Continuous oxygen monitoring to enhance ex-vivo organ machine perfusion and reconstructive surgery.

Continuous oxygenation monitoring of machine-perfused organs or transposed autologous tissue is not currently implemented in clinical practice. Oxygenation is a critical parameter that could be used to verify tissue viability and guide corrective interventions, such as perfusion machine parameters or surgical revision. This work presents an innovative technology based on oxygen-sensitive, phosphorescent metalloporphyrin allowing continuous and non-invasive oxygen monitoring of ex-vivo perfused vascularized fasciocutaneous flaps. The method comprises a small, low-energy optical transcutaneous oxygen sensor applied on the flap's skin paddle as well as oxygen sensing devices placed into the tubing. An intermittent perfusion setting was designed to study the response time and accuracy of this technology over a total of 54 perfusion cycles. We further evaluated correlation between the continuous oxygen measurements and gold-standard perfusion viability metrics such as vascular resistance, with good agreement suggesting potential to monitor graft viability at high frequency, opening the possibility to employ feedback control algorithms in the future. This proof-of-concept study opens a range of research and clinical applications in reconstructive surgery and transplantation at a time when perfusion machines undergo rapid clinical adoption with potential to improve outcomes across a variety of surgical procedures and dramatically increase access to transplant medicine.

AlphaFold2 for Protein Structure Prediction: Best Practices and Critical Analyses.

AlphaFold2 (AF2) has emerged in recent years as a groundbreaking innovation that has revolutionized several scientific fields, in particular structural biology, drug design, and the elucidation of disease mechanisms. Many scientists now use AF2 on a daily basis, including non-specialist users. This chapter is aimed at the latter. Tips and tricks for getting the most out of AF2 to produce a high-quality biological model are discussed here. We suggest to non-specialist users how to maintain a critical perspective when working with AF2 models and provide guidelines on how to properly evaluate them. After showing how to perform our own structure prediction using ColabFold, we list several ways to improve AF2 models by adding information that is missing from the original AF2 model. By using software such as AlphaFill to add cofactors and ligands to the models, or MODELLER to add disulfide bridges between cysteines, we guide users to build a high-quality biological model suitable for applications such as drug design, protein interaction, or molecular dynamics studies.

Program VUE: analysing distributions of cryo-EM projections using uniform spherical grids.

Three-dimensional cryo electron microscopy reconstructions are obtained by extracting information from a large number of projections of the object. These projections correspond to different 'views' or 'orientations', i.e. directions in which these projections show the reconstructed object. Uneven distribution of these views and the presence of dominating preferred orientations may distort the reconstructed spatial images. This work describes the program VUE (views on uniform grids for cryo electron microscopy), designed to study such distributions. Its algorithms, based on uniform virtual grids on a sphere, allow an easy calculation and accurate quantitative analysis of the frequency distribution of the views. The key computational element is the Lambert azimuthal equal-area projection of a spherical uniform grid onto a disc. This projection keeps the surface area constant and represents the frequency distribution with no visual bias. Since it has multiple tunable parameters, the program is easily adaptable to individual needs, and to the features of a particular project or of the figure to be produced. It can help identify problems related to an uneven distribution of views. Optionally, it can modify the list of projections, distributing the views more uniformly. The program can also be used as a teaching tool.

VCA supercooling in a swine partial hindlimb model.

Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia-reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at - 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.

Methylation of ESCRT-III components regulates the timing of cytokinetic abscission.

Abscission is the final stage of cytokinesis, which cleaves the intercellular bridge (ICB) connecting two daughter cells. Abscission requires tight control of the recruitment and polymerization of the Endosomal Protein Complex Required for Transport-III (ESCRT-III) components. We explore the role of post-translational modifications in regulating ESCRT dynamics. We discover that SMYD2 methylates the lysine 6 residue of human CHMP2B, a key ESCRT-III component, at the ICB, impacting the dynamic relocation of CHMP2B to sites of abscission. SMYD2 loss-of-function (genetically or pharmacologically) causes CHMP2B hypomethylation, delayed CHMP2B polymerization and delayed abscission. This is phenocopied by CHMP2B lysine 6 mutants that cannot be methylated. Conversely, SMYD2 gain-of-function causes CHMP2B hypermethylation and accelerated abscission, specifically in cells undergoing cytokinetic challenges, thereby bypassing the abscission checkpoint. Additional experiments highlight the importance of CHMP2B methylation beyond cytokinesis, namely during ESCRT-III-mediated HIV-1 budding. We propose that lysine methylation signaling fine-tunes the ESCRT-III machinery to regulate the timing of cytokinetic abscission and other ESCRT-III dependent functions.

Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia.

BACKGROUND: Warm ischemia time (WIT) and ischemia-reperfusion injury are limiting factors for vascularized composite allograft (VCA) transplantation. Subnormothermic machine perfusion (SNMP) has demonstrated the potential to extend WIT in organ transplantation. This study evaluates the effect of SNMP on VCA viability after prolonged WIT. METHODS: Rat hindlimbs underwent WIT for 30, 45, 60, 120, 150, or 210 min, followed by 3-h SNMP. Monitoring of perfusion parameters and outflow determined the maximum WIT compatible with limb viability after SNMP. Thereafter, 2 groups were assessed: a control group with inbred transplantation (Txp) after 120 min of WIT and an experimental group that underwent WIT + SNMP + Txp. Graft appearance, blood gas, cytokine levels, and histology were assessed for 21 d. RESULTS: Based on potassium levels, the limit of WIT compatible with limb viability after SNMP is 120 min. Before this limit, SNMP reduces potassium and lactate levels of WIT grafts to the same level as fresh grafts. In vivo, the control group presented 80% graft necrosis, whereas the experimental group showed no necrosis, had better healing (P = 0.0004), and reduced histological muscle injury (P = 0.012). Results of blood analysis revealed lower lactate, potassium levels, and calcium levels (P = 0.048) in the experimental group. Both groups presented an increase in interleukin (IL)-10 and IL-1b/IL-1F2 with a return to baseline after 7 to 14 d. CONCLUSIONS: Our study establishes the limit of WIT compatible with VCA viability and demonstrates the effectiveness of SNMP in restoring a graft after WIT ex vivo and in vivo, locally and systemically.

Exploring Iodide and Hydrogen Sulfide as ROS Scavengers to Delay Acute Rejection in MHC-Defined Vascularized Composite Allografts.

Vascularized composite allografts (VCA) face ischemic challenges due to their limited availability. Reperfusion following ischemia triggers oxidative stress and immune reactions, and scavenger molecules could mitigate ischemia-reperfusion injuries and, therefore, immune rejection. We compared two scavengers in a myocutaneous flap VCA model. In total, 18 myocutaneous flap transplants were performed in Major histocompatibility complex (MHC)-defined miniature swine. In the MATCH group (n = 9), donors and recipients had minor antigen mismatch, while the animals were fully mismatched in the MISMATCH group (n = 9). Grafts were pretreated with saline, sodium iodide (NaI), or hydrogen sulfide (H2S), stored at 4 °C for 3 h, and then transplanted. Flaps were monitored until clinical rejection without immunosuppression. In the MATCH group, flap survival did not significantly differ between the saline and hydrogen sulfide treatments (p = 0.483) but was reduced with the sodium iodide treatment (p = 0.007). In the MISMATCH group, survival was similar between the saline and hydrogen sulfide treatments (p = 0.483) but decreased with the sodium iodide treatment (p = 0.007). Rhabdomyolysis markers showed lower but non-significant levels in the experimental subgroups for both the MATCH and MISMATCH animals. This study provides insightful data for the field of antioxidant-based approaches in VCA and transplantation.

Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation.

The current gold standard for preserving vascularized composite allografts (VCA) is 4°C static cold storage (SCS), albeit muscle vulnerability to ischemia can be described as early as after 2 h of SCS. Alternatively, machine perfusion (MP) is growing in the world of organ preservation. Herein, we investigated the outcomes of oxygenated acellular subnormothermic machine perfusion (SNMP) for 24-h VCA preservation before allotransplantation in a swine model. Six partial hindlimbs were procured on adult pigs and preserved ex vivo for 24 h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14 days. Clinical monitoring was carried out twice daily, and graft biopsies and blood samples were regularly collected. Two blinded pathologists assessed skin and muscle samples. Overall survival was higher in the SNMP group. Early euthanasia of 2 animals in the SCS group was linked to significant graft degeneration. Analyses of the grafts showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group 2 weeks after allotransplantation. Therefore, this 24-h SNMP protocol using a modified Steen solution generated better clinical and histological outcomes in allotransplantation when compared to time-matched SCS.

Acute Rejection Rates in Vascularized Composite Allografts: A Systematic Review of Case Reports.

INTRODUCTION: Vascularized Composite Allografts (VCA) are usually performed in a full major histocompatibility complex mismatch setting, with a risk of acute rejection depending on factors such as the type of immunosuppression therapy and the quality of graft preservation. In this systematic review, we present the different immunosuppression protocols used in VCA and point out relationships between acute rejection rates and possible factors that might influence it. METHODS: This systematic review was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We systematically searched Medline (PubMed), Embase, and The Cochrane Library between November 2022 and February 2023, using following Mesh Terms: Transplant, Transplantation, Hand, Face, Uterus, Penis, Abdominal Wall, Larynx, and Composite Tissue Allografts. All VCA case reports and reviews describing multiple case reports were included. RESULTS: We discovered 211 VCA cases reported. The preferred treatment was a combination of antithymocyte globulins, mycophenolate mofetil (MMF), tacrolimus, and steroids; and a combination of MMF, tacrolimus, and steroids for induction and maintenance treatment, respectively. Burn patients showed a higher acute rejection rate (P = 0.073) and were administered higher MMF doses (P = 0.020). CONCLUSIONS: In contrast to previous statements, the field of VCA is not rapidly evolving, as it has encountered challenges in addressing immune-related concerns. This is highlighted by the absence of a standardized immunosuppression regimen. Consequently, more substantial data are required to draw more conclusive results regarding the immunogenicity of VCAs and the potential superiority of one immunosuppressive treatment over another. Future efforts should be made to report the VCA surgeries comprehensively, and muti-institutional long-term prospective follow-up studies should be performed to compare the number of acute rejections with influencing factors.

Analytical Formalism for Data Representation and Object Detection with 2D LiDAR: Application in Mobile Robotics.

In mobile robotics, LASER scanners have a wide spectrum of indoor and outdoor applications, both in structured and unstructured environments, due to their accuracy and precision. Most works that use this sensor have their own data representation and their own case-specific modeling strategies, and no common formalism is adopted. To address this issue, this manuscript presents an analytical approach for the identification and localization of objects using 2D LiDARs. Our main contribution lies in formally defining LASER sensor measurements and their representation, the identification of objects, their main properties, and their location in a scene. We validate our proposal with experiments in generic semi-structured environments common in autonomous navigation, and we demonstrate its feasibility in multiple object detection and identification, strictly following its analytical representation. Finally, our proposal further encourages and facilitates the design, modeling, and implementation of other applications that use LASER scanners as a distance sensor.

Metabolic profiling of murine radiation-induced lung injury with Raman spectroscopy and comparative machine learning.

Radiation-induced lung injury (RILI) is a dose-limiting toxicity for cancer patients receiving thoracic radiotherapy. As such, it is important to characterize metabolic associations with the early and late stages of RILI, namely pneumonitis and pulmonary fibrosis. Recently, Raman spectroscopy has shown utility for the differentiation of pneumonitic and fibrotic tissue states in a mouse model; however, the specific metabolite-disease associations remain relatively unexplored from a Raman perspective. This work harnesses Raman spectroscopy and supervised machine learning to investigate metabolic associations with radiation pneumonitis and pulmonary fibrosis in a mouse model. To this end, Raman spectra were collected from lung tissues of irradiated/non-irradiated C3H/HeJ and C57BL/6J mice and labelled as normal, pneumonitis, or fibrosis, based on histological assessment. Spectra were decomposed into metabolic scores via group and basis restricted non-negative matrix factorization, classified with random forest (GBR-NMF-RF), and metabolites predictive of RILI were identified. To provide comparative context, spectra were decomposed and classified via principal component analysis with random forest (PCA-RF), and full spectra were classified with a convolutional neural network (CNN), as well as logistic regression (LR). Through leave-one-mouse-out cross-validation, we observed that GBR-NMF-RF was comparable to other methods by measure of accuracy and log-loss (p > 0.10 by Mann-Whitney U test), and no methodology was dominant across all classification tasks by measure of area under the receiver operating characteristic curve. Moreover, GBR-NMF-RF results were directly interpretable and identified collagen and specific collagen precursors as top fibrosis predictors, while metabolites with immune and inflammatory functions, such as serine and histidine, were top pneumonitis predictors. Further support for GBR-NMF-RF and the identified metabolite associations with RILI was found as CNN interpretation heatmaps revealed spectral regions consistent with these metabolites.

Optimized Decellularization of a Porcine Fasciocutaneaous Flap.

Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps.

Molecular dynamics of the human RhD and RhAG blood group proteins.

Introduction: Blood group antigens of the RH system (formerly known as "Rhesus") play an important role in transfusion medicine because of the severe haemolytic consequences of antibodies to these antigens. No crystal structure is available for RhD proteins with its partner RhAG, and the precise stoichiometry of the trimer complex remains unknown. Methods: To analyse their structural properties, the trimers formed by RhD and/or RhAG subunits were generated by protein modelling and molecular dynamics simulations were performed. Results: No major differences in structural behaviour were found between trimers of different compositions. The conformation of the subunits is relatively constant during molecular dynamics simulations, except for three large disordered loops. Discussion: This work makes it possible to propose a reasonable stoichiometry and demonstrates the potential of studying the structural behaviour of these proteins to investigate the hundreds of genetic variants relevant to transfusion medicine.

Impact of Patient-Reported Allergies on Post-operative Complications and Healthcare Utilization Following Carpal Tunnel Release.

INTRODUCTION: Open carpal tunnel release (O-CTR) is associated with high patient satisfaction and low complication rates. Risk factors for complications are well-established. Recent studies have found that patient-reported allergies (PRAs) and psychiatric comorbidities may be associated with increased complication rates. The impact of these factors after elective hand surgery has not been evaluated. This study sought to identify whether PRAs and psychiatric comorbidities are associated with complications after O-CTR and to evaluate their association with prolonged follow-up and the need for post-operative occupational therapy (OT). METHODS: Patient demographics, PRAs, Patient Health Questionnaire-2 score, Charlson Comorbidity Index, Carpal Tunnel Symptoms-6 score, postoperative complications, OT utilization, and time to final follow-up were recorded for patients who underwent elective O-CTR between 2014 and 2022. Multivariable binomial logistic regression analysis was used to determine pre-operative variables associated with increased risk for complication. RESULTS: About 250 patients met the inclusion criteria. Fifty-one (20.4%) patients developed minor complications, including scar tenderness (N=34, 13.6%), superficial wound dehiscence (N=9, 3.6%), and superficial infection (N=8, 3.2%). There were no major complications. Independent risk factors for complications included PRAs (OR 1.80, p<0.01) and PHQ-2 score (OR 1.39, p=0.04). Five or more PRAs and PHQ-2 score ≥3 are significant independent risk factors for increased post-operative complications. Increased PRAs and PHQ-2 scores were associated with longer follow-up (p=0.01 and p<0.01, respectively) but not increased OT utilization. CONCLUSION: An increased number of PRAs and higher PHQ-2 scores are significant, independent risk factors for minor complications following O-CTR. Risk adjustment and peri-operative counseling should incorporate and account for these variables.

A Swine Burn Model for Investigating the Healing Process in Multiple Depth Burn Wounds.

Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face significant challenges. Among these challenges, the extent of the burned soft tissue can evolve in the early phase, creating a delicate balance between conservative treatments and necrosing tissue removal. Thermal burns are the most common type, and burn depth varies depending on multiple parameters, such as temperature and exposure time. Burn depth also varies in time, and the secondary aggravation of the "shadow zone" remains a poorly understood phenomenon. In response to these challenges, several innovative treatments have been studied, and more are in the early development phase. Nanoparticles in modern wound dressings and artificial skin are examples of these modern therapies still under evaluation. Taken together, both burn diagnosis and burn treatments need substantial advancements, and research teams need a reliable and relevant model to test new tools and therapies. Among animal models, swine are the most relevant because of their strong similarities in skin structure with humans. More specifically, Yucatan minipigs show interesting features such as melanin pigmentation and slow growth, allowing for studying high phototypes and long-term healing. This article aims to describe a reliable and reproducible protocol to study multi-depth burn wounds in Yucatan minipigs, enabling long-term follow-up and providing a relevant model for diagnosis and therapeutic studies.

Smartphone-based thermography in flap surgery: A systematic review and meta-analysis of perforator identification.

Background: Thermography can be used in pre-operative planning of free perforator flap surgeries. Thermography assesses skin temperature by measuring the quantity of infrared radiation observed. In this meta-analysis, authors assess the sensitivity of smartphone-based thermal imaging (SBTI) in the detection of perforators and analyze the difference between static and dynamic imaging. Materials and methods: Authors followed the PRISMA guidelines for systematic reviews and meta-analyses. The meta package in R was used to conduct the meta-analysis. The "metaprop" function was used to calculate the overall sensitivity estimate and 95% confidence interval. The "metaprop.one" function was used to calculate subgroup estimates for static and dynamic study types. The "metareg" function was used to conduct meta-regression analyses to explore sources of heterogeneity. Results: This study includes seven articles with 1429 perforators being evaluated. The overall proportion of the sensitivities was estimated to be 0.8754 (95% CI: 0.7542; 0.9414) using a random effects model. The heterogeneity of the studies was high, as indicated by the tau^2 value of 1.2500 (95% CI: 0.4497; 8.4060) and the I^2 value of 92.6% (95% CI: 88.1%; 95.4%). The pooled sensitivity for static imaging was 0.8636 (95%CI: 0.6238-0.9603) with a tau^2 of 2.0661 and a tau of 1.4374, while the pooled sensitivity for dynamic imaging was slightly higher (p = 0.7016) at 0.8993 (95%CI: 0.7412-0.9653) with a smaller tau^2 of 0.8403 and a tau of 0.9167. Conclusion: Further studies need to confirm that SBTI is a reliable and convenient technique for detecting perforators for the pre-operative planning of free perforator flap surgeries.

Alternative Flap Options for Upper Extremity Reconstruction.

For major upper limb defects, a wide range of established pedicled and free flap options can be used. These include the latissimus dorsi/thoracodorsal artery perforator, lateral arm, posterior interosseous artery, rectus abdominis, gracilis, and anterolateral thigh flaps. Technical proficiency is essential, and favorable success rates in terms of functional and esthetic outcomes can be achieved. Herein, alternative flap options (both pedicled and free) are introduced and discussed through a few illustrative case examples.

Nonsurgical Treatment of Postburn Hypopigmentation: A Literature Review.

The treatment of postburn hypopigmentation was primarily surgical before the advent of new technologies. Medical devices and therapies are emerging to manage scar sequelae that can be disfiguring and associated with severe psychosocial impact. These innovations have been poorly investigated for hypopigmentation, but they represent a real hope. We reviewed all articles published on Pubmed up to June 2022. Included studies had to specifically focus on treating postburn hypopigmented scars. All articles evaluating transient solutions such as make-up, and articles describing inflammation-linked hypopigmentation with no etiological details or no burn injury history were excluded. Through this review, we have highlighted 6 different types of nonsurgical treatments reported in postburn leukoderma potentially allowing definitive results. Electrophoto-biomodulation or E light (combining intensive pulsed light, radiofrequency, and cooling), topical daylight psoralen UVA therapy, and lasers (fractional lasers using pulse energies or CO2FL devices, lasers-assisted drug delivery as local bimatoprost and tretinoin or pimecrolimus) have been explored with encouraging results in hypopigmented burns. Finally, other promising medical strategies include using FK506, a nonsteroidal anti-inflammatory drug, to induce melanogenesis or using melanocyte-stimulating hormones with fractional laser-assisted drug deliveries, which are expected to emerge soon.