Exosomal proteomic signatures correlate with drug resistance and carboplatin treatment outcome in a spontaneous model of canine osteosarcoma.

BACKGROUND: Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. METHODS: In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. RESULTS: Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of > 300 days, and dogs with < 100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient's exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. CONCLUSIONS: The findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

Congenital Hypothyroidism.

Congenital hypothyroidism (CH) is a disorder of thyroid hormone deficiency which develops secondary to incomplete thyroid development or inadequate thyroid hormone production. State-mandated newborn screening throughout the United States has increased the detection rate of CH, allowing for early intervention. Although the overall mortality rate of CH is low, delayed or omitted treatment can lead to devastating neurocognitive outcomes. As such, CH is regarded as the leading cause of preventable intellectual disability in children. Early identification, facilitated by astute neonatal nursing and medical care, is contingent upon an active working knowledge of the disease process and awareness of the limitations of the newborn screen.

Antibody gene transfer with adeno-associated viral vectors as a method for HIV prevention.

Broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus (HIV) show great promise in HIV prevention as they are capable of potently neutralizing a considerable breadth of genetically diverse strains. Passive transfer of monoclonal bNAb proteins can confer protection in animal models of HIV infection at modest concentrations, inspiring efforts to develop an HIV vaccine capable of eliciting bNAb responses. However, these antibodies demonstrate high degrees of somatic mutation and other unique characteristics that may hinder the ability of conventional approaches to consistently and effectively produce bNAb analogs. As an alternative strategy, we and others have proposed vector-mediated gene transfer to generate long-term, systemic production of bNAbs in the absence of immunization. Herein, we review the use of adeno-associated virus (AAV) vectors for delivery of HIV bNAbs and antibody-like proteins and summarize both the advantages and disadvantages of this strategy as a method for HIV prevention.

School Nurses Practices Promoting Self-Management and Healthcare Transition Skills for Adolescents with Chronic Conditions in Urban Public Schools: A Mixed Methods Study.

Background: Adolescents and young adults (AYA) with chronic conditions should acquire self-management skills as part of their healthcare transition (HCT) from pediatric to adult-focused care. HCT/self-management skills have the potential to help mitigate health disparities among minority AYA with chronic conditions. This study investigated school nurses' practices promoting HCT/self-management skills in urban public schools. Methods: Seventy-nine nurses from three urban school districts in Massachusetts completed a survey of 32 Likert-type questions on HCT/self-management skills, eight demographic questions, and five open-ended practice questions assessing how often they have asked students with chronic conditions about HCT/self-management skills, based on the UNC TRxANSITION IndexTM. Results: Among the 79 school nurses who participated (response rate 76%), 67% never or rarely assessed students' knowledge of HCT/self-management, and 90% would use a tool that promotes/measures HCT/self-management skills. Conclusion: In our study sample, most school nurses acknowledged the importance of assessing HCT/self-management skills. The majority favored using a tool to promote these skills.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis.

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell-cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation.

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of ß-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.

Exosomes from Osteosarcoma and normal osteoblast differ in proteomic cargo and immunomodulatory effects on T cells.

BACKGROUND: Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant-derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells. RESULTS: Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF-ß, α fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree. CONCLUSIONS: Osteosarcoma-derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast-derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype.

Anisometry of Medial Patellofemoral Ligament Reconstruction in the Setting of Increased Tibial Tubercle-Trochlear Groove Distance and Patella Alta.

PURPOSE: To assess the impact elevated tibial tubercle-trochlear groove (TT-TG) distance and patella height, as measured by the Caton-Deschamps Index (CDI), have on the isometry of a reconstructed medial patellofemoral ligament (MPFL). METHODS: Nine fresh-frozen cadaveric knees were placed on a custom testing fixture, with a fixed femur and a mobile tibia. A suture fixed to the MPFL origin on the patella and free to move at the Schöttle point on the femur represented a reconstructed MPFL. A local coordinate system was established, and retroreflective markers attached to the suture quantified MPFL length changes by use of a 3-dimensional motion capture system. The tubercle was transferred to create TT-TG distances of 20 mm and 25 mm and CDIs of 1.2 and 1.4 (patella alta). Recordings of the MPFL suture length change as the knee was brought through a range of motion were made using all combinations of tubercle anatomy in a randomized order for each specimen. A generalized estimating equation modeling technique was used to analyze and control for the clustered nature of the data. RESULTS: Knees with native tibial tubercle anatomy showed MPFL isometry through 20° to 70° range of motion. Tibial tubercle lateralization (increased TT-TG distance) significantly altered MPFL isometry with a TT-TG distance of 20 mm (P < .0001). Patella alta significantly altered MPFL isometry with a CDI of 1.2 (P = .0182). The interaction of tibial tubercle lateralization combined with patella alta significantly increased the amount of anisometry seen in the reconstructed MPFL (P < .001). CONCLUSIONS: Increased tibial tubercle lateralization and patella alta produce anisometry in an MPFL reconstruction using currently recommended landmarks, leading to potentially increased graft tension and potential failure. CLINICAL RELEVANCE: Tibial tubercle transfer should be considered when performing an MPFL reconstruction for recurrent patellofemoral instability in the setting of significant patella alta and an elevated TT-TG distance-especially when both are present-because an isolated MPFL reconstruction will be prone to failure given the anisometry shown in this study.

Magnetic Resonance Imaging Validation of Tibial Tubercle Transfer Distance in the Fulkerson Osteotomy: A Clinical and Cadaveric Study.

PURPOSE: To validate the medialization and anteriorization distances, and the osteotomy angle of anteromedialization tibial tubercle osteotomies using postoperative axial imaging. METHODS: From March 2004 to August 2015, 117 consecutive patients who underwent anteromedialization osteotomies of the tibial tubercle by a single surgeon were identified. Only patients with pre- and postoperative magnetic resonance imaging (MRI) studies were included. Using MRI multiplanar reformats, distances that the tibial tubercle was translated medially (medialization) and anteriorly (anteriorization) were measured. In addition, the osteotomy angle was measured on the postoperative MRI. The measured values were compared with intraoperative estimates. Tibial tubercle osteotomies were then performed on 3 cadaveric knee specimens and imaged with pre- and postprocedure MRIs to correlate intraoperative measurements with MRI findings. RESULTS: A total of 40 patients (41 knees) (34.2%) had both pre- and postoperative MRIs and were included. Compared with intraoperative assessment, MRI measured medialization values average 94.7% (standard deviation [SD] 37.7) of dictated values (P = .1). MRI measured anteriorization averaged less than half of dictated values (48.9%, SD 18.2%, P < .0001). MRI measured osteotomy angles averaged 67.2% of dictated values (SD 50.3%, P < .0001). The steepest osteotomy angle that could be performed without violating the posterior cortex and/or endangering the posterior neurovascular structures was 46.3°. CONCLUSIONS: Surgeons often overestimate both the anteriorization distance and the osteotomy angle in anteromedialization tibial tubercle osteotomies. The steepest osteotomy angle is less than the 60° described in the literature. Modifications should be considered when more anteriorization is desired with tubercle transfers. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Wearable Inertial Sensors Allow for Quantitative Assessment of Shoulder and Elbow Kinematics in a Cadaveric Knee Arthroscopy Model.

PURPOSE: To develop a model using wearable inertial sensors to assess the performance of orthopaedic residents while performing a diagnostic knee arthroscopy. METHODS: Fourteen subjects performed a diagnostic arthroscopy on a cadaveric right knee. Participants were divided into novices (5 postgraduate year 3 residents), intermediates (5 postgraduate year 4 residents), and experts (4 faculty) based on experience. Arm movement data were collected by inertial measurement units (Opal sensors) by securing 2 sensors to each upper extremity (dorsal forearm and lateral arm) and 2 sensors to the trunk (sternum and lumbar spine). Kinematics of the elbow and shoulder joints were calculated from the inertial data by biomechanical modeling based on a sequence of links connected by joints. Range of motion required to complete the procedure was calculated for each group. Histograms were used to compare the distribution of joint positions for an expert, intermediate, and novice. RESULTS: For both the right and left upper extremities, skill level corresponded well with shoulder abduction-adduction and elbow prono-supination. Novices required on average 17.2° more motion in the right shoulder abduction-adduction plane than experts to complete the diagnostic arthroscopy (P = .03). For right elbow prono-supination (probe hand), novices required on average 23.7° more motion than experts to complete the procedure (P = .03). Histogram data showed novices had markedly more variability in shoulder abduction-adduction and elbow prono-supination compared with the other groups. CONCLUSIONS: Our data show wearable inertial sensors can measure joint kinematics during diagnostic knee arthroscopy. Range-of-motion data in the shoulder and elbow correlated inversely with arthroscopic experience. Motion pattern-based analysis shows promise as a metric of resident skill acquisition and development in arthroscopy. CLINICAL RELEVANCE: Wearable inertial sensors show promise as metrics of arthroscopic skill acquisition among residents.

The Tibial Tubercle-to-Trochlear Groove Distance Is Reliable in the Setting of Trochlear Dysplasia, and Superior to the Tibial Tubercle-to-Posterior Cruciate Ligament Distance When Evaluating Coronal Malalignment in Patellofemoral Instability.

PURPOSE: To determine best practices for consistent and accurate evaluation of coronal alignment in patients with patellofemoral (PF) instability. METHODS: Six reviewers examined 239 knee magnetic resonance images (MRIs) in patients with PF instability and anterior cruciate ligament (ACL) rupture. Measurements included tibial tubercle-to-trochlear groove (TT-TG) distance measured at the most proximal and distal portions of the trochlea, tibial tubercle-to-PCL (TT-PCL) distance, and Dejour classification of trochlear dysplasia. RESULTS: Interrater reliability was low for Dejour classification (k = 0.289), but improved to moderate (k = 0.448) when patients were separated into normal/Dejour A and Dejour B/C/D. Interrater reliability was high for proximal and distal TT-TG measurements (interclass correlation coefficients [ICCs] = 0.807 and 0.936, respectively). TT-PCL was moderately reliable (ICC = 0.625), and correlated with TT-TG (r = 0.457, P < .001 proximal and r = 0.451, P < .001 distal). No significant difference was found between the proximal and distal measurements of TT-TG in each patient, though the PF group exhibited higher values than the ACL group (P < .001 for both). TT-PCL was significantly higher for the PF group than the ACL group (P = .015), but this difference lost significance when the group was divided by the TT-PCL cutoff of 24 mm (P = .371). CONCLUSIONS: The proximal and distal techniques for measuring the TT-TG distance are similar to each other, and reliable despite level of reviewer training or presence of dysplasia. The TT-TG distance was predictive of patellofemoral instability. The TT-PCL distance was found to be less reliable than either method of measuring the TT-TG distance. Thus, this study demonstrated TT-TG to be superior to TT-PCL as a measurement of coronal malalignment. Given the variability in Dejour classification in this and other studies, a more reliable classification system for trochlear dysplasia as defined on cross-sectional imaging is warranted. LEVEL OF EVIDENCE: Level III, retrospective clinical trial.

HEMATOLOGY AND PLASMA BIOCHEMISTRY INTERVALS FOR CAPTIVE-BORN CALIFORNIA TIGER SALAMANDERS (AMBYSTOMA CALIFORNIENSE).

Hematology and plasma biochemistry parameters were determined for 34 captive-born California tiger salamanders ( Ambystoma californiense ). The animals were manually restrained for general examination and venipuncture. This is the first comprehensive report of hematology and plasma biochemistry parameters in apparently healthy California tiger salamanders and may serve as a reference for clinical assessment and future study of this species.

Structural basis for Rab1 de-AMPylation by the Legionella pneumophila effector SidD.

The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.

MED23-associated intellectual disability in a non-consanguineous family.

Intellectual disability (ID) is a heterogeneous condition arising from a variety of environmental and genetic factors. Among these causes are defects in transcriptional regulators. Herein, we report on two brothers in a nonconsanguineous family with novel compound heterozygous, disease-segregating mutations (NM_015979.3: [3656A > G];[4006C > T], NP_057063.2: [H1219R];[R1336X]) in MED23. This gene encodes a subunit of the Mediator complex that modulates the expression of RNA polymerase II-dependent genes. These brothers, who had profound ID, spasticity, congenital heart disease, brain abnormalities, and atypical electroencephalography, represent the first case of MED23-associated ID in a non-consanguineous family. They also expand upon the clinical features previously reported for mutations in this gene.

A Curricular Model for Simulation Within Orthopaedic Residency Training.

Introduction: American Board of Orthopaedic Surgery/American Council on GraduateMedical Education Residency Review Committee training requirements have necessitated the need for the adoption of simulation education into existing programmatic requirements. Current guidelines focus only on interns at a potentially significant cost to programs; both in total dollar amount and time. Methods: The authors aim to provide a model that can maximize utility for all resident levels, manage cost by maximizing the use of cadaveric material, and allow integration of varied industry support. Results: The Oregon Health & Science University Orthopaedic education program has developed a high-fidelity training curriculum that (1) is applicable to both junior and senior residents (2) has minimized the cost per resident with the reuse of cadaveric specimens and (3) has nurtured partnerships with industry stakeholders to reduce bias in training by collaborating with most major industry representatives. Conclusion: The simulation curriculum outlined in this manuscript may serve as a reference for other programs and institutions to develop their own residency educational curriculum models.

Resident Performance on the Fundamentals of Arthroscopic Surgery Training (FAST) Workstation Does Not Predictably Improve With Postgraduate Year.

Purpose: To identify differences in performance on the Fundamentals of Arthroscopic Surgery Training (FAST) workstation between residents across different postgraduate years and training sites. Methods: During the 2018-2019 academic year, 102 orthopaedic surgery residents from 4 training sites completed 6 FAST modules. Failure was defined as either completion time exceeding benchmark time or commission of task-specific errors. With the exception of knot tying, each module was completed by participants twice-once with each hand serving as the camera hand. Time to completion (except for knot tying) and errors were recorded for each of the modules. Completion times and failure rates were compared between postgraduate years, seniority groups, and training sites. Results: In all modules for which time was recorded, except for the suture-passage module, there was no significant difference in time to completion based on seniority (P < .01 for suture passage and P > .05 for all others). Significant differences in completion time were observed between sites for all modules except for the suture-passage module (P = .957 for suture passage and P < .05 for all others). Site predicted failure by at least 1 measure (time or technical error) for all modules (P < .05) except for number probing and suture passage. Failure rate across training years varied for each module. Conclusions: Time to completion and rate of failure did not predictably decrease with level of training. Training site proved to be a significant predictor of performance. Factors such as hand dominance and familiarity with the equipment proved to be important considerations for some modules. Clinical Relevance: Objective assessment of arthroscopic skills among orthopaedic trainees is difficult. Using reproducible methodology to assess trainees on specific skills at all postgraduate years and at multiple training sites may provide important information about orthopaedic training.

HIV broadly neutralizing antibody escapability drives the therapeutic efficacy of vectored immunotherapy.

Broadly neutralizing antibodies (bNAbs) have shown great promise for prevention and treatment of HIV infection. Breadth of bNAb neutralization, measured in vitro across panels of diverse viral isolates, is often used as a predictor of clinical potential. However, recent prevention studies demonstrate that the clinical efficacy of a broad and potent bNAb (VRC01) is undermined by neutralization resistance of circulating strains. Using HIV-infected humanized mice, we find that therapeutic efficacy of bNAbs delivered as Vectored ImmunoTherapy (VIT) is a function of both the fitness cost and resistance benefit of mutations that emerge during viral escape, which we term 'escapability'. Applying this mechanistic framework, we find that the sequence of the envelope V5-loop alters the resistance benefits of mutants that arise during escape, thereby impacting the therapeutic efficacy of VIT-mediated viral suppression. We also find that an emtricitabine-based antiretroviral drug regimen dramatically enhances the efficacy of VIT, by reducing the fitness of mutants along the escape path. Our findings demonstrate that bNAb escapability is a key determinant to consider in the rational design of antibody regimens with maximal efficacy and illustrates a tractable means of minimizing viral escape from existing bNAbs.

Antibody-mediated prevention of vaginal HIV transmission is dictated by IgG subclass in humanized mice.

HIV broadly neutralizing antibodies (bNAbs) are capable of both blocking viral entry and driving innate immune responses against HIV-infected cells through their Fc region. Vaccination or productive infection results in a polyclonal mixture of class-switched immunoglobulin G (IgG) antibodies composed of four subclasses, each encoding distinct Fc regions that differentially engage innate immune functions. Despite evidence that innate immunity contributes to protection, the relative contribution of individual IgG subclasses is unknown. Here, we used vectored immunoprophylaxis in humanized mice to interrogate the efficacy of individual IgG subclasses during prevention of vaginal HIV transmission by VRC07, a potent CD4-binding site-directed bNAb. We find that VRC07 IgG2, which lacks Fc-mediated functionality, exhibited substantially reduced protection in vivo relative to other subclasses. Low concentrations of highly functional VRC07 IgG1 yielded substantial protection against vaginal challenge, suggesting that interventions capable of eliciting modest titers of functional IgG subclasses may provide meaningful benefit against infection.

Evaluation of a Tool to Measure the Demands of Immigration.

BACKGROUND: The Demands of Immigration (DI) scale consists of six subscales and is designed to measure demands that negatively affect emotional health of immigrants. PURPOSE: The purpose of this review is to (a) examine the DI scale's language versions and translation procedures; and (b) evaluate reliability and validity of the DI scale based on studies subsequent to its initial development. METHODS: Published studies were identified through CINAHL Complete, MEDLINE, Social Sciences Premium Collection and PsycInfo. RESULTS: Seventeen quantitative studies reported the DI scale's reliability (internal consistency and test-retest) and/or validity (content and construct). Correlations among the DI subscales and key variables (e.g., job satisfaction, acculturation, depression, perceived support, and resilience) support its construct validity. CONCLUSION: The DI scale is a reliable and valid tool for measuring demands or challenges that immigrants face.