A Workflow for Meaningful Interpretation of Classification Results from Handheld Ambient Mass Spectrometry Analysis Probes.

While untargeted analysis of biological tissues with ambient mass spectrometry analysis probes has been widely reported in the literature, there are currently no guidelines to standardize the workflows for the experimental design, creation, and validation of molecular models that are utilized in these methods to perform class predictions. By drawing parallels with hurdles that are faced in the field of food fraud detection with untargeted mass spectrometry, we provide a stepwise workflow for the creation, refinement, evaluation, and assessment of the robustness of molecular models, aimed at meaningful interpretation of mass spectrometry-based tissue classification results. We propose strategies to obtain a sufficient number of samples for the creation of molecular models and discuss the potential overfitting of data, emphasizing both the need for model validation using an independent cohort of test samples, as well as the use of a fully characterized feature-based approach that verifies the biological relevance of the features that are used to avoid false discoveries. We additionally highlight the need to treat molecular models as "dynamic" and "living" entities and to further refine them as new knowledge concerning disease pathways and classifier feature noise becomes apparent in large(r) population studies. Where appropriate, we have provided a discussion of the challenges that we faced in our development of a 10 s cancer classification method using picosecond infrared laser mass spectrometry (PIRL-MS) to facilitate clinical decision-making at the bedside.

Metabolic Lipids in Melanoma Enable Rapid Determination of Actionable BRAF-V600E Mutation with Picosecond Infrared Laser Mass Spectrometry in 10 s.

Rapid molecular profiling of biological tissues with picosecond infrared laser mass spectrometry (PIRL-MS) has enabled the detection of clinically important histologic types and molecular subtypes of human cancers in as little as 10 s of data collection and analysis time. Utilizing an engineered cell line model of actionable BRAF-V600E mutation, we observed statistically significant differences in 10 s PIRL-MS molecular profiles between BRAF-V600E and BRAF-wt cells. Multivariate statistical analyses revealed a list of mass-to-charge (m/z) values most significantly responsible for the identification of BRAF-V600E mutation status in this engineered cell line that provided a highly controlled testbed for this observation. These metabolites predicted BRAF-V600E expression in human melanoma cell lines with greater than 98% accuracy. Through chromatography and tandem mass spectrometry analysis of cell line extracts, a 30-member "metabolite array" was characterized for determination of BRAF-V600E expression levels in subcutaneous melanoma xenografts with an average sensitivity and specificity of 95.6% with 10 s PIRL-MS analysis. This proof-of-principle work warrants a future large-scale study to identify a metabolite array for 10 s determination of actionable BRAF-V600E mutation in human tissue to guide patient care.

Picosecond Infrared Laser Mass Spectrometry Identifies a Metabolite Array for 10 s Diagnosis of Select Skin Cancer Types: A Proof-of-Concept Feasibility Study.

Currently, a large number of skin biopsies are taken for each true skin cancer case detected, creating a need for a rapid, high sensitivity, and specificity skin cancer detection tool to reduce the number of unnecessary biopsies taken from benign tissue. Picosecond infrared laser mass spectrometry (PIRL-MS) using a hand-held sampling probe is reported to detect and classify melanoma, squamous cell carcinoma, and normal skin with average sensitivity and specificity values of 86-95% and 91-98%, respectively (at a 95% confidence level) solely requiring 10 s or less of total data collection and analysis time. Classifications are not adversely affected by specimen's quantity of melanin pigments and are mediated by a number of metabolic lipids, further identified herein as potential biomarkers for skin cancer-type differentiation, 19 of which were sufficient here (as a fully characterized metabolite array) to provide high specificity and sensitivity classification of skin cancer types. In situ detection was demonstrated in an intradermal melanoma mouse model wherein in vivo sampling did not cause significant discomfort. PIRL-MS sampling is further shown to be compatible with downstream gross histopathologic evaluations despite loss of tissue from the immediate laser sampling site(s) and can be configured using selective laser pulses to avoid thermal damage to normal skin. Therefore, PIRL-MS may be employed as a decision-support tool to reduce both the subjectivity of clinical diagnosis and the number of unnecessary biopsies currently required for skin cancer screening.

Lipid Profiling in Cancer Diagnosis with Hand-Held Ambient Mass Spectrometry Probes: Addressing the Late-Stage Performance Concerns.

Untargeted lipid fingerprinting with hand-held ambient mass spectrometry (MS) probes without chromatographic separation has shown promise in the rapid characterization of cancers. As human cancers present significant molecular heterogeneities, careful molecular modeling and data validation strategies are required to minimize late-stage performance variations of these models across a large population. This review utilizes parallels from the pitfalls of conventional protein biomarkers in reaching bedside utility and provides recommendations for robust modeling as well as validation strategies that could enable the next logical steps in large scale assessment of the utility of ambient MS profiling for cancer diagnosis. Six recommendations are provided that range from careful initial determination of clinical added value to moving beyond just statistical associations to validate lipid involvements in disease processes mechanistically. Further guidelines for careful selection of suitable samples to capture expected and unexpected intragroup variance are provided and discussed in the context of demographic heterogeneities in the lipidome, further influenced by lifestyle factors, diet, and potential intersect with cancer lipid pathways probed in ambient mass spectrometry profiling studies.

Breaking Through the Barrier: Regulatory Considerations Relevant to Ambient Mass Spectrometry at the Bedside.

Rapid characterization of tissue disorder using ambient mass spectrometry (MS) techniques, requiring little to no preanalytical preparations of sampled tissues, has been shown using a variety of ion sources and with many disease classes. A brief overview of ambient MS in clinical applications, the state of the art in regulatory affairs, and recommendations to facilitate adoption for use at the bedside are presented. Unique challenges in the validation of untargeted MS methods and additional safety and compliance requirements for deployment within a clinical setting are further discussed. Development of a harmonized validation strategy for ambient MS methods is emphasized.

Mass Spectrometry Imaging Reveals a Gradient of Cancer-like Metabolic States in the Vicinity of Cancer Not Seen in Morphometric Margins from Microscopy.

Spatially resolved ambient mass spectrometry imaging methods have gained popularity to characterize cancer sites and their borders using molecular changes in the lipidome. This utility, however, is predicated on metabolic homogeneity at the border, which would create a sharp molecular transition at the morphometric borders. We subjected murine models of human medulloblastoma brain cancer to mass spectrometry imaging, a technique that provides a direct readout of tissue molecular content in a spatially resolved manner. We discovered a distance-dependent gradient of cancer-like lipid molecule profiles in the brain tissue within 1.2 mm of the cancer border, suggesting that a cancer-like state progresses beyond the histologic border, into the healthy tissue. The results were further corroborated using orthogonal liquid chromatography and mass spectrometry (LC-MS) analysis of selected tissue regions subjected to laser capture microdissection. LC-MS/MS analysis for robust identification of the affected molecules implied changes in a number of different lipid classes, some of which are metabolized from the essential docosahexaenoic fatty acid (DHA) present in the interstitial fluid. Metabolic molecular borders are thus not as sharp as morphometric borders, and mass spectrometry imaging can reveal molecular nuances not observed with microscopy. Caution must be exercised in interpreting multimodal imaging results stipulated on a coincidental relationship between metabolic and morphometric borders of cancer, at least within animal models used in preclinical research.

Chronic caloric restriction maintains a youthful phosphoproteome in aged skeletal muscle.

Caloric restriction (CR) can prolong aged skeletal muscle function, yet the molecular mechanisms are not completely understood. We performed phosphoproteomic analysis on muscle from young and old mice fed an ad libitum diet, and old mice fed a CR diet. CR promoted a youthful phosphoproteomic signature, suppressing several known "pro-aging" pathways including Protein kinase A (PKA). This study validates global signaling changes in skeletal muscle during CR.

Exercise protects against cardiac and skeletal muscle dysfunction in a mouse model of inflammatory arthritis.

Rheumatoid arthritis (RA) is a systemic inflammatory arthritis impacting primarily joints and cardiac and skeletal muscle. RA's distinct impact on cardiac and skeletal muscle tissue is suggested by studies showing that new RA pharmacologic agents strongly improve joint inflammation, but have little impact on RA-associated mortality, cardiovascular disease, and sarcopenia. Thus, the objective is to understand the distinct effects of RA on cardiac and skeletal muscle, and to therapeutically target these tissues through endurance-based exercise as a way to improve RA mortality and morbidity. We utilize the well-characterized RA mouse model, the K/BxN mouse, to investigate cardiac and skeletal muscle pathologies, including the use of wheel-running exercise to mitigate these pathologies. Strikingly, we found that K/BxN mice, like patients with RA, also exhibit both cardiac and skeletal muscle myopathies that were correlated with circulating IL-6 levels. Three months of wheel-running exercise significantly improved K/BxN joint swelling and reduced systemic IL-6 concentrations. Importantly, there were morphological, gene expression, and functional improvements in both the skeletal muscle and cardiac myopathies with exercise. The K/BxN mouse model of RA recapitulated important RA clinical comorbidities, including altered joint, cardiac and skeletal muscle function. These morphological, molecular, and functional alterations were mitigated with regular exercise, thus suggesting exercise as a potential therapeutic intervention to lessen disease activity in the joint and the peripheral tissues, including the heart and skeletal muscle.NEW & NOTEWORTHY RA, even when controlled, is associated with skeletal muscle weakness and greater risk of cardiovascular disease (CVD). Using exercise as a therapeutic against, the progression of RA is often avoided due to fear of worsening RA pathology. We introduce the K/BxN mouse as an RA model to study both myocardial and skeletal muscle dysfunction. We show that endurance exercise can improve joint, cardiac, and skeletal muscle function in K/BxN mice, suggesting exercise may be beneficial for patients with RA.

Potential impact of tissue molecular heterogeneity on ambient mass spectrometry profiles: a note of caution in choosing the right disease model.

This review provides a summary of known molecular alterations in commonly used cancer models and strives to stipulate how they may affect ambient mass spectrometry profiles. Immortalized cell lines are known to accumulate mutations, and xenografts derived from cell lines are known to contain tumour microenvironment elements from the host animal. While the use of human specimens for mass spectrometry profiling studies is highly encouraged, patient-derived xenografts with low passage numbers could provide an alternative means of amplifying material for ambient MS research when needed. Similarly, genetic preservation of patient tissue seen in some organoid models, further verified by qualitative proteomic and transcriptomic analyses, may argue in favor of organoid suitability for certain ambient profiling studies. However, to choose the appropriate model, pre-evaluation of the model's molecular characteristics in the context of the research question(s) being asked will likely provide the most appropriate strategy to move research forward. This can be achieved by performing comparative ambient MS analysis of the disease model of choice against a small amount of patient tissue to verify concordance. Disease models, however, will continue to be useful tools to orthogonally validate metabolic states of patient tissues through controlled genetic alterations that are not possible with patient specimens.

Predictive validity of the adult tobacco dependence index: Findings from waves 1 and 2 of the Population Assessment of Tobacco and Health (PATH) study.

BACKGROUND AND AIMS: Building on published work1 establishing concurrent validity of a self-report tobacco dependence (TD) index among users of different tobacco products in Wave 1 (W1) of the Population Assessment of Tobacco and Health (PATH) Study, the current study examines prospective relationships with tobacco use behaviors to establish predictive validity of the TD index. Hypotheses suggested high levels of W1 TD would be associated with persistent tobacco use at Wave 2 (W2). PARTICIPANTS: A U.S. nationally representative sample of 32,320 adult W1 and W2 interviews focused on 11,615 W1 adults who were current established tobacco users and completed the W2 interview. FINDINGS: Higher TD scores and greater changes in TD scores were associated with greater quantity and frequency of tobacco use at the W2 interview for Cigarette Only (n = 7068), Smokeless (smokeless or snus pouches) Only (n = 772), Cigarette plus E-Cigarette (n = 592), and Multiple Products (n = 1866) users, although not significantly so for E-Cigarette Only (n = 367), Cigar Only (traditional, cigarillo, or filtered) (n = 584), or Hookah Only (n = 366) users. Higher TD was associated with decreased odds of successful quitting for Cigarette and Multiple Product users. Higher TD was associated with increased odds of a quit attempt for those in the Hookah and Multiple Products user groups and was not associated with quit attempts or deceased odds of quit success among exclusive E-Cigarette, Cigar, Smokeless and Cigarette plus E-Cigarette users. CONCLUSION: Support for the predictive validity of the PATH Study measures of adult TD will enable regulatory investigations of TD across several tobacco products.

A Scoping Review on the Attributes of Cluster Randomized Controlled Trials in Long-Term Care Facilities.

Cluster randomized trial design, where groups of participants are randomized instead of individual participants, is increasingly being used in long-term care research. The purpose of this review was to determine the characteristics of cluster randomized trials in long-term care facilities. A medical librarian conducted the literature search. Two independent reviewers reviewed each paper. Studies were included if the design was cluster randomized and participants were from long-term care facilities. For each included study, two independent data extractors captured data on study attributes, including: journal, location, year published, author discipline, funding, methodology, number of participants, and intervention target. The literature search yielded 7,679 unique studies, with 195 studies meeting the selection criteria and being included for data extraction. The included studies were published between 1976 and 2017, with 53% of studies published after 2009. The term cluster randomized was in the title of only 45% of the studies. The studies were conducted worldwide; the United States had the largest number of studies (23%), followed by the United Kingdom (18%). Ten percent of studies were published in journals with an impact factor >10. The most frequent discipline of the first and last authors was medicine (34%), followed by nursing (17%). Forty-nine percent of the studies had government funding, while only 20% had medical industry funding. In studies with <1000 residents, 85% of the studies obtained consent from the resident and/or their proxy, while in studies with ≥ 1000 residents, it was 31%. The most frequent intervention targets were infection (13%), falls/fracture (13%), and behavior/physical restraint (13%). Cluster randomized controlled trials in long-term care have a unique set of characteristics. Results of this review will provide guidance to researchers conducting studies in long-term care facilities.

Comprehensive Rehabilitation for a Permanent Tooth Anodontia Patient Using an Integrated Digital Approach.

Agenesis of the permanent dentition is rare. This report describes a 20-year-old woman with 19 deciduous teeth, a single permanent mandibular premolar, and other physical traits associated with ectodermal dysplasia. The patient demonstrated esthetic parameters associated with maxillomandibular alveolar insufficiency, and her chief complaints were directed toward esthetics and the potential impact of restorative choices on function. Three typical options for restoration include overdentures, removable partial dentures, or implant-supported prostheses replacing her natural dentition. This report illustrates a fully integrated digital approach to treatment planning, the fabrication of a computer-aided design/computer-assisted manufacture surgical guide and provisional restoration, guided implant placement, and definitive restoration using monolithic zirconia implant-supported fixed dental prostheses. The lifelong management of this rehabilitation is an acknowledged challenge.

Dual Laser and Desorption Electrospray Ionization Mass Spectrometry Imaging Using the Same Interface.

For a more comprehensive characterization of molecular heterogeneities of matter, multimodal mass spectrometry imaging must be developed to take advantage of the complementarity of information available through different ionization mechanisms. We report the design, implementation, and performance validation of a laser desorption imaging interface composed of add-on components that adapt a commercial Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging interface for dual imaging of Picosecond Infrared Laser Mass Spectrometry (PIRL-MS) with DESI-MS. The interface utilizes hardware elements and data analysis pipelines already established for DESI-MS imaging, and was further validated in cancer margin assessments using human medulloblastoma cancers. The PIRL-MS images were robust and reproducible across multiple experimental runs on independently prepared xenograft tumors, and could be segmented into cancer and healthy regions in concordance with pathology using a variety of supervised and unsupervised clustering methods. The spectral quality and complexity obtained with this interface were examined with infiltrating and noninfiltrating tumors, and were comparable to other mass spectrometry analysis interfaces. The average PIRL-MS spectra from spatially resolved images could be used for robust cancer m/z model building to classify medulloblastoma cancer from healthy tissue without any misclassifications, an observation that held true over close to 70 sampling data points. While the unsupervised spectral analysis methods suggested a slight suppression of signal in the phospholipid range compared to the hand-held configuration, these changes were insufficient to hamper utility in cancer margin assessment with spatially resolved data obtained with our interface. Dual PIRL-MS and DESI-MS imaging of consecutive sections, as suggested by multivariate loading plots, revealed highly complementary molecular information with m/z values identifiable with one desorption method sufficient to reveal cancer regions being absent in another, further emphasizing the need for effective hardware and software interfaces for dual mass spectrometry imaging.

Structured Literacy Intervention for Students With Dyslexia: Focus on Growing Morphological Skills.

Purpose Structured literacy (SL) is an umbrella term used by the International Dyslexia Association that refers to evidence-based instructional approaches that incorporate all aspects of spoken language into the teaching of reading, spelling, and writing (International Dyslexia Association, 2016). SL has gained prominence in the field of reading but is less familiar to speech-language pathologists. This tutorial seeks to describe SL with specific attention to the morphological component. Using current research literature combined with descriptions of specific therapeutic practices, this tutorial offers research-informed, clinical strategies for facilitating the development of morphological skills in students with spoken and written language impairments including dyslexia. Method In this tutorial, the authors focus on the research literature and clinical applications related to the topics of (a) spoken and written language impairments, including dyslexia; (b) SL intervention; (c) intervention in the areas of morphological awareness and analysis; and (d) the promotion of academic success in students who struggle with language and literacy. Conclusions SL is a term used to unify and describe evidence-based principles and components that should be included in all effective reading and writing instructions. Among other linguistic skills, morphology holds a prominent place in SL. It is critical that speech-language pathologists become familiar with SL and the evidence-based practices for growing these students' morphological awareness skills in order to promote language and literacy success.

In situ tissue pathology from spatially encoded mass spectrometry classifiers visualized in real time through augmented reality.

Integration between a hand-held mass spectrometry desorption probe based on picosecond infrared laser technology (PIRL-MS) and an optical surgical tracking system demonstrates in situ tissue pathology from point-sampled mass spectrometry data. Spatially encoded pathology classifications are displayed at the site of laser sampling as color-coded pixels in an augmented reality video feed of the surgical field of view. This is enabled by two-way communication between surgical navigation and mass spectrometry data analysis platforms through a custom-built interface. Performance of the system was evaluated using murine models of human cancers sampled in situ in the presence of body fluids with a technical pixel error of 1.0 ± 0.2 mm, suggesting a 84% or 92% (excluding one outlier) cancer type classification rate across different molecular models that distinguish cell-lines of each class of breast, brain, head and neck murine models. Further, through end-point immunohistochemical staining for DNA damage, cell death and neuronal viability, spatially encoded PIRL-MS sampling is shown to produce classifiable mass spectral data from living murine brain tissue, with levels of neuronal damage that are comparable to those induced by a surgical scalpel. This highlights the potential of spatially encoded PIRL-MS analysis for in vivo use during neurosurgical applications of cancer type determination or point-sampling in vivo tissue during tumor bed examination to assess cancer removal. The interface developed herein for the analysis and the display of spatially encoded PIRL-MS data can be adapted to other hand-held mass spectrometry analysis probes currently available.

Management of Implant/Prosthodontic Complications.

Dental implants continue to grow in popularity because they are a predictable treatment to replace missing teeth. They have a high success rate; however, they are still associated with some clinical complications. This article discusses a diverse range of complications related to the restorative and mechanical aspects of dental implants and the management of such complications, as well as potential factors contributing to them.

Longitudinal e-Cigarette and Cigarette Use Among US Youth in the PATH Study (2013-2015).

BACKGROUND: Evidence is accumulating that youth who try Electronic Nicotine Delivery Systems (ENDS, e-cigarettes) may go on to try cigarettes. This analysis examines the bidirectional patterns of ENDS and cigarette use among US youth over one year and uses propensity score matching (PSM) to examine frequency of ENDS use on changes in cigarette smoking. METHODS: Our analysis included 11 996 participants who had two waves of available data (Wave 1 [W1] 2013-2014; Wave 2 [W2] 2014-2015) drawn from the longitudinal Population Assessment of Tobacco and Health Study. Cross-sectional weighted prevalence estimates are reported for cigarettes and ENDS. We used PSM to estimate the likelihood of ENDS use at W1 and to draw matched analytic samples, then used regression (logistic or linear) models to examine the effect of W1 ENDS use on W2 cigarette smoking. All statistical tests were two-sided. RESULTS: In weighted analyses, 69.3% of W1 past-30-day cigarette smokers exhibited past-30-day smoking at W2; 42.2% of W1 past-30-day ENDS users were using ENDS at W2. W1 ever use of either product was similarly associated with W2 new use of the other product. Unweighted PSM models indicated W1 cigarette-naïve ENDS use was associated with W2 ever-cigarette smoking (n = 676; adjusted odds ratio = 3.21, 95% confidence interval [CI] = 1.95 to 5.45, P < .001); W1 ever-ENDS use did not affect change in cigarette frequency at W2 (n = 1020, beta = 0.31, 95% CI = -0.76 to 1.39, P = .57); 1-5 days ENDS use compared with ever, no past-30-day ENDS use was associated with a statistically significant decrease of W2 smoking days (n = 256, beta = -2.64, 95% CI = -4.96 to -0.32; P = .03); and W1 6+ day ENDS users did not show a decrease in frequency of cigarette smoking. CONCLUSIONS: Ever-ENDS use predicts future cigarette smoking, and frequency of ENDS use has a differential impact on subsequent cigarette smoking uptake or reduction. These results suggest that both cigarettes and ENDS should be targeted in early tobacco prevention efforts with youth.

The impact of breast reduction surgery on breastfeeding: Systematic review of observational studies.

BACKGROUND: Almost half a million breast reduction surgeries are performed internationally each year, yet it is unclear how this type of surgery impacts breastfeeding. This is particularly important given the benefits of breastfeeding. OBJECTIVES: To determine if breast reduction surgery impacts breastfeeding success and whether different surgical techniques differentially impact breast feeding success. METHODS: Databases were searched up to September 5, 2017. Studies were included if they reported the number of women successful at breastfeeding or lactation after breast reduction surgery, and if they reported either the total number of women who had children following breast reduction surgery, or the total number of women who attempted to breastfeed following surgery. RESULTS: Of 1,212 studies, 51 studies met the inclusion criteria; they were located worldwide and had 31 distinct breast reduction techniques. The percentage of breastfeeding success among studies was highly variable. However, when analyzed by the preservation of the column of parenchyma from the nipple areola complex to the chest wall (subareolar parenchyma), a clear pattern emerged. The median breastfeeding success was 4% (interquartile range (IQR) 0-38%) for techniques with no preservation, compared to 75% (IQR 37-100%) for techniques with partial preservation and 100% (IQR 75-100%) for techniques with full preservation. CONCLUSIONS: Techniques that preserve the column of subareolar parenchyma appear to have a greater likelihood of successful breastfeeding. The preservation of the column of subareolar parenchyma should be disclosed to women prior to surgery. Guidelines on the best breast reduction techniques to be used in women of child bearing years may be advantageous to ensure women have the greatest potential for successful breastfeeding after breast reduction surgery.

Electronic nicotine delivery devices, and their impact on health and patterns of tobacco use: a systematic review protocol.

INTRODUCTION: E-cigarettes or electronic nicotine delivery systems (ENDS) have recently attracted considerable attention. Among some individuals there is strong debate and a polarisation of views about the public health benefits versus harms of ENDS. With little regulation, the ENDS market is evolving, and new products are introduced and marketed constantly. Rapid developments in manufacturing, marketing and consumer domains related to ENDS will warrant frequent re-evaluation, based on the state of the evolving science. The purpose of this article is to describe a protocol for an ongoing comprehensive review of the published scientific literature on ENDS. METHODS AND ANALYSIS: We will undertake a systematic review of published empirical research literature on ENDS using the National Library of Medicine's PubMed electronic database to search for relevant articles. Data from included studies will be extracted into a standardised form, tables with study details and key outcomes for each article will be created, and studies will be synthesised qualitatively. ETHICS AND DISSEMINATION: This review synthesises published literature and presents no primary data. Therefore, no ethical approval is required for this study. Subsequent papers will provide greater detail on results, within select categories, that represent gaps in the literature base.

Balanced intervention for adolescents and adults with language impairment: a clinical framework.

Providing effective intervention services for adolescents and adults who struggle with spoken and written language presents a variety of unique challenges. This article discusses the 5S Framework (skills, strategies, school, student buy-in, and stakeholders) for designing and implementing balanced spoken and written language interventions for adolescents and adults. An in-depth case illustration highlights the usefulness of the framework for targeting the language and literacy skills of adolescents and young adults. By describing and illustrating the five key components of the intervention framework, the article provides a useful clinical tool to help guide clinicians and educators who serve the needs of adolescents and adults who struggle with spoken and written language.