Discovery of a novel 2-spiroproline steroid mimetic scaffold for the potent inhibition of 11ß-HSD1.

11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues, resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11ß-HSD1, particularly in adipose tissues, has been associated with a variety of ailments including metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11ß-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a 3-point pharmacophore for 11ß-HSD1 that was utilized to design a 2-spiroproline derivative as a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of several leads, such as compounds 39 and 51. Importantly, deleterious hERG inhibition and pregnane X receptor induction were mitigated by the introduction of a 4-hydroxyl group to the proline ring system.

Discovery of 1'-(1-phenylcyclopropane-carbonyl)-3H-spiro[isobenzofuran-1,3'-pyrrolidin]-3-one as a novel steroid mimetic scaffold for the potent and tissue-specific inhibition of 11ß-HSD1 using a scaffold-hopping approach.

11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11ß-HSD1, particularly in adipose tissues, has been associated with metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11ß-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a three-point pharmacophore for 11ß-HSD1 that was utilized to design a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of INCB13739. Clinical evaluation of INCB13739 confirmed for the first time that tissue-specific inhibition of 11ß-HSD1 in patients with type 2 diabetes mellitus was efficacious in controlling glucose levels and reducing cardiovascular risk factors.

Surgical Technique: Abductor Reconstruction With Gluteus Maximus and Tensor Fascia Lata in Revision Total Hip Arthroplasty.

BACKGROUND: Abductor deficiency in revision total hip arthroplasty (THA) is a common problem that can lead to pain, limping, and instability. Repair and reconstruction of the abductors is challenging, with a high rate of failure reported in the literature. The purpose of this study is to describe a simplified technique of abductor repair augmented with the transfer of gluteus maximus (Gmax) and the tensor fascia lata (TFL). METHODS: We describe a novel abductor reconstruction with transfer of the anterior 30% of Gmax and the posterior 70% of TFL to the vastus lateralis origin. These transfers can be used in isolation or to augment repair of torn abductors to the greater trochanter. The technique is simple and quick to perform via a lateral approach, requiring dissection of only two muscle slips and minimal additional soft tissue dissection. RESULTS: We describe the use and outcomes of this technique on three patients undergoing revision THA with severe and irreparable abductor deficiency. Although these patients reported improved function after the reconstruction, there was persistence of mild to moderate limping. CONCLUSION: Abductor reconstruction with partial transfers of Gmax and TFL is a promising approach to manage abductor deficiency in revision THA. Larger series are required to determine the efficacy of this technique for restoring abductor function and improving patient reported outcomes.

Primary cilia on muscle stem cells are critical to maintain regenerative capacity and are lost during aging.

During aging, the regenerative capacity of muscle stem cells (MuSCs) decreases, diminishing the ability of muscle to repair following injury. We found that the ability of MuSCs to regenerate is regulated by the primary cilium, a cellular protrusion that serves as a sensitive sensory organelle. Abolishing MuSC cilia inhibited MuSC proliferation in vitro and severely impaired injury-induced muscle regeneration in vivo. In aged muscle, a cell intrinsic defect in MuSC ciliation was associated with the decrease in regenerative capacity. Exogenous activation of Hedgehog signaling, known to be localized in the primary cilium, promoted MuSC expansion, both in vitro and in vivo. Delivery of the small molecule Smoothened agonist (SAG1.3) to muscles of aged mice restored regenerative capacity leading to increased strength post-injury. These findings provide fresh insights into the signaling dysfunction in aged MuSCs and identify the ciliary Hedgehog signaling pathway as a potential therapeutic target to counter the loss of muscle regenerative capacity which accompanies aging.

AP-1 is a temporally regulated dual gatekeeper of reprogramming to pluripotency.

Somatic cell transcription factors are critical to maintaining cellular identity and constitute a barrier to human somatic cell reprogramming; yet a comprehensive understanding of the mechanism of action is lacking. To gain insight, we examined epigenome remodeling at the onset of human nuclear reprogramming by profiling human fibroblasts after fusion with murine embryonic stem cells (ESCs). By assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing we identified enrichment for the activator protein 1 (AP-1) transcription factor c-Jun at regions of early transient accessibility at fibroblast-specific enhancers. Expression of a dominant negative AP-1 mutant (dnAP-1) reduced accessibility and expression of fibroblast genes, overcoming the barrier to reprogramming. Remarkably, efficient reprogramming of human fibroblasts to induced pluripotent stem cells was achieved by transduction with vectors expressing SOX2, KLF4, and inducible dnAP-1, demonstrating that dnAP-1 can substitute for exogenous human OCT4. Mechanistically, we show that the AP-1 component c-Jun has two unexpected temporally distinct functions in human reprogramming: 1) to potentiate fibroblast enhancer accessibility and fibroblast-specific gene expression, and 2) to bind to and repress OCT4 as a complex with MBD3. Our findings highlight AP-1 as a previously unrecognized potent dual gatekeeper of the somatic cell state.

Real-world Outcomes With Rituximab-based Therapy for Posttransplant Lymphoproliferative Disease Arising After Solid Organ Transplant.

BACKGROUND: Optimal upfront therapy for posttransplant lymphoproliferative disease (PTLD) arising after solid organ transplant remains contentious. Rituximab monotherapy (R-Mono) in unselected patients has shown a lack of durable remissions. Cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP)-based chemotherapy confers improved response rates, although concerns exist about toxicity. METHODS: This multicenter retrospective study reports outcomes for adults with biopsy-proven B-cell PTLD treated initially with R-Mono or Rituximab plus CHOP (R-CHOP). Selection of therapy was made according to physician preference. RESULTS: Among 101 patients, 41 received R-Mono and 60 had R-CHOP. Most (93%) had undergone renal or liver transplantation. R-CHOP showed a trend toward improved complete (53% versus 71%; P = 0.066) and overall (75% versus 90%; P = 0.054) response rates. In the R-Mono group, 13 of 41 (32%) subsequently received chemotherapy, while 25 of 41 (61%) remained progression-free without further therapy. With median follow-up of 47 months, overall survival (OS) was similar for R-Mono and R-CHOP, with 3-year OS of 71% and 63%, respectively (P = 0.722). Non-PTLD mortality was 3 of 41 (7%) and 4 of 60 (7%) within 12 months of R-Mono or R-CHOP, respectively. The International Prognostic Index was statistically significant, with low- (0-2 points) and high-risk (≥3 points) groups exhibiting 3-year OS of 78% and 54%, respectively (P = 0.0003). In low-risk PTLD, outcomes were similar between therapies. However, in high-risk disease R-Mono conferred an inferior complete response rate (21% versus 68%; P = 0.006), albeit with no impact on survival. CONCLUSIONS: Our data support R-Mono as initial therapy for PTLD arising after renal or liver transplantation. However, upfront R-CHOP may benefit selected high-risk cases in whom rapid attainment of response is desirable.

Non-uniform in vivo Expansion of Epstein-Barr Virus-Specific T-Cells Following Donor Lymphocyte Infusion for Post-transplant Lymphoproliferative Disease.

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is a life-threatening complication of T-lymphocyte deplete allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with PTLD refractory to Rituximab, donor lymphocyte infusion (DLI) is established as a successful option for salvage therapy. However, although in vivo lymphocyte expansion has been correlated with good clinical outcome following DLI, the specificity and functional characteristics of EBV-specific T-cell responses remain poorly characterized. Here we describe two patients with Rituximab-refractory PTLD complicating T-cell deplete allo-HSCT, both of whom were successfully rescued with 1 × 106/Kg unselected stem cell donor-derived DLI. Prospective analyses revealed that complete clinical and radiological responses were associated with in vivo expansion of T and NK cells. Furthermore, EBV MHC tetramer, and interferon gamma analyses revealed a marked increase in EBV-specific T-cell frequency from 4 weeks after DLI. Reactivity was demonstrated against a range of EBV latent and lytic antigens, including those detected in tumor biopsy material. The immunodominant EBV-specific T cell response expanding in vivo following infusion matched the dominant response present in the DLI preparations prior to administration. Furthermore, differences in the repertoire of subdominant antigen-specific T-cells were also detected, suggesting that antigen-encounter in vivo can shape the immune response. These results demonstrate the value of prospectively studying in vivo T-cell responses, by facilitating the identification of important specificities required for clinical efficacy. Applying this approach on a larger scale promises to yield data which may be essential for the optimization of future adoptive immunotherapeutic strategies for PTLD.

Glenoid component positioning and guidance techniques in anatomic and reverse total shoulder arthroplasty: A systematic review and meta-analysis.

BACKGROUND: Positioning of the glenoid component is one of the most challenging steps in shoulder arthroplasty, and prosthesis longevity as well as functional outcomes is considered highly dependent on accurate positioning. This review considers the evidence supporting surgical navigation and patient-specific instruments for glenoid implant positioning in anatomic and reverse total shoulder arthroplasty. METHODS: A systematic literature search was performed for studies assessing glenoid implant positioning accuracy as measured by cross-sectional imaging on live subjects or cadaver models. Meta-analysis of controlled studies was performed to estimate the primary effects of navigation and patient-specific instruments on glenoid implant positioning error. Meta-analysis of absolute positioning outcomes was also performed for each group incorporating data from controlled and uncontrolled studies. RESULTS: Nine studies, four controlled and five uncontrolled, with 258 total subjects were included in the analysis. Meta-analysis of controlled studies supported that both navigation and patient-specific instruments had a moderate statistically significant effect on improving glenoid implant positioning outcomes. Meta-analysis of absolute positioning outcomes demonstrates glenoid implant positioning with standard instrumentation results in a high rate of malposition. DISCUSSION: Navigation and patient-specific instruments improve glenoid positioning outcomes. Whether the improvement in positioning outcomes achieved translate to better clinical outcomes is unknown.

Multivariate multiple regression models of poly(ethylene-terephthalate) film degradation under outdoor and multi-stressor accelerated weathering exposures.

Developing materials for use in photovoltaic (PV) systems requires knowledge of their performance over the warranted lifetime of the PV system. Poly(ethylene-terephthalate) (PET) is a critical component of PV module backsheets due to its dielectric properties and low cost. However, PET is susceptible to environmental stressors and degrades over time. Changes in the physical properties of nine PET grades were modeled after outdoor and accelerated weathering exposures to characterize the degradation process of PET and assess the influence of stabilizing additives and weathering factors. Multivariate multiple regression (MMR) models were developed to quantify changes in color, gloss, and haze of the materials. Natural splines were used to capture the non-linear relationship between predictors and responses. Model performance was evaluated via adjusted-R2 and root mean squared error values from leave-one-out cross validation analysis. All models described over 85% of the variation in the data with low relative error. Model coefficients were used to assess the influence of weathering stressors and material additives on the property changes of films. Photodose was found to be the primary degradation stressor and moisture was found to increase the degradation rate of PET. Direct moisture contact was found to impose more stress on the material than airbone moisture (humidity). Increasing the concentration of TiO2 was found to generally decrease the degradation rate of PET and mitigate hydrolytic degradation. MMR models were compared to physics-based models and agreement was found between the two modeling approaches. Cross-correlation of accelerated exposures to outdoor exposures was achieved via determination of cross-correlation scale factors. Cross-correlation revealed that direct moisture contact is a key factor for reliable accelerated weathering testing and provided a quantitative method to determine when accelerated exposure results can be made more aggressive to better approximate outdoor exposure conditions.

Saturated Salt Solution Cadaver-Embalming Method Improves Orthopaedic Surgical Skills Training.

BACKGROUND: Human cadaver surgical skills training offers the highest-fidelity simulation of the operative environment, with the potential to enhance surgeon training and to reduce operative risks to patients. Embalming extends the duration that a cadaveric specimen may be used for surgical skills training and reduces the risk of disease transmission, but it can alter the properties of the cadaver tissue, reducing the simulation fidelity and training quality. The purpose of this controlled laboratory study was to evaluate 3 embalming methods, formaldehyde solution, alcohol-glycol solution, and saturated salt solution, and to compare their relative performance in a 2-week orthopaedic surgical skills training course. METHODS: The 3 embalming methods were applied to 3 cadavers each. Joint range of motion and joint stiffness were measured prior to dissection. The cadavers were assessed by 4 orthopaedic surgeons on the first and last days of the training course in 4 domains: tissue visual fidelity, tissue tactile fidelity, odor, and overall suitability for surgical skills training using 7-point Likert scales. Sterile specimens for bacterial and fungal culture were obtained from cadaver muscle tissue on the fourth and fifteenth days of use. RESULTS: All cadaver groups were successfully preserved without putrefaction and were utilized over the duration of the training course. The saturated salt solution cadavers had supple joints with excellent motion and were rated highly across all domains. The saturated salt solution cadaver joint motion, stiffness, visual and tactile tissue fidelity, odor, and suitability for surgical skills training were significantly superior (p < 0.05) to the formaldehyde solution and alcohol-glycol solution cadavers. CONCLUSIONS: The saturated salt embalming method is inexpensive and logistically straightforward and preserves human cadavers at room temperature in a state suitable for high-fidelity orthopaedic surgical skills training.

Shoulder physiotherapy exercise recognition: machine learning the inertial signals from a smartwatch.

OBJECTIVE: Participation in a physical therapy program is considered one of the greatest predictors of successful conservative management of common shoulder disorders. However, adherence to these protocols is often poor and typically worse for unsupervised home exercise programs. Currently, there are limited tools available for objective measurement of adherence in the home setting. The goal of this study was to develop and evaluate the potential for performing home shoulder physiotherapy monitoring using a commercial smartwatch. APPROACH: Twenty healthy adult subjects with no prior shoulder disorders performed seven exercises from an evidence-based rotator cuff physiotherapy protocol, while 6-axis inertial sensor data was collected from the active extremity. Within an activity recognition chain (ARC) framework, four supervised learning algorithms were trained and optimized to classify the exercises: k-nearest neighbor (k-NN), random forest (RF), support vector machine classifier (SVC), and a convolutional recurrent neural network (CRNN). Algorithm performance was evaluated using 5-fold cross-validation stratified first temporally and then by subject. MAIN RESULTS: Categorical classification accuracy was above 94% for all algorithms on the temporally stratified cross validation, with the best performance achieved by the CRNN algorithm (99.4%). The subject stratified cross validation, which evaluated classifier performance on unseen subjects, yielded lower accuracies scores again with CRNN performing best (88.9%). SIGNIFICANCE: This proof of concept study demonstrates the technical feasibility of a smartwatch device and supervised machine learning approach to more easily monitor and assess the at-home adherence of shoulder physiotherapy exercise protocols.

Low-dose lithium regimen enhances endochondral fracture healing in osteoporotic rodent bone.

Osteoporotic bone fractures are highly prevalent and involve lengthy recovery. Lithium, commonly used in psychiatric medicine, inhibits glycogen synthase kinase-3ß in the Wnt/ß-catenin pathway, leading to up-regulation of osteogenesis. Our recent preclinical work demonstrated that a 20 mg/kg lithium dose administered beginning 7 days post-fracture for 14 days optimally improved femoral fracture healing in healthy rats at 4 weeks post fracture (46% higher torsional strength). In this study, lithium treatment was evaluated for healing of osteoporotic bone fractures. Six-month-old ovariectomized rats were subjected to closed, load-drop induced femoral diaphyseal fracture. Two regimens involving treatment initiation on day 7 and day 10, respectively, 20 mg/kg/day oral dose and 14 days duration were evaluated. Femurs of lithium- vs. saline- treated rats were analyzed at 4 weeks (for day 7 onset regimen) or 6 weeks (for day 10 onset regimen) post-fracture by stereology and torsional mechanical testing. Initiation on day 10 led to a significant 50% higher maximum yield torque (primary outcome measure) at 6 weeks (309 vs. 206 N-mm, p = 0.005; n = 7, 7). Initiation on day 7 suggested a trend toward a more modest improvement in maximum yield torque (13%) evaluated at 4 weeks post-fracture (234 vs. 206 N-mm, p = 0.10; n = 10, 13). Qualitatively, lithium-treated femurs demonstrated better periosteal and mineralized callus bridging in the day 10 initiation group. Lithium is a widely-available, orally administered, low-cost drug, which represents a feasible pharmacological intervention for both healthy and osteoporotic fracture healing. This study provides important guidelines for future clinical evaluation of lithium in osteoporotic fracture patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1783-1789, 2018.

Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.

Diagnosis of Engaging Bipolar Bone Defects in the Shoulder Using 2-Dimensional Computed Tomography: A Cadaveric Study.

BACKGROUND: Anatomic studies have demonstrated that bipolar glenoid and humeral bone loss have a cumulative effect on shoulder instability and that these defects may engage in functional positions depending on their size and location, potentially resulting in failure of stabilization procedures. Determining which lesions pose a risk for engagement remains challenging, with arthroscopic assessments and a 3-dimensional computed tomography (CT)-based glenoid track method being accepted approaches at this time. PURPOSE: The purpose was to investigate the interaction of humeral and glenoid bone defects on shoulder engagement in a cadaveric model. Two alternative approaches to predicting engagement were evaluated: (1) CT of the shoulder in abduction and external rotation (ABER) and (2) measurement of the glenoid lesion width and measurement of a novel parameter, the intact anterior articular angle (IAAA), on conventional 2-dimensional multiplane reformats. STUDY DESIGN: Controlled laboratory study. METHODS: Hill-Sachs and glenoid defects of varying sizes were created in 12 cadaveric upper limbs, producing 45 bipolar defect combinations. The defect characteristics were assessed using CT with the shoulder in a neutral position. ABER CT was performed with the shoulder positioned in 60° of glenohumeral abduction (corresponding to 90° of abduction relative to the trunk) and 90° of external rotation. The IAAA was measured as the cartilage arc angle anterior to the Hill-Sachs defect on the axial slice bisecting the humeral head. The performance of the ABER CT and IAAA approaches to predicting engagement were compared with the glenoid track method. RESULTS: Of the 45 defect combinations, 24 (53%) were classified as engaging using the glenoid track method. ABER CT predicted engagement accurately in 43 of 45 (96%), with a sensitivity and specificity of 92% and 100%, respectively. A logistic model based on the glenoid defect width and IAAA provided a prediction accuracy of 87%, with a sensitivity and specificity of 92% and 81%, respectively. CONCLUSION/CLINICAL RELEVANCE: Bipolar lesions at risk for engagement can be identified accurately using an ABER CT scan or by performing 2-dimensional measurements of the glenoid defect width and IAAA on conventional CT multiplane reformats. This information will be useful for surgical planning in the setting of bipolar bone defects before shoulder stabilization.

Memory B-cell reconstitution following allogeneic hematopoietic stem cell transplantation is an EBV-associated transformation event.

Allogeneic stem cell transplantation (allo-HSCT) provides a unique opportunity to track Epstein-Barr virus (EBV) infection in the context of the reconstituting B-cell system. Although many allo-HSCT recipients maintain low or undetectable levels of EBV DNA posttransplant, a significant proportion exhibit elevated and rapidly increasing EBV loads which, if left untreated, may lead to potentially fatal EBV-associated posttransplant lymphoproliferative disease. Intriguingly, this high-level EBV reactivation typically arises in the first 3 months posttransplant, at a time when the peripheral blood contains low numbers of CD27+ memory cells which are the site of EBV persistence in healthy immunocompetent donors. To investigate this apparent paradox, we prospectively monitored EBV levels and B-cell reconstitution in a cohort of allo-HSCT patients for up to 12 months posttransplant. In patients with low or undetectable levels of EBV, the circulating B-cell pool consisted predominantly of transitional and naive cells, with a marked deficiency of CD27+ memory cells which lasted >12 months. However, among patients with high EBV loads, there was a significant increase in both the proportion and number of CD27+ memory B cells. Analysis of sorted CD27+ memory B cells from these patients revealed that this population was preferentially infected with EBV, expressed EBV latent transcripts associated with B-cell growth transformation, had a plasmablastic phenotype, and frequently expressed the proliferation marker Ki-67. These findings suggest that high-level EBV reactivation following allo-HSCT may drive the expansion of latently infected CD27+ B lymphoblasts in the peripheral blood.

Changing rates of adenocarcinoma of the lung.

Over the past several decades, adenocarcinoma of the lung has been increasing as a fraction of all lung cancer. Examination of the available evidence led the 2014 Report of the Surgeon General to conclude that the increases in the rates of adenocarcinoma among smokers in the U.S. were a result of changes in cigarette design and composition over the past 6 decades. While a causal link to design and composition changes as a whole is clear, the changes that have been implemented over the past several decades are not uniformly applied across all cigarette brands in the current market, raising questions about differences in risks among users of different cigarette brands. Recognition of the increased risks resulting from design and composition changes offers a corollary opportunity to reduce current disease risks by identifying and regulating the specific compositional and design changes that produced the increase in risk.

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells.

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.