Global warming and Bergmann’s rule: do central European passerines adjust their body size to rising temperatures?

Recent climate change has caused diverse ecological responses in plants and animals. However, relatively little is known about homeothermic animals’ ability to adapt to changing temperature regimes through changes in body size, in accordance with Bergmann’s rule. We used fluctuations in mean annual temperatures in south-west Germany since 1972 in order to look for direct links between temperature and two aspects of body size: body mass and flight feather length. Data from regionally born juveniles of 12 passerine bird species were analysed. Body mass and feather length varied significantly among years in eight and nine species, respectively. Typically the inter-annual changes in morphology were complexly non-linear, as was inter-annual variation in temperature. For six (body mass) and seven species (feather length), these inter-annual fluctuations were significantly correlated with temperature fluctuations. However, negative correlations consistent with Bergmann’s rule were only found for five species, either for body mass or feather length. In several of the species for which body mass and feather length was significantly associated with temperature, morphological responses were better predicted by temperature data that were smoothed across multiple years than by the actual mean breeding season temperatures of the year of birth. This was found in five species for body mass and three species for feather length. These results suggest that changes in body size may not merely be the result of phenotypic plasticity but may hint at genetically based microevolutionary adaptations.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Nest site competition between cavity nesting passerines and golden paper wasps Polistes fuscatus

Nest boxes provide sheltered nesting sites for both passerines and paper wasps. Although neither wasps nor birds appear to evict the other once one is fully established, it is unclear which is the dominant competitor at the onset of the breeding season. Using wire mesh, we excluded birds but not golden paper wasps Polistesfuscatus from alternating boxes along a transect through edge habitat in North Carolina from 2006 – 2008. If wasps dominate Carolina chickadees Poecile carolinensis and eastern bluebirds Sialia sialis during the early spring (all have similar nest initiation dates), we would expect wasps to settle in both box types at equal frequencies. However, if birds dominate wasps, we would expect wasp nests to be concentrated in “bird‐proof” boxes. We found wasps in bird‐proof boxes significantly more often than in bird‐accessible boxes, indicating that secondary‐cavity nesting birds are able to exclude wasps from available nest sites. Additionally, we found that during the period of nest initiation, birds usurp wasps more often than vice versa.     

Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting.     

Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.     

First experiences with semi-autonomous robotic harvesting of protein crystals

The demonstration unit of the Universal Micromanipulation Robot (UMR) capable of semi-autonomous protein crystal harvesting has been tested and evaluated by independent users. We report the status and capabilities of the present unit scheduled for deployment in a high-throughput protein crystallization center. We discuss operational aspects as well as novel features such as micro-crystal handling and drip-cryoprotection, and we extrapolate towards the design of a fully autonomous, integrated system capable of reliable crystal harvesting. The positive to enthusiastic feedback from the participants in an evaluation workshop indicates that genuine demand exists and the effort and resources to develop autonomous protein crystal harvesting robotics are justified.     

A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway – Adaptation to HIV Envelope Processing

The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.     

A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum,Zeamine-Related Antibiotics

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.     

Activity alters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways that grow to link synaptic sites

In partially denervated rodent muscle, terminal Schwann cells (TSCs) located at denervated end plates grow processes, some of which contact neighboring innervated end plates. Those processes that contact neighboring synapses (termed "bridges") appear to initiate nerve terminal sprouting and to guide the growth of the sprouts so that they reach and reinnervate denervated end plates. Studies conducted prior to knowledge of this potential involvement of Schwann cells showed that direct muscle stimulation inhibits terminal sprouting following partial denervation (Brown and Holland, 1979). We have investigated the possibility this inhibition results from an alteration in the growth of TSC processes. We find that stimulation of partially denervated rat soleus muscle does not alter the length or number of TSC processes but does reduce the number of TSC bridges. Stimulation also reduces the number of TSC bridges that form between end plates during reinnervation of a completely denervated muscle. The nerve processes ("escaped fibers") that normally grow onto TSC processes during reinnervation are also reduced in length. Therefore, stimulation alters at least two responses to denervation in muscles: (1) the ability of TSC processes to form or maintain bridges with innervated synaptic sites, and (2) the growth of axons along processes extended by TSCs.