Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Sequential Collinear Photofragmentation and Atomic Absorption Spectroscopy for Online Laser Monitoring of Triatomic Metal Species.

Industrial chemical processes are struggling with adverse effects, such as corrosion and deposition, caused by gaseous alkali and heavy metal species. Mitigation of these problems requires novel monitoring concepts that provide information on gas-phase chemistry. However, selective optical online monitoring of the most problematic diatomic and triatomic species is challenging due to overlapping spectral features. In this work, a selective, all-optical, in situ gas-phase monitoring technique for triatomic molecules containing metallic atoms was developed and demonstrated with detection of PbCl2. Sequential collinear photofragmentation and atomic absorption spectroscopy (CPFAAS) enables determination of the triatomic PbCl2 concentration through detection of released Pb atoms after two consecutive photofragmentation processes. Absorption cross-sections of PbCl2, PbCl, and Pb were determined experimentally in a laboratory-scale reactor to enable calibration-free quantitative determination of the precursor molecule concentration in an arbitrary environment. Limit of detection for PbCl2 in the laboratory reactor was determined to be 0.25 ppm. Furthermore, the method was introduced for in situ monitoring of PbCl2 concentration in a 120 MWth power plant using demolition wood as its main fuel. In addition to industrial applications, the method can provide information on chemical reaction kinetics of the intermediate species that can be utilized in reaction simulations.

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host.

Chemical elemental analysis of single acoustic-levitated water droplets by laser-induced breakdown spectroscopy.

Laser-induced breakdown spectroscopy is presented for trace element detection of liquid samples by analyzing a single droplet levitated by ultrasonic waves. A single liquid droplet is placed in the node of a standing acoustic wave produced by a uniaxial levitator for further chemical analysis. The acoustic levitator consists of a commercial Langevin-type transducer, attached to a concave mechanical amplifier, and a concave reflector. A micro-syringe was used to manually place individual liquid droplet samples in the acoustic levitation system. For chemical analysis, a laser-induced plasma is produced by focusing a single laser pulse on the levitated water droplet after it partially dries. The performance of the acoustic levitator on micron-sized droplets is discussed, and the detection of Ba, Cd, Hg, and Pb at parts per million (milligrams/liter) and sub-parts per million levels is reported. The process, starting from placing the sample in the acoustic levitator and ending on the chemical identification of the traces, takes a few minutes. The approach is particularly interesting in applications demanding limited volumes of liquid samples and relative simple and inexpensive techniques.

Propofol pharmacokinetic and pharmacodynamic profile and its electroencephalographic interaction with remifentanil in children.

BACKGROUND: Propofol and remifentanil are commonly combined during total intravenous anesthesia. The impact of remifentanil in this relationship is poorly quantified in children. Derivation of an integrated pharmacokinetic and pharmacodynamic propofol model, containing remifentanil pharmacodynamic interaction information, enables propofol effect-site target-controlled infusion in children with a better prediction of its hypnotic effect when both drugs are combined. AIMS: We designed this study to derive an integrated propofol pharmacokinetic-pharmacodynamic model in children and to describe the pharmacodynamic interaction between propofol and remifentanil on the electroencephalographic bispectral index effect. METHODS: Thirty children (mean age: 5.45 years, range 1.3-11.9; mean weight: 23.5 kg, range 8.5-61) scheduled for elective surgery with general anesthesia were studied. After sevoflurane induction, maintenance of anesthesia was based on propofol and remifentanil. Blood samples to measure propofol concentration were collected during anesthesia maintenance and up to 6 hours in the postoperative period. Bispectral index data were continuously recorded throughout the study. A pharmacokinetic-pharmacodynamic model was developed using population modeling. The Greco model was used to examine the pharmacokinetic-pharmacodynamic interaction between propofol and remifentanil for BIS response RESULTS: Propofol pharmacokinetic data from a previous study in 53 children were pooled with current data and simultaneously analyzed. Propofol pharmacokinetics were adequately described by a three-compartment distribution model with first-order elimination. Theory-based allometric relationships based on TBW improved the model fit. The Greco model supported an additive interaction between propofol and remifentanil. Remifentanil showed only a minor effect in BIS response. CONCLUSION: We have developed an integrated propofol pharmacokinetic-pharmacodynamic model that can describe the pharmacodynamic interaction between propofol and remifentanil for BIS response. An additive interaction was supported by our modeling analysis.

Enhanced self-mixing interferometry based on volume Bragg gratings and laser diodes emitting at 405-nm wavelengths.

Self-mixing interferometry (SMI) represents a robust, self-aligned technique for metrology applications. Recently, it has been shown that the detection of the frequency-modulated (FM) signal enhances the conventional SMI signal based on the amplitude modulation. Here, an all-optical, simple and effective alternative approach to detect the FM self-mixing signal is presented. We demonstrate the enhanced self-mixing approach using a laser diode emitting at 405 nm and a volume Bragg grating (VBG) to map frequency to intensity modulations for further optical detection. Our approach overcomes the spectral range limitation of the edge filtering approach based on molecular absorption filters, since the VBGs can be fabricated at any spectral range.

Detection of single microparticles in airflows by edge-filter enhanced self-mixing interferometry.

A laser Doppler velocimetry (LDV) sensor using the edge-filter enhanced self-mixing interferometry (ESMI) is presented based on speed measurements of single microparticles. The ESMI detection utilizes an acetylene edge-filter that maps the frequency modulation of a semiconductor laser into an intensity modulation as the laser wavelength is tuned to the steep edge of the absorption profile. In this work, the ESMI signal was analyzed for aerosol particles of different sizes from 1 µm to 10 µm at a distance of 2.5 m. At this operation range, the signal from single particles of all sizes was successfully acquired enabling particle velocity measurements through the Doppler shifted frequency along the beam axis. For the particular case of 10 µm particles, single aerosol particles were still detected at an unprecedented range of 10 m. A theoretical treatment describing the relation between Mie scattering theory and the self-mixing phenomenon on single-particle detection is presented supporting the experimental results. The results show that the edge-filter enhanced self-mixing technique opens new possibilities for self-mixing detection where longer ranges, lower backscattering laser powers and higher velocities are involved. For example, it can be used as a robust and inexpensive anemometer for LDV applications for airflows with low-number density of microparticles.

Edge filter enhanced self-mixing interferometry.

Self-mixing interferometry (SMI) represents a simple, robust, and self-aligned technique for metrology applications. Still, its applicability on diffuse targets is limited to distances up to few meters. We present an enhanced approach based on the detection of the frequency-modulated (FM) self-mixing signal. The FM signal detection is achieved using an acetylene edge filter that maps laser frequency variations into intensity variations as the laser wavelength is tuned to the edge of the steep absorption profile. An experimental comparison between the enhanced and the conventional SMI approach is presented. The new approach yields to about two orders of magnitude larger signal-to-noise ratio and extends the applicability of SMI into new fields allowing longer detection ranges and lower backscattering signals.

Enhanced standing-wave acoustic levitation using high-order transverse modes in phased array ultrasonic cavities.

Airborne acoustic trapping by ultrasonic phased arrays has seen great advances in recent years, and yet the manipulation of objects with different shapes and sizes or heavy particles remains challenging. Here, we demonstrate that the manipulation capabilities of a standing-wave acoustic levitator can be extended by introducing intracavity high-order transverse (HOT) modes in the azimuthal direction, enabling the simultaneous trapping of several objects within a wide range of shapes and sizes with positional and rotational stability, including objects with sizes larger than one wavelength and weights in the scale of millinewtons. The conditions to generate different HOT modes are theoretically analyzed and experimentally implemented. We numerically calculate the pressure distributions, exhibiting good qualitative agreement with the experimental pressure distributions obtained with schlieren images. In addition, we calculate the acoustic force field for several examples of HOT modes and different particle sizes, which leads to a qualitative understanding of the experimental observations.

Simulation-Based Training Program for Peripherally Inserted Central Catheter Placement: Randomized Comparative Study of in-Person Training With Synchronous Feedback Versus Distance Training With Asynchronous Feedback.

INTRODUCTION: Simulation training that includes deliberate practice is effective for procedural skill training. Delivering feedback remotely and asynchronously has been examined for more cost-efficient training. This prospective randomized study aimed to compare 2 feedback techniques for simulation training: synchronous direct feedback versus asynchronous distance feedback (ASYNC). METHODS: Forty anesthesia and internal medicine residents were recruited after study approval by the institutional ethics committee. Residents reviewed instructional material on an online platform and performed a pretraining assessment (PRE) for peripherally inserted central catheter (PICC) placement. Each resident was then randomly assigned to 1 of 2 training types, practice with synchronous direct feedback (SYNC) or practice with ASYNC. Training consisted of four, 1-hour practice sessions; each was conducted once per week. Both groups underwent posttraining evaluation (POST). The PRE and POST assessments were videotaped and evaluated by 2 independent, blinded reviewers using a global rating scale. RESULTS: Thirty-five residents completed the training program and both evaluations. Both groups had significantly improved global rating scale scores after 4 sessions. The SYNC group improved from 28 to 45 points (P < 0.01); the ASYNC group improved from 26.5 to 46 points (P < 0.01). We found no significant between-group differences for the PRE (P = 0.42) or POST assessments (P = 0.13). CONCLUSION: This simulation-based training program significantly improved residents' peripherally inserted central venous catheter placement skills using either modality. With these results, we are unable to demonstrate the superiority of synchronous feedback over ASYNC. Asynchronous feedback training modality represents a new, innovative approach for health care procedural skills training.

Oral infectivity through carnivorism in murine model of Trypanosoma cruzi infection.

Introduction: Oral transmission of T. cruzi is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat from animals infected with T. cruzi may be responsible for transmitting the infection. Therefore, the general objective of this study was to investigate host-pathogen interactions between the parasite and gastric mucosa and the role of meat consumption from infected animals in the oral transmission of T. cruzi. Methods: Cell infectivity assays were performed on AGS cells in the presence or absence of mucin, and the roles of pepsin and acidic pH were determined. Moreover, groups of five female Balb/c mice were fed with muscle tissue obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by a parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacities. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed, and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies and by LC-MS/MS and bioinformatics analysis. Results: Trypomastigote migration in the presence of mucin was reduced by approximately 30%, whereas in the presence of mucin and pepsin at pH 3.5, only a small proportion of parasites were able to migrate (∼6%). Similarly, the ability of TCTs to infect AGS cells in the presence of mucin is reduced by approximately 20%. In all cases, 60-100% of the animals were fed meat from mice infected in the acute phase or infected with trypomastigotes or amastigotes developed high parasitemia, and 80% died around day 40 post-infection. The adhesion assay showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS and bioinformatics analysis, also confirmed that transialidase, cysteine proteinases, and gp63 may be involved in TCTs attachment or invasion of human stomach cells because they can potentially interact with different proteins in the human stomach mucosa. In addition, several human gastric mucins have cysteine protease cleavage sites. Discussion: Then, under our experimental conditions, consuming meat from infected animals in the acute phase allows the T. cruzi infection. Similarly, trypomastigotes and amastigotes could infect mice when administered orally, whereas cysteinyl proteinases and trans-sialidase appear to be relevant molecules in this infective process.

Effectiveness of Teaching Mini Handball through Non-Linear Pedagogy in Different Socioeconomic Contexts: A Pilot Study.

Mini handball is among the sports included as part of school physical education in Chile to improve children's motor skills and to motivate their adherence to a healthy and active lifestyle in response to concerns about this country's high level of childhood obesity. To this end, non-linear pedagogy (NLP) has been used to develop motor skills through mini handball in the school context. However, socioeconomic differences that influence the development of children's motor skills have not been considered to determine whether the methodology applies to everyone. The aim of the present observational study is to describe and compare the effectiveness of the previously applied NLP methodology in two contrasting socioeconomic contexts to determine whether it helps to develop motor skills through mini handball in both school contexts. The Levine test was used to determine the homogeneity of the variances (p < 0.05), as the distribution of the data was not normal. The Kruskal-Wallis H statistical test was used to analyse within-group data. Additionally, the Mann-Whitney U test was applied for comparisons between groups. The results show significant improvements in the acquisition of the expected motor skills specific to mini handball. Additionally, a shortening of the gap was evidenced between the groups during the training process, with no significant differences at the end of the progression. Therefore, the investigated NLP is equally as effective for schoolchildren in two opposite socioeconomic contexts.

Adjusting single-axis acoustic levitators in real time using rainbow schlieren deflectometry.

Acoustic levitation uses focused high-intensity airborne ultrasound to hold particles in mid-air. It is becoming an important tool for experiments in spectrometry, lab-on-a-droplet, and display technologies. Nowadays, arrays of multiple small transducers can be used to build acoustic levitators; however, their performance depends on the optimal alignment. This work describes a simple method capable of visualizing a 2D projection of the acoustic field in real time using rainbow schlieren deflectometry. Good agreement was found between the images obtained with this technique and simulations of the acoustic pressure. It was also found that the maximum amplitudes of the field were obtained with the levitator aligned so that the power consumption was minimum, showing another simple and affordable way to adjust the levitators. As a result of the alignment optimization, it was possible for the first time to levitate steel and mercury in a levitator constructed with off-the-shelf components. The schlieren technique was applied to the TinyLev acoustic levitation system, but it can be applied to visualize the acoustic potential produced by different types of levitation systems.

Characterization of pectin extracted under mild conditions from tempesquistle (Sideroxylon palmeri) fruit at two maturity stages.

BACKGROUND: le tree (Sideroxylon palmeri) belongs to family Sapotaceae, and its fruits contain hydrophilic and hydrophobic gums. Aim of this study was to develop an extraction method for pectin, a hydrophilic gum, from green and ripe tempesquistle fruit. The extraction method was gentle to minimize potential structural changes in pectin, a maceration was used, and then pectin precipitation was induced with acetone at room temperature. Pectin characterization was carried out in two steps. The first step consisted of qualitative tests of Molisch, Fehling and Lugol. The second step consisted of quantitative tests to determine esterification degree (ED) by ATR-FTIR and D-galacturonic acid (D-GalA) content with a colorimetric assay. ATR-FTIR spectroscopic method revealed that green and ripe tempesquistle fruit have an ED of 0% and 30.4%, respectively. Both fruit have a low ED pectin. The content of D-GalA in green and ripe pectins was 18.8 ±2.7% and 20.2 ±0.6%, respectively. The yield obtained in green and ripe fruit samples was 1.6 ±0.2% and 3.0 ±0.1%, respectively. The extraction method allowed two types of pectins obtained in function of maturity stage of tempesquistle fruit distinguishable by. METHODS: istle tree (Sideroxylon palmeri) belongs to family Sapotaceae, and its fruits contain hydrophilic and hydrophobic gums. Aim of this study was to develop an extraction method for pectin, a hydrophilic gum, from green and ripe tempesquistle fruit. The extraction method was gentle to minimize potential structural changes in pectin, a maceration was used, and then pectin precipitation was induced with acetone at room temperature. Pectin characterization was carried out in two steps. The first step consisted of qualitative tests of Molisch, Fehling and Lugol. The second step consisted of quantitative tests to determine esterification degree (ED) by ATR-FTIR and D-galacturonic acid (D-GalA) content with a colorimetric assay. ATR-FTIR spectroscopic method revealed that green and ripe tempesquistle fruit have an ED of 0% and 30.4%, respectively. Both fruit have a low ED pectin. The content of D-GalA in green and ripe pectins was 18.8 ±2.7% and 20.2 ±0.6%, respectively. The yield obtained in green and ripe fruit samples was 1.6 ±0.2% and 3.0 ±0.1%, respectively. The extraction method allowed two types of pectins obtained in function of maturity stage of tempesquistle fruit distinguishable by ATR. RESULTS: istle tree (Sideroxylon palmeri) belongs to family Sapotaceae, and its fruits contain hydrophilic and hydrophobic gums. Aim of this study was to develop an extraction method for pectin, a hydrophilic gum, from green and ripe tempesquistle fruit. The extraction method was gentle to minimize potential structural changes in pectin, a maceration was used, and then pectin precipitation was induced with acetone at room temperature. Pectin characterization was carried out in two steps. The first step consisted of qualitative tests of Molisch, Fehling and Lugol. The second step consisted of quantitative tests to determine esterification degree (ED) by ATR-FTIR and D-galacturonic acid (D-GalA) content with a colorimetric assay. ATR-FTIR spectroscopic method revealed that green and ripe tempesquistle fruit have an ED of 0% and 30.4%, respectively. Both fruit have a low ED pectin. The content of D-GalA in green and ripe pectins was 18.8 ±2.7% and 20.2 ±0.6%, respectively. The yield obtained in green and ripe fruit samples was 1.6 ±0.2% and 3.0 ±0.1%, respectively. The extraction method allowed two types of pectins obtained in function of maturity stage of tempesquistle fruit distinguishable by ATR-FTIR.

Incorporating the Patient Voice Into Practice Improvement: A Role for Medical Trainees.

BACKGROUND AND OBJECTIVES: Many primary care clinics rely on providers and staff to generate quality improvement (QI) ideas without explicitly including patients. However, without understanding patient perspectives, clinics may miss areas for improvement. We identified practice improvement opportunities using a medical student-driven pilot of QI design that incorporates the patient voice and explored provider/staff perceptions of patient perspectives. METHODS: One medical student interviewed eight patients for their perspectives on QI opportunities at a university-based primary care practice. Two trainees independently coded interview transcripts using directed content analysis (final codebook: 11 themes) and determined frequency of mentions for each theme. We surveyed 11 providers/staff by asking them to select 5 of the 11 themes and place them in rank order based on their perceptions of patient concerns; their surveys were aggregated into one ranked list. RESULTS: Patients most frequently identified the following themes as QI opportunities: relationship (ie, feeling of personal connection with providers/staff), specialty care, convenience, sustainability, and goal follow-up. While patients frequently identified relationship (rank=1) and goal follow-up (rank=3) as QI opportunities, the provider/staff top five list did not include relationship (rank=10) or goal follow-up (rank=7). CONCLUSIONS: Our study demonstrates two things: (1) there are areas of discordance between provider/staff perceptions and patient perspectives regarding practice improvement opportunities; and (2) medical students can participate meaningfully in the QI process. By harnessing patient perspectives with the help of medical trainees, clinics may better understand patient concerns and avoid potential QI blind spots.

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes.

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.

[Thrombotic and haemorrhagic complications in patients with cerebral aneurysms treated by endovascular approach and their association with the use of antiplatelet agents: Descriptive evaluation]. / Complicaciones trombóticas y hemorrágicas en pacientes con aneurismas cerebrales tratados por vía endovascular y su relación con el uso de antiagregantes plaquetarios: evaluación descriptiva.

OBJECTIVE: The protocol for optimal antiplatelet therapy to prevent thrombotic complications following brain aneurysm embolisation is not clear. Our objective is to describe the characteristics of patients presenting with thrombotic or haemorrhagic complications secondary to endovascular treatment. METHODS: A cross sectional descriptive study was performed, which included all patients that required endovascular treatment for brain aneurysm at San Ignacio University Hospital from November 2007 to January 2016. Thrombotic and haemorrhagic complications over six months of follow-up were assessed, considering the premedication regimen with antiplatelet agents, location, size of the aneurysm and embolisation technique performed. RESULTS: 122 patients were evaluated, on whom 130 procedures were performed for endovascular treatment of brain aneurysms. Thrombotic complications were more frequent in patients who did not receive premedication (25%) compared to those who did receive an antiplatelet treatment regimen (standard dose 3.87% or loading dose 8.70%), and this difference was statistically significant (P=.043). CONCLUSIONS: Thromboembolic events are the most common complication of brain aneurysm embolisation. Both our study and the literature suggest that the use of dual antiplatelet therapy with aspirin and clopidogrel lowers the rate of symptomatic thromboembolic complications, regardless of the administration protocol.