An easy‐to‐run method for routine analysis of eumelanin and pheomelanin in pigmented tissues

A procedure for analysis of melanin‐pigmented tissues based on alkaline hydrogen peroxide degradation coupled with high‐performance liquid chromatography (HPLC) ultraviolet determination of pyrrole‐2,3,5‐tricarboxylic acid (PTCA) for eumelanin and 6‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA) and 1,3‐thiazole‐2,4,5‐tricarboxylic acid for pheomelanin was recently developed. Despite advantages related to the degradation conditions and sample handling, a decrease of the reproducibility and resolution was observed after several chromatographic runs. We report herein an improved chromatographic methodology for simultaneous determination of PTCA and BTCA as representative markers of eumelanin and pheomelanin, respectively, based on the use of an octadecylsilane column with polar end‐capping with 1% formic acid (pH 2.8)/methanol as the eluant. The method requires conventional HPLC equipments and gives very good peak shapes and resolution, without need of ion pair reagents or high salt concentrations in the mobile phase. The intra‐assay precision of the analytical runs was satisfactory with CV values ≤4.0% (n = 5) for the two markers which did not exceed 8% after 50 consecutive injections on the column over 1 week. The peak area ratios at 254 and 280 nm (A₂₈₀/A₂₅₄: PTCA = 1.1, BTCA = 0.6) proved a valuable parameter for reliable identification of the structural markers even in the most complex degradation mixtures. The method can be applied to various eumelanin and pheomelanin pigmented tissues, including mammalian hair, skin and irides, and is amenable to be employed in population screening studies.     

Homeodomain Interacting Protein Kinase 2: A Target for Alzheimer's Beta Amyloid Leading to Misfolded p53 and Inappropriate Cell Survival

Background

Homeodomain interacting protein kinase 2 (HIPK2) is an evolutionary conserved serine/threonine kinase whose activity is fundamental in maintaining wild-type p53 function, thereby controlling the destiny of cells when exposed to DNA damaging agents. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer''s Disease (AD) that led to an impaired and dysfunctional response to stressors.

Methodology/Principal Findings

Here we examined the molecular mechanisms underlying the impairment of p53 activity in two cellular models, HEK-293 cells overexpressing the amyloid precursor protein and fibroblasts from AD patients, starting from recent findings showing that p53 conformation may be regulated by HIPK2. We demonstrated that beta-amyloid 1–40 induces HIPK2 degradation and alters HIPK2 binding activity to DNA, in turn regulating the p53 conformational state and vulnerability to a noxious stimulus. Expression of HIPK2 was analysed by western blot experiments, whereas HIPK2 DNA binding was examined by chromatin immunoprecipitation analysis. In particular, we evaluated the recruitment of HIPK2 onto some target promoters, including hypoxia inducible factor-1α and metallothionein 2A.

Conclusions/Significance

These results support the existence of a novel amyloid-based pathogenetic mechanism in AD potentially leading to the survival of injured dysfunctional cells.     

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

The Orai1 Ca²⁺ permeable ion channel is an important component of store operated Ca²⁺ entry (SOCE) in cells. It’s over-expression in basal molecular subtype breast cancers has been linked with poor prognosis, making it a potential target for drug development. We pharmacologically characterised a number of reported inhibitors of SOCE in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader (FLIPR) assay, and show that the rank order of their potencies in this assay is the same as those reported in a wide range of published assays. The assay was also used in a screening project seeking novel inhibitors. Following a broad literature survey of classes of calcium channel inhibitors we used simplified ligand structures to query the ZINC on-line database, and following two iterations of refinement selected a novel Orai1-selective dichlorophenyltriazole hit compound. Analogues of this were synthesized and evaluated in the FLIPR assay to develop structure–activity relationships (SAR) for the three domains of the hit; triazole (head), dichlorophenyl (body) and substituted phenyl (tail). For this series, the results suggested the need for a lipophilic tail domain and an out-of-plane twist between the body and tail domains.     

Effects of Shift Work on the Postural and Psychomotor Performance of Night Workers

The purpose of the study was to investigate the effects of shift work on the psychomotor and postural performance of night workers. The study included 20 polysomnography technicians working schedule of 12-h night shift by 36-h off. On the first day of protocol, the body mass and height were measured, and an actigraph was placed on the wrist of each participant. On the second day of protocol, sleepiness by Karolinska Sleepiness Scale, postural control by force platform (30 seconds) and psychomotor performance by Psychomotor Vigilance Task (10 minutes) were measured before and after 12-h night work. Results showed that after 12-h night work, sleepiness increased by 59% (p<0.001), postural control variables increased by 9% (p = 0.048), and 14% (p = 0.006). Mean reaction time, and the number of lapses of attention increased by 13% (p = 0.006) and 425% (p = 0.015), respectively, but the mean reciprocal reaction time decreased by 7%. In addition, there were correlations between sleepiness and postural control variables with opened eyes (r = 0.616, 95% confidence interval [CI] = 0.361–0.815; r = 0.538; 95% CI = 0.280–0.748) and closed eyes (r = 0.557; 95% CI = 0.304–0.764, r = 0497; 95% CI = 0.325–0.715) and a pronounced effect of sleepiness on postural sway (R² = 0.393; 95% CI = 0.001–0.03). Therefore, 12-h night work system and sleepiness showed a negative impact in postural and psychomotor vigilance performance of night workers. As unexpected, the force platform was feasibility to detect sleepiness in this population, underscoring the possibility of using this method in the workplace to prevent occupational injuries and accidents.     

Ca-Dependent Folding of Human Calumenin

Human calumenin (hCALU) is a six EF-hand protein belonging to the CREC family. As other members of the family, it is localized in the secretory pathway and regulates the activity of SERCA2a and of the ryanodine receptor in the endoplasmic reticulum (ER). We have studied the effects of Ca²⁺ binding to the protein and found it to attain a more compact structure upon ion binding. Circular Dichroism (CD) measurements suggest a major rearrangement of the protein secondary structure, which reversibly switches from disordered at low Ca²⁺ concentrations to predominantly alpha-helical when Ca²⁺ is added. SAXS experiments confirm the transition from an unfolded to a compact structure, which matches the structural prediction of a trilobal fold. Overall our experiments suggest that calumenin is a Ca²⁺ sensor, which folds into a compact structure, capable of interacting with its molecular partners, when Ca²⁺ concentration within the ER reaches the millimolar range.     

Investigating the effect of increasing robot group sizes on the human psychophysiological state in the context of human–swarm interaction

We study the psychophysiological state of humans when exposed to robot groups of varying sizes. In our experiments, 24 participants are exposed sequentially to groups of robots made up of 1, 3 and 24 robots. We measure both objective physiological metrics (skin conductance level and heart rate), and subjective self-reported metrics (from a psychological questionnaire). These measures allow us to analyse the psychophysiological state (stress, anxiety, happiness) of our participants. Our results show that the number of robots to which a human is exposed has a significant impact on the psychophysiological state of the human and that higher numbers of robots provoke a stronger response.     

Spatial coding of the predicted impact location of a looming object

Avoiding or intercepting looming objects implies a precise estimate of both time until contact and impact location. In natural situations, extrapolating a movement trajectory relative to some egocentric landmark requires taking into account variations in retinal input associated with moment-to-moment changes in body posture. Here, human observers predicted the impact location on their face of an approaching stimulus mounted on a robotic arm, while we systematically manipulated the relation between eye, head, and trunk orientation. The projected impact point on the observer's face was estimated most accurately when the target originated from a location aligned with both the head and eye axes. Eccentric targets with respect to either axis resulted in a systematic perceptual bias ipsilateral to the trajectory's origin. We conclude that (1) predicting the impact point of a looming target requires combining retinal information with eye position information, (2) that this computation is accomplished accurately for some, but not all, possible combinations of these cues, (3) that the representation of looming trajectories is not formed in a single, canonical reference frame, and (4) that the observed perceptual biases could reflect an automatic adaptation for interceptive/defensive actions within near peripersonal space.