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Abstract Background Monitoring the time course of mortality by cause is a key public health issue. However, several mortality data production changes may affect cause-specific time trends, thus altering the interpretation. This paper proposes a statistical method that detects abrupt changes ("jumps") and estimates correction factors that may be used for further analysis. Methods The method was applied to a subset of the AMIEHS (Avoidable Mortality in the European Union, toward better Indicators for the Effectiveness of Health Systems) project mortality database and considered for six European countries and 13 selected causes of deaths. For each country and cause of death, an automated jump detection method called Polydect was applied to the log mortality rate time series. The plausibility of a data production change associated with each detected jump was evaluated through literature search or feedback obtained from the national data producers. For each plausible jump position, the statistical significance of the between-age and between-gender jump amplitude heterogeneity was evaluated by means of a generalized additive regression model, and correction factors were deduced from the results. Results Forty-nine jumps were detected by the Polydect method from 1970 to 2005. Most of the detected jumps were found to be plausible. The age- and gender-specific amplitudes of the jumps were estimated when they were statistically heterogeneous, and they showed greater by-age heterogeneity than by-gender heterogeneity. Conclusion The method presented in this paper was successfully applied to a large set of causes of death and countries. The method appears to be an alternative to bridge coding methods when the latter are not systematically implemented because they are time- and resource-consuming.

Advances in arthroscopy have contributed toward improved understanding and management of diverse pathological conditions in the shoulder. As a result, arthroscopy is often preferred by both patients and surgeons. However, surgery can be complicated by limited visualization. Techniques to improve visualization include patient and portal positioning, mechanical débridement, radiofrequency ablation, epinephrine added to irrigation fluid, tranexamic acid administration, and controlled hypotensive anesthesia. Despite published literature on each, a thorough understanding of the evidence supporting these techniques and adjuvants is essential to interpret the clinical utility of each., (Copyright © 2024 by the American Academy of Orthopaedic Surgeons.)

Case: A 58-year-old, very active man sustained bilateral isolated teres major (TM) tendon tears when his arms were forcefully elevated overhead while wakeboarding. Staged surgical repair was performed. A progressive rehabilitation protocol was followed, and he returned to high-level activities 7 months postoperatively. At 1-year follow-up, outcome measures for bilateral shoulders were DASH 0, SST 12, ASES 100, and EQ-5D 1.0., Conclusion: Despite literature supporting conservative treatment, this case demonstrates that operative treatment of acute, isolated TM tears can result in highly successful outcomes for motivated active patients., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSCC/C386)., (Copyright © 2024 by The Journal of Bone and Joint Surgery, Incorporated.)

Purpose: With a lack of standardization among outcome measures in fracture literature, cross-study comparisons remain limited. This systematic review aimed to identify trends in outcome measures reported by studies of the treatment of humeral shaft fractures., Methods: A systematic review was performed of studies reporting clinical outcomes of humeral shaft fractures indexed in PubMed. Extracted data included demographics, fracture characteristics, treatment modalities, outcomes, patient reported outcome measures (PROMs), and journal characteristics. Cochran-Armitage tests and linear regressions were used to identify data trends. Pearson chi-square and Kruskal-Wallis tests were used for comparisons between studies., Results: This review included 197 studies with outcomes of 15,445 humeral shaft fractures. 126 studies reported PROMs and 37 different PROMs were used. The Constant Score was most commonly reported (34% of studies), followed by ASES Score (21%), MEPS (21%), and DASH Score (20%). There was a significant increase in PROM usage over time (p = 0.016) and in articles using three or more PROMs (p = 0.005). The number of PROMs were significantly greater in prospective cohort studies and RCTs (p = 0.012) compared to retrospective cohort studies and case series (p = 0.044 for both). Post-treatment shoulder motion was reported in 43% of studies and 34% reported elbow motion. 86% of studies reported complications as an outcome parameter. Time to union and nonunion rate were published in 69% and 88% of studies, respectively., Conclusion: This study identified increasing PROM usage over time and disparities in the reporting of outcomes in humeral shaft fracture literature requiring further validation and standardization of available outcome measures., (© 2024. The Author(s), under exclusive licence to Springer-Verlag France SAS, part of Springer Nature.)

Background: Humeral implant designs for anatomic total shoulder arthroplasty (aTSA) focus on anatomic reconstruction of the articular segment. Likewise, the pathoanatomy of advanced glenohumeral osteoarthritis often results in humeral head deformity. We hypothesized the anatomic reconstruction of the humeral head in aTSA risks overstuffing the glenohumeral joint., Methods: Ninety-seven cases (52 females) of primary glenohumeral osteoarthritis in patients treated with aTSA were evaluated. Preoperative computed tomography scans were used to classify glenoid morphology according to the Walch classification. Coronal plane images in the plane of the humerus were used to determine the anatomic best-fit circle as described by Youderian et al. Humeral head thinning was determined as the distance from the center of rotation of the best-fit circle to the nearest point along the humeral articular surface. aTSA was modeled with a predicted anatomic humeral head and a simulated 4-mm polyethylene glenoid component. The change in the position of the native humerus was determined. Wilcoxon Rank Sum tests were used to evaluate differences in humeral head thinning and humeral lateralization between monoconcave and biconcave glenoid morphologies. Spearman's rank correlation coefficients were used to assess the relationship between humeral head thinning with preoperative active forward elevation and external rotation., Results: The mean radius of the best-fit circle was 25.0 ± 2.1 mm. There was a mean thinning of 2.4 ± 2.0 mm (range -1.7 to 8.3). The mean percent thinning of the humeral head was 9.4% ± 7.7%. The mean humeral lateralization was 6.4 ± 2.0 mm. Humeral head thinning was not significantly associated with active forward elevation (r = -0.15, P = .14) or active external rotation (r = -0.12, P = .25). There were no significant differences in the percentage of humeral head thinning (P = .324) or humeral lateralization (P = .350) between concentric and eccentric glenoid wear patterns., Conclusions: Utilization of the best-fit circle as a guide in aTSA may risk excessive lateralization of the humerus and overstuffing the glenohumeral joint. This may have implications for subscapularis repair and healing, as well as glenoid implant and rotator cuff longevity. These findings call into question whether recreation of normal glenohumeral anatomy in aTSA is appropriate for all patients. Humeral head reconstruction in aTSA should account for glenohumeral joint volume and soft tissue contracture., (Copyright © 2024. Published by Elsevier Inc.)

Case: A 71-year-old woman presented with post-traumatic arthritis 11 months after open reduction and internal fixation for a left proximal humerus fracture (PHF) dislocation. After revision to reverse total shoulder arthroplasty (rTSA), the patient's left upper extremity was found to be avascular. An emergent thrombectomy was performed with restoration of arterial flow after removal of an acute-on-chronic axillary artery thrombus., Conclusion: Although rare, as rTSA becomes more common for management of PHF, incidence of associated vascular injuries is likely to rise. Screening methods and clinical vigilance in diagnosis are advised for patients with anterior PHF dislocations and arterial injury risk factors., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSCC/C346)., (Copyright © 2024 by The Journal of Bone and Joint Surgery, Incorporated.)

Case: A 40-year-old man with shoulder pain secondary to severe bilateral glenoid hypoplasia without posterior instability was treated successfully with bilateral posterior glenoid bone graft augmentation., Conclusion: While glenoid hypoplasia is associated with a variety of patient presentations, treatment of the stable and nonarthritic shoulder is rarely described. A posterior glenoid bone graft can be used to augment deficient posterior glenoids, increasing the articulating surface area, restoring function, and potentially facilitating future shoulder arthroplasty., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSCC/C245)., (Copyright © 2023 by The Journal of Bone and Joint Surgery, Incorporated.)

Introduction: Reported rates of nerve injury after upper extremity (UE) gunshot wound (GSW) range from 15% to 45%. Many surgeons prefer initial observation; however, this may delay diagnosis of neurotmesis and surgical treatment. We aimed to determine the incidence of nerve transection in adults after upper extremity GSWs., Methods: This was a retrospective chart review approved by the institutional review board. Operative records of five orthopedic surgeons between 2014 to 2019 were filtered for ICD-10 and CPT codes cross-referenced to include both UE GSW assault and nerve injuries. Inclusion criteria consisted of age greater than 18 at time of injury, neurologic deficit on presentation, and surgical exploration nerve procedure. Records were reviewed for degree of nerve injury, procedure performed, and complications. Postoperative outcomes included nerve recovery, complications, and reoperation rate., Results: Of the 17 patients that fit the inclusion criteria, the incidence of complete nerve transection was 64.7% and the incidence of a complete or partial nerve transection was 70.6%. The most common location of GSWs was the hand (70.5%). Average time from date of injury to surgery was 26.1 days. There were 14 identifiable nerve injuries-complete transection in 11, partial transection in one, and contusion in two patients. Of patients with nerve transection, 72.7% sustained a fracture. Postoperative complications included stiffness, chronic pain, and wound infection. The re-operation rate was 29.4%. The average postoperative follow-up was 4.4 months. There was longer follow-up among nerve transection patients (5.3 months) compared to neuropraxia patients (2.68 months)., Conclusion: This study demonstrates a higher incidence of nerve transection in upper extremity GSW patients than previously reported. Predictors of nerve transection are GSWs to the hand and associated fracture.

Each year there are nearly 57 million deaths worldwide, with over 2.7 million in the United States. Timely, accurate and complete death reporting is critical for public health, especially during the COVID-19 pandemic, as institutions and government agencies rely on death reports to formulate responses to communicable diseases. Unfortunately, determining the causes of death is challenging even for experienced physicians. The novel coronavirus and its variants may further complicate the task, as physicians and experts are still investigating COVID-related complications. To assist physicians in accurately reporting causes of death, an advanced Artificial Intelligence (AI) approach is presented to determine a chronically ordered sequence of conditions that lead to death (named as the causal sequence of death), based on decedent's last hospital discharge record. The key design is to learn the causal relationship among clinical codes and to identify death-related conditions. There exist three challenges: different clinical coding systems, medical domain knowledge constraint, and data interoperability. First, we apply neural machine translation models with various attention mechanisms to generate sequences of causes of death. We use the BLEU (BiLingual Evaluation Understudy) score with three accuracy metrics to evaluate the quality of generated sequences. Second, we incorporate expert-verified medical domain knowledge as constraints when generating the causal sequences of death. Lastly, we develop a Fast Healthcare Interoperability Resources (FHIR) interface that demonstrates the usability of this work in clinical practice. Our results match the state-of-art reporting and can assist physicians and experts in public health crisis such as the COVID-19 pandemic.

Orthopaedic surgeons are among the highest prescribers of opioids. This study explores the effect of an educational intervention on orthopaedic surgery residents' opioid knowledge and prescribing practices. Orthopaedic residents were surveyed at three urban academic institutions. A pre-survey was administered to residents prior to an educational lecture and case-based session. This included background on the opioid epidemic, multimodal analgesia, opioid consumption in common orthopaedic procedures, and state laws regulating prescribing. Following this intervention, residents were given a post-survey to complete. There was a significant increase in resident confidence concerning their opioid prescribing training (p = 0.03) and their knowledge of alternative pain management therapies (p = 0.03). This was accompanied by an objective improvement in knowledge of state prescribing laws and of metrics regarding the opioid epidemic. Hypothetical opioid pills prescribed after common orthopaedic procedures decreased between the pre- and post-tests. The educational session significantly improved orthopaedic surgery residents' knowledge about opioids and prescribing habits. Formal resident education on opioid knowledge and evidence-based prescribing strategies is an area of potential improvement to combat the opioid crisis. (Journal of Surgical Orthopaedic Advances 31(1):030-033, 2022).

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication., (© 2021 Hoffman et al.; Published by Cold Spring Harbor Laboratory Press.)

Plasmacytoid dendritic cells (pDC), a highly specialized class of innate immune cells that serve as rapid sensors of danger signals in circulation or in lymphoid tissue are well studied. However, there remains knowledge gaps about age-dependent changes of pDC function in the intestinal mucosa. Here, we report that under homeostatic conditions, the proportion of pDC expressing C-C chemokine receptor 9 (CCR9) in the intestinal intraepithelial cell (iIEC) population is comparable between young (2-4 months) and aged (18-24 months) mice, but the absolute numbers of iIEC and pDC are significantly lower in aged mice. Employing the classic model of acute endotoxemia induced by lipopolysaccharide (LPS), we found a decrease in the proportion and absolute number of intraepithelial pDC in both young and aged mice despite the LPS-induced increased expression of the chemokine C-C ligand 25 (CCL25), the ligand of CCR9, in the intestinal mucosa of young mice. In adoptive transfer experiments, a significantly lower number of pDC was retained into the intestinal layer of aged host mice after LPS administration. This was associated with recoverable pDC numbers in the intestinal lumen. Furthermore, co-adoptive transfer of young and aged pDC into young hosts also showed significantly lower retention of aged pDC in the epithelial layer compared to the co-transferred young pDC. Collectively, these data show age-associated changes in mucosal CCL25 gene expression and in pDC number. These may underlie the reported inadequate responses to gastrointestinal pathogens during chronologic aging., Competing Interests: Competing Interests Authors have no financial conflicts of interest., (copyright: © 2021 Hoffman et al.)

MUC16, a sialomucin that contains the ovarian cancer biomarker CA125, binds at low abundance to leucocytes via the immune receptor, Siglec-9. Conventional fluorescence-based imaging techniques lack the sensitivity to assess this low-abundance event, prompting us to develop a novel "digital" optical cytometry technique for qualitative and quantitative assessment of CA125 binding to peripheral blood mononuclear cells (PBMC). Plasmonic nanoparticle labeled detection antibody allows assessment of CA125 at the near-single molecule level when bound to specific immune cell lineages that are simultaneously identified using multiparameter fluorescence imaging. Image analysis and deep learning were used to quantify CA125 per each cell lineage. PBMC from treatment naïve ovarian cancer patients (N = 14) showed higher cell surface abundance of CA125 on the aggregate PBMC population as well as on NK ( p = 0.013), T ( p < 0.001) and B cells ( p = 0.024) compared to circulating lymphocytes of healthy donors (N = 7). Differences in CA125 binding to monocytes or NK-T cells between the two cohorts were not significant. There was no correlation between the PBMC-bound and serum levels of CA125, suggesting that these two compartments are not in stoichiometric equilibrium. Understanding where and how subset-specific cell-bound surface CA125 takes place may provide guidance towards a new diagnostic biomarker in ovarian cancer.

Steinernema nematodes and their Xenorhabdus symbionts are a malleable model system to study mutualistic relations. One of the advantages they possess is their ability to be disassociated under in vitro rearing conditions. Various in vitro methods have been developed to produce symbiont colonized and aposymbiotic (symbiont-free) nematodes. Until now, there has been no investigation on how in vitro rearing conditions may have an impact on the storage ability and the protein content of the infective juvenile at different storage temperatures. Thus, in this study, we investigated how infective juvenile longevity and protein content are impacted when the nematodes were reared with two in vitro methods (lipid and liver kidney agar) considering colonized and uncolonized nematodes, and under two different temperatures: 15 °C and 20 °C (mild stress). Infective juveniles reared in vitro (with or without their symbionts) had lower 8-week survival rates. No in vitro reared, colonized IJs survived to the desired 16-week time point. Survival of infective juveniles stored under mild stress temperature (20 °C) was lower than that observed at 15 °C. However, when comparing the interaction between rearing condition and storage temperature, there were not significant differences. With respect to protein content, in vivo, colonized infective juveniles maintained a static protein content over time, suggesting symbiont colonization may influence protein metabolism and/or turnover in infective juveniles., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The Ebola virus disease (EVD) epidemic that occurred in West Africa between 2014-16 resulted in over 28,000 cases and 11,000 deaths - one of the deadliest to date. A generalized model of the spatiotemporal progression of EVD for Liberia, Guinea, and Sierra Leone in 2014-16 remains elusive. There is also a disconnect in the literature on which interventions are most effective in curbing disease progression. To solve these two key issues, we designed a hybrid agent-based and compartmental model that switches from one paradigm to the other on a stochastic threshold. We modeled disease progression with promising accuracy using WHO datasets.

One pressing need in the area of public health is timely, accurate, and complete reporting of deaths and the diseases or conditions leading up to them. Fast Healthcare Interoperability Resources (FHIR) is a new HL7 interoperability standard for electronic health record, while Sustainable Medical Applications and Reusable Technologies (SMART)-on-FHIR enables third-party app development that can work "out of the box." This paper demonstrates the feasibility of developing SMART-on-FHIR applications that enables medical professionals to perform timely and accurate death reporting within multiple different USA State jurisdictions. We explored how the information on a standard certificate of death can be mapped to resources defined in the FHIR standard Draft Standard for Trial Use Version 2 and common profiles. We also demonstrated analytics for potentially improving the accuracy and completeness of mortality reporting data.

Accurate reporting of causes of death on death certificates is essential to formulate appropriate disease control, prevention and emergency response by national health-protection institutions such as Center for disease prevention and control (CDC). In this study, we utilize knowledge from publicly available expert-formulated rules for the cause of death to determine the extent of discordance in the death certificates in national mortality data with the expert knowledge base. We also report the most commonly occurring invalid causal pairs which physicians put in the death certificates. We use sequence rule mining to find patterns that are most frequent on death certificates and compare them with the rules from the expert knowledge based. Based on our results, 20.1% of the common patterns derived from entries into death certificates were discordant. The most probable causes of these discordance or invalid rules are missing steps and non-specific ICD-10 codes on the death certificates.

Background: Surgical site infection (SSI) remains a concern in shoulder surgery, especially during arthroplasty. While many studies have explored the characteristics and efficacy of different sterilizing solutions, no study has evaluated the method of application. The purpose of this study was to compare two popular pre-surgical preparatory applications (two 4 x 4 cm gauze sponges and applicator stick) in their ability to cover the skin of the shoulder., Methods: Two orthopedic surgeons simulated the standard pre-surgical skin preparation on 22 shoulders of volunteer subjects. Each surgeon alternated between an applicator stick and two sterile 4x4 cm gauze sponges. Skin preparation was performed with a commercially available solution that can be illuminated under UV-A light. Advanced image-analysis software was utilized to determine un-prepped areas. A two-tailed paired t-test was performed to compare percentage of un-prepped skin., Results: The applicator stick method resulted in a significantly higher percentage of un-prepped skin (27.25%, Range 10-49.3) than the gauze sponge method (15.37%, Range 5-32.8, P=0.002). Based on image evaluation, most un-prepped areas were present around the axilla., Conclusion: Based on our findings, the use of simple gauze sponges for pre-surgical preparatory application of sterilization solution may result in a lower percent of un-prepped skin than commercially available applicator stick. Orthopaedic surgeons and operating room staff should be careful during the pre-surgical sterile preparation of the shoulder, especially the region around the axilla, in order to reduce the potential risk of surgical site infection. Level of evidence: III.

Chronic wounds are increasing in prevalence and are a costly problem for the US healthcare system and throughout the world. Typically outcomes studies in the field of wound care have been limited to small clinical trials, comparative effectiveness cohorts and attempts to extrapolate results from claims databases. As a result, outcomes in real world clinical settings may differ from these published studies. This study presents a modified intent-to-treat framework for measuring wound outcomes and measures the consistency of population based outcomes across two distinct settings. In this retrospective observational analysis, we describe the largest to date, cohort of patient wound outcomes derived from 626 hospital based clinics and one academic tertiary care clinic. We present the results of a modified intent-to-treat analysis of wound outcomes as well as demographic and descriptive data. After applying the exclusion criteria, the final analytic sample includes the outcomes from 667,291 wounds in the national sample and 1,788 wounds in the academic sample. We found a consistent modified intent to treat healing rate of 74.6% from the 626 clinics and 77.6% in the academic center. We recommend that a standard modified intent to treat healing rate be used to report wound outcomes to allow for consistency and comparability in measurement across providers, payers and healthcare systems., (© 2017 by the Wound Healing Society.)

Various cell populations expressing NK1.1 contribute to innate host defense and systemic inflammatory responses, but their role in hemorrhagic shock and trauma remains uncertain. NK1.1 + cells were depleted by i.p. administration of anti-NK1.1 (or isotype control) on two consecutive days, followed by hemorrhagic shock with resuscitation and peripheral tissue trauma (HS/T). The plasma levels of IL-6, MCP-1, alanine transaminase (ALT), and aspartate aminotransferase (AST) were measured at 6 and 24 h. Histology in liver and gut were examined at 6 and 24 h. The number of NK cells, NKT cells, neutrophils, and macrophages in liver, as well as intracellular staining for TNF-α, IFN-γ, and MCP-1 in liver cell populations were determined by flow cytometry. Control mice subjected to HS/T exhibited end organ damage manifested by marked increases in circulating ALT, AST, and MCP-1 levels, as well as histologic evidence of hepatic necrosis and gut injury. Although NK1.1 + cell-depleted mice exhibited a similar degree of organ damage as nondepleted animals at 6 h, NK1.1 + cell depletion resulted in marked suppression of both liver and gut injury by 24 h after HS/T. These findings indicate that NK1.1 + cells contribute to the persistence of inflammation leading to end organ damage in the liver and gut., (© Society for Leukocyte Biology.)

Histone post-translational modifications play vital roles in a variety of nuclear processes, including DNA repair. It has been previously shown that histone H3K79 methylation is important for the cellular response to DNA damage caused by ultraviolet (UV) radiation, with evidence that specific methylation states play distinct roles in UV repair. Here, we report that H3K79 methylation is reduced in response to UV exposure in Saccharomyces cerevisiae This reduction is specific to the dimethylated state, as trimethylation levels are minimally altered by UV exposure. Inhibition of this reduction has a deleterious effect on UV-induced sister chromatid exchange, suggesting that H3K79 dimethylation levels play a regulatory role in UV repair. Further evidence implicates an additional role for H3K79 dimethylation levels in error-free translesion synthesis, but not in UV-induced G1/S checkpoint activation or double-stranded break repair. Additionally, we find that H3K79 dimethylation levels are influenced by acetylatable lysines on the histone H4 N-terminal tail, which are hyperacetylated in response to UV exposure. Preclusion of H4 acetylation prevents UV-induced reduction of H3K79 dimethylation, and similarly has a negative effect on UV-induced sister chromatid exchange. These results point to the existence of a novel histone crosstalk pathway that is important for the regulation of UV-induced DNA damage repair., (Copyright © 2017 by the Genetics Society of America.)

We developed a fully automated procedure for analyzing data from LED pulses and multilevel bead sets to evaluate backgrounds and photoelectron scales of cytometer fluorescence channels. The method improves on previous formulations by fitting a full quadratic model with appropriate weighting and by providing standard errors and peak residuals as well as the fitted parameters themselves. Here we describe the details of the methods and procedures involved and present a set of illustrations and test cases that demonstrate the consistency and reliability of the results. The automated analysis and fitting procedure is generally quite successful in providing good estimates of the Spe (statistical photoelectron) scales and backgrounds for all the fluorescence channels on instruments with good linearity. The precision of the results obtained from LED data is almost always better than that from multilevel bead data, but the bead procedure is easy to carry out and provides results good enough for most purposes. Including standard errors on the fitted parameters is important for understanding the uncertainty in the values of interest. The weighted residuals give information about how well the data fits the model, and particularly high residuals indicate bad data points. Known photoelectron scales and measurement channel backgrounds make it possible to estimate the precision of measurements at different signal levels and the effects of compensated spectral overlap on measurement quality. Combining this information with measurements of standard samples carrying dyes of biological interest, we can make accurate comparisons of dye sensitivity among different instruments. Our method is freely available through the R/Bioconductor package flowQB. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

One pressing need in the area of public health is timely, accurate, and complete reporting of deaths and the conditions leading up to them. Fast Healthcare Interoperability Resources (FHIR) is a new HL7 interoperability standard for electronic health record (EHR), while Sustainable Medical Applications and Reusable Technologies (SMART)-on-FHIR enables third-party app development that can work "out of the box". This research demonstrates the feasibility of developing SMART-on-FHIR applications to enable medical professionals to perform timely and accurate death reporting within multiple different jurisdictions of US. We explored how the information on a standard certificate of death can be mapped to resources defined in the FHIR standard (DSTU2). We also demonstrated analytics for potentially improving the accuracy and completeness of mortality reporting data.

Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions and cautions are provided for common terms to alert the reader to critical distinctions in meaning. This unit presents extensive background on particle types and cautions and describes practical aspects of methods to standardize and calibrate instruments. Procedures are provided to characterize performance in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity. Finally, suggestions follow for analyzing particles used for calibration. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection.

Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument-independent across all fluorescence channels. This unit details a procedure for quantifying surface and intracellular biomarkers by calibrating the output of a multicolor flow cytometer in units of antibody bound per cell (ABC). The procedure includes (1) quality control of the flow cytometer, (2) fluorescence intensity calibration using hard dyed microspheres assigned with fluorescence intensity values, (3) compensation for fluorescence spillover between adjacent fluorescence channels, and (4) application of a biological reference calibrator to establish an ABC scale. The unit also points out current efforts for quantifying biomarkers in a manner that is independent of instrument platforms and reagent differences., (Copyright © 2016 John Wiley & Sons, Inc.)

Photoacoustic tomography has emerged as a promising alternative to MRI and X-ray scans in the clinical setting due to its ability to afford high-resolution images at depths in the cm range. However, its utility has not been established in the basic research arena owing to a lack of analyte-specific photoacoustic probes. To this end, we have developed acoustogenic probes for copper(II)-1 and -2 (APC-1 and APC-2, a water-soluble congener) for the chemoselective visualization of Cu(II), a metal ion which plays a crucial role in chronic neurological disorders such as Alzheimer's disease. To detect Cu(II), we have equipped both APCs with a 2-picolinic ester sensing module that is readily hydrolyzed in the presence of Cu(II) but not by other divalent metal ions. Additionally, we designed APC-1 and APC-2 explicitly for ratiometric photoacoustic imaging by using an aza-BODIPY dye scaffold exhibiting two spectrally resolved NIR absorbance bands which correspond to the 2-picolinic ester capped and uncapped phenoxide forms. The normalized ratiometric turn-on responses for APC-1 and APC-2 were 89- and 101-fold, respectively.

Objective: This study aims to compare radiofrequency (RF) heat lesion size across electrodes and generator settings available for interventional pain management., Methods: Monopolar lesions are generated ex vivo in animal tissue using sharp cannulae with tip diameters 23, 22, 20, 18, 16 gauge; tip lengths 5, 6, 10, 15 mm; set temperatures 60, 70, 80, 90°C; set times 1, 1.5, 2, 3, 5, 10 minutes. Lesions are generated using the RRE electrode, cooled RF, and parallel-tip bipolar RF for comparison. Lesion sizes are assessed by automated photographic temperature inference from over 400 lesions, using multiple lesions per configuration., Results: Monopolar lesion width and length increase with each factor (P < 0.001). Increasing cannula diameter from 22 to 16 gauge increases average lesion width 58-65% (3-4 mm) at 80°C and 2 minutes. Increasing temperature from 60°C to 90°C increases lesion width 108-152% at 2 minutes. Although dimensions grow most rapidly over the first minute, average lesion width is 11-20% larger at 2 minutes, and 23-32% larger at 3 minutes, compared with 1 minute. Lesion length extends distal and proximal to the tip, and exceeds tip length by 1-5 mm at 80°C and 2 minutes. Conventional 16 gauge cannulae at 80-90°C for 2-3 minutes generate lesions of average width similar to that produced by the cooled RF configuration proposed for sacroiliac joint denervation. Bipolar RF between parallel cannulae produces a rounded brick-shaped lesion of comparable shape to three sequential monopolar lesions generated using the same cannulae and generator settings., Conclusions: Tip gauge, tip length, temperature, and time substantially affect RF lesion size., (Wiley Periodicals, Inc.)

Chronic rejection is the primary cause of long-term failure of transplanted organs and is often viewed as an antibody-dependent process. Chronic rejection, however, is also observed in mice and humans with no detectable circulating alloantibodies, suggesting that antibody-independent pathways may also contribute to pathogenesis of transplant rejection. Here, we have provided direct evidence that chronic rejection of vascularized heart allografts occurs in the complete absence of antibodies, but requires the presence of B cells. Mice that were deficient for antibodies but not B cells experienced the same chronic allograft vasculopathy (CAV), which is a pathognomonic feature of chronic rejection, as WT mice; however, mice that were deficient for both B cells and antibodies were protected from CAV. B cells contributed to CAV by supporting splenic lymphoid architecture, T cell cytokine production, and infiltration of T cells into graft vessels. In chimeric mice, in which B cells were present but could not present antigen, both T cell responses and CAV were markedly reduced. These findings establish that chronic rejection can occur in the complete absence of antibodies and that B cells contribute to this process by supporting T cell responses through antigen presentation and maintenance of lymphoid architecture.

Computational analysis of histopathological whole slide images (WSIs) has emerged as a potential means for improving cancer diagnosis and prognosis. However, an open issue relating to the automated processing of WSIs is the identification of biological regions such as tumor, stroma, and necrotic tissue on the slide. We develop a method for classifying WSI portions (512x512-pixel tiles) into biological regions by (1) extracting a set of 461 image features from each WSI tile, (2) optimizing tile-level prediction models using nested cross-validation on a small (600 tile) manually annotated tile-level training set, and (3) validating the models against a much larger (1.7x10 6 tile) data set for which ground truth was available on the whole-slide level. We calculated the predicted prevalence of each tissue region and compared this prevalence to the ground truth prevalence for each image in an independent validation set. Results show significant correlation between the predicted (using automated system) and reported biological region prevalences with p < 0.001 for eight of nine cases considered.

Automated processing of digital histopathology slides has the potential to streamline patient care and provide new tools for cancer classification and grading. Before automatic analysis is possible, quality control procedures are applied to ensure that each image can be read consistently. One important quality control step is color normalization of the slide image, which adjusts for color variances (batch-effects) caused by differences in stain preparation and image acquisition equipment. Color batch-effects affect color-based features and reduce the performance of supervised color segmentation algorithms on images acquired separately. To identify an optimal normalization technique for histopathological color segmentation applications, five color normalization algorithms were compared in this study using 204 images from four image batches. Among the normalization methods, two global color normalization methods normalized colors from all stain simultaneously and three stain color normalization methods normalized colors from individual stains extracted using color deconvolution. Stain color normalization methods performed significantly better than global color normalization methods in 11 of 12 cross-batch experiments (p<;0.05). Specifically, the stain color normalization method using k-means clustering was found to be the best choice because of high stain segmentation accuracy and low computational complexity.

Background: Toll-like receptors (TLRs) detect endogenous ligands released after trauma and contribute to the proinflammatory response to injury. Posttraumatic mortality correlates with the extent of the immunoinflammatory response to injury that is composed of a complex regulation of innate and adaptive immune responses. Although TLRs are known to modulate innate immune responses, their role in the suppression of lymphocyte responses following traumatic tissue injury is unclear., Methods: This study used a murine model of severe peripheral tissue injury, involving muscle crush injury and injection of fracture components, to evaluate the roles of TLR2, TLR4, and TLR9 in the early and delayed immunoinflammatory phenotype. Posttraumatic immune dysfunction was measured in our trauma model using the following parameters: ex vivo splenocyte proliferation, TH1 cytokine release, and iNOS (inducible nitric oxide synthase) induction within splenic myeloid-derived suppressor cells. Systemic inflammation and liver damage were determined by circulating interleukin 6 levels and hepatocellular injury., Results: Suppression of splenocyte responses after injury was dependent on TLR4 and TLR9 signaling as was posttraumatic iNOS upregulation in splenic myeloid-derived suppressor cells. TLR2 was found to have only a partial role through contribution to inhibition of splenocyte proliferation. This study also reveals the involvement of TLR2 and TLR4 in the initial systemic inflammatory response to traumatic tissue injury; however, this response was found to be TLR9 independent., Conclusion: These findings demonstrate the previously unidentified role of TLR2, TLR4, and TLR9 in the T cell-associated immune dysfunction following traumatic tissue injury. Importantly, this study also illustrates that TLRs play differing and selective roles in both the initial proinflammatory response and adaptive immune response after trauma. Furthermore, results in TLR9-deficient mice establish that the upregulation of early proinflammatory markers do not always correlate with the extent of sustained immune dysfunction. This suggests potential for targeted therapies that could limit immune dysfunction through selective inhibition of receptor function following injury.

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Inbreeding depression and lack of genetic diversity in inbred mice could mask unappreciated causes of graft failure or remove barriers to tolerance induction. To test these possibilities, we performed heart transplantation between outbred or inbred mice. Unlike untreated inbred mice in which all allografts were rejected acutely (6-16 days posttransplantation), untreated outbred mice had heterogeneous outcomes, with grafts failing early (<4 days posttransplantation), acutely (6-24 days) or undergoing chronic rejection (>75 days). Blocking T cell costimulation induced long-term graft acceptance in both inbred and outbred mice, but did not prevent the early graft failure observed in the latter. Further investigation of this early phenotype established that it is dependent on the donor, and not the recipient, being outbred and that it is characterized by hemorrhagic necrosis and neutrophilic vasculitis in the graft without preformed, high titer antidonor antibodies in the recipient. Complement or neutrophil depletion prevented early failure of outbred grafts, whereas transplanting CD73-deficient inbred hearts, which are highly susceptible to ischemia-reperfusion injury, recapitulated the early phenotype. Therefore, outbred mice could provide broader insight into donor and recipient determinants of allograft outcomes but their hybrid vigor and genetic diversity do not constitute a uniform barrier to tolerance induction., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue™), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads., (Copyright © 2012 International Society for Advancement of Cytometry.)

Music has been shown to be a useful adjunct for many forms of exercise and has been observed to improve athletic performance in some settings. Nonetheless, because of the limited availability of practical applications of sound conduction in water, there are few studies of the effects of music on swimming athletes. The SwiMP3 is a novel device that uses bone conduction as a method to circumvent the obstacles to transmitting high fidelity sound in an aquatic environment. Thus, we studied the influence of music on swimming performance and enjoyment using the SwiMP3. Twenty-four competitive swimmers participated in a randomized crossover design study in which they completed timed swimming trials with and without the use of music delivered via bone conduction with the SwiMP3. Each participant swam four 50-m trials and one 800-m trial and then completed a physical enjoyment survey. Statistically significant improvements in swimming performance times were found in both the 50-m (0.32 seconds; p = 0.013) and 800-m (6.5 seconds; p = 0.031) trials with music using the SwiMP3. There was no significant improvement in physical enjoyment with the device as measured by a validated assessment tool. Bone-conducted music appears to have a salutary influence on swimming performance in a practice environment among competitive adult swimmers.

Objective: To assess the use of an ultra-high-resolution (UHR) optical coherence tomography (OCT) as an adjuvant diagnostic tool in distinguishing ocular surface squamous neoplasia (OSSN) and pterygia., Design: Prospective case series., Participants: Thirty-four eyes of 34 patients with conjunctival lesions clinically suspicious for OSSN or pterygia., Methods: All patients were photographed and then imaged with a custom-built UHR OCT device. Subsequently, each patient underwent excisional or incisional biopsy with histopathologic diagnosis., Main Outcome Measures: Comparison of preoperative UHR OCT images and the corresponding histopathologic specimen; comparison of epithelial thickness between the 2 groups as measured by UHR OCT., Results: Preoperative UHR OCT images of OSSN demonstrated similarities to the histopathologic specimens. Both optical and pathologic specimens showed a thickened layer of epithelium, often with an abrupt transition from normal to neoplastic tissue. Likewise, preoperative UHR OCT images of patients with pterygia were well correlated with the histopathologic specimens. As opposed to OSSN, both UHR OCT and pathologic images of pterygia demonstrated a normal thin epithelium, with underlying thickening of the subepithelial mucosal layers. Differences in the measured epithelial thickness on UHR OCT between OSSN and pterygia were statistically significant, with an average epithelial thickness of 346 μm (standard deviation [SD], 167) in OSSN patients and 101 μm (SD, 22) in pterygium patients (P<0.001). By receiver operating characteristic curve, the sensitivity and specificity of UHR OCT for differentiating between OSSN and pterygia was found to be 94% and 100%, respectively, using a cutoff value of 142 μm., Conclusions: Ultra-high-resolution OCT may show promise as a noninvasive diagnostic tool to evaluate ocular surface lesions. In addition to a statistically significant difference in epithelial thickness, a significant degree of morphologic correlation with the histopathologic results demonstrates its potential in evaluating ocular surface squamous neoplasia and pterygia., (Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Background: Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here, we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T-cell migration to vascularized allografts., Methods: CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3 and wildtype B6 mice and cotransferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hr later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues., Results: We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype versus CXCR3 CD4 and CD8 memory T cells to allografts could be detected in wildtype or aly/aly hosts. In the latter, wildtype and CXCR3 memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a chemokine receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3 memory T cells., Conclusions: CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell-homing to transplanted organs may be distinct from those required for their migration to sites of infection., (© 2011 by Lippincott Williams & Wilkins)