Infection After Lower-Limb Osseointegration: A Single-Center Retrospective Evaluation of Pathogens, Management, and Outcomes.

PURPOSE: Osseointegration (OI) is a novel alternative to traditional socket-suspended prostheses for lower-limb amputees, eliminating the socket-skin interface and allowing for weight bearing directly on the skeletal system. However, the stoma through which the implant attaches to the external prosthesis creates an ingress route for bacteria, and infection rates as high as 66% have been reported. The aims of this study are to classify infection management and long-term outcomes in this patient population to maximize implant salvage. METHODS: An institutional review board-approved retrospective analysis was performed on all patients who underwent lower-limb OI at our institution between 2017 and 2022. Demographic, operative, and outcome data were collected for all patients. Patients were stratified by the presence and severity of infection. Chi-square and t tests were performed on categorical and continuous data, respectively, using an alpha of 0.05. RESULTS: One hundred two patients met our study criteria; 62 had transfemoral OI and 40 had transtibial OI. Patients were followed for 23.8 months on average (range, 3.5-63.7). Osteomyelitis was more likely than soft tissue infection to be polymicrobial in nature (71% vs 23%, P < 0.05). Infections at the stoma were mostly (96%) managed with oral antibiotics alone, whereas deeper soft tissue infections also required intravenous antibiotics (75%) or operative washout (19%). Osteomyelitis was managed with intravenous antibiotics and required operative attention; 5 (71%) underwent washout and 2 (29%) underwent explantation. Both implants were replaced an average of 3.5 months after explantation. There was no correlation between history of soft tissue infection and development of osteomyelitis (P > 0.05). The overall implant salvage rate after infection was 96%. CONCLUSIONS: This study describes our institution's experience managing infection after OI and soft tissue reconstruction. Although infections do occur, they are easily treatable and rarely require operative intervention. Explantation due to infection is rare and can be followed up with reimplantation, reaffirming that OI is a safe and effective treatment modality.

Real-world treatment patterns and outcomes in patients with primary hemophagocytic lymphohistiocytosis treated with emapalumab.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening, hyperinflammatory syndrome. Emapalumab, a fully human monoclonal antibody that neutralizes the proinflammatory cytokine interferon-gamma, is approved in the United States to treat primary HLH (pHLH) in patients with refractory, recurrent, progressive disease or intolerance with conventional HLH. REAL-HLH, a retrospective study, conducted across 33 US hospitals, evaluated real-world treatment patterns and outcomes in patients treated with ≥1 dose of emapalumab between November 20, 2018, and October 31, 2021. Forty-six patients met the pHLH classification criteria. Median (range) age at diagnosis was 1.0 (0.3-21.0) year. Emapalumab was initiated for treating refractory (19/46), recurrent (14/46), or progressive (7/46) pHLH. At initiation, 15/46 patients were in the intensive care unit and 35/46 had received prior HLH-related therapies. Emapalumab treatment resulted in normalization of key laboratory parameters, including chemokine ligand 9 (CXCL9) (24/33; 72.7%), ferritin (20/45; 44.4%), fibrinogen (37/38; 97.4%), platelets (39/46; 84.8%), and absolute neutrophil count (40/45; 88.9%). Forty-two (91.3%) patients were considered eligible for transplant. Pre-transplant survival was 38/42 (90.5%). Thirty-one (73.8%) transplant-eligible patients proceeded to transplant and 23/31 (74.2%) of those transplanted were alive at the end of the follow-up period. Twelve-month survival probability from emapalumab initiation for the entire cohort (n=46) was 73.1%. There were no discontinuations due to adverse events. In conclusion, results from the REAL-HLH study, which describes treatment patterns, effectiveness, and outcomes in patients with pHLH treated with emapalumab in real-world settings, are consistent with the emapalumab pivotal phase 2/3 pHLH trial.

Label-Free Quantitative Analysis of Pig Liver Proteome after Hepatitis E Virus Infection.

Hepatitis E represents an emerging zoonotic disease caused by the Hepatitis E virus (HEV), for which the main route of transmission is foodborne. In particular, infection in humans has been associated with the consumption of contaminated undercooked meat of pig origin. The aim of this study was to apply comparative proteomics to determine if porcine liver protein profiles could be used to distinguish between pigs seropositive and seronegative for HEV. Preliminarily, an ELISA was used to evaluate the presence of anti-HEV antibodies in the blood serum of 136 animals sent to slaughter. Among the analyzed samples, a seroprevalence of 72.8% was estimated, and it was also possible to identify 10 animals, 5 positive and 5 negative, coming from the same farm. This condition created the basis for the quantitative proteomics comparison between homogeneous animals, in which only the contact with HEV should represent the discriminating factor. The analysis of the proteome in all samples of liver exudate led to the identification of 554 proteins differentially expressed between the two experimental groups, with 293 proteins having greater abundance in positive samples and 261 more represented in negative exudates. The pathway enrichment analysis allowed us to highlight the effect of the interaction between HEV and the host biological system in inducing the potential enrichment of 69 pathways. Among these, carbon metabolism stands out with the involvement of 41 proteins, which were subjected to interactomic analysis. This approach allowed us to focus our attention on three enzymes involved in glycolysis: glucose-6-phosphate isomerase (GPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and fructose-bisphosphate aldolase A (ALDOA). It therefore appears that infection with HEV induced a strengthening of the process, which involves the breakdown of glucose to obtain energy and carbon residues useful for the virus's survival. In conclusion, the label-free LC-MS/MS approach showed effectiveness in highlighting the main differences induced on the porcine liver proteome by the interaction with HEV, providing crucial information in identifying a viral signature on the host metabolism.

International Prognostic Score for Nodular Lymphocyte-Predominant Hodgkin Lymphoma.

PURPOSE: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare cancer, and large international cooperative efforts are needed to evaluate the significance of clinical risk factors and immunoarchitectural patterns (IAPs) for all stages of pediatric and adult patients with NLPHL. METHODS: Thirty-eight institutions participated in the Global nLPHL One Working Group retrospective study of NLPHL cases from 1992 to 2021. We measured progression-free survival (PFS), overall survival (OS), transformation rate, and lymphoma-specific death rate. We performed uni- and multivariable (MVA) Cox regression stratified by management to select factors for the lymphocyte-predominant international prognostic score (LP-IPS) validated by five-fold cross-validation. RESULTS: We identified 2,243 patients with a median age of 37 years (IQR, 23-51). The median follow-up was 6.3 years (IQR, 3.4-10.8). Most had stage I to II (72.9%) and few B symptoms (9.9%) or splenic involvement (5.4%). IAP was scored for 916 (40.8%). Frontline management included chemotherapy alone (32.4%), combined modality therapy (30.5%), radiotherapy alone (24.0%), observation after excision (4.6%), rituximab alone (4.0%), active surveillance (3.4%), and rituximab and radiotherapy (1.1%). The PFS, OS, transformation, and lymphoma-specific death rates at 10 years were 70.8%, 91.6%, 4.8%, and 3.3%, respectively. On MVA, IAPs were not associated with PFS or OS, but IAP E had higher risk of transformation (hazard ratio [HR], 1.81; P < .05). We developed the LP-IPS with 1 point each for age ≥45 years, stage III-IV, hemoglobin <10.5 g/dL, and splenic involvement. Increasing LP-IPS was significantly associated with worse PFS (HR, 1.52) and OS (HR, 2.31) and increased risk of lymphoma-specific death (HR, 2.63) and transformation (HR, 1.41). CONCLUSION: In this comprehensive study of all ages of patients with NLPHL, we develop the LP-IPS to identify high-risk patients and inform upcoming prospective clinical trials evaluating de-escalation of therapy for patients with low LP-IPS scores (<2).

Kartograf: A Geometrically Accurate Atom Mapper for Hybrid-Topology Relative Free Energy Calculations.

Relative binding free energy (RBFE) calculations have emerged as a powerful tool that supports ligand optimization in drug discovery. Despite many successes, the use of RBFEs can often be limited by automation problems, in particular, the setup of such calculations. Atom mapping algorithms are an essential component in setting up automatic large-scale hybrid-topology RBFE calculation campaigns. Traditional algorithms typically employ a 2D subgraph isomorphism solver (SIS) in order to estimate the maximum common substructure. SIS-based approaches can be limited by time-intensive operations and issues with capturing geometry-linked chemical properties, potentially leading to suboptimal solutions. To overcome these limitations, we have developed Kartograf, a geometric-graph-based algorithm that uses primarily the 3D coordinates of atoms to find a mapping between two ligands. In free energy approaches, the ligand conformations are usually derived from docking or other previous modeling approaches, giving the coordinates a certain importance. By considering the spatial relationships between atoms related to the molecule coordinates, our algorithm bypasses the computationally complex subgraph matching of SIS-based approaches and reduces the problem to a much simpler bipartite graph matching problem. Moreover, Kartograf effectively circumvents typical mapping issues induced by molecule symmetry and stereoisomerism, making it a more robust approach for atom mapping from a geometric perspective. To validate our method, we calculated mappings with our novel approach using a diverse set of small molecules and used the mappings in relative hydration and binding free energy calculations. The comparison with two SIS-based algorithms showed that Kartograf offers a fast alternative approach. The code for Kartograf is freely available on GitHub (https://github.com/OpenFreeEnergy/kartograf). While developed for the OpenFE ecosystem, Kartograf can also be utilized as a standalone Python package.

The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.

Impact of the COVID-19 Pandemic on Emergency Department Encounters in a Major Metropolitan Area.

BACKGROUND: The end of 2019 marked the emergence of the COVID-19 pandemic. Public avoidance of health care facilities, including the emergency department (ED), has been noted during prior pandemics. OBJECTIVE: This study described pandemic-related changes in adult and pediatric ED presentations, acuity, and hospitalization rates during the pandemic in a major metropolitan area. METHODS: The study was a cross-sectional analysis of ED visits occurring before and during the pandemic. Sites collected daily ED patient census; monthly ED patient acuity, as the Emergency Severity Index (ESI) score; and disposition. Prepandemic ED visits occurring from January 1, 2019 through December 31, 2019 were compared with ED visits occurring during the pandemic from January 1, 2020 through March 31, 2021. The change in prepandemic and pandemic ED volume was found using 7-day moving average of proportions. RESULTS: The study enrolled 83.8% of the total ED encounters. Pandemic adult and pediatric visit volume decreased to as low as 44.7% (95% CI 43.1-46.3%; p < 0.001) and 22.1% (95% CI 19.3-26.0%; p < 0.001), respectively, of prepandemic volumes. There was also a relative increase in adult and pediatric acuity (ESI level 1-3) and the admission percentage for adult (20.3% vs. 22.9%; p < 0.01) and pediatric (5.1% vs. 5.6%; p < 0.01) populations. CONCLUSIONS: Total adult and pediatric encounters were reduced significantly across a major metropolitan area. Patient acuity and hospitalization rates were relatively increased. The development of strategies for predicting ED avoidance will be important in future pandemics.

Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices.

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.

Navigating Practice Issues Related to the Unsatisfactory Cervicovaginal Papanicolaou Test: Survey Results of Laboratories Participating in the 2020 College of American Pathologists PAP Education Program.

CONTEXT.­: Unsatisfactory Papanicolaou (Pap) tests pose a unique set of challenges to the laboratory with regard to their processing, review, reporting, and performance of human papillomavirus (HPV) testing. There are no standardized guidelines for the review process and handling of unsatisfactory Pap tests. OBJECTIVE.­: To assess the current practice patterns regarding various aspects of the unsatisfactory Pap test, from processing to reporting, across laboratories worldwide. DESIGN.­: A supplemental questionnaire was mailed to laboratories participating in the 2020 College of American Pathologists (CAP) Gynecologic Cytopathology (PAP Education) Program, requesting data regarding the unsatisfactory Pap test. RESULTS.­: Of 1520 participating laboratories, 619 (40.7%) responded, and the responses of 577 laboratories were included for further analysis. Only 64.6% (373 of 577) laboratories used the unsatisfactory Pap test criteria as specified by the 2014 Bethesda System. About three-quarters of the respondents (433 of 576; 75.2%) routinely rescreened unsatisfactory Pap tests. Routine repreparation of such Pap tests was performed by 54.9% (316 of 576) of laboratories, and 52.0% (293 of 563) used glacial acetic acid for repreparing excessively bloody specimens. HPV test results were reported for unsatisfactory Pap tests, always or sometimes, by 62.4% (353 of 566) of respondents. CONCLUSIONS.­: This CAP survey reveals important information regarding the practice patterns pertaining to several aspects of the unsatisfactory Pap test. It also provides valuable insight into the quality assurance measures that can be implemented for such tests. Future studies can further aid in the standardization of all components of the handling of unsatisfactory Pap tests for overall quality improvement.

Women 50 Years and Older With Negative Pap Test and Positive Human Papillomavirus Test for Genotypes Other Than 16 and 18-Follow-up Outcomes.

OBJECTIVE: A follow-up of women 50 years or older with concomitant positive high-risk human papillomavirus (HPV) genotypes other than 16 and 18 (hrHPVO) and negative Pap test (NILMPap) was conducted to better understand the implications of hrHPVO positivity on potential risk of developing significant high-grade lesions. MATERIAL AND METHODS: A retrospective review of 2014 cytology data of patients with co-testing (Pap test and HPV DNA) identified 85 women 50 years or older with NILMPap and hrHPVO+. RESULTS: Most patients (63) had repeat co-testing on next follow-up. Of these, 41 patients with persistent hrHPVO+ status, 3 developed cervical intraepithelial neoplasia 2 (CIN2), and 1 CIN3. Nineteen patients were followed with biopsies. Of these, 7 biopsies were abnormal, 5 of which showed low-grade (CIN1) and 2 high-grade (CIN3) histology; none progressed on further follow-up. Three patients were followed with Pap test only, all had NILMPap, and none progressed on further follow-up. In summary, of the 85 patients, 26 developed abnormal histology during follow-up, 6 of whom had high-grade histology (CIN2 and CIN3, 3 each).The 5-year risk of CIN1+ in this cohort was 43.8% and for CIN2+ was 12.3%. The risk of abnormal histology did not differ significantly by prior history of Pap tests, histology, and/or HPV results. CONCLUSIONS: A persistent positivity for hrHPVO indicated higher likelihood to develop a lesion, and this risk was not reduced for patients 50 and older compared with the published screening population risk.

How We Approach Suppressive Antibiotic Therapy Following Debridement, Antibiotics, and Implant Retention for Prosthetic Joint Infection.

The optimal treatment of prosthetic joint infection (PJI) remains uncertain. Patients undergoing debridement, antibiotics, and implant retention (DAIR) receive extended antimicrobial treatment, and some experts leave patients at perceived highest risk of relapse on suppressive antibiotic therapy (SAT). In this narrative review, we synthesize the literature concerning the role of SAT to prevent treatment failure following DAIR, attempting to answer 3 key questions: (1) What factors identify patients at highest risk for treatment failure after DAIR (ie, patients with the greatest potential to benefit from SAT), (2) Does SAT reduce the rate of treatment failure after DAIR, and (3) What are the rates of treatment failure and adverse events necessitating treatment discontinuation in patients receiving SAT? We conclude by proposing risk-benefit stratification criteria to guide use of SAT after DAIR for PJI, informed by the limited available literature.

Oral Carbon Monoxide Enhances Autophagy Modulation in Prostate, Pancreatic, and Lung Cancers.

Modulation of autophagy, specifically its inhibition, stands to transform the capacity to effectively treat a broad range of cancers. However, the clinical efficacy of autophagy inhibitors has been inconsistent. To delineate clinical and epidemiological features associated with autophagy inhibition and a positive oncological clinical response, a retrospective analysis of patients is conducted treated with hydroxychloroquine, a known autophagy inhibitor. A direct correlation between smoking status and inhibition of autophagy with hydroxychloroquine is identified. Recognizing that smoking is associated with elevated circulating levels of carbon monoxide (CO), it is hypothesized that supplemental CO can amplify autophagy inhibition. A novel, gas-entrapping material containing CO in a pre-clinical model is applied and demonstrated that CO can dramatically increase the cytotoxicity of autophagy inhibitors and significantly inhibit the growth of tumors when used in combination. These data support the notion that safe, therapeutic levels of CO can markedly enhance the efficacy of autophagy inhibitors, opening a promising new frontier in the quest to improve cancer therapies.

OpenMM 8: Molecular Dynamics Simulation with Machine Learning Potentials.

Machine learning plays an important and growing role in molecular simulation. The newest version of the OpenMM molecular dynamics toolkit introduces new features to support the use of machine learning potentials. Arbitrary PyTorch models can be added to a simulation and used to compute forces and energy. A higher-level interface allows users to easily model their molecules of interest with general purpose, pretrained potential functions. A collection of optimized CUDA kernels and custom PyTorch operations greatly improves the speed of simulations. We demonstrate these features in simulations of cyclin-dependent kinase 8 (CDK8) and the green fluorescent protein chromophore in water. Taken together, these features make it practical to use machine learning to improve the accuracy of simulations with only a modest increase in cost.

Individual Prediction of Optimal Treatment Allocation Between Electroconvulsive Therapy or Ketamine using the Personalized Advantage Index.

Introduction: Electroconvulsive therapy (ECT) and ketamine are two effective treatments for depression with similar efficacy; however, individual patient outcomes may be improved by models that predict optimal treatment assignment. Here, we adapt the Personalized Advantage Index (PAI) algorithm using machine learning to predict optimal treatment assignment between ECT and ketamine using medical record data from a large, naturalistic patient cohort. We hypothesized that patients who received a treatment predicted to be optimal would have significantly better outcomes following treatment compared to those who received a non-optimal treatment. Methods: Data on 2526 ECT and 235 mixed IV ketamine and esketamine patients from McLean Hospital was aggregated. Depressive symptoms were measured using the Quick Inventory of Depressive Symptomatology (QIDS) before and during acute treatment. Patients were matched between treatments on pretreatment QIDS, age, inpatient status, and psychotic symptoms using a 1:1 ratio yielding a sample of 470 patients (n=235 per treatment). Random forest models were trained and predicted differential patientwise minimum QIDS scores achieved during acute treatment (min-QIDS) scores for ECT and ketamine using pretreatment patient measures. Analysis of Shapley Additive exPlanations (SHAP) values identified predictors of differential outcomes between treatments. Results: Twenty-seven percent of patients with the largest PAI scores who received a treatment predicted optimal had significantly lower min-QIDS scores compared to those who received a non-optimal treatment (mean difference=1.6, t=2.38, q<0.05, Cohen's D=0.36). Analysis of SHAP values identified prescriptive pretreatment measures. Conclusions: Patients assigned to a treatment predicted to be optimal had significantly better treatment outcomes. Our model identified pretreatment patient factors captured in medical records that can provide interpretable and actionable guidelines treatment selection.

The anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of primary brain tumors and brain metastases. Phenothiazines (PTZs) have demonstrated anti-cancer properties through a variety of mechanisms. However, it remains unclear whether these effects are entirely separate from their activity as dopamine and serotonin receptor (DR/5-HTR) antagonists. Methods: In this study, we evaluated the anti-cancer efficacy of a novel PTZ analog, CWHM-974, that was shown to be 100-1000-fold less potent against DR/5-HTR than its analog fluphenazine (FLU). Results: CWHM-974 was more potent than FLU against a panel of cancer cell lines, thus clearly demonstrating that its anti-cancer effects were independent of DR/5-HTR signaling. Our results further suggested that calmodulin (CaM) binding may be necessary, but not sufficient, to explain the anti-cancer effects of CWHM-974. While both FLU and CWHM-974 induced apoptosis, they induced distinct effects on the cell cycle (G0/G1 and mitotic arrest respectively) suggesting that they may have differential effects on CaM-binding proteins involved in cell cycle regulation. Discussion: Altogether, our findings indicated that the anti-cancer efficacy of the CWHM-974 is separable from DR/5-HTR antagonism. Thus, reducing the toxicity associated with phenothiazines related to DR/5-HTR antagonism may improve the potential to repurpose this class of drugs to treat brain tumors and/or brain metastasis.

OpenMM 8: Molecular Dynamics Simulation with Machine Learning Potentials.

Machine learning plays an important and growing role in molecular simulation. The newest version of the OpenMM molecular dynamics toolkit introduces new features to support the use of machine learning potentials. Arbitrary PyTorch models can be added to a simulation and used to compute forces and energy. A higher-level interface allows users to easily model their molecules of interest with general purpose, pretrained potential functions. A collection of optimized CUDA kernels and custom PyTorch operations greatly improves the speed of simulations. We demonstrate these features on simulations of cyclin-dependent kinase 8 (CDK8) and the green fluorescent protein (GFP) chromophore in water. Taken together, these features make it practical to use machine learning to improve the accuracy of simulations at only a modest increase in cost.