Exploring relationships among multi-disciplinary assessments for knee joint health in service members with traumatic unilateral lower limb loss: a two-year longitudinal investigation.

Motivated by the complex and multifactorial etiologies of osteoarthritis, here we use a comprehensive approach evaluating knee joint health after unilateral lower limb loss. Thirty-eight male Service members with traumatic, unilateral lower limb loss (mean age = 38 yr) participated in a prospective, two-year longitudinal study comprehensively evaluating contralateral knee joint health (i.e., clinical imaging, gait biomechanics, physiological biomarkers, and patient-reported outcomes); seventeen subsequently returned for a two-year follow-up visit. For this subset with baseline and follow-up data, outcomes were compared between timepoints, and associations evaluated between values at baseline with two-year changes in tri-compartmental joint space. Upon follow-up, knee joint health worsened, particularly among seven Service members who presented at baseline with no joint degeneration (KL = 0) but returned with evidence of degeneration (KL ≥ 1). Joint space narrowing was associated with greater patellar tilt (r[12] = 0.71, p = 0.01), external knee adduction moment (r[13] = 0.64, p = 0.02), knee adduction moment impulse (r[13] = 0.61, p = 0.03), and CTX-1 concentration (r[11] = 0.83, p = 0.001), as well as lesser KOOSSport and VR-36General Health (r[16] = - 0.69, p = 0.01 and r[16] = - 0.69, p = 0.01, respectively). This longitudinal, multi-disciplinary investigation highlights the importance of a comprehensive approach to evaluate the fast-progressing onset of knee osteoarthritis, particularly among relatively young Service members with lower limb loss.

A Comprehensive, Multidisciplinary Assessment for Knee Osteoarthritis Following Traumatic Unilateral Lower Limb Loss in Service Members.

INTRODUCTION: Knee osteoarthritis (KOA) is a primary source of long-term disability and decreased quality of life (QoL) in service members (SM) with lower limb loss (LL); however, it remains difficult to preemptively identify and mitigate the progression of KOA and KOA-related symptoms. The objective of this study was to explore a comprehensive cross-sectional evaluation, at the baseline of a prospective study, for characterizing KOA in SM with traumatic LL. MATERIALS AND METHODS: Thirty-eight male SM with traumatic unilateral LL (23 transtibial and 15 transfemoral), 9.5 ± 5.9 years post-injury, were cross-sectionally evaluated at initial enrollment into a prospective, longitudinal study utilizing a comprehensive evaluation to characterize knee joint health, functionality, and QoL in SM with LL. Presences of medial, lateral, and/or patellofemoral articular degeneration within the contralateral knee were identified via magnetic resonance imaging(for medically eligible SM; Kellgren-Lawrence Grade [n = 32]; and Outerbridge classification [OC; n = 22]). Tri-planar trunk and pelvic motions, knee kinetics, along with temporospatial parameters, were quantified via full-body gait evaluation and inverse dynamics. Concentrations of 26 protein biomarkers of osteochondral tissue degradation and inflammatory activity were identified via serum immunoassays. Physical function, knee symptoms, and QoL were collected via several patient reported outcome measures. RESULTS: KOA was identified in 12 of 32 (37.5%; KL ≥ 1) SM with LL; however, 16 of 22 SM presented with patellofemoral degeneration (72.7%; OC ≥ 1). Service members with versus without KOA had a 26% reduction in the narrowest medial tibiofemoral joint space. Biomechanically, SM with versus without KOA walked with a 24% wider stride width and with a negative correlation between peak knee adduction moments and minimal medial tibiofemoral joint space. Physiologically, SM with versus without KOA exhibited elevated concentrations of pro-inflammatory biomarker interleukin-7 (+180%), collagen breakdown markers collagen II cleavage (+44%), and lower concentrations of hyaluronic acid (-73%) and bone resorption biomarker N-telopeptide of Type 1 Collagen (-49%). Lastly, there was a negative correlation between patient-reported contralateral knee pain severity and patient-reported functionality and QoL. CONCLUSIONS: While 37.5% of SM with LL had KOA at the tibiofemoral joint (KL ≥ 1), 72.7% of SM had the presence of patellofemoral degeneration (OC ≥ 1). These findings demonstrate that the patellofemoral joint may be more susceptible to degeneration than the medial tibiofemoral compartment following traumatic LL.

Non-invasive Imaging of Antisense Oligonucleotides in the Brain via In Vivo Click Chemistry.

PURPOSE: The treatment of complex neurological diseases often requires the administration of large therapeutic drugs, such as antisense oligonucleotide (ASO), by lumbar puncture into the intrathecal space in order to bypass the blood-brain barrier. Despite the growing number of ASOs in clinical development, there are still uncertainties regarding their dosing, primarily around their distribution and kinetics in the brain following intrathecal injection. The challenge of taking measurements within the delicate structures of the central nervous system (CNS) necessitates the use of non-invasive nuclear imaging, such as positron emission tomography (PET). Herein, an emergent strategy known as "pretargeted imaging" is applied to image the distribution of an ASO in the brain by developing a novel PET tracer, [18F]F-537-Tz. This tracer is able to undergo an in vivo "click" reaction, covalently binding to a trans-cyclooctene conjugated ASO. PROCEDURES: A novel small molecule tracer for pretargeted PET imaging of ASOs in the CNS is developed and tested in a series of in vitro and in vivo experiments, including biodistribution in rats and non-human primates. RESULTS: In vitro data and extensive in vivo rat data demonstrated delivery of the tracer to the CNS, and its successful ligation to its ASO target in the brain. In an NHP study, the slow tracer kinetics did not allow for specific binding to be determined by PET. CONCLUSION: A CNS-penetrant radioligand for pretargeted imaging was successfully demonstrated in a proof-of-concept study in rats, laying the groundwork for further optimization.

Differential Uptake of Antisense Oligonucleotides in Mouse Hepatocytes and Macrophages Revealed by Simultaneous Two-Photon Excited Fluorescence and Coherent Raman Imaging.

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time- and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex in vitro models and liver tissues to evaluate ASO distribution and activity.

Development and validation of a nomogram incorporating gene expression profiling and clinical factors for accurate prediction of metastasis in patients with cutaneous melanoma following Mohs micrographic surgery.

BACKGROUND: There is a need to improve prognostic accuracy for patients with cutaneous melanoma. A 31-gene expression profile (31-GEP) test uses the molecular biology of primary tumors to identify individual patient metastatic risk. OBJECTIVE: Develop a nomogram incorporating 31-GEP with relevant clinical factors to improve prognostic accuracy. METHODS: In an IRB-approved study, 1124 patients from 9 Mohs micrographic surgery centers were prospectively enrolled, treated with Mohs micrographic surgery, and underwent 31-GEP testing. Data from 684 of those patients with at least 1-year follow-up or a metastatic event were included in nomogram development to predict metastatic risk. RESULTS: Logistic regression modeling of 31-GEP results and T stage provided the simplest nomogram with the lowest Bayesian information criteria score. Validation in an archival cohort (n = 901) demonstrated a significant linear correlation between observed and nomogram-predicted risk of metastasis. The resulting nomogram more accurately predicts the risk for cutaneous melanoma metastasis than T stage or 31-GEP alone. LIMITATIONS: The patient population is representative of Mohs micrographic surgery centers. Sentinel lymph node biopsy was not performed for most patients and could not be used in the nomogram. CONCLUSIONS: Integration of 31-GEP and T stage can gain clinically useful prognostic information from data obtained noninvasively.

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides.

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.

Solid-Phase Separation of Toxic Phosphorothioate Antisense Oligonucleotide-Protein Nucleolar Aggregates Is Cytoprotective.

Phosphorothioate antisense oligonucleotides (PS-ASOs) interact with proteins and can localize to or induce the formation of a variety of subcellular PS-ASO-protein or PS-ASO-ribonucleoprotein aggregates. In this study, we show that these different aggregates that form with varying compositions at various concentrations in the cytosol, nucleus, and nucleolus may undergo phase separations in cells. Some aggregates can form with both nontoxic and toxic PS-ASOs, such as PS bodies, paraspeckles, and nuclear filaments. However, toxic PS-ASOs have been shown to form unique nucleolar aggregates that result in nucleolar dysfunction and apoptosis. These include liquid-like aggregates that we labeled "cloudy nucleoli" and solid-like perinucleolar filaments. Toxic nucleolar aggregates may undergo solid-phase separation and in the solid phase, protein mobility in and out of the aggregates is limited. Other aggregates appear to undergo liquid-phase separation, including paraspeckles and perinucleolar caps, in which protein mobility is negatively correlated with the binding affinity of the proteins to PS-ASOs. However, PS bodies and nuclear filaments are solid-like aggregates. Importantly, in cells that survived treatment with toxic PS-ASOs, solid-like PS-ASO aggregates accumulated, especially Hsc70-containing nucleolus-like structures, in which modest pre-rRNA transcriptional activity was retained and appeared to mitigate the nucleolar toxicity. This is the first demonstration that exogenous drugs, PS-ASOs, can form aggregates that undergo phase separations and that solid-phase separation of toxic PS-ASO-induced nucleolar aggregates is cytoprotective.

Acute on chronic back pain as the presentation of an occult foreign body bowel perforation with resultant epidural abscess, osteomyelitis and sepsis.

A 29-year-old male paratrooper presented to multiple emergency departments (EDs) and his primary provider multiple times over sequential days. Each time, the patient received the same diagnosis of acute on chronic back pain. The patient was treated conservatively and routine MRI of the lumbar spine was performed 5 days after the last visit. It revealed a lesion occupying nearly all the visualised portion of the spinal canal. The patient was immediately called back for further imaging. The patient was then diagnosed with a foreign body perforating the rectosigmoid colon, an epidural abscess and pelvic osteomyelitis. The patient was immediately taken to the ED where he was found to be septic. The foreign body was surgically removed and determined to be a swallowed toothpick. Urgent surgical decompression of epidural space was also performed. The patient then underwent a prolonged but near complete recovery.

MXD3 antisense oligonucleotide with superparamagnetic iron oxide nanoparticles: A new targeted approach for neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. The outcomes for aggressive forms of NB remain poor. The aim of this study was to develop a new molecular-targeted therapy for NB using an antisense oligonucleotide (ASO) and superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as a delivery vehicle, targeting the transcription regulator MAX dimerization protein 3 (MXD3). We previously discovered that MXD3 was highly expressed in high-risk NB, acting as an anti-apoptotic factor; therefore, it can be a good therapeutic target. In this study, we developed two ASO-NP complexes using electrostatic conjugation to polyethylenimine-coated SPIO NPs and chemical conjugation to amphiphilic polymers on amine-functionalized SPIO NPs. Both ASO-NP complexes demonstrated MXD3 knockdown, which resulted in apoptosis in NB cells. ASO chemically-conjugated NP complexes have the potential to be used in the clinic as they showed great efficacy with minimum NP-associated cytotoxicity.

Spatially Restricting Bioorthogonal Nucleoside Biosynthesis Enables Selective Metabolic Labeling of the Mitochondrial Transcriptome.

The cellular RNA pool in animals arises from two separate genomes stored in the nucleus and multiple mitochondria. Chemical methods to track nascent RNA synthesis are unable to distinguish between these two with stringency. Herein, we report that spatially restricting bioorthogonal nucleoside biosynthesis enables, for the first time, selective metabolic labeling of the RNA transcribed in the mitochondria. We envision that this approach could open the door for heretofore-impossible analyses of mitochondrial RNA. Beyond our results revealed herein, our approach provides a roadmap for researchers to begin to design strategies to examine biomolecules within subcellular compartments.

Peptidic inhibitors of insulin-degrading enzyme with potential for dermatological applications discovered via phage display.

Insulin-degrading enzyme (IDE) is an atypical zinc-metalloendopeptidase that hydrolyzes insulin and other intermediate-sized peptide hormones, many of which are implicated in skin health and wound healing. Pharmacological inhibitors of IDE administered internally have been shown to slow the breakdown of insulin and thereby potentiate insulin action. Given the importance of insulin and other IDE substrates for a variety of dermatological processes, pharmacological inhibitors of IDE suitable for topical applications would be expected to hold significant therapeutic and cosmetic potential. Existing IDE inhibitors, however, are prohibitively expensive, difficult to synthesize and of undetermined toxicity. Here we used phage display to discover novel peptidic inhibitors of IDE, which were subsequently characterized in vitro and in cell culture assays. Among several peptide sequences tested, a cyclic dodecapeptide dubbed P12-3A was found to potently inhibit the degradation of insulin (Ki = 2.5 ± 0.31 µM) and other substrates by IDE, while also being resistant to degradation, stable in biological milieu, and highly selective for IDE. In cell culture, P12-3A was shown to potentiate several insulin-induced processes, including the transcription, translation and secretion of alpha-1 type I collagen in primary murine skin fibroblasts, and the migration of keratinocytes in a scratch wound migration assay. By virtue of its potency, stability, specificity for IDE, low cost of synthesis, and demonstrated ability to potentiate insulin-induced processes involved in wound healing and skin health, P12-3A holds significant therapeutic and cosmetic potential for topical applications.

Protected pyrimidine nucleosides for cell-specific metabolic labeling of RNA.

RNA molecules can perform a myriad of functions, from the regulation of gene expression to providing the genetic blueprint for protein synthesis. Characterizing RNA expression dynamics, in a cell-specific manner, still remains a great challenge in biology. Herein we present a new set of protected alkynyl nucleosides for cell-specific metabolic labeling of RNA. We anticipate these analogs will find wide spread utility toward the goal of understanding RNA expression in complex cellular and tissue environments, even within living animals.

Understanding Mohs Micrographic Surgery: A Review and Practical Guide for the Nondermatologist.

The incidence and diagnosis of cutaneous malignancies are steadily rising. In addition, with the aging population and increasing use of organ transplant and immunosuppressive medications, subsets of patients are now more susceptible to skin cancer. Mohs micrographic surgery (MMS) has become the standard of care for the treatment of high-risk nonmelanoma skin cancers and is increasingly used to treat melanoma. Mohs micrographic surgery has the highest cure rates, spares the maximal amount of normal tissue, and is cost-effective for the treatment of cutaneous malignancies. As in other medical fields, appropriate use criteria were developed for MMS and have become an evolving guideline for determining which patients and tumors are appropriate for referral to MMS. Patients with cutaneous malignancies often require multidisciplinary care. With the changing landscape of medicine and the rapidly increasing incidence of skin cancer, primary care providers and specialists who do not commonly manage cutaneous malignancies will need to have an understanding of MMS and its role in patient care. This review better familiarizes the medical community with the practice of MMS, its utilization and capabilities, differences from wide excision and vertical section pathology, and cost-effectiveness, and it guides practitioners in the process of appropriately evaluating and determining when patients with skin cancer might be appropriate candidates for MMS.

EC-tagging allows cell type-specific RNA analysis.

Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.

Cell-Selective Bioorthogonal Metabolic Labeling of RNA.

Stringent chemical methods to profile RNA expression within discrete cellular populations remains a key challenge in biology. To address this issue, we developed a chemical-genetic strategy for metabolic labeling of RNA. Cell-specific labeling of RNA can be profiled and imaged using bioorthogonal chemistry. We anticipate that this platform will provide the community with a much-needed chemical toolset for cell-type specific profiling of cell-specific transcriptomes derived from complex biological systems.

Metabolic Incorporation of Azide Functionality into Cellular RNA.

Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2'- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.

Radiographic Changes and Fracture in Patients Having Received Bisphosphonate Therapy for ≥ 5 Years at a Single Institution.

INTRODUCTION: Bisphosphonates have been shown to reduce the incidence of vertebral fractures, as well as intertrochanteric and femoral neck fractures; however, data also demonstrate the association of prolonged use and atypical femur fractures. We intend to report the incidence of atypical femur fractures and cortical irregularities in patients from a single institution having received prolonged bisphosphonate therapy. METHODS: A retrospective review of the pharmaceutical database was performed for all patients who were prescribed bisphosphonates at a single institution from 2002 to 2012. People taking bisphosphonates for ≥ 5 years were included. Those identified as having adequate radiographs with visualization of at least 5-cm distal to the lesser trochanter were selected. Radiographs were reviewed for evidence of atypical femur fractures or lateral cortical beaking. Medical records were retrospectively reviewed to determine any prodromal symptoms or clinical risk factors. RESULTS: A total of 7,671 patients were identified between the years of 2002 and 2012 as having been prescribed bisphosphonates at a single institution. Of these, 1,684 were using bisphosphonates for ≥ 5 years (2002-2007). 396 patients taking bisphosphonates for greater than 5 years had adequate radiographs. In total, 8 patients (2.02%) had positive findings; 7 were females. More specifically, 2 (0.51%) patients had incomplete subtrochanteric fractures while 6 (1.52%) demonstrated lateral cortical beaking. DISCUSSION/CONCLUSION: Over 2% of patients at our institution having received bisphosphonate therapy for ≥ 5 years demonstrated cortical irregularities or have already experienced an atypical femur fracture. These findings could call into question the need for radiographic screening for patients on prolonged bisphosphonate therapy.

Diffusion-weighted magnetic resonance imaging of an intramedullary epidermoid cyst with dorsal dermal sinus tract in a toddler.

We report the use of diffusion-weighted magnetic resonance imaging to diagnose and manage a rare case of a symptomatic thoracic intramedullary congenital epidermoid cyst with associated dermal sinus in a girl. Congenital intramedullary epidermoid cysts with associated dermal sinuses are very rare occurrences and seldom present symptomatically in very young children. We present a case of a 32-month old with a draining dimpled skin lesion. Magnetic resonance images demonstrated an intramedullary epidermoid with a dorsal dermal sinus tract opening to the skin surface which was confirmed surgically. The patient was treated with debulking to prevent recurrent infection and progression of neurological symptoms. This case demonstrates the use of diffuse-weighted MRI to assist in the diagnosis and surgical management of an atypical presentation of a rare developmental abnormality, which is not well documented in the pediatric radiological literature. Failure to diagnosis may have significant neurological permanent debilitating consequences.

An atypical symptomatic discoid lateral meniscus in a toddler.

Meniscal tears and meniscal instability are rare complications of a discoid lateral meniscus in a very young child. We report a case of a 32-month-old male who presented with a limp and limited extension of his right knee. Magnetic resonance and sonographic images demonstrated a discoid meniscus with a posterior horn tear and unstable anterior horn, confirmed by arthroscopy. The patient was treated with saucerization of the lateral discoid meniscus, debridement of the posterior horn tear, and repair of the unstable anterior horn. This case shows a rare complication of variant anatomy that is not well documented in the pediatric radiologic literature, but nonetheless should be considered in the pediatric population.