Construct validity of nine new inanimate exercises for robotic surgeon training using a standardized setup

Background

As more surgeons choose to complete procedures robotically, validated training tools are needed so that they can acquire and maintain the technical skills required to proficiently use robotic systems. The purpose of this study was to show construct validity of nine new inanimate training exercises for robot-assisted surgery. The inanimate training exercises were designed to span several core technical skills required to use a robotic system.

Methods

New (n = 30) and experienced (n = 11) robotic surgeons participated in the study. New robotic surgeons had not yet completed their first robotic surgery case and participated in this study before attending their robotic certification course. Experienced robotic surgeons had completed more than 200 robotic surgery cases. The raw scores from the exercises were reported so that other research groups could easily define custom proficiency levels. Example normalized scores that could be used in proficiency-based curricula were computed. These normalized scores balanced efficiency (completion time) and accuracy (exercise-specific errors) to measure performance. Finally, the setup was standardized using a custom docking model, which enabled consistent and repeatable completion of the inanimate exercises across surgeons.

Results

For all nine exercises, experienced robotic surgeons completed the exercises significantly faster than new robotic surgeons (p < 0.01). Similarly, experienced robotic surgeons achieved higher normalized scores than new robotic surgeons for all nine exercises (p < 0.01). Finally, consistent robot setup was achieved using the custom docking model based on an analysis of the robot kinematic data.

Conclusions

In summary, all nine inanimate exercises showed construct validity. The results suggest that the inanimate exercises along with the custom docking model can be used as part of proficiency-based curricula to improve robotic surgeon training.     

Breakdown of Phosphatidylserine Asymmetry Following Treatment of Erythrocytes with Lumefantrine

Background: Lumefantrine, a commonly used antimalarial drug, inhibits hemozoin formation in parasites. Several other antimalarial substances counteract parasitemia by triggering suicidal death or eryptosis of infected erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), formation of ceramide, oxidative stress and/or activation of p38 kinase, protein kinase C (PKC), or caspases. The present study explored, whether lumefantrine stimulates eryptosis. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species from 2'',7''-dichlorodihydrofluorescein-diacetate fluorescence, content of reduced glutathione (GSH) from mercury orange fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to lumefantrine (3 µg/mL) was followed by a significant increase of annexin-V-binding without significantly altering forward scatter, [Ca²⁺]_i, ROS formation, reduced GSH, or ceramide abundance. The annexin-V-binding following lumefantrine treatment was not significantly modified by p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM), PKC inhibitor staurosporine (1 µM) or pancaspase inhibitor zVAD (1 or 10 µM). Conclusions: Lumefantrine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca²⁺, ceramide formation, ROS formation, glutathione content, p38 kinase, PKC or caspases.     

Impaired polysaccharide responsiveness without agammaglobulinaemia in three patients with hypomorphic mutations in Bruton Tyrosine Kinase—No detection by newborn screening for primary immunodeficiencies

Hypomorphic mutations in the gene encoding Bruton tyrosine kinase (BTK) may result in milder phenotypes and delayed diagnosis of B-cell related immunodeficiencies due to residual BTK function. Newborn screening for kappa-deleting-recombination-excision circles (KRECs) reliably identifies classical X-linked agammaglobulinaemia (XLA) patients with profound B-cell lymphopenia at birth but has not been evaluated in patients with residual BTK function. We aimed to evaluate clinical findings, BTK function and KREC copy numbers in three patients with BTK mutations presenting with impaired polysaccharide responsiveness without agammaglobulinaemia. One patient had an invasive pneumococcal infection at the age of 4 years. All three patients (two brothers) had visible tonsils, normal to slightly decreased immunoglobulin G levels, undetectable pneumococcal antibodies despite pneumococcal conjugate vaccinations, no antibody response after a diagnostic polysaccharide vaccination as well as profound B-cell lymphopenia with residual B-cell differentiation. BTK mutations were identified by Sanger sequencing. BTK staining and phosphorylation assays were performed on peripheral B cells. KREC copy numbers were determined from dried blood spots obtained within the first week of life as well as once at the age of 8, 6 and 3 years, respectively. BTK staining showed residual protein expression. Also, residual BTK activity could be demonstrated. KREC copy numbers from dried blood spots were above the threshold set for detection of patients with profound B-cell lymphopenia. Male patients with impaired polysaccharide responsiveness should be evaluated for B-cell lymphopenia followed by BTK analyses irrespective of immunoglobulin levels or tonsil size.     

Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction

Although transplantation of adipose-derived stem cells (ADSC) in chronic myocardial infarction (MI) models is associated with functional improvement, its therapeutic value is limited due to poor long-term cell engraftment and survival. Thus, the objective of this study was to examine whether transplantation of collagen patches seeded with ADSC could enhance cell engraftment and improve cardiac function in models of chronic MI. With that purpose, chronically infarcted Sprague–Dawley rats (n = 58) were divided into four groups and transplanted with media, collagen scaffold (CS), rat ADSC, or CS seeded with rat ADSC (CS-rADSC). Cell engraftment, histological changes, and cardiac function were assessed 4 months after transplantation. In addition, Göttingen minipigs (n = 18) were subjected to MI and then transplanted 2 months later with CS or CS seeded with autologous minipig ADSC (CS-pADSC). Functional and histological assessments were performed 3 months post-transplantation. Transplantation of CS-rADSC was associated with increased cell engraftment, significant improvement in cardiac function, myocardial remodeling, and revascularization. Moreover, transplantation of CS-pADSC in the pre-clinical swine model improved cardiac function and was associated with decreased fibrosis and increased vasculogenesis. In summary, transplantation of CS-ADSC resulted in enhanced cell engraftment and was associated with a significant improvement in cardiac function and myocardial remodeling.     

Clinical Heterogeneity of Immunodysregulation,Polyendocrinopathy, Enteropathy,X-linked: Pulmonary Involvement as a Non-Classical Disease Manifestation

Purpose

IPEX (Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked) is a rare X-linked recessive life-threatening disorder characterized by autoimmunity and early death. Pulmonary complication related with IPEX has not been elucidated exactly. Here, we report 4 IPEX patients, 3 of which died from severe pulmonary disease.

Methods

Clinical data and laboratory findings including autoantibodies, immunoglobulin levels as well as number of T, B and NK cells were evaluated. FOXP3 expression and T reg activity were analyzed. The FOXP3 gene was sequenced and RNA analysis was performed.

Results

Patient I (PI) presented with nephrotic syndrome at 3 years of age and then developed autoimmune hepatitis without eczema, enteropathy or high IgE and died at 9 years of age due to acute respiratory distress syndrome (ARDS). Two cousins of PI had the same hypomorphic splice site mutation leading to a deletion of 27 amino acids, but normal FOXP3 protein expression and normal suppressive capacity of T reg in a proliferation inhibition assay. However, they exhibited typical symptoms such as eczema, diabetes and enteropathy with eosinophilia at early age (PII, PIII) and were transplanted in infancy. One of them had severe respiratory distress right after birth (PIII). Patient IV from another family presented with chronic diarrhea without autoimmune manifestations and died due to ARDS.

Conclusion

Lung disease related to IPEX syndrome has not been reported before and this entity could be a critical factor in disease outcome.     

Nanoscale magnetic and charge anisotropies at manganite interfaces

Strong correlated manganites are still under intense research owing to their complex phase diagrams in terms of Sr-doping and their sensitivity to intrinsic and extrinsic structural deformations. Here, we performed X-ray absorption spectroscopy measurements of manganite bilayers to explore the effects that a local Sr-doping gradient produce on the charge and antiferromagnetic anisotropies. In order to gradually tune the Sr-doping level along the axis perpendicular to the samples we have grown a series of bilayers with different thicknesses of low-doped manganites (from 0 nm to 6 nm) deposited over a La_0.7Sr_0.3MnO₃ metallic layer. This strategy permitted us to resolve with high accuracy the thickness region where the charge and spin anisotropies vary and the critical thickness t_c over which the out of plane orbital asymmetry does not have any further modifications. We found that the antiferromagnetic spin axis points preferentially out of the sample plane regardless the capping layer thickness. However, it tilts partially into the sample plane far from this critical thickness, owing to the combined contributions of the external structural strain and electron doping. Furthermore, we found that the doping level of the capping layer strongly affects the critical thickness, giving clear evidence of the influence exerted by the electron doping on the orbital and magnetic configurations. These anisotropic changes induce subtle modifications on the domain reorientation of La_0.7Sr_0.3MnO₃, as evidenced from the magnetic hysteresis cycles.

Nanoscale variation of antiferromagnetic and charge anisotropies has been found at manganite interfaces with an artificially created Sr-doping.     

A microdevice for parallelized pulmonary permeability studies

We describe a compartmentalized microdevice specifically designed to perform permeability studies across a model of lung barrier. Epithelial cell barriers were reproduced by culturing Calu-3 cells at the air-liquid interface (AIC) in 1 mm² microwells made from a perforated glass slide with an embedded porous membrane. We created a single basolateral reservoir for all microwells which eliminated the need to renew the growth medium during the culture growth phase. To perform drug permeability studies on confluent cell layers, the cell culture slide was aligned and joined to a collection platform consisting in 35 μL collection reservoirs connected at the top and bottom with microchannels. The integrity and functionality of the cell barriers were demonstrated by measurement of trans-epithelial electrical resistance (TEER), confocal imaging and permeability assays of ¹⁴C-sucrose. Micro-cell barriers were able to form confluent layers in 1 week, demonstrating a similar bioelectrical evolution as the Transwell systems used as controls. Tight junctions were observed throughout the cell-cell interfaces, and the low permeability coefficients of ¹⁴C-sucrose confirmed their functional presence, creating a primary barrier to the diffusion of solutes. This microdevice could facilitate the monitoring of biomolecule transport and the screening of formulations promoting their passage across the pulmonary barrier, in order to select candidates for pulmonary administration to patients.     

Impact of the Omicron variant on SARS-CoV-2 reinfections in France,March 2021 to February 2022

Since the first reports in summer 2020, SARS-CoV-2 reinfections have raised concerns about the immunogenicity of the virus, which will affect SARS-CoV-2 epidemiology and possibly the burden of COVID-19 on our societies in the future. This study provides data on the frequency and characteristics of possible reinfections, using the French national COVID-19 testing database. The Omicron variant had a large impact on the frequency of possible reinfections in France, which represented 3.8% of all confirmed COVID-19 cases since December 2021.     

Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization

Therapeutic strategies directed at the tumor surfaceome (TS), including checkpoint inhibitor blocking antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cells, provide a new armament to fight cancer. However, a remaining bottleneck is the lack of strategies to comprehensively interrogate patient tumors for potential TS targets. Here, we have developed a platform (tumor surfaceome mapping [TS-MAP]) integrated with a newly curated TS classifier (SURFME) that allows profiling of primary 3D cultures and intact patient glioma tumors with preserved tissue architecture. Moreover, TS-MAP specifically identifies proteins capable of endocytosis as tractable targets for ADCs and other modalities requiring toxic payload internalization. In high-grade gliomas that remain among the most aggressive forms of cancer, we show that cellular spatial organization (2D vs. 3D) fundamentally transforms the surfaceome and endocytome (e.g., integrins, proteoglycans, semaphorins, and cancer stem cell markers) with general implications for target screening approaches, as exemplified by an ADC targeting EGFR. The TS-MAP platform was further applied to profile the surfaceome and endocytome landscape in a cohort of freshly resected gliomas. We found a highly diverse TS repertoire between patient tumors, not directly associated with grade and histology, which highlights the need for individualized approaches. Our data provide additional layers of understanding fundamental to the future development of immunotherapy strategies, as well as procedures for proteomics-based target identification and selection. The TS-MAP platform should be widely applicable in efforts aiming at a better understanding of how to harness the TS for personalized immunotherapy.

Cell-surface proteins have a key role in drug development, and approximately two-thirds of approved human drugs target a cell-surface protein (1). Recently, tumor cell–surface proteins integrated with the plasma membrane (tumor surfaceome [TS]) have attracted considerable attention as targets for immunotherapies in cancer. Immune checkpoint-blocking antibodies (e.g., ipilimumab and nivolumab), antibody drug conjugates (ADCs, e.g., trastuzumab emtansin), radioimmunotherapy (RIT, e.g., ⁹⁰Y ibritumomab tiuxetan), and chimeric antigen receptor T (CAR-T) cells are all directed at the TS and currently revolutionize cancer treatment (2–6). With the impressive development of creative methods for antibody and T cell engineering, a remaining challenge is the lack of strategies to comprehensively map potential TS target antigens for the design of more rational, individualized treatments (7). Although advancements in DNA and RNA sequencing provide high throughput data for prediction algorithms, e.g., personalized peptide vaccine trials (8, 9), the predicted proteome derived from these platforms is not necessarily expressed and available for targeting. Moreover, proteomics-based strategies involve analysis of the bulk from disintegrated tumor tissue, resulting in loss of spatial information and limited coverage of the less abundant and hydrophobic TS proteins (10, 11). Of particular relevance, ADC, RIT, and other intracellular drug delivery strategies rely on TS targets that functionally engage in endocytic internalization (12). Clearly, despite its great targeting potential in cancer immunotherapy, the TS remains an elusive treasure for further discovery.Procedures for unbiased mapping of the TS and target identification should include specific labeling of the TS in freshly resected patient tumors with preserved tissue architecture. Enrichment of TS proteins and reduction of noise from intracellular proteins as well as abundant extracellular matrix collagens and glycoproteins would greatly improve downstream mass spectrometry analysis. Moreover, the approach should allow functional and dynamic profiling of TS internalization in an intact tissue environment. With the aim to address these challenges and to provide insight into the complexity of the TS, we have developed a versatile technology for TS mapping (TS-MAP). As proof of concept, we focused on primary brain tumors that remain among the most aggressive forms of cancer and for which attempts to conquer the most common variant, glioblastoma (GBM) (World Health Organization [WHO] grade IV) have failed so far (13). TS-MAP is compatible with spheroids from primary human stem cell–like GBM cultures, as well as mouse and patient brain tumors, and separately profiles surface resident and internalized TS proteins. Moreover, a TS classifier (SURFME) was curated for filtering and categorization of bona fide membrane proteins exposed to the extracellular space. We find significant differences in the TS between the 2D and 3D spheroid format, which underlines the importance of cellular spatial organization. In strong support of the need of individualized approaches, our findings suggest substantial intertumoral heterogeneity in the relative abundance of TS proteins in a cohort of freshly resected patient gliomas.