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Objectives: The objective of this study was to assess whether zinc-doped fluorapatite (ZnFA) could serve as an effective antimicrobial dental bone filler for bone regeneration compared to autografts., Methods: FA and 2 % zinc-doped FA (2ZnFA) were synthesized and characterized in-house. Compressed and sintered FA and 2ZnFA disks were incubated with bacteria to assess antimicrobial properties. Adipose-derived stem cells were cultured on these discs to evaluate the surfaces' ability to support cell growth and promote osteogenic differentiation. Surfaces exhibiting the highest expressions of the bone markers osteopontin and osteocalcin were selected for an in vivo study in a rat mandibular defect model. Twenty rats were divided into 5 groups, equally, and a 5 mm surgical defect of the jaw was left untreated or filled with 2ZnFA, FA, autograft, or demineralized bone matrix (DBM). At 12 weeks, the defects and surrounding tissues were harvested and subjected to microCT and histological evaluations., Results: Standard techniques such as FTIR, ICP-MS, fluoride probe, and XRD revealed the sintered FA and ZnFA's chemical compositions and structures. Bacterial studies revealed no significant differences in surface bacterial adhesion properties between FA and 2ZnFA, but significantly fewer bacterial loads than control titanium discs (p < 0.05). Cell culture data confirmed that both surfaces could support cell growth and promote the osteogenic differentiation of stem cells. MicroCT analysis confirmed statistical similarities in bone regeneration within FA, 2ZnFA, and autograft groups., Conclusion: The data suggests that both FA and 2ZnFA could serve as alternatives to autograft materials, which are the current gold standard. Moreover, these bone fillers outperformed DBM, an allograft material commonly used as a dental bone void filler., Clinical Significance: The use of FA or 2ZnFA for treating mandibular defects led to bone regeneration statistically similar to autograft repair and significantly outperformed the widely used dental bone filler, DBM. Additional translational research may confirm FA-based materials as superior substitutes for existing synthetic bone fillers, ultimately enhancing patient outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Patients implanted with osseointegrated (OI) prosthetic systems have reported vastly improved upper and lower extremity prosthetic function compared with their previous experience with socket-suspension systems. However, OI systems have been associated with superficial and deep-bone infections and implant loosening due, in part, to a failure of the osseointegration process. Although monitoring the osseointegration using circulating biomarkers has clinical relevance for understanding the progression of osseointegration with these devices, it has yet to be established. Ten patients were enrolled in this study. Blood samples were collected at pre-selected times, starting before implantation surgery, and continuing to 12 months after the second surgery. Bone formation markers, bone resorption markers, and circulating amino acids were measured from blood samples. A linear mixed model was generated for each marker, incorporating patient ID and age with the normalized marker value as the response variable. Post hoc comparisons were made between 1 week before Stage 1 Surgery and all subsequent time points for each marker, followed by multiple testing corrections. Serial radiographic imaging of the residual limb containing the implant was obtained during follow-up, and the cortical index (CI) was calculated for the bone at the porous region of the device. Two markers of bone formation, specifically bone-specific alkaline phosphatase (Bone-ALP) and amino-terminal propeptide of type I procollagen (PINP), exhibited significant increases when compared with the baseline levels of unloaded residual bone prior to the initial surgery, and they subsequently returned to their baseline levels by the 12-month mark. Patients who experienced clinically robust osseointegration experienced increased cortical bone thickness at the porous coated region of the device. A medium correlation was observed between Bone-ALP and the porous CI values up to PoS2-M1 (p = .056), while no correlation was observed for PINP. An increase in bone formation markers and the lack of change observed in bone resorption markers likely reflect increased cortical bone formation induced by the end-loading design of the Utah OI device used in this study. A more extensive study is required to validate the correlation observed between Bone-ALP and porous CI values., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Hydroxyapatite (HA)-based materials are widely used as bone substitutes due to their inherent biocompatibility, osteoconductivity, and bio-absorption properties. However, HA scaffolds lack compressive strength when compared to autograft bone. It has been shown that the fluoridated form of HA, fluorapatite (FA), can be sintered to obtain this desired strength as well as slower degradation properties. Also, FA surfaces have been previously shown to promote stem cell differentiation toward an osteogenic lineage. Thus, it was hypothesized that FA, with and without stromal vascular fraction (SVF), would guide bone healing to an equal or better extent than the clinical gold standard. The regenerative potentials of these scaffolds were tested in 32 Lewis rats in a femoral condylar defect model with untreated (negative), isograft (positive), and commercial HA as controls. Animals were survived for 12 weeks post-implantation. A semi-quantitative micro-CT analysis was developed to quantify the percent new bone formation within the defects. Our model showed significantly higher (p < .05) new bone depositions in all apatite groups compared to the autograft group. Overall, the FA group had the most significant new bone deposition, while the differences between HA, FA, and FA + SVF were insignificant (p > .05). Histological observations supported the micro-CT findings and highlighted the presence of healthy bone tissues without interposing capsules or intense immune responses for FA groups. Most importantly, the regenerating bone tissue within the FA + SVF scaffolds resembled the architecture of the surrounding trabecular bone, showing intertrabecular spaces, while the FA group presented a denser cortical bone-like architecture. Also, a lower density of cells was observed near FA granules compared to HA surfaces, suggesting a reduced immune response. This first in vivo rat study supported the tested hypothesis, illustrating the utility of FA as a bone scaffold material., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

An amine-containing molecule called Compound A has been reported by a group from Bristol-Myers Squibb to act as a positive allosteric modulator (PAM) at the dopamine D1 receptor. We synthesized the more active enantiomer of Compound A (BMS-A1) and compared it with the D1 PAMs DETQ and MLS6585, which are known to bind to intracellular loop 2 and the extracellular portion of transmembrane helix 7, respectively. Results from D1/D5 chimeras indicated that PAM activity of BMS-A1 tracked with the presence of D1 sequence in the N-terminal/extracellular region of the D1 receptor, a unique location compared with either of the other PAMs. In pairwise combinations, BMS-A1 potentiated the small allo-agonist activity of each of the other PAMs, while the triple PAM combination (in the absence of dopamine) produced a cAMP response about 64% of the maximum produced by dopamine. Each of the pairwise PAM combinations produced a much larger leftward shift of the dopamine EC 50 than either single PAM alone. All three PAMs in combination produced a 1000-fold leftward shift of the dopamine curve. These results demonstrate the presence of three non-overlapping allosteric sites that cooperatively stabilize the same activated state of the human D1 receptor. SIGNIFICANCE STATEMENT: Deficiencies in dopamine D1 receptor activation are seen in Parkinson disease and other neuropsychiatric disorders. In this study, three positive allosteric modulators of the dopamine D1 receptor were found to bind to distinct and separate sites, interacting synergistically with each other and dopamine, with the triple combination causing a 1000-fold leftward shift of the response to dopamine. These results showcase multiple opportunities to modulate D1 tone and highlight new pharmacological approaches for allosteric modulation of G-protein-coupled receptors., (Copyright © 2023 by The Author(s).)

Staphylococcus aureus causes the majority of implant-related infections. These infections present as biofilms, in which bacteria adhere to the surface of foreign materials and form robust communities that are resilient to the human immune system and antibiotic drugs. The heavy use of broad-spectrum antibiotics against these pathogens disturbs the host's microbiome and contributes to the growing problem of antibiotic-resistant infections. The use of bacteriophages as antibacterial agents is a potential alternative therapy. In this study, bioluminescent strains of S. aureus were grown to form 48-h biofilms on polyether ether ketone (PEEK), a material used to manufacture orthopaedic implants, in either static or dynamic growth conditions. Biofilms were treated with vancomycin, staphylococcal phage, or a combination of the two. We showed that vancomycin and staph phages were able to independently reduce the total bacterial load. Most phage-antibiotic combinations produced greater log reductions in surviving bacteria compared to single-agent treatments, suggesting antimicrobial synergism. In addition to demonstrating the efficacy of combining vancomycin and staph phage, our results demonstrate the importance of growth conditions in phage-antibiotic combination studies. Dynamic biofilms were found to have a substantial impact on apparent treatment efficacy, as they were more resilient to combination treatments than static biofilms.

Three structurally closely related dopamine D1 receptor positive allosteric modulators (D1 PAMs) based on a tetrahydroisoquinoline (THIQ) scaffold were profiled for their CYP3A4 induction potentials. It was found that the length of the linker at the C5 position greatly affected the potentials of these D1 PAMs as CYP3A4 inducers, and the level of induction correlated well with the activation of the pregnane X receptor (PXR). Based on the published PXR X-ray crystal structures, we built a binding model specifically for these THIQ-scaffold-based D1 PAMs in the PXR ligand-binding pocket via an ensemble docking approach and found the model could explain the observed CYP induction disparity. Combined with our previously reported D1 receptor homology model, which identified the C5 position as pointing toward the solvent-exposed space, our PXR-binding model coincidentally suggested that structural modifications at the C5 position could productively modulate the CYP induction potential while maintaining the D1 PAM potency of these THIQ-based PAMs.

Objective: Evaluate the safety and efficacy of a novel press-fit bone-anchored prosthesis in an FDA approved study., Design: Single-center, prospective 1-year follow-up cohort study of a percutaneous bone anchored prosthesis., Setting: Veterans Health Administration Hospital., Participants: Ten male Veterans with unilateral transfemoral amputation that occurred at least 6 months prior to enrollment and was not the result of dysvascular disease (N=10)., Interventions: All participants received the novel press-fit Percutaneous Osseointegrated Prosthesis (POP) and a minimum of 10 days supervised rehabilitation therapy., Outcome Measures: Adverse events and radiographs were collected to assess device safety. Temporal assessments of bone density, stomal skin, prosthetic don/doff, functional ambulation, and patient-reported outcome compared our POP to a socket suspension system., Results: Ten male participants mean age 48.8±12.1 years (range, 32-68 y) with mean time since amputation of 9.4± years (range 1-18 y) completed a two-staged implantation protocol and progressed to ambulation with an assistive device by post-operative day 14. Eight of 10 completed all study procedures. One implant loosened at 5 weeks, requiring removal. A second was removed after periprosthetic fracture from a non-device-related fall at 28 weeks. One patient required oral antibiotics for superficial infection. There were significant (P<.05) increases in bone density in the lumbar spine and adjacent to the distal porous coating with no radiographic evidence of bone resorption. Compared to the socket system, the use of POP significantly (P<.05) reduced prosthetic don and doff times and patient-reported prosthetic problem scores. Significant improvements (P<.05) in mean mobility, global health, and walking test scores were also observed., Conclusions: Improvements in bone density, function, and patient reported outcomes were observed with the POP device when compared to a socket suspension system. This Early Feasibility Study established initial safety and effectiveness of the POP device, supporting expanded investigation as an alternative to socket prostheses., (Published by Elsevier Inc.)

Background: The US Department of Veterans Affairs has created a portfolio of educational programs to train primary care providers (PCPs) in the evaluation and management of common musculoskeletal (MSK) conditions. Appropriate resource utilization for evaluation of knee pain, including limiting unnecessary magnetic resonance imaging (MRI) studies, is an important theme of these initiatives. The objective of this study was to report the utilization of knee MRI by PCP providers before and after the MSK education program and to determine the appropriateness of these MRI orders., Methods: Twenty-six PCPs participated in the MSK Mini-Residency educational program held in Salt Lake City between April 2012 and October 2014. Knee MRI orders submitted by these providers 12 months before and 12 months after their participation were reviewed. Magnetic resonance imaging orders were categorized as "inappropriate," "probably inappropriate," or "possibly appropriate," based on accepted guidelines for knee MRI utilization. Differences in the numbers of precourse and postcourse MRI orders for each of these categories were compared using Student t test., Results: Following our program, MRI orders decreased from 130 (precourse) to 93 (postcourse), a reduction of 28% ( p = 0.04). This reduction was observed entirely within the "inappropriate" and "probably inappropriate" categories; the number of orders categorized as "possibly appropriate" increased, but not significantly., Conclusions: The MSK Mini-Residency training program was a successful educational intervention and was associated with a reduction in inappropriate knee MRI utilization for some participants, while keeping appropriate MRI utilization stable., (Copyright © 2022 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a “work of the United States Government” for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Aims: While the benefits of direct skeletal attachment of artificial limbs are well recognized, device failure due to infection and insufficient osseointegration remain obstacles to obtaining consistently successful outcomes. Currently, the potential for device failure is assessed by subjective pain, clinical function scores, radiographic evidence of bone atrophy, and the presence of radiolucent lines at the bone-implant interface, and subjective pain and function scores. Our hypothesis is that measurable biological indices might add another objective means to assess trends toward bone and stomal healing. This longitudinal cohort study was undertaken to identify potential serological biomarkers suggestive of bone remodeling and the presence of stomal tissue inflammation., Methods: Ten unilateral transfemoral amputee veterans, who were implanted with a percutaneous osseointegrated (OI) skeletal limb docking system, were recruited to participate in this IRB-approved study. Venous blood samples were obtained from before the Stage 1 Surgery up to 1 year following the Stage 2 Surgery. Whole-blood RNA was extracted, sequenced, mapped, and analyzed. Of the significant differentially expressed (DEGs) genes (p<0.05) identified, four genes of interest (IL12B, IL33, COL2A1, and SOST) were validated using qPCR. Enrichment analysis was performed to identify significant (p<0.01) Gene Ontology (GO) terms., Results: Most differentially expressed genes were only detected at PoS1 immediately after the first surgery. Of the significant genes identified, IL12B and IL33 were related to inflammation, and COL2A1 and SOST were associated with bone remodeling. These four genes were identified with greater than 20 log fold-change., Conclusion: Whole-blood RNA-seq data from 10 patients who previously underwent percutaneous osseointegrated lower limb implantation revealed four genes of interest that are known to be involved in inflammation or bone remodeling. If verified in future studies, these genes may serve as markers for predicting optimal bone remodeling and stomal tissue healing following OI device implantation., Competing Interests: Drs Jay Agarwal, Sujee Jeyapalina and James Peter Beck currently own patents for percutaneous implant systems with respect to the design used in the current study. Also, Drs. Jay Agarwal and James Peter Beck were design surgeons for the DJO Surgical (POP) devices used in the associated clinical trial. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The human pregnane X receptor (hPXR) regulates the expression of major drug metabolizing enzymes. A wide range of drug candidates bind and activate hPXR, and hence are at risk of increasing drug-drug interactions and reducing clinical efficacy. hPXR structural features that function as hot spots for ligand binding are identified and highlighted in this concise review. Based on literature structure-activity relationship data as case studies, structure-based strategies to mitigate hPXR transactivation are summarized for medicinal chemists., Competing Interests: All the authors are employees of Eli Lilly and Company., (This journal is © The Royal Society of Chemistry.)

Purpose: Hydroxyapatite (HA) scaffolds are common replacement materials used in the clinical management of critical-sized bone defects. This study was undertaken to examine the potential benefits of fluoridated derivatives of hydroxyapatite, fluorapatite (FA), and fluorohydroxyapatite (FHA) as bone scaffolds in conjunction with adipose-derived stem cells (ADSCs). If FHA and FA surfaces could drive the differentiation of stem cells to an osteogenic phenotype, the combination of these ceramic scaffolds with ADSCs could produce materials with mechanical strength and remodeling potential comparable to autologous bone. This study was designed to investigate the ability of the apatite surfaces HA, FA, and FHA produced at different sintering temperatures to drive ADSCs toward osteogenic lineages., Methods: HA, FHA, and FA surfaces sintered at 1150 °C and 1250 °C were seeded with ADSCs and evaluated for cell growth and gene and protein expression of osteogenic markers at 2 and 10 days post-seeding., Results: In vitro, ADSC cells were viable on all surfaces; however, differentiation of these cells into osteoblastic lineage only observed in apatite surfaces. ADSCs seeded on FA and FHA expressed genes and proteins related to osteogenic differentiation markers to a greater extent by Day 2 when compared to HA and cell culture controls. By day 10, HA, FA, and FHA all expressed more bone differentiation markers compared to cell culture controls., Conclusion: FA and FHA apatite scaffolds may promote the differentiation of ADSCs at an earlier time point than HA surfaces. Combining apatite scaffolds with ADSCs has the potential to improve bone regeneration following bone injury., (Published by Elsevier Ltd.)

The beta-site APP cleaving enzyme 1, known as BACE1, has been a widely pursued Alzheimer's disease drug target owing to its critical role in the production of amyloid-beta. We have previously reported the clinical development of LY2811376 and LY2886721. LY2811376 advanced to Phase I before development was terminated due to nonclinical retinal toxicity. LY2886721 advanced to Phase II, but development was halted due to abnormally elevated liver enzymes. Herein, we report the discovery and clinical development of LY3202626, a highly potent, CNS-penetrant, and low-dose BACE inhibitor, which successfully addressed these key development challenges.

Purpose: A retrospective analysis of clinical outcomes and complication rates of patients treated with the latest implant design of the so-called Endo-Exo-Femoral Prosthesis (EEFP) was performed. The aim is to gain specific information on long-term complications of this treatment-method., Methods: In January 2019, data of all transfemoral amputees who were treated with TOPS at an acute clinic in Schleswig-Holstein from 2010 to 2016 were retrospectively analysed. This was done with special consideration of postoperative complications. For this purpose, all examination findings from routine clinical follow-up examinations were used. The complications were divided into stoma problems, orthopaedic-technical (OT) problems, fractures and explantations. All EEFPs had the same implant design (3rd generation). This implant is currently the only TOPS in Germany that is clinically used. Descriptive statistics as well as ratio information about occurred complications were calculated., Results: A total of 68 implantations were performed during this period. Average observation time was 6.32 years (±2.16 years). The mean age of the patients was 51.84 years±12.12 years. Cause of amputation was mainly trauma (82,35%). Stoma-associated problems had the highest incidence (7%) among all observed patient-related complications and posed the greatest challenges during the rehabilitation process. Looking only at surgical complications, 81% had no complications at all. In total, 15% had technical problems, 6% had peri-prosthetic fractures, 7% had stoma problems and 3% had to be explanted due to infection., Conclusion: The analysis of collected data shows that TOPS (here the 3rd generation EEFP) can be a successful alternative treatment method to shaft prostheses after transfemoral amputation. The indication should only be given after the failure of a shaft-prosthesis and contraindications must be comprehensively excluded. The greatest challenges in the rehabilitation process are the avoidance of stoma complications, infections and OT-problems. The rehabilitation of amputees treated with TOPS therefore requires an interdisciplinary, specialized rehabilitation team and lifelong rehabilitative care., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Epidermal downgrowth around percutaneous devices produce sinus tracts, which then accumulate bacteria becoming foci of infection. This mode to failure is epidermal-centric, and is accelerated by changes in the chemokines and cytokines of the underlying periprosthetic granulation tissue (GT). In order to more fully comprehend the mechanism of downgrowth, in this 28-day study, percutaneous devices were placed in 10 Zucker diabetic fatty rats; 5 animals were induced with diabetes mellitus II (DM II) prior to the surgery and 5 animals served as a healthy, nondiabetic cohort. At necropsy, periprosthetic tissues were harvested, and underwent histological and polymerase chain reaction (PCR) studies. After isolating GTs from the surrounding tissue and extracting ribonucleic acids, PCR array and quantitative-PCR (qPCR) analyses were carried-out. The PCR array for 84 key wound-healing associated genes showed a five-fold or greater change in 31 genes in the GTs of healthy animals compared to uninjured healthy typical skin tissues. Eighteen genes were overexpressed and these included epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). Thirteen genes were underexpressed. When GTs of DM II animals were compared to healthy animals, there were 8 genes overexpressed and 25 genes underexpressed; under expressed genes included EGF and EGFR. The qPCR and immunohistochemistry data further validated these observations. Pathway analysis of genes up-regulated 15-fold or more indicated two, EGFR and interleukin-10, centric clustering effects. It was concluded that EGFR could be a key player in exacerbating the epidermal downgrowth, and might be an effective target for preventing downgrowth., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Inhibition of BACE1 has become an important strategy in the quest for disease modifying agents to slow the progression of Alzheimer's disease. We previously reported the fragment-based discovery of LY2811376, the first BACE1 inhibitor reported to demonstrate robust reduction of human CSF Aβ in a Phase I clinical trial. We also reported on the discovery of LY2886721, a potent BACE1 inhibitor that reached phase 2 clinical trials. Herein we describe the preparation and structure activity relationships (SAR) of a series of BACE1 inhibitors utilizing trans-cyclopropyl moieties as conformational constraints. The design, details of the stereochemically complex organic synthesis, and biological activity of these BACE1 inhibitors is described., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Percutaneous osseointegrated (OI) prostheses (POPs) are used to skeletally attach artificial limbs in amputees. While any permanent percutaneous interface is at risk of becoming infected by the resident microbiota colonizing the stoma, most of these patients remain infection-free. Avoidance of infection likely depends upon a mechanically and/or biologically stable skin-to-implant interface. The ultimate question remains, "why do some stomata become infected while others do not?" The answer might be found in the dynamic bacterial communities of the patient and within the stomal site itself. This study is an appendix to the first Food and Drug Administration approved prospective early feasibility study of OI prosthetic docking, in which, 10 transfemoral amputees were implanted with a unique POP device. In this analytical, longitudinal cohort study, each patient's skin and stomal microbiota were analyzed from the initial surgery to 1 year following the second-stage surgery. During each follow-up visit, three swab samples-stomal, device thigh skin and contralateral thigh skin-were obtained. DNA was extracted, and bacterial 16S ribosomal RNA (rRNA) genes were amplified and sequenced to profile microbial communities. The stomal microbiota were distinct from the microbiota on the adjacent thigh skin and the skin of the contralateral thigh, with a significantly increased abundance of Staphylococcus aureus within the stoma. Early on stomal microbiota were characterized by high diversity and high relative abundance of obligate anaerobes. Over time, the stomal microbiota shifted and stabilized in communities of lower diversity dominated by Streptococcus, Corynebacterium, and/or Staphylococcus spp. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2645-2654, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Percutaneous osseointegrated (OI) devices for amputees are metallic endoprostheses, that are surgically implanted into the residual stump bone and protrude through the skin, allowing attachment of an exoprosthetic limb. In contrast to standard socket suspension systems, these percutaneous OI devices provide superior attachment platforms for artificial limbs. However, bone adaptation, which includes atrophy and/or hypertrophy along the extent of the host bone-endoprosthetic interface, is seen clinically and depends upon where along the bone the device ultimately transfers loading forces to the skeletal system. The goal of this study was to determine if a percutaneous OI device, designed with a porous coated distal region and an end-loading collar, could promote and maintain stable bone attachment. A total of eight, 18 to 24-month old, mixed-breed sheep were surgically implanted with a percutaneous OI device. For 24-months, the animals were allowed to bear weight as tolerated and were monitored for signs of bone remodelling. At necropsy, the endoprosthesis and the surrounding tissues were harvested, radiographically imaged, and histomorphometrically analyzed to determine the periprosthetic bone adaptation in five animals. Bone growth into the porous coating was achieved in all five animals. Serial radiographic data showed stress-shielding related bone adaptation occurs based on the placement of the endoprosthetic stem. When collar placement and achieved end-bearing against the transected bone, distal bone conservation/hypertrophy was observed. The results supported the use of a distally loading and distally porous coated percutaneous OI device to achieve distal host bone maintenance., Competing Interests: The authors have declared that no competing interests exist.

Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 ( 3 ) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

The wound healing process in the soft tissues adjacent to percutaneous implants induces "epithelial downgrowth", and subsequently, a sinus tract around the device. This provides an optimal environment for bacterial colonization and proliferation. In an attempt to arrest downgrowth and achieve epithelial attachment to a device surface, we have sought to mimic the most common and successful percutaneous organ, the tooth. Since teeth are composed of partially and fully fluoridated forms of hydroxyapatite (HA), it was hypothesized that the surface properties of fluoridated apatites, fluorohydroxyapatite (FHA) and fluorapatite (FA), would improve epithelial cellular adhesion and differentiation when compared to HA and titanium (Ti) surfaces. In this study, the apatites (HA, FHA, and FA) were synthesized and characterized. Following a high-temperature sintering treatment of these apatites, keratinocyte and fibroblast adhesion and differentiation properties were analyzed in vitro, revealing a statistically significant increase in keratinocyte adhesion and terminal differentiation on FA surfaces sintered at 1050-1150 °C as compared to Ti or HA. Moreover, fibroblasts displayed enhanced adhesion on FHA surfaces. This data suggests that percutaneous devices coated with, or fabricated from, fluoridated apatites may induce improved epithelial cellular adhesion and differentiation, potentially limiting deeply penetrating epithelial downgrowth and resultant bacterial ingress., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the last 10 years tremendous progress has been made in the development of artificial pancreas (AP) systems for people with type 1 diabetes (T1D). The pan-European consortium CLOSE (Automated Glu cose Contro l at H ome for People with Chronic Disea se) is aiming to develop integrated AP solutions (APplus) tailored to the needs of people with type 2 diabetes (T2D). APplus comprises a product and service package complementing the AP system by obligatory training as well as home visits and telemedical consultations on demand. Outcome predictors and performance indicators shall help to identify people who could benefit most from AP usage and facilitate the measurement of AP impact in diabetes care. In a first step CLOSE will establish a scalable APplus model case working at the interface between patients, homecare service providers, and payers in France. CLOSE will then scale up APplus by pursuing geographic distribution, targeting additional audiences, and enhancing AP functionalities and interconnectedness. By being part of the European Institute of Innovation and Technology (EIT) Health public-private partnership, CLOSE is committed to the EIT "knowledge triangle" pursuing the integrated advancement of technology, education, and business creation. Putting stakeholders, education, and impact into the center of APplus advancement is considered key for achieving wide AP use in T2D care.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1 S ,3 R )-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1 H )-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [ N -(6- tert -butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β 2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β 2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously., (Copyright © 2018 The Author(s).)

DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder., (Copyright © 2017 Eli Lilly and Company. Published by Elsevier Ltd.. All rights reserved.)

Percutaneous osseointegrated (OI) prostheses directly connect an artificial limb to the residual appendicular skeleton via a permanently implanted endoprosthesis with a bridging connector that protrudes through the skin. The resulting stoma produces unique medical and biological challenges. Previously, a study using a large animal amputation model indicated that infection could be largely prevented, for at least a 12-month period, but the terminal epithelium continued to downgrow. The current study was undertaken to test the longer-term efficacy of this implant construct to maintain a stable skin-implant interface for 24 months. Using the previously successful amputation and implantation surgical procedure, a total of eight sheep were fitted with a percutaneous OI prosthesis. Two animals were removed from the study due to early complications. Of the remaining six sheep, one (16.7%) became infected at 15-months post-implantation and five remained infection-free for the intended 24 months. The histological data of the remaining animals further confirmed the grossly observable epithelial downgrowth. Albeit a receding interface, it was clear that all animals that survived to the end of the study had residual fibrous soft-tissue ingrowth into, and debris within, the exposed titanium porous-coated surface. Overall, the data demonstrated that the porous coated subdermal barrier offered initial protection against infection. However, the fibrous skin attachment was continuously lysed over time by the down-growing epithelium.

Background: We developed two objective structured clinical examinations (OSCEs) to educate and evaluate trainees in the evaluation and management of shoulder and knee pain. Our objective was to examine the evidence for validity of these OSCEs., Methods: A multidisciplinary team of content experts developed checklists of exam maneuvers and criteria to guide rater observations. Content was proposed by faculty, supplemented by literature review, and finalized using a Delphi process. One faculty simulated the patient, another rated examinee performance. Two faculty independently rated a portion of cases. Percent agreement was calculated and Cohen's kappa corrected for chance agreement on binary outcomes. Examinees' self-assessment was explored by written surveys. Responses were stratified into 3 categories and compared with similarly stratified OSCE scores using Pearson's coefficient., Results: A multi-disciplinary cohort of 69 examinees participated. Examinees correctly identified rotator cuff and meniscal disease 88% and 89% of the time, respectively. Inter-rater agreement was moderate for the knee (87%; k = 0.61) and near perfect for the shoulder (97%; k = 0.88). No correlation between stratified self-assessment and OSCE scores were found for either shoulder (0.02) or knee (-0.07)., Conclusions: Validity evidence supports the continuing use of these OSCEs in educational programs addressing the evaluation and management of shoulder and knee pain. Evidence for validity includes the systematic development of content, rigorous control of the response process, and demonstration of acceptable interrater agreement. Lack of correlation with self-assessment suggests that these OSCEs measure a construct different from learners' self-confidence.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a K b of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists., (Copyright © 2016 The Author(s).)

Introduction: A cost-effective professional development program enhancing musculoskeletal (MSK) skills of physicians and allied health providers working in primary care settings has been reported at a single site. This article describes the first 2 years of the national expansion and implementation of a 3-day "MSK Mini-residency.", Methods: Faculty from Veterans Affairs (VA) medical centers worked in partnership with national program faculty from the Salt Lake City VA to present an intensive, integrated, multidisciplinary program to strengthen the skills of primary care providers in evaluating and managing MSK conditions common in primary care. Course assessments included written surveys and a two-station observed structured clinical examination (OSCE) evaluating the physical examination of the shoulder and knee., Results: In the first 2 years of the program, 13 VA facilities participated. Two hundred twenty-seven health care providers, including 135 physicians, were trained. Two hundred seven participants (91 %) completed all pre- and post-course written assessments and the two-station OSCE., Discussion: The MSK Mini-residency program is an effective and well-received mixed-method educational initiative to strengthen the skills of primary care physicians and other health care providers in evaluating and managing patients with MSK complaints and to document their competence in performing physical examinations of the shoulder and knee. The 2-year experience in implementation suggests that this model of educational partnerships is a feasible approach to disseminating innovative educational programs in a way that preserves curricular consistency yet is adaptable to local needs., Competing Interests: All authors declare that they have no conflicts of interest and no competing interests.

Background: Musculoskeletal (MSK) problems are common, and a recent US Bone and Joint Initiative calls for new models of education and professional collaboration. Evidence of feasibility and acceptability of innovative methods are needed., Objective: We assessed if an experimental immersion interdisciplinary MSK curriculum would be acceptable to residents from different specialties, be feasible within existing rotations, and be effective in strengthening clinical skills., Methods: Through funding from the Veterans Affairs Office of Academic Affiliations and the Office of Specialty Care, we developed a Center of Excellence in MSK Care and Education. A core element is the monthly MSK Education Week, which teaches skills and provides opportunities to apply these in clinical settings. Participants include internal medicine, physical medicine and rehabilitation, and orthopaedic surgery residents, as well as students and residents from other health professions programs. All were assigned to the MSK week in lieu of other clinical experiences. Faculty encompassed primary care, rheumatology, endocrinology, orthopaedics, and physical medicine and rehabilitation. Assessments include surveys and a 2-station objective structured clinical examination (OSCE)., Results: Since 2012, a total of 176 trainees have participated. Percentage of trainees reporting ability to evaluate and manage MSK complaints increased (9% to 87% for shoulder; 18% to 86% for knee), and confidence performing MSK injections increased from 10% to 70%. Competency in evaluation of shoulder and knee pain was confirmed by OSCEs., Conclusions: The MSK week program was accepted by residents from the 3 specialties, with learners reporting improved ability to perform shoulder and knee examinations, as demonstrated by OSCEs.

The BACE1 enzyme is a key target for Alzheimer's disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6-8 is described., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Integral-Leg-Prosthesis (ILP) is a comparatively new attachment system that allows direct skeletal docking of artificial limbs. Between January 1999 and December 2013, 69 patients with transfemoral amputation were fitted with ILPs by a single German surgeon. Device design iterations and surgical techniques evolved during these years. For the purposes of comparison, patients receiving the first two designs and procedure iterations were placed in group 1 and the patients fitted with the final design were placed in group 2. Infection rate and planned and unplanned surgical interventions were statistically compared using Fisher exact test. Data demonstrated that the high rate of stoma-associated infections seen in group 1 was dramatically reduced in group 2. Of the 39 patients with 42 implants in group 2, none had operative interventions secondary to infection. All group 2 patients remained infection-free without the use of antibiotics by following a simple but defined wound-hygiene protocol. We concluded that the final iteration of the osseointegrated intramedullary device with a low energy surface at the soft tissue and prosthesis interface allowed a biologically stable skin stoma that remained infection-free without chronic use of antibiotics. The reduction in the infection rate was attributed to the clinically based, empirically driven changes in design and surgical techniques.

Background: Percutaneous osseointegrated prosthetic (POP) devices have been used clinically in Europe for decades. Unfortunately, their introduction into the United States has been delayed, in part due to the lack of data documenting the progression of osseointegration and mechanical stability., Questions/purposes: We determined the progression of bone ingrowth into porous-coated POP devices and established the interrelationship with mechanical stability., Methods: After amputation, 64 skeletally mature sheep received a custom porous-coated POP device and were then randomized into five time groups, with subsequent measurement of percentage of bone ingrowth into the available pore spaces (n = 32) and the mechanical pullout force (n = 32)., Results: Postimplantation, there was an accelerated progression of bone ingrowth (~48% from 0 to 3 months) producing a mean pullout force of 5066 ± 1543 N. Subsequently, there was a slower but continued progression of bone ingrowth (~23% from 3 to 12 months) culminating with a mean pullout force of 13,485 ± 1855 N at 12 months postimplantation. There was a high linear correlation (R = 0.94) between the bone ingrowth and mechanical pullout stability., Conclusions: This weightbearing model shows an accelerated progression of bone ingrowth into the porous coating; the amount of ingrowth observed at 3 months after surgery within the porous-coated POP devices was sufficient to generate mechanical stability., Clinical Relevance: The data document progression of bone ingrowth into porous-coated POP devices and establish a strong interrelationship between ingrowth and pullout strength. Further human data are needed to validate these findings.

Background: Percutaneous osseointegrated prostheses (POPs) are being investigated as an alternative to conventional socket suspension and require a radiographic followup in translational studies to confirm that design objectives are being met., Questions/purposes: In this 12-month animal study, we determined (1) radiographic signs of osseointegration and (2) radiographic signs of periprosthetic bone hypertrophy and resorption (adaptation) and (3) confirmed them with the histologic evidence of host bone osseointegration and adaptation around a novel, distally porous-coated titanium POP with a collar., Methods: A POP device was designed to fit the right metacarpal bone of sheep. Amputation and implantation surgeries (n = 14) were performed, and plane-film radiographs were collected quarterly for 12 months. Radiographs were assessed for osseointegration (fixation) and bone adaptation (resorption and hypertrophy). The cortical wall and medullary canal widths were used to compute the cortical index and expressed as a percentage. Based on the cortical index changes and histologic evaluations, bone adaptation was quantified., Results: Radiographic data showed signs of osseointegration including those with incomplete seating against the collar attachment. Cortical index data indicated distal cortical wall thinning if the collar was not seated distally. When implants were bound proximally, bone resorbed distally and the diaphyseal cortex hypertrophied., Conclusions: Histopathologic evidence and cortical index measurements confirmed the radiographic indications of adaptation and osseointegration. Distal bone loading, through collar attachment and porous coating, limited the distal bone resorption., Clinical Relevance: Serial radiographic studies, in either animal models or preclinical trials for new POP devices, will help to determine which designs are likely to be safe over time and avoid implant failures.

The structures of Cl(-)-(Methanol)1,2 clusters have been unraveled combining Infrared Predissociation (IR-PD) experiments and DFT-based molecular dynamics simulations (DFT-MD) at 100 K. The dynamical IR spectra extracted from DFT-MD provide the initial 600 cm(-1) large anharmonic red-shift of the O-H stretch from uncomplexed methanol (3682 cm(-1)) to Cl(-)-(Methanol)1 complex (3085 cm(-1)) as observed in the IR-PD experiment, as well as the subtle supplementary blue- and red-shifts of the O-H stretch in Cl(-)-(Methanol)2 depending on the structure. The anharmonic vibrational calculations remarkably provide the 100 cm(-1) O-H blue-shift when the two methanol molecules are simultaneously organized in the anion first hydration shell (conformer 2A), while they provide the 240 cm(-1) O-H red-shift when the second methanol is in the second hydration shell of Cl(-) (conformer 2B). RRKM calculations have also shown that 2A/2B conformers interconvert on a nanosecond time-scale at the estimated 100 K temperature of the clusters formed by evaporative cooling of argon prior to the IR-PD process., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Infrared Predissociation (IRPD) spectra of Cl(-)(NMA)1(H2O)0-2Ar2 combined with Born-Oppenheimer Molecular Dynamics (BOMD) IR spectra have been acquired, providing the structure and dynamics of these systems. We show that the chloride ion is bound to the hydrogen of the amide N-H group, forming a strong ionic hydrogen bond, weakening the N-H stretch, and shifting it to lower frequency. The presence of water molecules enhances the ionic hydrogen bond by binding to the amide carbonyl oxygen of NMA and shifts the N-H stretch further to lower frequency. The BOMD IR spectra can recapture all, but about 100 cm(-1), of the 600 to 700 cm(-1) shifts due to the strong N-H stretch anharmonicities observed in experiments. This residual error was found to be due to the lack of zero point energy in the classical treatment of motion in the BOMD method.

Percutaneous medical devices are indispensable in contemporary clinical practice, but the associated incidence of low to moderate mortality infections represents a significant economic and personal cost to patients and healthcare providers. Percutaneous osseointegrated prosthetics also suffer from a similar risk of infection, limiting their clinical acceptance and usage in patients with limb loss. We hypothesized that transepidermal water loss (TEWL) management at the skin-implant interface may improve and maintain a stable skin-to-implant interface. In this study, skin reactions in a 3-month, pig dorsum model were assessed using standard histology, immunohistochemistry, and quantitative image analysis. Immunohistochemical analysis of peri-implant tissue explants showed evidence of: continuous healing (cytokeratin 6+), hypergranulation tissue (procollagen+), hyper-vascularity (collagen 4+), and the presence of fibrocytes (CD45+ and procollagen type 1+). Importantly, the gross skin response was correlated to a previous load-bearing percutaneous osseointegrated prosthetic sheep study conducted in our lab. The skin responses of the two models indicated a potentially shared mechanism of wound healing behavior at the skin-implant interface. Although TEWL management did not reduce skin migration at the skin-implant interface, the correlation of qualitative and quantitative measures validated the pig dorsum model as a high-throughput platform for translational science based percutaneous interface investigations in the future.

A percutaneous, osseointegrated (OI) prosthetics are alternative docking systems for upper- and lower-extremity prostheses. Persistent inflammation and micro-motion are known to cause negative soft-tissue adaptation in wound healing and may also be detrimental to implant longevity. In this study, a unique single-stage sheep amputation and implantation model was developed to assess the efficacy of a porous coated sub-dermal fixation surface in the prevention of skin regression around a percutaneous osseointegrated prosthetic implant. Porous coated and smooth sub-dermal fixation surface prosthetics were implanted in the right forelimb of skeletally mature sheep for up to 12 months. Skin regression kinetics and sub-dermal fixation surface coverage were measured from histological samples. Quantitative measurements of porous coated surfaces yielded skin migration rates of 0.90 ± 0.23, 0.56 ± 0.15, 0.44 ± 0.22 mm/month for the 6, 9, and 12 month animals, respectively. In addition, three load dependent regions of skin adaptation were identified; an interface, a transition, and a stress absorbance region. Immediate post-implantation immobilization of the skin may foster improved load-bearing percutaneous device outcomes. The skin adaptations reported here will aid in informing the design and optimization of future percutaneous, OI devices intended for the treatment of upper- and lower-extremity amputees., (Copyright © 2012 Wiley Periodicals, Inc.)

Novel 4-phenyl tetrahydroisoquinolines that inhibit both dopamine and norepinephrine transporters were designed and prepared. In this Letter, we describe the synthesis, in vitro activity and associated structure-activity relationships of this series. We also report the ex vivo NET occupancy of a representative compound, 41., (Copyright © 2012 Elsevier Ltd. All rights reserved.)