Rapid Sampling of Molecules via Skin for Diagnostic and Forensic Applications

Skin provides an excellent portal for diagnostic monitoring of a variety of entities; however, there is a dearth of reliable methods for patient-friendly sampling of skin constituents. This study describes the use of low-frequency ultrasound as a one-step methodology for rapid sampling of molecules from the skin. Sampling was performed using a brief exposure of 20 kHz ultrasound to skin in the presence of a sampling fluid. In vitro sampling from porcine skin was performed to assess the effectiveness of the method and its ability to sample drugs and endogenous epidermal biomolecules from the skin. Dermal presence of an antifungal drug—fluconazole and an abused substance, cocaine—was assessed in rats. Ultrasonic sampling captured the native profile of various naturally occurring moisturizing factors in skin. A high sampling efficiency (79 ± 13%) of topically delivered drug was achieved. Ultrasound consistently sampled greater amounts of drug from the skin compared to tape stripping. Ultrasonic sampling also detected sustained presence of cocaine in rat skin for up to 7 days as compared to its rapid disappearance from the urine. Ultrasonic sampling provides significant advantages including enhanced sampling from deeper layers of skin and high temporal sampling sensitivity

High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol

DIGE Proteome Analysis Reveals Suitability of Ischemic Cardiac In Vitro Model for Studying Cellular Response to Acute Ischemia and Regeneration

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy

A Systematic Analysis of Eluted Fraction of Plasma Post Immunoaffinity Depletion: Implications in Biomarker Discovery

Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins. However, depletion of these abundant proteins can result in concomitant removal of low abundant proteins. Although there are some reports suggesting the removal of non-targeted proteins, the predominant view is that number of such proteins is small. In this study, we identified proteins that are removed along with the targeted high abundant proteins. Three plasma samples were depleted using each of the three MARS (Hu-6, Hu-14 and Proteoprep 20) cartridges. The affinity bound fractions were subjected to gelC-MS using an LTQ-Orbitrap instrument. Using four database search algorithms including MassWiz (developed in house), we selected the peptides identified at <1% FDR. Peptides identified by at least two algorithms were selected for protein identification. After this rigorous bioinformatics analysis, we identified 101 proteins with high confidence. Thus, we believe that for biomarker discovery and proper quantitation of proteins, it might be better to study both bound and depleted fractions from any MARS depleted plasma sample

Classroom Emotions in Civic Education: A Multilevel Approach to Antecedents and Effects

Classroom emotions are major predictors of student learning and academic outcomes. Emotions might be of particular significance in civic education, where oftentimes highly controversial and heated debates take place. We examined antecedents and effects of classroom emotions in civic education through the lens of the control-value theory, where emotions are categorized in terms of valence (positive/negative) and arousal (activating/deactivating). Specifically, we investigated the classroom climate during discussions of political and social issues as an antecedent of students’ enjoyment, shame, anxiety, and boredom, in addition to a possible mediation effect of these emotions on political knowledge and participation as core outcomes in this domain. We tested our assumptions based on data from 1,117 students at vocational schools (grades 10–13). Multilevel structural equation modeling revealed different results based on the valence of emotions, showing a positive relation between an open classroom climate and enjoyment and negative relations with anxiety and boredom. No support was provided for the relation to shame. Enjoyment related positively, and all negative emotions (shame, anxiety, boredom) negatively to achievement on the knowledge test. In terms of arousal, all activating emotions (enjoyment, shame, anxiety) related positively to intended political participation. Furthermore, enjoyment mediated the association between classroom climate and intended political participation at the student level. Thus, this study strongly supports the theoretical assumptions of the control-value theory. Pedagogically, the results imply that fostering a classroom context that is open to diverse opinions can prevent the experience of negative emotions and increase students’ experience of enjoyment

Practice and results of retrograde intra-renal surgery using the Uscope PU3022a single-use flexible ureteroscope and Holmium laser in the management of upper urinary tract stones: observation of a reference centre in Guadeloupe, France

International audienceBackground:With the advancement of technology, single-use flexible ureteroscopes (suf-URS) are beginning to occupy an important place in the management of lithiasis disease. In addition to the satisfactory outcome they offer, they have the advantage of bypassing the traditional problems posed by reusable flexible ureteroscopes (rf-URS). Recently, a 9.2 FR single-use flexible ureteroscope (Uscope PU3022a) was introduced to the market by PUSENTM. The purpose of this study was to report our practice and outcome with the Uscope PU3022a using a Holmium laser in the management of upper urinary tract stones.Methods:From January 2021 to June 2022, 109 patients meeting the criteria were included in the study. Patient characteristics, stone characteristics, procedure, outcome, and complications were studied.Results:The mean age of the patients at the time of surgery was 62 years. The sex ratio was in favour of men (64.2%). More than half of the patients had a history of calculus (52.3%). The mean size of the stone was 13 mm (4–28 mm). The mean density was 1 112 HU (300– 1 900 HU). The average procedure time was 70 minutes (30–130 minutes). In the majority of cases, the stone was solitary (n = 72; 66.1%). The location of the stone was caliceal in 51.4% of cases. A double JJ catheter was placed in 96 (88.1%) patients before the suf-URS. A ureteral access sheath (UAS) was used in 95.4% of cases. Perioperative difficulties were encountered in 16.5% of the procedures.Conclusion:In our series, retrograde intrarenal surgery (RIRS) using the Uscope PU3022a in the management of upper urinary tract stones gives a good stone-free rate at the first session. It can be used for stones larger than 20 mm. Further large randomised studies are needed to assess the use of this type of suf-URS. The stone-free rate was 73.4%. Postoperative complications were reported in 5.5% of patients