A Light Insensitive Method for Contrast Enhancement of Insect Neurons Filled with a Cobalt-Lysine Complex

Modified protocols for cobalt-filling and silver intensification of neurons in the larval and adult stages of the moth, Manduca sexta, have led to improved neuronal visualization and minimal background staining. In particular, long distance projecting multisegmental in-terneurons. originating in the pterothoracic or terminal abdominal ganglion, were best visualized when a cobalt:lysine complex was used to fill hemi-connectives for several days at 4 C. Ganglia closest to the placement of tracer, which became flooded with cobalt:lysine during the filling period. were removed from the insect. This step eliminated the artifactual filling of neurons that may have taken up the tracer from such pooled regions. This led to a more accurate assessment of whether a multisegmental interneuron projected through the full length of nerve cord to the original site of tracer placement. The protocol for light insensitive silver intensification of cobalt-filled neurons was modified to include an important pH adjustment. NaOH was used to alter the pH of the protective colloid, sodium tungstate, to 10.4 or greater in solution. Especially in larvae. our techniques produced intensely stained cobalt-filled neurons within ganglia that remained transparent and relatively free of nonspecific silver deposition.     

Unequal binding of estrogens in human endometrium

Binding activity characteristics in human endometrium of estradiol-17-beta (E2), estrone (E1), estriol (E3), and 17-alpha-ethinylestradiol-17-beta (EE2) were determined in cytosol extracts. Unequal binding was observed. A lower affinity of E3 binding in endometrial cytosol when compared to E2 was parallel to a slower rate of association and to a higher rate of dissociation. For EE2, the slightly higher affinity was parallel to a higher rate of association and a slower dissociation rate. For E1, the association rate constant was 1/2 of that for E2 when the total number of binding sites able to bind E2 was considered in the calculation. Association rate constant was only 23% of that for E2 when the total number of binding sites able to bind E1 was considered in the calculation, and the dissociation rate was neglected. The dissociation rate of E1 receptor complexes was 20 times higher at both 0 and 25 degrees centigrade than the E2 receptor complexes. An unequal number of binding sites was measured for E2 and E1, an unexpected finding. Also observed was an unequal binding for E2 and E1 which varied during the menstrual cycle, and it is suggested that progesterone may be the regulatory factor since E1 and E2 receptors increased during the luteal phase.     

Strong Discrepancies between Local Temperature Mapping and Interpolated Climatic Grids in Tropical Mountainous Agricultural Landscapes

Bridging the gap between the predictions of coarse-scale climate models and the fine-scale climatic reality of species is a key issue of climate change biology research. While it is now well known that most organisms do not experience the climatic conditions recorded at weather stations, there is little information on the discrepancies between microclimates and global interpolated temperatures used in species distribution models, and their consequences for organisms’ performance. To address this issue, we examined the fine-scale spatiotemporal heterogeneity in air, crop canopy and soil temperatures of agricultural landscapes in the Ecuadorian Andes and compared them to predictions of global interpolated climatic grids. Temperature time-series were measured in air, canopy and soil for 108 localities at three altitudes and analysed using Fourier transform. Discrepancies between local temperatures vs. global interpolated grids and their implications for pest performance were then mapped and analysed using GIS statistical toolbox. Our results showed that global interpolated predictions over-estimate by 77.5±10% and under-estimate by 82.1±12% local minimum and maximum air temperatures recorded in the studied grid. Additional modifications of local air temperatures were due to the thermal buffering of plant canopies (from −2.7°K during daytime to 1.3°K during night-time) and soils (from −4.9°K during daytime to 6.7°K during night-time) with a significant effect of crop phenology on the buffer effect. This discrepancies between interpolated and local temperatures strongly affected predictions of the performance of an ectothermic crop pest as interpolated temperatures predicted pest growth rates 2.3–4.3 times lower than those predicted by local temperatures. This study provides quantitative information on the limitation of coarse-scale climate data to capture the reality of the climatic environment experienced by living organisms. In highly heterogeneous region such as tropical mountains, caution should therefore be taken when using global models to infer local-scale biological processes.     

Dilute-and-shoot analysis of therapeutic monoclonal antibody variants in fermentation broth: a method capability study

Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient therapeutic agents for various medical conditions, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drugs. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N-glycosylation and truncation variants of the expressed mAb are identified at the intact protein level. Simultaneously, absolute quantification of mAb content in fermentation broth is achieved. The whole workflow features excellent robustness as well as retention time and peak area stability. Additional enzymatic removal of N-glycans enables determination of mAb glycation levels, which are subsequently considered in relative N-glycoform quantification to correct for isobaric galactosylation. Several molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production, as well as targeted biosimilar development.     

Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation

Stimulation of iNKT cells by ??-galactosyl ceramide (??-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells’ response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in ??-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of ??-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation.     

Backdoor opening mechanism in acetylcholinesterase based on X-ray crystallography and molecular dynamics simulations

The transient opening of a backdoor in the active‐site wall of acetylcholinesterase, one of nature's most rapid enzymes, has been suggested to contribute to the efficient traffic of substrates and products. A crystal structure of Torpedo californica acetylcholinesterase in complex with the peripheral‐site inhibitor aflatoxin is now presented, in which a tyrosine at the bottom of the active‐site gorge rotates to create a 3.4‐Å wide exit channel. Molecular dynamics simulations show that the opening can be further enlarged by movement of Trp84. The crystallographic and molecular dynamics simulation data thus point to the interface between Tyr442 and Trp84 as the key element of a backdoor, whose opening permits rapid clearance of catalysis products from the active site. Furthermore, the crystal structure presented provides a novel template for rational design of inhibitors and reactivators, including anti‐Alzheimer drugs and antidotes against organophosphate poisoning.     

Strength of response suppression to distracter stimuli determines attentional-filtering performance in primate prefrontal neurons

Neurons in the primate dorsolateral prefrontal cortex (dlPFC) filter attended [corrected] targets distinctly from distracters through their response rates. The extent to which this ability correlates with the organism's performance, and the neural processes underlying it, remain unclear. We trained monkeys to attend to a visual target that differed in rank along a color-ordinal scale from that of a distracter. The animals' performance at focusing attention on the target and filtering out the distracter improved as ordinal distance between the stimuli increased. Importantly, dlPFC neurons also improved their filtering performance with increasing ordinal target-distracter distance; they built up their response rate in anticipation of the target-distracter onset, and then units encoding target representations increased their firing rate by similar amounts, whereas units encoding distracter representations gradually suppressed their rates as the interstimulus ordinal distance increased. These results suggest that attentional-filtering performance in primates relies upon dlPFC neurons' ability to suppress distracter representations.     

Distinct cerebrospinal fluid proteomes differentiate post-treatment lyme disease from chronic fatigue syndrome

Background

Neurologic Post Treatment Lyme disease (nPTLS) and Chronic Fatigue (CFS) are syndromes of unknown etiology. They share features of fatigue and cognitive dysfunction, making it difficult to differentiate them. Unresolved is whether nPTLS is a subset of CFS.

Methods and Principal Findings

Pooled cerebrospinal fluid (CSF) samples from nPTLS patients, CFS patients, and healthy volunteers were comprehensively analyzed using high-resolution mass spectrometry (MS), coupled with immunoaffinity depletion methods to reduce protein-masking by abundant proteins. Individual patient and healthy control CSF samples were analyzed directly employing a MS-based label-free quantitative proteomics approach. We found that both groups, and individuals within the groups, could be distinguished from each other and normals based on their specific CSF proteins (p<0.01). CFS (n = 43) had 2,783 non-redundant proteins, nPTLS (n = 25) contained 2,768 proteins, and healthy normals had 2,630 proteins. Preliminary pathway analysis demonstrated that the data could be useful for hypothesis generation on the pathogenetic mechanisms underlying these two related syndromes.

Conclusions

nPTLS and CFS have distinguishing CSF protein complements. Each condition has a number of CSF proteins that can be useful in providing candidates for future validation studies and insights on the respective mechanisms of pathogenesis. Distinguishing nPTLS and CFS permits more focused study of each condition, and can lead to novel diagnostics and therapeutic interventions.     

"Cost" of virginity in wild Drosophila melanogaster females

Laboratory studies have revealed a significant "cost of mating" to Drosophila melanogaster females in the form of reduced longevity. The effect is attributable to nonsperm components of the ejaculate. Female D. melanogaster are known to mate up to six times in nature, and given that they do not typically remate daily, it raises the question as to the extent to which the longevity of wild mated females is reduced. Here I addressed this question by comparing the longevity of wild virgin females, collected as they emerged from rotting fruit, to the longevity of randomly collected mature females at the same site. Because the randomly collected females all were inseminated and were fully pigmented at the time of collection, they already were older than the virgins when the experiment began. Contrary to expectations from laboratory studies, the older, mated females lived significantly longer than the virgins. Rather than a "cost of mating," there appears to be a "cost of virginity" to female D. melanogaster in the wild.     

Attenuation of carrageenan-induced hind paw edema and plasma TNF-α level by Philippine stingless bee (Tetragonula biroi Friese) propolis

Despite decades-long existence of the Philippine stingless bee industry, the biological activity of propolis from this native bee species (Tetragonula biroi Friese) remains poorly understood and sparingly investigated. Herein, we examined the potential anti-inflammatory efficacy of Philippine stingless bee propolis using the lambda (λ)-carrageenan-induced mice model of hind paw edema. Thirty (30), six-week-old, male ICR mice were randomly assigned into three treatment groups (n=10/group) as follows: distilled water group, diclofenac sodium group (10 mg/kg), and propolis group (100 mg/kg). All treatment were administered an hour prior to the injection of the phlogistic agent. As observed at 3 h post-injection, λ-carrageenan remarkably evoked the classical signs of hind paw edema exemplified grossly by swelling and hyperemia. The ameliorative effect of propolis became apparent at the onset of 6 h post-injection with a statistically significant finding evident at the 24-h period. This gross attenuation histologically correlated to a considerable and specific reduction of the dermal edema, which mirrored those of the diclofenac sodium group. Furthermore, both propolis and diclofenac sodium significantly attenuated the λ-carrageenan-induced increase in the protein expression levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) depicting more than two-fold decrement relative to the distilled water group. Altogether, these suggest that Philippine stingless bee propolis also exhibited a promising in vivo anti-inflammatory property, which can be partly mediated through the inhibition of TNF-α.