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Here, we present a protocol for monitoring phagocytosis by M2-type macrophages using automated counting of phagocytic events with an imaging cytometer. We describe steps for isolating and differentiating peripheral blood mononuclear cell (PBMC)-derived monocytes into M2-like macrophages, preparing cancer cells expressing a green fluorescence marker, labeling with a pH-sensitive dye, and co-culturing with macrophages. We then outline procedures for enumerating phagocytic events using an imaging cytometer. For complete details on the use and execution of this protocol, please refer to Mishra et al.1.

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Cervical traction is a common and effective treatment for degenerative disk diseases and pain in the cervical spine. However, the manual or mechanical methods of applying traction to the head-neck are limited due to variability in the applied forces and orientation of the head-neck relative to the shoulder during the procedure. Current robotic neck braces are not designed to provide independent rotation angles and independent vertical translation, or traction, to the brace end-effector connected to the head, making them unsuitable for traction application. This work proposes a novel architecture of a robotic neck brace, which can provide vertical traction to the head while keeping the head in a prescribed orientation, with flexion and lateral bending angles. In this paper, the kinematics of the end-effector attached to the head relative to a coordinate frame on the shoulders are described as well as the velocity kinematics and force control. The paper also describes benchtop experiments designed to validate the position control and the ability of the brace to provide a vertical traction force. It was shown that the maximum achievable end-effector orientations are 16° in flexion, 13.9° in extension, and ± 6.5° in lateral bending. The kinematic model of the active brace was validated using an independent motion capture system with a maximum root mean square error of 2.41°. In three different orientations of the end-effector, neutral, flexed, and laterally bent, the brace was able to provide a consistent upward traction force during intermittent force application. In these configurations, the force error has standard deviations of 0.55, 0.29, and 0.07N, respectively. This work validates the mechanism's ability to achieve a range of head orientations and provide consistent upward traction force in these orientations, making it a promising intervention tool in cases of cervical disk degeneration.

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BACKGROUND: Autosomal dominant neovascular inflammatory vitreoretinopathy (NIV), formerly called "ADNIV," is a rare autoinflammatory condition mainly of adulthood caused by mutations in calcium-activated calpain-5 protease (CAPN5). Our aim is to report the treatment and visual outcomes of children newly diagnosed with NIV after systemic treatment. METHODS: We reviewed charts of patients ≤18 years old with CAPN5 gene mutation, ocular findings consistent with NIV, and treated with systemic immunosuppression for a minimum of 6 months. Treatment response was based on ophthalmic examination, ultra-widefield fluorescein-angiography (UWFFA), and optical coherence tomography (OCT). RESULTS: Eight children (16 eyes) were diagnosed with NIV at a median age of 14 (Range [R] 9-16) years, with a median follow-up of 18 months (R6-20). At diagnosis, one patient had impaired visual acuity (VA > 0.4), eight had vascular leakage, two had neovascularization, and three had macular edema. All responded to oral or local glucocorticoids but was not sustained. Systemic immunosuppression was started in seven patients with methotrexate and infliximab after a median time from diagnosis of 1.5 months (R0.5-2) and 3.2 months (R2.5-3.1), respectively. Infliximab was discontinued in all after a median time of 7 months (R3.5-10) for ineffectiveness, and 5/7 switched to tocilizumab and 1 to adalimumab. Five failed to respond (4 tocilizumab, 1 adalimumab) and one had a minimal response to tocilizumab. CONCLUSIONS: We report on the systemic treatment response of seven children with ADNIV treated with methotrexate, infliximab, and tocilizumab. None were able to control disease. Further studies are needed to understand long-term outcomes and the utility of systemic immunosuppression.

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Glioblastoma (GBM) is the most aggressive and frequently occurring type of malignant brain tumor in adults. The initiation, progression, and recurrence of malignant tumors are known to be driven by a small subpopulation of cells known as tumor-initiating cells or cancer stem cells (CSCs). GBM CSCs play a pivotal role in orchestrating drug resistance and tumor relapse. As a prospective avenue for GBM intervention, the targeted suppression of GBM CSCs holds considerable promise. In this study, we found that rocaglates, compounds which are known to inhibit translation via targeting of the DEAD-box helicase eIF4A, exert a robust, dose-dependent cytotoxic impact on GBM CSCs with minimal killing of nonstem GBM cells. Subsequent optimization identified novel rocaglate derivatives (rocaglate acyl sulfamides or Roc ASFs) that selectively inhibit GBM CSCs with nanomolar EC50 values. Furthermore, comparative evaluation of a lead CSC-optimized Roc ASF across diverse mechanistic and target profiling assays revealed suppressed translation inhibition relative to that of other CSC-selective rocaglates, with enhanced targeting of the DEAD-box helicase DDX3X, a recently identified secondary target of rocaglates. Overall, these findings suggest a promising therapeutic strategy for targeting GBM CSCs.

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MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi.

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Goal functions make virtual goal-oriented motor tasks easier to analyze and manipulate by explicitly linking movement to outcome. However, they have only been used to study constrained (e.g., planar) upper limb movements. We present a design framework for integrating goal functions with unconstrained postural and upper limb movements in a virtual reality (VR) device. VR tasks designed with the framework can mimic unconstrained natural motions and thus train a range of functional movements yet remain analytically tractable. We created three in-place VR motor tasks: a bow-and-arrow, a reach-and-strike, and a punching bag task. Each task was adjusted to subject-specific workspace limits and anthropometrics. We studied the effects of 3 days of practice and 3 reach/lean distances on task performance in 12 healthy adults. Subjects performed all tasks on day 1 with moderate proficiency and improved with practice at all reach/lean distances. Task-specific results showed that performance decreased and movement variability increased near the edge of the reaching workspace; viewing angles and the imperfect depth cues in VR likely led to biases in performance and practice could attenuate the former effect; in reach-and-strike, subjects learned movement patterns similar to those seen in a real-world striking sport. These results show that our framework can deliver tasks useful for analyzing and training motor performance and can guide future in-place motor training. Post-hoc, we demonstrated the feasibility of generalizable methods that adjust required movement speeds and task difficulty for impaired populations.

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PURPOSE: Evaluate quantitative leakage parameters on ultra-widefield fluorescein angiography (UWF-FA) images and explore their association with Diabetic Retinopathy Severity Scale (DRSS), predominantly peripheral lesions (PPLs), visual acuity, and clinical characteristics. DESIGN: A post-hoc analysis of baseline UWF-FA images in the DRCR Retina Network observational study Protocol AA. PARTICIPANTS: N = 575 eyes from 384 adults across 38 sites in the US and Canada with gradable UWF-FA. METHODS: A machine learning-enhanced feature extraction platform provided initial leakage segmentation of UWF-FA images sequentially reviewed and corrected by two certified readers for segmentation accuracy. UWF-FA leakage was measured in five retinal zones: panretinal (whole retina), central macular (3-disc diameter fovea-centered circle), posterior pole (6-disc diameter fovea-centered circle), peripheral (outside 6-disc diameter circle) and widefield far peripheral (outside 9-disc diameter circle); associations with clinical factors were evaluated with marginal beta regression models. MAIN OUTCOME MEASURES: UWF-FA leakage index, calculated as the area with leakage divided by the analyzable retinal area. RESULTS: The mean quantitative leakage index was 3.5% for panretinal, 6.6% for macular, 4.8% for posterior pole, 3.3% for peripheral and 2.8% for wide-field far peripheral retinal zones. Panretinal leakage was associated with DRSS (mean 2.2% for no to mild NPDR, 3.4% for moderate NPDR, 4.2% for moderately severe NPDR, 4.8% for severe NPDR and 5.1% for PDR; P<.001), HbA1c (3.2% for HbA1c <8% vs. 3.8% for HbA1c ≥8%; P=.01 for continuous HbA1c), visual acuity (3.3% for 20/25 or better vs. 4.7% for 20/32 or worse; continuous P<.001), and UWF-FA-PPL types of IRMA (4.3% vs. 3.3%; P=.005) or NVE (5.7% vs. 3.4%; P=.003). DRSS was also statistically significant for leakage within all retinal zones (P<.001); eyes with non-central DME versus no DME had higher mean leakage in the central macular (11.2% vs. 5.9%; P=0.005) and posterior pole regions (9.2% vs. 4.2%; P=.002). CONCLUSION: Quantitative UWF-FA leakage analysis identified associations between leakage and DRSS, visual acuity, and presence of DME. In the future, quantitative UWF-FA leakage parameters may be explored as potential biomarkers for disease progression risk.

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Moral injury in health care is characterized as the lasting psychological, biological, and social impact on providers that occurs following an adverse patient outcome. Moral injury can contribute to second victim syndrome and lasting psychological harm. Although many surgeons face moral injury due to patient acuity and the potential for intraoperative or postoperative complications, the transplant ecosystem compounds the impact of moral injury. Institutional blame placed on the transplant surgeon following a posttransplant death or graft loss is magnified by public reporting. Centers whose outcomes fall below threshold levels are subject to regulatory citation and financial loss. Moral injury can also result in risk aversion, limiting access to transplants for higher-risk candidates and reducing acceptance of marginal organs hurting donor families. Strategies to increase resilience, reduce accusation and blame, and focus on system quality improvement are vital to mitigate the impact of moral injury on transplant professionals. The transplant community must proactively work to reduce moral injury to protect surgeons, ensure access to life-saving transplant procedures, and avoid unnecessary organ offer declines.

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Malaria, caused by Plasmodium falciparum, remains a significant health burden. One major barrier for developing antimalarial drugs is the ability of the parasite to rapidly generate resistance. We previously demonstrated that salinipostin A (SalA), a natural product, potently kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism that results in a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a small library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent antiparasitic potencies that enabled the identification of therapeutically relevant targets. The active compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor orlistat and shows synergistic killing with orlistat. Our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are promising, synthetically tractable antimalarials.

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Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) T. brucei. This editing, directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs and may developmentally control respiration. Canonical editing by gRNAs that specify protein-encoding mRNA sequences occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression at a major early checkpoint in mRNA ND7, involving helicase KREH2-dependent opposite modulation of canonical and non-canonical 'terminator' gRNA utilization. Terminator-programmed editing derails canonical editing and installs proposed repressive structure in 30% of the ND7 transcriptome. BSF-to-PCF differentiation in vitro recreated this negative control. Remarkably, KREH2-RNAi knockdown relieved repression and increased editing progression by reverting canonical/terminator gRNA utilization. ND7 transcripts lacking early terminator-directed editing in PCF exhibited similar negative editing control along the mRNA sequence, suggesting global modulation of gRNA utilization fidelity. The terminator is a 'moonlighting' gRNA also associated with mRNA COX3 canonical editing, so the gRNA transcriptome seems multifunctional. Thus, KREH2 is the first identified repressor in developmental editing control. This and our prior work support a model whereby KREH2 activates or represses editing in a stage and substrate-specific manner. KREH2's novel dual role tunes mitochondrial gene expression in either direction during development.

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BACKGROUND: Treatment for infective endocarditis (IE) is usually medical, with surgery reserved for those failing medical management or developing complications. Currently, 25%-50% of patients undergo surgery for IE with a 70%-80% immediate survival rate. However, there is controversy over the timing of surgery following cerebrovascular events, which occur in 15%-30% of IE patients. This study aimed to investigate whether surgical management is superior to medical management in patients with IE and to determine the optimal timing for surgery following the development of neurological symptoms. METHODS: Data were collected retrospectively between 2012 and 2018 from 436 patients diagnosed with IE and treated at our tertiary teaching hospital. The authors analysed the type of treatment, the timing of surgery, and the outcomes of these including mortality, IE recurrence, and length of hospital stay. RESULTS: A total of 421 patients were included in the analysis. More than two-thirds (69.1%) of patients underwent surgical intervention. The survival rate of patients having surgery for IE was 77.2%, compared to 50.7% in patients who did not undergo surgical intervention. 6.8% of patients presented with neurological symptoms; 73.3% of these patients had surgery within 14 days with a 90.9% survival. CONCLUSION: This study finds surgery to be safe with a seemingly higher survival rate compared to medical management alone, although this may be confounded by patients in the medical group being less likely to have surgery. Surgery in patients presenting with neurological symptoms is safe within 2 weeks from presentation with excellent outcomes.

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PURPOSE: Severe obesity is a barrier to listing for kidney transplantation due to concern for poor outcomes. This study aims to compare bariatric surgery with medical weight loss as a means of achieving weight loss and subsequent listing for renal transplant. We hypothesize that bariatric surgery will induce greater frequency of listing for transplant within 18 months of study initiation. MATERIALS AND METHODS: We performed a randomized study of metabolic bariatric surgery (MBS) vs medical weight loss (MM) in patients on dialysis with a body mass index (BMI) of 40-55 kg/m2. The primary outcome was suitability for renal transplant within 18 months of initiating treatment. Secondary outcomes included weight loss, mortality, and complications. RESULTS: Twenty patients enrolled, only 9 (5 MBS, 4 MM) received treatment. Treated groups did not differ in age, gender, or race (P ≥ .44). There was no statistically significant difference in the primary endpoint: 2 MBS (40%) and 1 MM (25%) listed for transplant ≤18 months (P = 1.00). With additional time, 100% MBS and 25% MM patients achieved listing status (P = .048); 100% of MBS and 0 MM received kidney transplants to date (P = .008). Weight, weight loss, and BMI trajectories differed between the groups (P ≤ .002). One death from COVID-19 occurred in the MM group, and 1 MBS patient had a myocardial infarction 3.75 years after baseline evaluation. CONCLUSION: These results suggest MBS is superior to MM in achieving weight loss prior to listing for kidney transplantation. Larger studies are needed to ensure the safety profile is acceptable in patients with ESRD undergoing bariatric surgery.

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We explore the interaction between corneal immunity and mesenchymal stem/stromal cells (MSCs) and their potential in treating corneal and ocular surface disorders. We outline the cornea's immune privilege mechanisms and the immunomodulatory substances involved. In this realm, MSCs are characterized by their immunomodulatory properties and regenerative potential, making them promising for therapeutic application. Therefore, we focus on the role of MSCs in immune-mediated corneal diseases such as dry eye disease, corneal transplantation rejection, limbal stem cell deficiency, and ocular graft-versus-host disease. Preclinical and clinical studies demonstrate MSCs' efficacy in promoting corneal healing and reducing inflammation in these conditions. Overall, we emphasize the potential of MSCs as innovative therapies in ophthalmology, offering promising solutions for managing various ocular surface pathologies.

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BACKGROUND: The primary function of dental cement is to seal and support prosthodontic restorative materials. Proper selection of the dental cement contributes to the clinical success of the restoration. METHODS: A total of 166 molar tooth samples were prepared to simulate the type of tooth commonly found in prosthodontic practice. Each sample was restored using one of the tested dental cement materials employing a prestabilized methodology. The performance of resin-modified glass ionomer cement (RMGIC) (GC Fuji PLUS Capsule, GC America, Alsip, IL), zinc phosphate cement (ZPC) (Dentsply Sirona, Charlotte, NC), and resin cement (RC) (RelyX ARC, 3M ESPE, Saint Paul, MN) in bonding strength, marginal adaptation, and microleakage was evaluated and compared. The bonding strength, marginal adaptation, and microgroove were tested using specific established methodologies. The outcomes were then analyzed using statistical analyses for means and standard deviations to compare different types of dental cement. RESULTS: The total outcome shows that the highest bonding strength with the highest mean was the resin cement, rating 24.8 MPa, followed by RMGIC and ZPC at 20.5 and 18.9 MPa, respectively. The marginal adaptation scores indicate that RC had the highest score at a mean of 4, followed by ZPC at 3.2 and RMGIC at 2.5. The dye penetration measurements in millimeters revealed that ZPC had a penetration of 0.31 mm, RMGIC had a penetration of 0.25 mm, and RC had the least penetration at 0.20 mm. The results of the statistical data analysis show significant differences between the dental cements in bonding strength and marginal adaptation. CONCLUSION: In conclusion, resin cement demonstrated superior performance in bonding strength, marginal adaptation, and resistance to microleakage compared to RMGIC and zinc phosphate cement. These findings highlight the importance of selecting resin cement for achieving optimal clinical outcomes in prosthodontic restorations.

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Importance: Noninfectious uveitis is a leading cause of visual impairment with an unmet need for additional treatment options. Objective: To assess the efficacy and safety of filgotinib, a Janus kinase 1 (JAK1) preferential inhibitor, for the treatment of noninfectious uveitis. Design, Setting, and Participants: The HUMBOLDT trial was a double-masked, placebo-controlled, phase 2, randomized clinical trial conducted from July 2017 to April 2021 at 26 centers in 7 countries. Eligible participants (aged ≥18 years) had active noninfectious intermediate uveitis, posterior uveitis, or panuveitis despite at least 2 weeks of treatment with oral prednisone (10-60 mg per day). Interventions: Participants were randomly assigned 1:1 to receive filgotinib, 200 mg, or placebo orally once daily for up to 52 weeks. Main Outcomes and Measures: The primary end point was the proportion of participants experiencing treatment failure by week 24. Treatment failure was a composite end point represented by assessment of the presence of chorioretinal and/or retinal vascular lesions, best-corrected visual acuity, and anterior chamber cell and vitreous haze grades. Safety was assessed in participants who received at least 1 dose of study drug or placebo. Results: Between July 26, 2017, and April 22, 2021, 116 participants were screened, and 74 (mean [SD] age, 46 [16] years; 43 female [59.7%] of 72 participants, as 2 participants did not receive treatment doses) were randomly assigned to receive filgotinib (n = 38) or placebo (n = 36). Despite early termination of the trial for business reasons ahead of meeting enrollment targets, a significantly reduced proportion of participants who received filgotinib experienced treatment failure by week 24 vs placebo (12 of 32 participants [37.5%] vs 23 of 34 participants [67.6%]; difference vs placebo -30.1%; 95% CI, -56.2% to -4.1%; P = .006). Business reasons were unrelated to efficacy or safety. Adverse events were reported in 30 of 37 participants (81.1%) who received filgotinib and in 24 of 35 participants (68.6%) who received placebo. Serious adverse events were reported in 5 of 37 participants (13.5%) in the filgotinib group and in 2 of 35 participants (5.7%) in the placebo group. No deaths were reported during the trial. Conclusions and Relevance: Results of this randomized clinical trial show that filgotinib lowered the risk of treatment failure in participants with active noninfectious intermediate uveitis, posterior uveitis, or panuveitis vs placebo. Although the HUMBOLDT trial provided evidence supporting the efficacy of filgotinib in patients with active noninfectious uveitis, the premature termination of the trial prevented collection of additional safety or efficacy information of this JAK1 preferential inhibitor. Trial Registration: ClinicalTrials.gov Identifier: NCT03207815.

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Insufficient proton conductivity and oxidative stability of sulfonated hydrocarbons hinder their applicability as proton exchange membrane electrolytes in fuel cells. In this regard, fabrication of proton conducting mixed-matrix membranes (PC-MMMs) can be a superior approach to obtain desirable properties. In this work, a triazole ligand (1H-1,2,4 triazole) was coordinated to a zinc metal node to create a 3D metal-organic framework (MOF) and incorporated as an additive in a sulfonated poly(ether ether ketone) matrix at 1, 3, and 5 weight percentage to fabricate PC-MMMs by the casting process. Several characterization tools such as electrochemical impedance spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy were used to characterise these membranes and study their potential application as electrolyte(s) in PEMFCs (proton exchange membrane fuel cells). Membranes were also tested for water uptake, ion-exchange capacity and oxidative stability in Fenton's reagent. The performance of the polymeric composite membrane containing a 3 wt% MOF was then assessed in a H2/O2 single cell as it demonstrated the highest proton conductivity of 0.04 S cm-1 among all the compositions and a maximum current density of 1191 mA cm-2. The membrane was also subjected to an OCV hold test for 12 hours to study the chemical durability over a period of time. This report establishes that the inclusion of a triazole based MOF enhances the proton conductivity, performance, and thermal and chemical durability of composite membranes which can be considered as a promising electrolyte material at intermediate temperatures after a proper optimisation of different cell parameters.

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Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration (AMD) that leads to progressive and irreversible vision loss. Identifying patients with greatest risk of GA progression is important for targeted utilization of emerging therapies. This study aimed to comprehensively evaluate the role of shape-based fractal dimension features ( F fd ) of sub-retinal pigment epithelium (sub-RPE) compartment and texture-based radiomics features ( F t ) of Ellipsoid Zone (EZ)-RPE and sub-RPE compartments for risk stratification for subfoveal GA (sfGA) progression. This was a retrospective study of 137 dry AMD subjects with a 5-year follow-up. Based on sfGA status at year 5, eyes were categorized as Progressors and Non-progressors. A total of 15 shape-based F fd of sub-RPE surface and 494 F t from each of sub-RPE and EZ-RPE compartments were extracted from baseline spectral domain-optical coherence tomography scans. The top nine features were identified from F fd and F t feature pool separately using minimum Redundancy maximum Relevance feature selection and used to train a Random Forest (RF) classifier independently using three-fold cross validation on the training set ( S t , N = 90) to distinguish between sfGA Progressors and Non-progressors. Combined F fd and F t was also evaluated in predicting risk of sfGA progression. The RF classifier yielded AUC of 0.85, 0.79 and 0.89 on independent test set ( S v , N = 47) using F fd , F t , and their combination, respectively. Using combined F fd and F t , the improvement in AUC was statistically significant on S v with p-values of 0.032 and 0.04 compared to using only F fd and only F t , respectively. Combined F fd and F t appears to identify high-risk patients. Our results show that FD and texture features could be potentially used for predicting risk of sfGA progression and future therapeutic response.

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