Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Rhodnius prolixus and Rhodnius robustus–like (Hemiptera, Reduviidae) wing asymmetry under controlled conditions of population density and feeding frequency

Habitat change in Rhodnius spp may represent an environmental challenge for the development of the species, particularly when feeding frequency and population density vary in nature. To estimate the effect of these variables in stability on development, the degree of directional asymmetry (DA) and fluctuating asymmetry (FA) in the wing size and shape of R. prolixus and R. robustus–like were measured under laboratory controlled conditions. DA and FA in wing size and shape were significant in both species, but their variation patterns showed both inter-specific and sexual dimorphic differences in FA of wing size and shape induced by nutrition stress. These results suggest different abilities of the genotypes and sexes of two sylvatic and domestic genotypes of Rhodnius to buffer these stress conditions. However, both species showed non-significant differences in the levels of FA between treatments that simulated sylvan vs domestic conditions, indicating that the developmental noise did not explain the variation in wing size and shape found in previous studies. Thus, this result confirm that the variation in wing size and shape in response to treatments constitute a plastic response of these genotypes to population density and feeding frequency.     

RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes

Here, we examine tRNA-aminoacyl synthetase (ARS) localization in protein synthesis. Proteomics reveals that ten of the twenty cytosolic ARSs associate with ribosomes in sucrose gradients: phenylalanyl-RS (FRS), and the 9 ARSs that form the multi-ARS complex (MSC). Using the ribopuromycylation method (RPM) for localizing intracellular translation, we show that FRS and the MSC, and to a lesser extent other ARSs, localize to translating ribosomes, most strikingly when translation is restricted to poxvirus or alphavirus factories in infected cells. Immunoproximity fluorescence indicates close proximity between MSC and the ribosome. Stress induced-translational shutdown recruits the MSC to stress-granules, a depot for mRNA and translation components. MSC binding to mRNA provides a facile explanation for its delivery to translating ribosomes and stress granules. These findings, along with the abundance of the MSC (9 × 10(6) copies per cell, roughly equimolar with ribosomes), is consistent with the idea that MSC specificity, recently reported to vary with cellular stress (Netzer, N., Goodenbour, J. M., David, A., Dittmar, K. A., Jones, R. B., Schneider, J. R., Boone, D., Eves, E. M., Rosner, M. R., Gibbs, J. S., Embry, A., Dolan, B., Das, S., Hickman, H. D., Berglund, P., Bennink, J. R., Yewdell, J. W., and Pan, T. (2009) Nature 462, 522-526) can be modulated at the level of individual mRNAs to modify decoding of specific gene products.     

Comparison of Partial Volume Effects in Arterial and Venous Contrast Curves in CT Brain Perfusion Imaging

Purpose

In brain CT perfusion (CTP), the arterial contrast bolus is scaled to have the same area under the curve (AUC) as the venous outflow to correct for partial volume effects (PVE). This scaling is based on the assumption that large veins are unaffected by PVE. Measurement of the internal carotid artery (ICA), usually unaffected by PVE due to its large diameter, may avoid the need for partial volume correction. The aims of this work are to examine i) the assumptions behind PVE correction and ii) the potential of selecting the ICA obviating correction for PVE.

Methods

The AUC of the ICA and sagittal sinus were measured in CTP datasets from 52 patients. The AUCs were determined by i) using commercial CTP software based on a Gaussian curve-fitting to the time attenuation curve, and ii) by simple integration of the time attenuation curve over a time interval. In addition, frames acquired up to 3 minutes after first bolus passage were used to examine the ratio of arterial and venous enhancement. The impact of selecting the ICA without PVE correction was illustrated by reporting cerebral blood volume (CBV) measurements.

Results

In 49 of 52 patients, the AUC of the ICA was significantly larger than that of the sagittal sinus (p = 0.017). Measured after the first pass bolus, contrast enhancement remained 50% higher in the ICA just after the first pass bolus, and 30% higher 3 minutes later. CBV measurements were significantly lowered when the ICA was used without PVE correction.

Conclusions

Contradicting the assumptions underlying PVE correction, contrast in the ICA was significantly higher than in the sagittal sinus, even 3 minutes after the first pass of the contrast bolus. PVE correction might lead to overestimation of CBV if the CBV is calculated using the AUC of the time attenuation curves.     

Nuclear translation visualized by ribosome-bound nascent chain puromycylation

Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.     

Terminal deoxynucleotidyl transferase establishes and broadens antiviral CD8+ T cell immunodominance hierarchies

The action of TdT on mouse TCR genes accounts for approximately 90% of T cell repertoire diversity. We report that in TdT-/- mice, total T(CD8+) responses to influenza and vaccinia viruses are reduced by approximately 30% relative to wild-type mice. We find that T(CD8+) responses to three subdominant influenza virus determinants are reduced to background values in TdT-/- mice while responses to three immunodominant determinants undergo a major reshuffling. A similar reshuffling occurs in T(CD8+) responses to immunodominant vaccinia virus determinants, and is clearly based on broad differences in TCR family usage and CDR3 length between wild-type and TdT-/- mice. These findings demonstrate that TdT plays a critical role in the magnitude and breadth of anti-viral T(CD8+) responses toward individual determinants and suggests that germline TCR repertoire bias toward the most dominant determinants is a major factor in establishing immunodominance hierarchies.     

A Serological Survey of Ruminant Livestock in Kazakhstan During Post-Soviet Transitions in Farming and Disease Control

The results of a serological survey of livestock in Kazakhstan, carried out in 1997–1998, are reported. Serum samples from 958 animals (cattle, sheep and goats) were tested for antibodies to foot and mouth disease (FMD), bluetongue (BT), epizootic haemorrhagic disease (EHD), rinderpest (RP) and peste des petits ruminants (PPR) viruses, and to Brucella spp. We also investigated the vaccination status of livestock and related this to changes in veterinary provision since independence in 1991. For the 2 diseases under official surveillance (FMD and brucellosis) our results were similar to official data, although we found significantly higher brucellosis levels in 2 districts and widespread ignorance about FMD vaccination status. The seroprevalence for BT virus was 23%, and seropositive animals were widespread suggesting endemicity, despite the disease not having being previously reported. We found a few seropositives for EHDV and PPRV, which may suggest that these diseases are also present in Kazakhstan. An hierarchical model showed that seroprevalence to FMD and BT viruses were clustered at the farm/village level, rather than at a larger spatial scale. This was unexpected for FMD, which is subject to vaccination policies which vary at the raion (county) level.     

Randomised controlled trial of gabapentin in Complex Regional Pain Syndrome type 1 [ISRCTN84121379]

Background  

Complex Regional Pain Syndrome type one (CRPS I) or formerly Reflex Sympathetic Dystrophy (RSD) is a disabling syndrome, in which a painful limb is accompanied by varying symptoms. Neuropathic pain is a prominent feature of CRPS I, and is often refractory to treatment. Since gabapentin is an anticonvulsant with a proven analgesic effect in various neuropathic pain syndromes, we sought to study the efficacy of the anticonvulsant gabapentin as treatment for pain in patients with CRPS I.     

Use of dydrogesterone as a progestogen for oral contraception

Dydrogesterone is a potent orally active progestogen that has been used in clinical practice for over 40 years. Chemically, it belongs to the class of retrosteroids. Dydrogesterone is closely related to endogenous progesterone. It differs from most other synthetic progestogens in that it has no estrogenic, androgenic, glucocorticoid, or anabolic effects. The use of progestogens such as dydrogesterone is indicated in all cases of relative or absolute endogenous progesterone deficiency. Nowadays, dydrogesterone is mainly used in hormone replacement therapy (HRT). The present pilot study explored whether dydrogesterone could also be used as a progestogen for oral contraception. Given its highly favorable safety and tolerability profile, it would provide improvement over existing progestogens currently used in oral contraceptives (OCs). The results of this study indicate that dydrogesterone might indeed be a suitable candidate for use in oral contraception. This concept is currently being investigated further in two open-label phase II trials.     

Heat-aggregated noninfectious influenza virus induces a more balanced CD8(+)-T-lymphocyte immunodominance hierarchy than infectious virus

CD8(+)-T-cell (T(CD8+)) responses to infectious viruses are characterized by an immunodominance hierarchy in which the majority of T(CD8+) respond to one or a few immunodominant determinants, with a minority of T(CD8+) responding to a number of subdominant determinants. It is now well established that exogenous antigens are capable of inducing T(CD8+) to such immunodominant determinants, but the diversity of the response and the nature of the immunodominance hierarchy have not been examined. We addressed this issue by characterizing T(CD8+) responses to influenza virus preparations rendered inert by incubation for 10 min at 100 degrees C, as first reported by Speidel et al. (Eur. J. Immunol. 27:2391-2399, 1997). Extending these findings, we show that the primary T(CD8+) response to boiled virus can be sufficiently robust to be detected ex vivo by intracellular cytokine staining and that the response encompasses many of the peptides recognized by T(CD8+) induced by infectious virus. Importantly, the immunodominance hierarchy elicited was leveled, and we were unable to detect T(CD8+) that were specific for boiled virus. We used peritoneal exudate cells as antigen-presenting cells in vitro, and a number of observations indicated that boiled virus is processed via a phagocytic route that is likely to be endosomal in nature. These findings suggest that the repertoires of immunogenic peptides generated by endosomes and cytosolic processes overlap to a surprising degree. Furthermore, they demonstrate that the form of antigen administered can influence immunodominance hierarchies and that exogenous-antigen vaccines can induce broad and balanced T(CD8+) responses.