Digital wearable insole-based identification of knee arthropathies and gait signatures using machine learning.

Gait is impaired in musculoskeletal conditions, such as knee arthropathy. Gait analysis is used in clinical practice to inform diagnosis and monitor disease progression or intervention response. However, clinical gait analysis relies on subjective visual observation of walking as objective gait analysis has not been possible within clinical settings due to the expensive equipment, large-scale facilities, and highly trained staff required. Relatively low-cost wearable digital insoles may offer a solution to these challenges. In this work, we demonstrate how a digital insole measuring osteoarthritis-specific gait signatures yields similar results to the clinical gait-lab standard. To achieve this, we constructed a machine learning model, trained on force plate data collected in participants with knee arthropathy and controls. This model was highly predictive of force plate data from a validation set (area under the receiver operating characteristics curve [auROC] = 0.86; area under the precision-recall curve [auPR] = 0.90) and of a separate, independent digital insole dataset containing control and knee osteoarthritis subjects (auROC = 0.83; auPR = 0.86). After showing that digital insole-derived gait characteristics are comparable to traditional gait measurements, we next showed that a single stride of raw sensor time-series data could be accurately assigned to each subject, highlighting that individuals using digital insoles can be identified by their gait characteristics. This work provides a framework for a promising alternative to traditional clinical gait analysis methods, adds to the growing body of knowledge regarding wearable technology analytical pipelines, and supports clinical development of at-home gait assessments, with the potential to improve the ease, frequency, and depth of patient monitoring.

The way we walk ­ our 'gait' ­ is a key indicator of health. Gait irregularities like limping, shuffling or a slow pace can be signs of muscle or joint problems. Assessing a patient's gait is therefore an important element in diagnosing these conditions, and in evaluating whether treatments are working. Gait is often assessed via a simple visual inspection, with patients being asked to walk back and forth in a doctor's office. While quick and easy, this approach is highly subjective and therefore imprecise. 'Objective gait analysis' is a more accurate alternative, but it relies on tests being conducted in specialised laboratories with large-scale, expensive equipment operated by highly trained staff. Unfortunately, this means that gait laboratories are not accessible for everyday clinical use. In response, Wipperman et al. aimed to develop a low-cost alternative to the complex equipment used in gait laboratories. To do this, they harnessed wearable sensor technologies ­ devices that can directly measure physiological data while embedded in clothing or attached to the user. Wearable sensors have the advantage of being cheap, easy to use, and able to provide clinically useful information without specially trained staff. Wipperman et al. analysed data from classic gait laboratory devices, as well as 'digital insoles' equipped with sensors that captured foot movements and pressure as participants walked. The analysis first 'trained' on data from gait laboratories (called force plates) and then applied the method to gait measurements obtained from digital insoles worn by either healthy participants or patients with knee problems. Analysis of the pressure data from the insoles confirmed that they could accurately predict which measurements were from healthy individuals, and which were from patients. The gait characteristics detected by the insoles were also comparable to lab-based measurements ­ in other words, the insoles provided similar type and quality of data as a gait laboratory. Further analysis revealed that information from just a single step could reveal additional information about the subject's walking. These results support the use of wearable devices as a simple and relatively inexpensive way to measure gait in everyday clinical practice, without the need for specialised laboratories and visits to the doctor's office. Although the digital insoles will require further analytical and clinical study before they can be widely used, Wipperman et al. hope they will eventually make monitoring muscle and joint conditions easier and more affordable.

A Multiplex Assay to Simultaneously Monitor Apoptosis and Necrosis Using the Cellaca® PLX Image Cytometer.

Apoptosis is the programmed cell death pathway that is critical for maintaining homeostasis, in which cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare different cancer therapeutics (i.e., small molecules, antibody drugs, cell therapies) that can initiate the process of apoptosis, enabling the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V, Caspase-3, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA). First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, where apoptosis and necrosis were assessed at 0, 1, 1.5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using image cytometry. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early- to late-stage apoptosis, and ultimately necrosis. A clear trend was observed analyzing apoptotic and necrotic populations during the first 1.5 h, showing differences of up to ~15% in single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis between apoptotic populations only, Annexin V+ only populations were higher than Caspase-3+ only populations by up to ~20% between 0 and 1.5 h. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image cytometric detection method may provide an effective and efficient tool for rapid and reliable simultaneous detection of early- late-stage apoptosis, and necrosis. Therefore, allowing researchers to better characterize and screen potential cancer therapeutic drug candidates for their treatment efficacy in a higher throughput manner.

Development of a high-throughput image cytometric screening method as a research tool for immunophenotypic characterization of patient samples from clinical studies.

Immunophenotyping has been the primary assay for characterization of immune cells from patients undergoing therapeutic treatments in clinical research, which is critical for understanding disease progression and treatment efficacy. Currently, flow cytometry has been the dominant methodology for characterizing surface marker expression for immunological research. Flow cytometry has been proven to be an effective and efficient method for immunophenotyping, however, it requires highly trained users and a large time commitment. Recently, a novel image cytometry system (Cellaca® PLX Image Cytometer, Revvity Health Sciences, Inc., Lawrence, MA) has been developed as a complementary method to flow cytometry for performing rapid and high-throughput immunophenotyping. In this work, we demonstrated an image cytometric screening method to characterize immune cell populations, streamlining the analysis of routine surface marker panels. The T cell, B cell, NK cell, and monocyte populations of 46 primary PBMC samples from subjects enrolled in autoimmune and oncological disease study cohorts were analyzed with two optimized immunophenotyping staining kits: Panel 1 (CD3, CD56, CD14) and Panel 2 (CD3, CD56, CD19). We validated the proposed image cytometry method by comparing the Cellaca® PLX and the AuroraTM flow cytometer (Cytek Biosciences, Fremont, CA). The image cytometry system was employed to generate bright field and fluorescent images, as well as scatter plots for multiple patient PBMC samples. In addition, the image cytometry method can directly determine cell concentrations for downstream assays. The results demonstrated comparable CD3, CD14, CD19, and CD56 cell populations from the primary PBMC samples, which showed an average of 5% differences between flow and image cytometry. The proposed image cytometry method provides a novel research tool to potentially streamline immunophenotyping workflow for characterizing patient samples in clinical studies.

Exogenous polyunsaturated fatty acids (PUFAs) influence permeability, antimicrobial peptide resistance, biofilm formation and membrane phospholipid structure in an A-layer and non-A-layer strain of Aeromonas salmonicida.

Aeromonas salmonicida is a Gram-negative bacterium that can infect a wide host range of fish populations, including salmonids and non-salmonids as well as freshwater and marine life. Some strains of A. salmonicida cause the disease furunculosis, which can cause lethargy, intestinal inflammation, ulcers, haemorrhaging and death. The infection is spread through fish-to-fish contact, and the presence of infection can have devastating effects on cultivated fish populations. The purpose of this study was to explore the ability of non-A-layer and A-layer A. salmonicida strains to incorporate polyunsaturated fatty acids (PUFAs) into their lipid profile and test the phenotypic effects thereof. Lipids were extracted from PUFA-exposed cultures and analysed for lipid modification by thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry, showing A. salmonicida, regardless of A-layer, capable of incorporating all seven of the PUFAs studied. Phenotypic effects were determined through the use of assays that tested for biofilm formation, membrane permeability and cyclic peptide susceptibility. Temperature-dependent effects on biofilm formation were observed, and PUFA exposure showed significant (p < .001) increases in membrane permeability as tested by the uptake of the hydrophobic compounds crystal violet and ethidium bromide. Additionally, some PUFAs elicited modest protection and vulnerability against the membrane-targeting cyclic peptides polymyxin B (PMB) and colistin. The diverse, strain-specific responses to exogenous PUFAs may allude to evolved adaptive strategies that enhance survival, persistence and virulence of non-pathogenic and pathogenic members of bacteria that oscillate between environmental and fish host niches.

Collagen fibrils from both positional and energy-storing tendons exhibit increased amounts of denatured collagen when stretched beyond the yield point.

Collagen molecules are the base structural unit of tendons, which become denatured during mechanical overload. We recently demonstrated that during tendon stretch, collagen denaturation occurs at the yield point of the stress-strain curve in both positional and energy-storing tendons. We were interested in investigating how this load is transferred throughout the collagen hierarchy, and sought to determine the onset of collagen denaturation when collagen fibrils are stretched. Fibrils are one level above the collagen molecule in the collagen hierarchy, allowing more direct probing of the effect of strain on collagen molecules. We isolated collagen fibrils from both positional and energy-storing tendon types and stretched them using a microelectromechanical system device to various levels of strain. We stained the fibrils with fluorescently labeled collagen hybridizing peptides that specifically bind to denatured collagen, and examined whether samples stretched beyond the yield point of the stress-strain curve exhibited increased amounts of denatured collagen. We found that collagen denaturation in collagen fibrils from both tendon types occurs at the yield point. Greater amounts of denatured collagen were found in post-yield positional fibrils than in energy-storing fibrils. This is despite a greater yield strain and yield stress in fibrils from energy-storing tendons compared to positional tendons. Interestingly, the peak modulus of collagen fibrils from both tendon types was the same. These results are likely explained by the greater crosslink density found in energy-storing tendons compared to positional tendons. The insights gained from this study could help management of tendon and other musculoskeletal injuries by targeting collagen molecular damage at the fibril level. STATEMENT OF SIGNIFICANCE: When tendons are stretched or torn, this can lead to collagen denaturation (damage). Depending on their biomechanical function, tendons are considered positional or energy-storing with different crosslink profiles. By stretching collagen fibrils instead of fascicles from both tendon types, we can more directly examine the effect of tensile stretch on the collagen molecule in tendons. We found that regardless of tendon type, collagen denaturation in fibrils occurs when they are stretched beyond the yield point of the stress-strain curve. This provides insight into how load affects different tendon sub-structures during tendon injuries and failure, which will help clinicians and researchers understand mechanisms of injuries and potentially target collagen molecular damage as a treatment strategy, leading to improved clinical outcomes following injury.

Collagen Molecular Damage is a Hallmark of Early Atherosclerosis Development.

Remodeling of extracellular matrix proteins underlies the development of cardiovascular disease. Herein, we utilized a novel molecular probe, collagen hybridizing peptide (CHP), to target collagen molecular damage during atherogenesis. The thoracic aorta was dissected from ApoE-/- mice that had been on a high-fat diet for 0-18 weeks. Using an optimized protocol, tissues were stained with Cy3-CHP and digested to quantify CHP with a microplate assay. Results demonstrated collagen molecular damage, inferred from Cy3-CHP fluorescence, was a function of location and time on the high-fat diet. Tissue from the aortic arch showed a significant increase in collagen molecular damage after 18 weeks, while no change was observed in tissue from the descending aorta. No spatial differences in fluorescence were observed between the superior and inferior arch tissue. Our results provide insight into the early changes in collagen during atherogenesis and present a new opportunity in the subclinical diagnosis of atherosclerosis.

Matrix anisotropy promotes angiogenesis in a density-dependent manner.

Angiogenesis is necessary for wound healing, tumorigenesis, implant inosculation, and homeostasis. In each situation, matrix structure and mechanics play a role in determining whether new vasculatures can establish transport to new or hypoxic tissues. Neovessel growth and directional guidance are sensitive to three-dimensional (3-D) matrix anisotropy and density, although the individual and integrated roles of these matrix features have not been fully recapitulated in vitro. We developed a tension-based method to align 3-D collagen constructs seeded with microvessel fragments in matrices of three levels of collagen fibril anisotropy and two levels of collagen density. The extent and direction of neovessel growth from the parent microvessel fragments increased with matrix anisotropy and decreased with density. The proangiogenic effects of anisotropy were attenuated at higher matrix densities. We also examined the impact of matrix anisotropy in an experimental model of neovessel invasion across a tissue interface. Matrix density was found to dictate the success of interface crossing, whereas interface curvature and fibril alignment were found to control directional guidance. Our findings indicate that complex configurations of matrix density and alignment can facilitate or complicate the establishment or maintenance of vascular networks in pathological and homeostatic angiogenesis. Furthermore, we extend preexisting methods for tuning collagen anisotropy in thick constructs. This approach addresses gaps in tissue engineering and cell culture by supporting the inclusion of large multicellular structures in prealigned constructs.NEW & NOTEWORTHY Matrix anisotropy and density have a considerable effect on angiogenic vessel growth and directional guidance. However, the current literature relies on 2-D and simplified models of angiogenesis (e.g., tubulogenesis and vasculogenesis). We present a method to align 3-D collagen scaffolds embedded with microvessel fragments to different levels of anisotropy. Neovessel growth increases with anisotropy and decreases with density, which may guide angiogenic neovessels across tissue interfaces such as during implant inosculation and tumorigenesis.

Glutaminase 2 knockdown reduces hyperammonemia and associated lethality of urea cycle disorder mouse model.

Amino acids, the building blocks of proteins in the cells and tissues, are of fundamental importance for cell survival, maintenance, and proliferation. The liver plays a critical role in amino acid metabolism and detoxication of byproducts such as ammonia. Urea cycle disorders with hyperammonemia remain difficult to treat and eventually necessitate liver transplantation. In this study, ornithine transcarbamylase deficient (Otcspf-ash ) mouse model was used to test whether knockdown of a key glutamine metabolism enzyme glutaminase 2 (GLS2, gene name: Gls2) or glutamate dehydrogenase 1 (GLUD1, gene name: Glud1) could rescue the hyperammonemia and associated lethality induced by a high protein diet. We found that reduced hepatic expression of Gls2 but not Glud1 by AAV8-mediated delivery of a short hairpin RNA in Otcspf-ash mice diminished hyperammonemia and reduced lethality. Knockdown of Gls2 but not Glud1 in Otcspf-ash mice exhibited reduced body weight loss and increased plasma glutamine concentration. These data suggest that Gls2 hepatic knockdown could potentially help alleviate risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle.

Tendons exhibit greater resistance to tissue and molecular-level damage with increasing strain rate during cyclic fatigue.

Musculoskeletal soft connective tissues are commonly injured due to repetitive use, but the evolution of mechanical damage to the tissue structure during repeated loading is poorly understood. We investigated the strain-rate dependence of mechanical denaturation of collagen as a form of structural microdamage accumulation during creep fatigue loading of rat tail tendon fascicles. We cycled tendons at three strain rates to the same maximum stress relative to their rate-dependent tensile strength. Collagen denaturation at distinct points during the fatigue process was measured by fluorescence quantification of collagen hybridizing peptide binding. The amount of collagen denaturation was significantly correlated with fascicle creep strain, independent of the cyclic strain rate, supporting our hypothesis that tissue level creep is caused by collagen triple-helix unfolding. Samples that were loaded faster experienced more creep strain and denaturation as a function of the number of loading cycles relative to failure. Although this increased damage capacity at faster rates may serve as a protective measure during high-rate loading events, it may also predispose these tissues to subsequent injury and indicate a mechanism of overuse injury development. These results build on evidence that molecular-level collagen denaturation is the fundamental mechanism of structural damage to tendons during tensile loading. STATEMENT OF SIGNIFICANCE: This study is the first to investigate the accumulation of denatured collagen in tendons throughout fatigue loading when the maximum stress is scaled with the applied strain rate. The amount of denatured collagen was correlated with creep strain, independent of strain rate, but samples that were cycled faster withstood greater amounts of denaturation before failure. Differential accumulation of collagen damage between fast and slow repetitive loading has relevance toward understanding the prevalence of overuse musculoskeletal injuries following sudden changes in activity level. Since collagen is a ubiquitous biological structural component, the basic patterns and mechanisms of loading-induced collagen damage in connective tissues are relevant for understanding injury and disease in other tissues, including those from the cardiovascular and pulmonary systems.

Collagen denaturation is initiated upon tissue yield in both positional and energy-storing tendons.

Tendons are collagenous soft tissues that transmit loads between muscles and bones. Depending on their anatomical function, tendons are classified as positional or energy-storing with differing biomechanical and biochemical properties. We recently demonstrated that during monotonic stretch of positional tendons, permanent denatured collagen begins accumulating upon departing the linear region of the stress-strain curve. However, it is unknown if this observation is true during mechanical overload of other types of tendons. Therefore, the purpose of this study was to investigate the onset of collagen denaturation relative to applied strain, and whether it differs between the two tendon types. Rat tail tendon (RTT) fascicles and rat flexor digitorum longus (FDL) tendons represented positional and energy-storing tendons, respectively. The samples were stretched to incremental levels of strain, then stained with fluorescently labeled collagen hybridizing peptides (CHPs); the CHP fluorescence was measured to quantify denatured collagen. Denatured collagen in both positional and energy-storing tendons began to increase at the yield strain, upon leaving the linear region of the stress-strain curve as the sample started to permanently deform. Despite significant differences between the two tendon types, it appears that collagen denaturation is initiated at tissue yield during monotonic stretch, and the fundamental mechanism of failure is the same for the two types of tendons. At tissue failure, positional tendons had double the percentage of denatured collagen compared to energy-storing tendons, with no difference between 0% control groups. These results help to elucidate the etiology of subfailure injury and rupture in functionally distinct tendons.

Accumulation of collagen molecular unfolding is the mechanism of cyclic fatigue damage and failure in collagenous tissues.

Overuse injuries to dense collagenous tissues are common, but their etiology is poorly understood. The predominant hypothesis that micro-damage accumulation exceeds the rate of biological repair is missing a mechanistic explanation. Here, we used collagen hybridizing peptides to measure collagen molecular damage during tendon cyclic fatigue loading and computational simulations to identify potential explanations for our findings. Our results revealed that triple-helical collagen denaturation accumulates with increasing cycles of fatigue loading, and damage is correlated with creep strain independent of the cyclic strain rate. Finite-element simulations demonstrated that biphasic fluid flow is a possible fascicle-level mechanism to explain the rate dependence of the number of cycles and time to failure. Molecular dynamics simulations demonstrated that triple-helical unfolding is rate dependent, revealing rate-dependent mechanisms at multiple length scales in the tissue. The accumulation of collagen molecular denaturation during cyclic loading provides a long-sought "micro-damage" mechanism for the development of overuse injuries.

Targeting cell membrane HDM2: A novel therapeutic approach for acute myeloid leukemia.

The E3 ligase human double minute 2 (HDM2) regulates the activity of the tumor suppressor protein p53. A p53-independent HDM2 expression has been reported on the membrane of cancer cells but not on that of normal cells. Herein, we first showed that membrane HDM2 (mHDM2) is exclusively expressed on human and mouse AML blasts, including leukemia stem cell (LSC)-enriched subpopulations, but not on normal hematopoietic stem cells (HSCs). Higher mHDM2 levels in AML blasts were associated with leukemia-initiating capacity, quiescence, and chemoresistance. We also showed that a synthetic peptide PNC-27 binds to mHDM2 and enhances the interaction of mHDM2 and E-cadherin on the cell membrane; in turn, E-cadherin ubiquitination and degradation lead to membrane damage and cell death of AML blasts by necrobiosis. PNC-27 treatment in vivo resulted in a significant killing of both AML "bulk" blasts and LSCs, as demonstrated respectively in primary and secondary transplant experiments, using both human and murine AML models. Notably, PNC-27 spares normal HSC activity, as demonstrated in primary and secondary BM transplant experiments of wild-type mice. We concluded that mHDM2 represents a novel and unique therapeutic target, and targeting mHDM2 using PNC-27 selectively kills AML cells, including LSCs, with minimal off-target hematopoietic toxicity.

Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor.

Here we describe TRACE (T7 polymerase-driven continuous editing), a method that enables continuous, targeted mutagenesis in human cells using a cytidine deaminase fused to T7 RNA polymerase. TRACE induces high rates of mutagenesis over multiple cell generations in genes under the control of a T7 promoter integrated in the genome. We used TRACE in a MEK1 inhibitor-resistance screen, and identified functionally correlated mutations.

Microplate assay for denatured collagen using collagen hybridizing peptides.

The purpose of this study was to develop a microplate assay for quantifying denatured collagen by measuring the fluorescence of carboxyfluorescein bound collagen hybridizing peptides (F-CHP). We have shown that F-CHP binds selectively with denatured collagen, and that mechanical overload of tendon fascicles causes collagen denaturation. Proteinase K was used to homogenize tissue samples after F-CHP staining, allowing fluorescence measurement using a microplate reader. We compared our new assay to our previous image analysis method and the trypsin-hydroxyproline assay, which is the only other available method to directly quantify denatured collagen. Relative quantification of denatured collagen was performed in rat tail tendon fascicles subjected to incremental tensile overload, and normal and ostoeoarthritic guinea pig cartilage. In addition, the absolute amount of denatured collagen was determined in rat tail tendon by correlating F-CHP fluorescence with percent denatured collagen as determined by the trypsin-hydroxyproline assay. Rat tail tendon fascicles stretched to low strains (<7.5%) exhibited minimal denatured collagen, but values rapidly increased at medium strains (7.5-10.5%) and plateaued at high strains (≥12%). Osteoarthritic cartilage had higher F-CHP fluorescence than healthy cartilage. Both of these outcomes are consistent with previous studies. With the calibration curve, the microplate assay was able to absolutely quantify denatured collagen in mechanically damaged rat tail tendon fascicles as reliably as the trypsin-hydroxyproline assay. Further, we achieved these results more efficiently than current methods in a rapid, high-throughput manner, with multiple types of collagenous tissue while maintaining accuracy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:431-438, 2019.

Adeno-associated virus gene delivery of broadly neutralizing antibodies as prevention and therapy against HIV-1.

Vectored gene delivery of HIV-1 broadly neutralizing antibodies (bNAbs) using recombinant adeno-associated virus (rAAV) is a promising alternative to conventional vaccines for preventing new HIV-1 infections and for therapeutically suppressing established HIV-1 infections. Passive infusion of single bNAbs has already shown promise in initial clinical trials to temporarily decrease HIV-1 load in viremic patients, and to delay viral rebound from latent reservoirs in suppressed patients during analytical treatment interruptions of antiretroviral therapy. Long-term, continuous, systemic expression of such bNAbs could be achieved with a single injection of rAAV encoding antibody genes into muscle tissue, which would bypass the challenges of eliciting such bNAbs through traditional vaccination in naïve patients, and of life-long repeated passive transfers of such biologics for therapy. rAAV delivery of single bNAbs has already demonstrated protection from repeated HIV-1 vaginal challenge in humanized mouse models, and phase I clinical trials of this approach are underway. Selection of which individual, or combination of, bNAbs to deliver to counter pre-existing resistance and the rise of escape mutations in the virus remains a challenge, and such choices may differ depending on use of this technology for prevention versus therapy.

SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function.

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs.

Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia.

Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR-ABL, which led to inhibition of the RAN-exportin-5-RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.

Impact and Cost-effectiveness of Selective Human Papillomavirus Vaccination of Men Who Have Sex With Men.

Background: Men who have sex with men (MSM) have a high lifetime risk of anogenital warts and cancers related to infection with human papillomavirus (HPV). They also benefit less from herd protection than heterosexual males in settings with female-only HPV vaccination. Methods: We evaluated the potential health impact and cost-effectiveness of offering vaccination to MSM who visit genitourinary medicine (GUM) clinics. We used a mathematical model of HPV 6/11/16/18 sexual transmission within an MSM population in England, parameterized with sexual behaviour, GUM attendance, HPV prevalence, HIV prevalence, warts, and cancer incidence data. Interventions considered were offering HPV vaccination to either HIV-positive MSM or MSM regardless of HIV status, for age bands 16-25, 16-30, 16-35, and 16-40 years. Results: Substantial declines in anogenital warts and male HPV-related cancer incidence are projected to occur following an offer of vaccination to MSM. MSM not attending GUM clinics will partially benefit from herd protection. Offering vaccination to HIV-positive MSM up to age 40 is likely to be cost-effective if vaccine procurement and administration costs are below £96.50 a dose. At £48 a dose, offering vaccination to all MSM up to age 40 is likely to be cost-effective. Conclusions: Quadrivalent HPV vaccination of MSM via GUM clinics is likely to be an effective and cost-effective way of reducing the burden of HPV-related disease in MSM.

Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor-treated CML stem cells.

Treatment of chronic myelogenous leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKI) fails to eliminate leukemia stem cells (LSC). Patients remain at risk for relapse, and additional approaches to deplete CML LSC are needed to enhance the possibility of discontinuing TKI treatment. We have previously reported that expression of the pivotal proinflammatory cytokine interleukin-1 (IL-1) is increased in CML bone marrow. We show here that CML LSC demonstrated increased expression of the IL-1 receptors, IL-1 receptor accessory protein and IL-1 receptor type 1 (IL-1R1), and enhanced sensitivity to IL-1-induced NF-κB signaling compared with normal stem cells. Treatment with recombinant IL-1 receptor antagonist (IL-1RA) inhibited IL-1 signaling in CML LSC and inhibited growth of CML LSC. Importantly, the combination of IL-1RA with TKI resulted in significantly greater inhibition of CML LSC compared with TKI alone. Our studies also suggest that IL-1 signaling contributes to overexpression of inflammatory mediators in CML LSC, suggesting that blocking IL-1 signaling could modulate the inflammatory milieu. We conclude that IL-1 signaling contributes to maintenance of CML LSC following TKI treatment and that IL-1 blockade with IL-1RA enhances elimination of TKI-treated CML LSC. These results provide a strong rationale for further exploration of anti-IL-1 strategies to enhance LSC elimination in CML.

Comparison of treatment for congenital nasolacrimal duct obstruction: a systematic review and meta-analysis.

OBJECTIVE: To conduct a systematic review and meta-analysis of randomized controlled trials comparing the success and complication rates among various congenital nasolacrimal duct obstruction (CNLDO) procedures, intervention times, and tubes types. DESIGN: Systematic review with quantitative meta-analysis. METHODS: Studies were identified by searching the PubMed, EMBASE, SCOPUS, and Cochrane databases. The comparisons between categorical variables were analyzed using the χ(2) test, and the dichotomous outcomes were reported as risk ratios. The precision of the effect size was based on the 95% confidence interval. RESULTS: Seven studies published between 2007 and 2013 were included. Immediate versus observation/deferred probing had similar rates of success (82.7% vs 81.8%). Balloon dacryocystoplasty and silicone intubation had similar rates of success (79.8% vs 77.8%). Monocanalicular and bicanalicular intubation had similar rates of success (88.3% vs 88.0%). The dislocation rates for monocanalicular versus bicanalicular intubation were 8.5% and 9.8%, respectively. CONCLUSIONS: Immediate and deferred probing do not differ in their success rates. No difference in success rates was observed between balloon dilation and intubation. Monocanalicular and bicanalicular intubation were similar in their success and dislocation rates. Therefore, the preference of surgeons on the treatment of CNLDO should be discussed with parents to ensure the best possible outcome.