Musculoskeletal Biorepository: Establishment, Sustainment, and Tips for Success.

A biorepository, also referred to as a "biobank," is a collection of biologic samples that are stored for laboratory research. With the emergence of precision medicine, the importance of leveraging individual patient biomolecular signatures to improve diagnosis, prognosis, and treatment is becoming increasingly recognized. Successful development and sustainment of a biorepository provides the potential for transformative preclinical research. Establishing a biobank requires a team approach with involvement of the institutions' research laboratory team and regulatory body. Execution of research activities requires a coordinated team approach for case identification, consent process, data and specimen collection, specimen processing, and storage and archiving. The advancing fields of precision medicine and orthobiologics provide incredible opportunities for institutions to generate novel lines of inquiry in musculoskeletal diseases through a multiomics approach (genomic, transcriptomic, proteomic, microbiomic). In addition, a biobank is an important component of post-market surveillance for the rapidly emerging field of orthobiologics.

The use of mobile thermal imaging and machine learning technology for the detection of early surgical site infections.

BACKGROUND: Surgical Site Infections (SSI) yield subtle, early signs that are not readily identifiable. This study sought to develop a machine learning algorithm that could identify early SSIs based on thermal images. METHODS: Images were taken of surgical incisions on 193 patients who underwent a variety of surgical procedures. Two neural network models were generated to detect SSIs, one using RGB images, and one incorporating thermal images. Accuracy and Jaccard Index were the primary metrics by which models were evaluated. RESULTS: Only 5 patients in our cohort developed SSIs (2.8%). Models were instead generated to demarcate the wound site. The models had 89-92% accuracy in predicting pixel class. The Jaccard indices for the RGB and RGB â€‹+ â€‹Thermal models were 66% and 64%, respectively. CONCLUSIONS: Although the low infection rate precluded the ability of our models to identify surgical site infections, we were able to generate two models to successfully segment wounds. This proof-of-concept study demonstrates that computer vision has the potential to support future surgical applications.

A Randomized Controlled Trial of Precision Nutrition Counseling for Service Members at Risk for Metabolic Syndrome.

INTRODUCTION: Metabolic syndrome (MetS) is a threat to the active component military as it impacts health, readiness, retention, and cost to the Military Health System. The most prevalent risk factors documented in service members' health records are high blood pressure (BP), low high-density lipoprotein cholesterol, and elevated triglycerides. Other risk factors include abdominal obesity and elevated fasting blood glucose. Precision nutrition counseling and wellness software applications have demonstrated positive results for weight management when coupled with high levels of participant engagement and motivation. MATERIALS AND METHODS: In this prospective randomized controlled trial, trained registered dietitians conducted nutrition counseling using results of targeted sequencing, biomarkers, and expert recommendations to reduce the risk for MetS. Upon randomization, the treatment arm initiated six weekly sessions and the control arm received educational pamphlets. An eHealth application captured diet and physical activity. Anthropometrics and BP were measured at baseline, 6 weeks, and 12 weeks, and biomarkers were measured at baseline and 12 weeks. The primary outcome was a change in weight at 12 weeks. Statistical analysis included descriptive statistics and t-tests or analysis of variance with significance set at P < .05. RESULTS: Overall, 138 subjects enrolled from November 2019 to February 2021 between two military bases; 107 completed the study. Demographics were as follows: 66% male, mean age 31 years, 66% married, and 49% Caucasian and non-Hispanic. Weight loss was not significant between groups or sites at 12 weeks. Overall, 27% of subjects met the diagnostic criteria for MetS on enrollment and 17.8% upon study completion. High deleterious variant prevalence was identified for genes with single-nucleotide polymorphisms linked to obesity (40%), cholesterol (38%), and BP (58%). Overall, 65% of subjects had low 25(OH)D upon enrollment; 45% remained insufficient at study completion. eHealth app had low adherence yet sufficient correlation with a valid reference. CONCLUSIONS: Early signs of progress with weight loss at 6 weeks were not sustained at 12 weeks. DNA-based nutrition counseling was not efficacious for weight loss.

An intrauterine genomic classifier reliably delineates the location of nonviable pregnancies.

OBJECTIVE: To compare the intrauterine gene expression signatures of women with surgically confirmed ectopic pregnancy (ECT) and those of women with miscarriage to inform the development of a genomic classifier for the reliable delineation of pregnancy location in women with clinically nonviable pregnancies of unknown location (NV-PULs). DESIGN: Discovery-based prospective cohort study. SETTING: Academic medical center. PATIENT(S): Women with clinically nonviable early pregnancy to include abnormal intrauterine pregnancy (AIUP), ECT, or NV-PUL. INTERVENTION(S): Endometrial (EM) pipelle sampling of the uterus was conducted at the time of scheduled surgery for clinically nonviable early pregnancy (dilation and curettage, manual vacuum aspiration, or laparoscopy). All pregnancy locations were surgically and/or histologically confirmed as intrauterine or ectopic. MAIN OUTCOME MEASURE(S): Gene expression profiles as determined by array hybridization, quantitative real-time polymerase chain reaction, and nCounter technology. RESULT(S): Intrauterine samples were obtained by EM pipelle from 27 women undergoing surgery for a clinically nonviable early pregnancy. Comparison of array-based global gene expression signatures from women with histologically confirmed ECT versus AIUP revealed 61 differentially expressed genes from which the 5 most informative were included in the pregnancy location classifier. All 5 genes (C20orf85, LRRC46, RSPH4A, WDR49, and ZBBX) were cilia-associated and showed increased expression in pipelle samples from women with ECT relative to expression in samples from women with AIUP. The 5-gene classifier demonstrated an average area under the receiver operator characteristic curve of 0.97 for the detection of ECT. In an external test set composed of publicly available EM pipelle-based gene expression data from a study with similar ECT and AIUP cohorts (n = 19), the classifier revealed an average area under the receiver operator characteristic curve of 0.84. CONCLUSION(S): Consistently increased expression of cilia-associated genes in the uterine cavity of women with ECT provides a reliable molecular signal for the delineation of pregnancy location in women with clinically assessed NV-PUL. A classifier consisting of the 5 most informative cilia-associated genes demonstrated 91% (42/46) accuracy in predicting the pregnancy location.

A Retrospective Cohort Study of Blood Lead Levels Among Special Operations Forces Soldiers Exposed to Lead at a Firing Range in Germany.

This report describes the results of testing for blood lead levels (BLLs) among special operations forces at a single installation in Germany where occupational exposures to lead were associated with use of a firing range. After recognition of elevated BLLs in some service members who used the firing range, a detailed industrial hygiene confirmation of lead exposures prompted mitigation measures undertaken by command authorities, facilities management, public health, and clinical occupational medicine. To assess the impact of the mitigation efforts, this study retrieved the results of all BLLs performed between 1 January 2016 and 30 September 2018 among SOF soldiers enrolled in an Occupational Safety and Health Administration (OSHA)-required medical surveillance program for lead exposure. Mitigation steps were taken during July-September 2017. BLLs from the periods before and after the mitigation efforts were compared. Among the 57 individuals who had levels measured both before and after the mitigation period, the range of BLL values fell from a range of 1-35 µg/dL to a range of 1-15 µg/dL. The number of individuals who had BLLs of greater than 20 µg/dL fell from 9 before, to 0 after the mitigation period. The various types of mitigation steps useful in reducing firing range-related lead exposure are described.

Are We Speaking the Same Language? Communicating Diagnostic Probability in the Radiology Report.

OBJECTIVE. The purpose of this study was to evaluate the level of agreement in diagnostic probability for selected phrases among radiologists and emergency medicine (EM) physicians. MATERIALS AND METHODS. A survey was distributed to the radiologists and EM physicians at our academic institution. Respondents selected the degree of diagnostic probability they believe was conveyed by 18 commonly used phrases chosen from studies in the radiology literature. Potential responses for the degree of diagnostic probability were < 10%, ≈ 25%, ≈ 50%, ≈ 75%, and > 90%. RESULTS. Seventy-eight percent (28/36) of EM residents and 56% (14/25) of EM attending physicians (combined fellows and attending physicians) completed the survey; 83% (15/18) of radiology residents and 81% (17/21) of radiology attending physicians completed the survey. There was a high degree of shared understanding for most phrases between the departments except for the phrase "compatible with," which was associated with a higher degree of diagnostic probability by radiologists than by EM physicians (p = .02). Although no term was significantly more specific than any other within the ≈ 50% category or below, "most likely" and "diagnostic of" were significantly more specific than other terms in the ≈ 75% and > 90% categories, respectively. CONCLUSION. The results of this study show a high degree of shared understanding between radiologists and EM physicians for most of the phrases (17/18) in the survey. The only phrase that showed a significant difference was "compatible with." These results can be used to generate diagnostic probability groups with suggested phrases that can be used when creating radiology reports, thereby improving communication with the emergency department.

Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response.

Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5ß1 and αvß3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvß3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.

Complexes of α6ß4 integrin and vimentin act as signaling hubs to regulate epithelial cell migration.

We find that clusters of ß4 integrin, organized into distinct puncta, localize along vimentin filaments within lamellipodia at the cell edge of A549 cells, as assessed by interferometric photoactivated localization microscopy. Moreover, puncta and vimentin filaments exhibit a dynamic interplay in live cells, as viewed by structured-illumination microscopy, with ß4 integrin puncta that associate with vimentin persisting for longer than those that do not. Interestingly, in A549 cells ß4 integrin regulates vimentin cytoskeleton organization. When ß4 integrin is knocked down there is a loss of vimentin filaments from lamellipodia. However, in these conditions, vimentin filaments instead concentrate around the nucleus. Although ß4 integrin organization is unaffected in vimentin-deficient A549 cells, such cells move in a less-directed fashion and exhibit reduced Rac1 activity, mimicking the phenotype of ß4 integrin-deficient A549 cells. Moreover, in vimentin-deficient cells, Rac1 fails to cluster at sites enriched in α6ß4 integrin heterodimers. The aberrant motility of both ß4 integrin and vimentin-deficient cells is rescued by expression of active Rac1, leading us to propose that complexes of ß4 integrin and vimentin act as signaling hubs, regulating cell motility behavior.

α6ß4 Integrin Regulates the Collective Migration of Epithelial Cells.

α6ß4 integrin is localized in a unique punctate distribution at the cell-substratum interface along the leading front of single, front-rear-polarized A549 cells. These puncta are interspersed between focal adhesions and lack association with the actin cytoskeleton. Knockdown of ß4 integrin in A549 cells inhibits their directed migration, with knockdown cells exhibiting large focal adhesions and reduced actin dynamics. Despite these changes, the speed of knockdown cells is equivalent to control cells. Interestingly, in such cells, α6 integrin retains its punctate distribution. Moreover, in ß4 integrin knockdown cells, we observe a loss of ß1 integrin from focal adhesions and an enhanced association with α6 integrin. We confirmed the switch in the ß integrin binding partner of α6 integrin in the knockdown cells by immunoprecipitation. We next investigated the role of ß4 integrin in collective cell migration. Wounded monolayers of ß4 integrin knockdown cells exhibit reduced collective migration compared with controls. When we forced expression of ß4 integrin in the leader cells of wounded monolayers, collective migration was restored. Similarly, forced expression of ß4 integrin in primary rat alveolar epithelial cells also promotes collective cell migration. In addition, we interrogated the pathway by which ß4 integrin regulates A549 cell-directed migration. Constitutively active Ras-related C3 botulinum toxin substrate 1 rescues motility defects resulting from ß4 integrin deficiency. Together, our results support the hypothesis that α6ß4 integrin is a positive regulator of collective cell migration of A549 cells through influence on signal pathways in leader cells.

Focusing super resolution on the cytoskeleton.

Super resolution imaging is becoming an increasingly important tool in the arsenal of methods available to cell biologists. In recognition of its potential, the Nobel Prize for chemistry was awarded to three investigators involved in the development of super resolution imaging methods in 2014. The availability of commercial instruments for super resolution imaging has further spurred the development of new methods and reagents designed to take advantage of super resolution techniques. Super resolution offers the advantages traditionally associated with light microscopy, including the use of gentle fixation and specimen preparation methods, the ability to visualize multiple elements within a single specimen, and the potential to visualize dynamic changes in living specimens over time. However, imaging of living cells over time is difficult and super resolution imaging is computationally demanding. In this review, we discuss the advantages/disadvantages of different super resolution systems for imaging fixed live specimens, with particular regard to cytoskeleton structures.

A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes.

During wound healing of the skin, keratinocytes disassemble hemidesmosomes and reorganize their actin cytoskeletons in order to exert traction forces on and move directionally over the dermis. Nonetheless, the transmembrane hemidesmosome component collagen XVII (ColXVII) is found in actin-rich lamella, situated behind the lamellipodium. A set of actin bundles, along which ColXVII colocalizes with actinin4, is present at each lamella. Knockdown of either ColXVII or actinin4 not only inhibits directed migration of keratinocytes but also relieves constraints on actin bundle retrograde movement at the site of lamella, such that actin bundle movement is enhanced more than 5-fold. Moreover, whereas control keratinocytes move in a stepwise fashion over a substrate by generating alternating traction forces, of up to 1.4 kPa, at each flank of the lamellipodium, ColXVII knockdown keratinocytes fail to do so. In summary, our data indicate that ColXVII-actinin4 complexes at the lamella of a moving keratinocyte regulate actin dynamics, thereby determining the direction of cell movement.-Hiroyasu, S., Colburn, Z. T., Jones, J. C. R. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes.

Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility.

The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton, and matrix adhesions. In this study, we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via a small hairpin RNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower compared with their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and ß4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration.