S-neurons and not L-neurons are the source of GABAergic action in the ocellar retina

Summary Electrophysiological evidence obtained with current- and voltage clamp experiments from single L-neurons of the ocellar nerve of locust (Locusta migratoria) questions a direct synaptic feedback from these neurons onto the photoreceptors. The synaptic currents recorded under voltage clamp reflected the photoresponse of the L-neuron, despite the fact it developed no synaptic activity under these conditions. This result is contrary to GABAergic feedback models proposed in the literature. Electrophysiological recordings, as well as immunocytochemistry revealing GABA and glutamate decarboxylase, indicated a possible contribution of S-neurons in such a feedback system. A population of probable S-neurons whose somas were in the pars intercerebralis adjacent to the ocellar nerve tracts was heavely labelled. About 10 fibres entered each tract and formed a dense network of fine arborizations within the ocellar plexiform layer. L-neurons showed no GABA-immunoreactivity. Based on these data a new model for GABAergic feedback is proposed and discussed.     

Survival of Trypanosoma brucei in the Tsetse Fly Is Enhanced by the Expression of Specific Forms of Procyclin

African trypanosomes are not passively transmitted, but they undergo several rounds of differentiation and proliferation within their intermediate host, the tsetse fly. At each stage, the survival and successful replication of the parasites improve their chances of continuing the life cycle, but little is known about specific molecules that contribute to these processes. Procyclins are the major surface glycoproteins of the insect forms of Trypanosoma brucei. Six genes encode proteins with extensive glutamic acid–proline dipeptide repeats (EP in the single-letter amino acid code), and two genes encode proteins with an internal pentapeptide repeat (GPEET). To study the function of procyclins, we have generated mutants that have no EP genes and only one copy of GPEET. This last gene could not be replaced by EP procyclins, and could only be deleted once a second GPEET copy was introduced into another locus. The EP knockouts are morphologically indistinguishable from the parental strain, but their ability to establish a heavy infection in the insect midgut is severely compromised; this phenotype can be reversed by the reintroduction of a single, highly expressed EP gene. These results suggest that the two types of procyclin have different roles, and that the EP form, while not required in culture, is important for survival in the fly.     

Instrumental and Non-Instrumental Evaluation of 4-Meter Walking Speed in Older Individuals

BackgroundManual measurement of 4-meter gait speed by a stopwatch is the gold standard test for functional assessment in older adults. However, the accuracy of this technique may be biased by several factors, including intra- and inter-operator variability. Instrumental techniques of measurement using accelerometers may have a higher accuracy. Studies addressing the concordance between these two techniques are missing. The aim of the present community-based observational study was to compare manual and instrumental measurements of 4-meter gait speed in older individuals and to assess their relationship with other indicators of physical performance.MethodsOne-hundred seventy-two (69 men, 103 women) non-disabled community-dwellers aged ≥65 years were enrolled. They underwent a comprehensive geriatric assessment including physical function by Short Physical Performance Battery (SPPB), hand grip strength, and 6-minute walking test (6MWT). Timed usual walking speed on a 4-meter course was assessed by using both a stopwatch (4-meter manual measurement, 4-MM) and a tri-axial accelerometer (4-meter automatic measurement, 4-MA). Correlations between these performance measures were evaluated separately in men and women by partial correlation coefficients.ResultsIn both genders, 4-MA was associated with 4-MM (men r = 0.62, p<0.001; women r = 0.73, p<0.001), handgrip strength (men r = 0.40, p = 0.005; women r = 0.29, p = 0.001) and 6MWT (men r = 0.50, p = 0.0004; women r = 0.22, p = 0.048). 4-MM was associated with handgrip strength and 6MWT in both men and women. Considering gait speed <0.6 m/s as diagnostic of dismobility syndrome, the two methods of assessment disagreed, with a different categorization of subjects, in 19% of men and 23% of women. The use of accelerometer resulted in 29 (13 M, 16 F) additional diagnoses of dismobility, compared with the 4-MM.ConclusionsIn an older population, the concordance of gait speeds manually or instrumentally assessed is not optimal. The results suggest that manual measures might lead to misclassification of a substantial number of subjects. However, longitudinal studies using standardized and validated procedures aimed at the comparison of different techniques are needed before recommending the use of accelerometers in comprehensive geriatric assessment.     

Photochromism of phenoxynaphthacenequinones: diabatic or adiabatic phenyl group transfer?

The photochromic reactions of 6-phenyloxy-5,12-naphthacenequinone (1) and of the 6,11-diphenyloxy derivative 2 were investigated by subpicosecond pump-probe, photoacoustic, and emission spectroscopies, and by nanosecond laser flash photolysis (LFP). The transformation of the trans-quinones 1 and 2 to their ana-isomers proceeds via short-lived triplet states of 1 and 2 (tau ca. 2 ns) and spiro-bridged biradical intermediates (ca. 6 ns). The long-lived (micros) ana-triplets that are observed by LFP of 1 and 2 are formed (predominantly) by reexcitation of the biradicals and ana-quinones, which appear during the laser pulse. The reverse reaction, ana-->trans, proceeds exclusively from the lowest pi,pi* singlet state of the ana-quinones.     

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.     

Morphometric differences in midgut epithelial cells between strains of female Aedes aegypti (L.) (Insecta,Diptera)

Midgut epithelial cells of female Aedes aegypti deriving from 2 different mosquito strains were compared morphometrically. One of the 2 strains was recently isolated from nature (East Africa). The other strain is an old laboratory strain, reared under laboratory conditions for about 30 years. The quantitative morphological comparison demonstrates, that the ultrastructural composition of the midqut epithelial cells corresponds generally in both strains. It can be shown, however, that in the old strain, compared to the new strain, the organelle amount of midgut epithelial cells is significantly reduced during the first 53 days of the mosquito's life. Parallel to decreased relative volumes of organelles and reduced surface densities of membrane systems an increase in lysosome-like structures is observed. These observations are interpreted as deterioration processes, possibly due to the long rearing in the laboratory. Therefore, care should be taken in the selection of an A.aegypti strain for any quantitative morphological or physiological investigation.     

Lack of statistical power as a major limitation in understanding MHC‐mediated immunocompetence in wild vertebrate populations

Disentangling the sources of variation in developing an effective immune response against pathogens is of major interest to immunoecology and evolutionary biology. To date, the link between immunocompetence and genetic variation at the major histocompatibility complex (MHC) has received little attention in wild animals, despite the key role of MHC genes in activating the adaptive immune system. Although several studies point to a link between MHC and immunocompetence, negative findings have also been reported. Such disparate findings suggest that limited statistical power might be affecting studies on this topic, owing to insufficient sample sizes and/or a generally small effect of MHC on the immunocompetence of wild vertebrates. To clarify this issue, we investigated the link between MHC variation and seven immunocompetence proxies in a large sample of barn owls and estimated the effect sizes and statistical power of this and published studies on this topic. We found that MHC poorly explained variation in immunocompetence of barn owls, with small‐to‐moderate associations between MHC and immunocompetence in owls (effect size: .1 ≥ r ≤ .3) similar to other vertebrates studied to date. Such small‐to‐moderate effects were largely associated with insufficient power, which was only sufficient (>0.8) to detect moderate‐to‐large effect sizes (r ≥ .3). Thus, studies linking MHC variation with immunocompetence in wild populations are underpowered to detect MHC effects, which are likely to be of generally small magnitude. Larger sample sizes (>200) will be required to achieve sufficient power in future studies aiming to robustly test for a link between MHC variation and immunocompetence.     

High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites

The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.