Radio‐transmitters have no impact on survival of pre‐fledged American Woodcocks

American Woodcocks (Scolopax minor) are a high priority species of conservation need across most of their breeding range due to long‐term population declines. Survival of juveniles may be key to understanding these population declines, but there have been few direct estimates of juvenile woodcock survival rates, and no recent assessment of the possible effect of radio‐tagging on juvenile survival. In 2011 and 2012, we radio‐tagged 73 juvenile American Woodcocks in west‐central Minnesota and compared survival rates of radio‐tagged (N = 58) and non‐radio‐tagged (N = 82) juveniles during the period from hatching to fledging. We compared survival rates of juveniles with known fates and used logistic‐exposure models to assess the potential impact of radio‐transmitters on survival. We evaluated variables related to juvenile survival including age, hatch date, maximum and minimum temperature, precipitation, and year to assess the possible effects of radio‐transmitters. The best‐supported model of survival rate of juvenile American Woodcocks included the interaction of age and year and a negative effect of precipitation (β = ?0.76, 85% CI: ?1.08 to ?0.43), but did not include a negative effect of transmitters. Our results suggest that radio‐transmitters did not impact survival of juvenile American Woodcocks and that transmitters are a reliable tool for studying survival of juvenile American Woodcocks, and perhaps other precocial shorebirds.     

Co‐evolution of multipartite interactions between an extended tmRNA tag and a robust Lon protease in Mycoplasma

Messenger RNAs that lack in‐frame stop codons promote ribosome stalling and accumulation of aberrant and potentially harmful polypeptides. The SmpB‐tmRNA quality control system has evolved to solve problems associated with non‐stop mRNAs, by rescuing stalled ribosomes and directing the addition of a peptide tag to the C‐termini of the associated proteins, marking them for proteolysis. In Escherichia coli, the ClpXP system is the major contributor to disposal of tmRNA‐tagged proteins. We have shown that the AAA+ Lon protease can also degrade tmRNA‐tagged proteins, but with much lower efficiency. Here, we present a unique case of enhanced recognition and degradation of an extended Mycoplasma pneumoniae (MP) tmRNA tag by the MP‐Lon protease. We demonstrate that MP‐Lon can efficiently and selectively degrade MP‐tmRNA‐tagged proteins. Most significantly, our studies reveal that the larger (27 amino acids long) MP‐tmRNA tag contains multiple discrete signalling motifs for efficient recognition and rapid degradation by Lon. We propose that higher‐affinity multipartite interactions between MP‐Lon and the extended MP‐tmRNA tag have co‐evolved from pre‐existing weaker interactions, as exhibited by Lon in E. coli, to better fulfil the function of MP‐Lon as the sole soluble cytoplasmic protease responsible for the degradation of tmRNA‐tagged proteins.     

A method for the automated long‐term monitoring of three‐spined stickleback Gasterosteus aculeatus shoal dynamics

This paper describes and evaluates a flexible, non‐invasive tagging system for the automated identification and long‐term monitoring of individual three‐spined sticklebacks Gasterosteus aculeatus. The system is based on barcoded tags, which can be reliably and robustly detected and decoded to provide information on an individual's identity and location. Because large numbers of fish can be individually tagged, it can be used to monitor individual‐ and group‐level dynamics within fish shoals.     

The Anaplasma phagocytophilum‐occupied vacuole selectively recruits Rab‐GTPases that are predominantly associated with recycling endosomes

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell‐derived vacuole. The A. phagocytophilum‐occupied vacuole (ApV) fails to mature along the endocytic pathway and is non‐fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)‐tagged Rab GTPases in A. phagocytophilum‐infected HL‐60 cells. GFP‐Rab4A, GFP‐Rab10, GFP‐Rab11A, GFP‐Rab14, RFP‐Rab22A and GFP‐Rab35, which regulate endocytic recycling, and GFP‐Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome‐associated GFP‐Rabs and acquisition of GFP‐Rab5, GFP‐Rab7, and the lysosomal marker, LAMP‐1. Wild‐type and GTPase‐ deficient versions, but not GDP‐restricted versions of GFP‐Rab1, GFP‐Rab4A and GFP‐Rab11A, localize to the ApV. Strikingly, GFP‐Rab10 recruitment to the ApV is guanine nucleotide‐independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.     

Range sizes and habitat use of non‐breeding Crested Caracaras in Florida

In Florida, habitats that include the breeding territories of Crested Caracaras (Caracara cheriway) are protected, but non‐breeding individuals may be vulnerable because they may occupy different areas and habitats. We captured and radio‐tagged 58 non‐breeding caracaras in Florida from July 2006–March 2009, determined their locations during weekly flights, and used GIS and compositional analysis to evaluate range sizes and habitat use. Non‐breeding caracaras (N = 58) ranged five times more widely during breeding seasons (N = 573 locations) than during non‐breeding seasons (N = 592 locations), and ranged >250 times more widely than breeding caracaras that defend territories year‐round. The large ranges of non‐breeders suggest they may be searching for and evaluating prospective territories or breeding opportunities (territory prospecting). Pasture occupied by cattle was the most used habitat relative to availability and was used more than pasture without cattle, likely because insects associated with cattle are an important food source for caracaras. Cattle numbers in Florida are declining and, because both breeding and non‐breeding caracaras primarily occupy pasture, this may present difficulties for long‐term management. Citrus groves were also used more than expected given availability by non‐breeding caracaras, but are rarely included in nesting territories. Because pasture and citrus were often adjacent, we suggest that citrus groves may function as refugia from socially dominant breeding caracaras. Conservation and recovery efforts for Florida's caracara population are needed throughout the range of non‐breeders, and should include management that ensures availability of habitat matrices of cattle pasture and citrus groves.     

Synthesis,Cytotoxicity and Antimicrobial Evaluation of New Coumarin‐Tagged β‐Lactam Triazole Hybrid

A series of coumarin‐tagged β‐lactam triazole hybrids ( 10a – 10o ) were synthesized and tested for their cytotoxic activity against MDA‐MB‐231 (triple negative breast cancer), MCF‐7 (estrogen receptor positive breast cancer (ER+)) and A549 (human lung carcinoma) cancer cell lines including one normal cell line, HEK‐293 (human embryonic kidney). Two compounds 10b and 10d exhibited substantial cytotoxic effect against MCF‐7 cancer cell lines with IC₅₀ values of 53.55 and 58.62 μm , respectively. More importantly, compounds 10b and 10d were non‐cytotoxic against HEK‐293 cell lines. Structure–activity relationship (SAR) studies suggested that the nitro and chloro group at the C‐3 position of phenyl ring are favorable for anticancer activity, particularly against MCF‐7 cell lines. Furthermore, antimicrobial evaluation of these compounds revealed modest inhibition of examined pathogenic strains with compounds 10c and 10i being the most promising antimicrobial agents against Pseudomonas aeruginosa and Candida albicans, respectively.     

Overexpression and one‐step renaturation‐purification of the tagged creatinine deiminase of Corynebacterium glutamicum in Escherichia coli cells

The codA gene of Corynebacterium glutamicum PCM 1945 coding for a creatinine deiminase (CDI) (EC 3.5.4.21) has been amplified and cloned. The recombinant strain of Escherichia coli that overproduces the (His)₆‐tagged inactive CDI of C. glutamicum as inclusion bodies has been constructed. After solubilization of inclusion bodies in the presence of 0.3% N‐lauroylsarcosine, the enzyme was renaturated and purified by a single‐step procedure using metal‐affinity chromatography. The yield of the (His)₆‐tagged CDI is ~30 mg from 1 L culture. The purified enzyme is sufficiently stable under the conditions designed and possesses an activity of 10–20 U/mg. The main characteristics of the tagged enzyme remained similar to that of the natural enzyme.     

Expression of Recombinant Potato leafroll virus Structural and Non‐structural Proteins for Antibody Production

Vector pMPM‐A4Ω and vectors pQE‐30 and pET‐45b(+) containing the 6x His‐tag sequence were used for expression of Potato leafroll virus (PLRV) structural and non‐structural proteins in Escherichia coli. Coat protein (CP) and RNA‐dependent RNA polymerase (RdRp)–fragments RdRp_43‐616 and RdRp_304‐537 were chosen for expression. A high level of CP and RdRp_304‐537 was obtained only in an expression system using pET‐45b(+) vector and E. coli Rosetta‐gami 2(DE3) cells. After purification, the His‐tagged PLRV proteins were used for immunization of rabbits.     

The apparent non‐host resistance of Ethiopian mustard to a radish‐infecting strain of Turnip mosaic virus is largely determined by the C‐terminal region of the P3 viral protein

Two different isolates of Turnip mosaic virus (TuMV: UK 1 and JPN 1) belonging to different virus strains were tested on three different Brassica species, namely turnip (Brassica rapa L.), Indian mustard (Brassica juncea L.) and Ethiopian mustard (Brassica carinata A. Braun). Although all three hosts were readily infected by isolate UK 1, isolate JPN 1 was able to establish a visible systemic infection only in the first two. Ethiopian mustard plants showed no local or systemic symptoms, and no virus antigens could be detected by enzyme‐linked immunosorbent assay (ELISA). Thus, this species looks like a non‐host for JPN 1, an apparent situation of non‐host resistance (NHR). Through an experimental approach involving chimeric viruses made by gene interchange between two infectious clones of both virus isolates, the genomic region encoding the C‐terminal domain of viral protein P3 was found to bear the resistance determinant, excluding any involvement of the viral fusion proteins P3N‐PIPO and P3N‐ALT in the resistance. A further determinant refinement identified two adjacent positions (1099 and 1100 of the viral polyprotein) as the main determinants of resistance. Green fluorescent protein (GFP)‐tagged viruses showed that the resistance of Ethiopian mustard to isolate JPN 1 is only apparent, as virus‐induced fluorescence could be found in discrete areas of both inoculated and non‐inoculated leaves. In comparison with other plant–virus combinations of extreme resistance, we propose that Ethiopian mustard shows an apparent NHR to TuMV JPN 1, but not complete immunity or extreme resistance.     

A new generation of cross‐linkers for selective detection by MALDI MS

We designed a new cross‐linker bearing a CHCA moiety. The use of the CHCA‐tagged cross‐linker JMV 3378 in conjunction with a neutral MALDI matrix α‐cyano‐4‐hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI‐TOF MS of cross‐link containing peptides. Discrimination between modified and non‐modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the α‐cyano‐4‐hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo‐myoglobine as model proteins.     

Radio‐transmitters do not affect seasonal productivity of female Golden‐winged Warblers

Investigating the potential effects of handling and marking techniques on study animals is important for correct interpretation of research results and to effect progress in data‐collection methods. Few investigators have compared the reproductive output of radio‐tagged and non‐radio‐tagged songbirds, and no one to date has examined the possible effect of radio‐tagging adult songbirds on the survival of their fledglings. In 2011 and 2012, we compared several parameters of reproductive output of two groups of female Golden‐winged Warblers (Vermivora chrysoptera) breeding in Minnesota, including 45 females with radio‐transmitters and 73 females we did not capture, handle, or mark. We found no difference between groups in clutch sizes, hatching success, brood sizes, length of incubation and nestling stages, fledging success, number of fledglings, or survival of fledglings to independence. Thus, radio‐tags had no measurable impact on the productivity of female Golden‐winged Warblers. Our results build upon previous studies where investigators have reported no effects of radio‐tagging on the breeding parameters of songbirds by also demonstrating no effect of radio‐tagging through the post‐fledging period and, therefore, the entire breeding season.     

The Chloroplast Function Database: a large‐scale collection of Arabidopsis Ds/Spm‐ or T‐DNA‐tagged homozygous lines for nuclear‐encoded chloroplast proteins,and their systematic phenotype analysis

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post‐translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor–mutator) transposon‐ or T‐DNA‐tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein‐coding regions. We systematically observed 3‐week‐old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild‐type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear‐encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/ . The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.     

Characterization and PCR multiplexing of polymorphic microsatellite loci in cashew (Anacardium occidentale L.) and their cross‐species utilization

Cashew (Anacardium occidentale L.) is the most economically important tropical nut crop in the world, and yet there are no sequence tagged site (STS) markers available for its study. Here we use an automated, high‐throughput system to isolate cashew microsatellites from a non‐enriched genomic library blotted onto membranes at high density for screening. Sixty‐five sequences contained a microsatellite array, of which 21 proved polymorphic among a closely related seed garden population of 49 genotypes. Twelve markers were suitable for multiplex analysis. Of these, 10 amplified in all three related tropical tree species tested: Anacardium microcarpum, Anacardium pumilum and Anacardium nanum.     

Rhodobacter capsulatus DprA is essential for RecA‐mediated gene transfer agent (RcGTA) recipient capability regulated by quorum‐sensing and the CtrA response regulator

Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best‐studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA‐mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA‐protecting protein A) homologue is essential for RcGTA‐mediated gene acquisition, and that dprA expression is induced by gtaI‐dependent quorum‐sensing and non‐phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C‐terminal region that resembles a dsDNA‐binding protein domain. Purified His‐tagged R. capsulatus DprA protein bound to both single‐stranded (ss)DNA and double‐stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single‐cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.     

Sex identification and PIT‐tagging: tools and prospects for studying intersexual differences in freshwater fishes

This study evaluated a technique to allow the long‐term monitoring of individual fishes of known sex in the wild using sex confirmation in close proximity to the reproductive period combined with individual tagging. Hundreds of partially migratory roach Rutilus rutilus were tagged with passive integrated transponders (PIT) following sex determination in spring and various performance measures were compared with fish tagged outside the reproductive period in autumn. Short‐term survival was >95% for R. rutilus sexed and tagged under natural field conditions. Total length (L_T) did not affect the probability of survival within the size range tagged (119–280 mm), nor were there differences in timing of migration the following season between individuals sexed and tagged in spring and individuals tagged in autumn (i.e. outside the reproductive period). Also, a similar per cent of R. rutilus sexed and tagged in spring and tagged in autumn migrated the following season (34·5 and 34·7%). Moreover, long‐term recapture data revealed no significant differences in body condition between R. rutilus individuals sexed and tagged in spring, individuals tagged in autumn and unmanipulated individuals. The observed sex ratio of recaptured fish did not differ from the expected values of equal recapture rates between males and females. Hence, there is no observable evidence for an adverse effect of tagging close to the reproductive period and therefore this method is suitable for studying intersexual differences and other phenotypic traits temporarily expressed during reproduction at the individual level in fishes.     

Characterization of an intact two‐component high‐affinity nitrate transporter from Arabidopsis roots

AtNRT2.1, a polypeptide of the Arabidopsis thaliana two‐component inducible high‐affinity nitrate transport system (IHATS), is located within the plasma membrane. The monomeric form of AtNRT2.1 has been reported to be the most abundant form, and was suggested to be the form that is active in nitrate transport. Here we have used immunological and transient protoplast expression methods to demonstrate that an intact two‐component complex of AtNRT2.1 and AtNAR2.1 (AtNRT3.1) is localized in the plasma membrane. A. thaliana mutants lacking AtNAR2.1 have virtually no IHATS capacity and exhibit extremely poor growth on low nitrate as the sole source of nitrogen. Near‐normal growth and nitrate transport in the mutant were restored by transformation with myc‐tagged AtNAR2.1 cDNA. Membrane fractions from roots of the restored myc‐tagged line were solubilized in 1.5% dodecyl‐β‐maltoside and partially purified in the first dimension by blue native gel electrophoresis. Using anti‐NRT2.1 antibodies, an oligomeric polypeptide (approximate molecular mass 150 kDa) was identified, but monomeric AtNRT2.1 was absent. This oligomer was also observed in the wild‐type, and was resolved, using SDS–PAGE for the second dimension, into two polypeptides with molecular masses of approximately 48 and 26 kDa, corresponding to AtNRT2.1 and myc‐tagged AtNAR2.1, respectively. This result, together with the finding that the oligomer is absent from NRT2.1 or NAR2.1 mutants, suggests that this complex, rather than monomeric AtNRT2.1, is the form that is active in IHATS nitrate transport. The molecular mass of the intact oligomer suggests that the functional unit for high‐affinity nitrate influx may be a tetramer consisting of two subunits each of AtNRT2.1 and AtNAR2.1.