these associations, but the magnitude of these factors can differ by spatial scales (i.e., local, regional and global). Consequently, to better understand global and regional drivers of avian‐haemosporidian associations, it is key to investigate these associations at smaller (local) spatial scales. Thus, here, we explore the effect of abiotic variables (e.g., temperature, forest structure, and anthropogenic disturbances) on haemosporidian prevalence and host–parasite networks on a horizontal spatial scale, comparing four fragmented forests and five localities within a continuous forest in Papua New Guinea. Additionally, we investigate if prevalence and host–parasite networks differ between the canopy and the understory (vertical stratification) in one forest patch. We found that the majority of Haemosporidian infections were caused by the genus Haemoproteus and that avian‐haemosporidian networks were more specialized in continuous forests. At the community level, only forest greenness was negatively associated with Haemoproteus infections, while the effects of abiotic variables on parasite prevalence differed between bird species. Haemoproteus prevalence levels were significantly higher in the canopy, and an opposite trend was observed for Plasmodium. This implies that birds experience distinct parasite pressures depending on the stratum they inhabit, likely driven by vector community differences. These three‐dimensional spatial analyses of avian‐haemosporidians at horizontal and vertical scales suggest that the effect of abiotic variables on haemosporidian infections are species specific, so that factors influencing community‐level infections are primarily driven by host community composition.     

The cytochromes c-550 of Paracoccus denitrificans and Thiosphaera pantotropha: a need for re-evaluation of the history of Paracoccus cultures

Abstract The c -type cytochrome and protein profiles were compared for a number of cultures of Paracoccus denitrificans obtained from a range of culture collections. The cultures fell into two groups corresponding to the two original isolates of this bacterial species. One group, which included NCIMB 8944, ATCC 13543, ATCC 17741, ATCC 19367, Pd 1222 and DSM 413, were similar or identical to LMD 22.21. The second group, including DSM 65 and LMG 4218, were similar or identical to LMD 52.44. These groupings were not compatible with the recorded history of culture deposition. Mass spectrometry and amino acid sequence comparisons showed that the cytochrome c -550 of the LMD 52.44 culture group differed by 16% from that of the LMD 22.21 group, and yet was only 1% different from the cytochrome c -550 of Thiosphaera pantotropha . These results suggest that consideration should be given to creation of a new species of Paracoccus pantotropha , which would include Thiosphaera pantotropha and Paracoccus denitrificans LMD 52.44.     

IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes

Following activation through high affinity IgE receptors (FcepsilonRI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. FcepsilonRI-induced degranulation is a SNARE (soluble N-ethylmaleimide attachment protein receptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through FcepsilonRI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (from Rab3D-associated kinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto- or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH(2)-terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.     

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity.     

Molecular epidemiology of nosocomial infection by extended-spectrum beta-lactamases-producing Klebsiella pneumoniae

Molecular epidemiology applied to the study of nosocomial infection has been fundamental in formulating and evaluating control methods. From patients in a level 3 Bogota hospital, Klebsiella pneumoniae samples were isolated that produced extended-spectrum beta-lactamases (ESBL). Each of 15 isolates was characterized microbiologically and by molecular characters realized by pulsed field gel electrophoresis (PFGE) and by repetitive-DNA sequences amplification (REP-PCR). Antimicrobial susceptibility and ESBL production was determined in accordance with NCCLS guidelines. The beta-lactamases were evaluated by isoelectric-focusing and PCR. Twelve (80%) of the isolates were associated with nosocomial infection; 11 of them were from intensive care units. The antibiotic susceptibility displayed 13 resistance patterns--87% presented co-resistance to amikacin, 53% to gentamicin, 33% to ciprofloxacin, 40% to cefepime, 67% to piperacillin/tazobactam, 60% to trimethoprim/sulfamethoxazole and 47% to chloranphenicol. All were sensitive to imipenem. Production of TEM and SHV beta-lactamases was detected simultaneously in most isolates by isoelectric focusing and 93.3% produced a ceftazidimase of pl 8.2 of the SHV-5 type. The 15 isolates were grouped into 11 and 12 electrophoretic patterns by PFGE and REP-PCR, respectively. The degree of genetic variability indicated an endogenous origin of the nosocomial infections.     

In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA

The number of protein structures solved using multiwavelength anomalous diffraction methods coupled with selenomethionine substitution has grown dramatically over the last years. We show using the outer membrane pyoverdin receptor FpvA that Pseudomonas aeruginosa can be used for producing proteins with a high level of selenomethionine incorporation. To circumvent problems encountered with mass spectroscopy analysis of purified membrane proteins, in-gel trypsin digestion of FpvA coupled with MALDI mass spectrometry analysis of the resulting peptides was used to determine the extent of selenomethionine incorporation. Selenomethionine incorporation greater than 95% was achieved using P. aeruginosa as an overexpression system.     

Measuring the size of biological nanostructures with spatially modulated illumination microscopy

Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads ( approximately 40-100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.