Single-cell morphodynamical trajectories enable prediction of gene expression accompanying cell state change.

Extracellular signals induce changes to molecular programs that modulate multiple cellular phenotypes, including proliferation, motility, and differentiation status. The connection between dynamically adapting phenotypic states and the molecular programs that define them is not well understood. Here we develop data-driven models of single-cell phenotypic responses to extracellular stimuli by linking gene transcription levels to "morphodynamics" - changes in cell morphology and motility observable in time-lapse image data. We adopt a dynamics-first view of cell state by grouping single-cell trajectories into states with shared morphodynamic responses. The single-cell trajectories enable development of a first-of-its-kind computational approach to map live-cell dynamics to snapshot gene transcript levels, which we term MMIST, Molecular and Morphodynamics-Integrated Single-cell Trajectories. The key conceptual advance of MMIST is that cell behavior can be quantified based on dynamically defined states and that extracellular signals alter the overall distribution of cell states by altering rates of switching between states. We find a cell state landscape that is bound by epithelial and mesenchymal endpoints, with distinct sequences of epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) intermediates. The analysis yields predictions for gene expression changes consistent with curated EMT gene sets and provides a prediction of thousands of RNA transcripts through extracellular signal-induced EMT and MET with near-continuous time resolution. The MMIST framework leverages true single-cell dynamical behavior to generate molecular-level omics inferences and is broadly applicable to other biological domains, time-lapse imaging approaches and molecular snapshot data.

Low-power laser manufacturing of copper tracks on 3D printed geometry using liquid polyimide coating.

Silver nanoparticle photoreduction synthesis by direct laser writing is a process that enables copper micro-track production on very specific polymers. However, some important 3D printing polymers, such as acrylonitrile butadiene styrene (ABS) and acrylates, do not accept this treatment on their surface. This work presents an approach to produce copper microcircuitry on 3D substrates from these materials by using direct laser writing at low power (32 mW CW diode laser). We show that by coating a thin layer of polyimide (PI) on a 3D-printed geometry, followed by a sequence of chemical treatments and low-power laser-induced photoreduction, copper tracks can be produced using silver as catalyst. The surface chemistry of the layer through the different stages of the process is monitored by FTIR and X-ray photoelectron spectroscopy. The copper tracks are selectively grown on the laser-patterned areas by electroless copper deposition, with conductivity (1.2 ± 0.7) × 107 S m-1 and a width as small as 28 µm. The patterns can be written on 3D structures and even inside cavities. The technique is demonstrated by integrating different circuits, including a LED circuit on 3D printed photopolymer acrylate and a perovskite solar cell on an ABS 3D curved geometry.

A multi-omic analysis of MCF10A cells provides a resource for integrative assessment of ligand-mediated molecular and phenotypic responses.

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.

Continuous Monitoring Using a Wearable Device Detects Activity-Induced Heart Rate Changes After Administration of Amphetamine.

Wearable digital devices offer potential advantages over traditional methods for the collection of health-related information, including continuous collection of dense data while study subjects are ambulatory or in remote settings. We assessed the utility of collecting continuous actigraphy and cardiac monitoring by deploying two US Food and Drug Administration (FDA) 510(k)-cleared devices in a phase I clinical trial of a novel compound, which included the use of an amphetamine challenge. The Phillips Actiwatch Spectrum Pro (Actiwatch) was used to assess mobility and sleep. The Preventice BodyGuardian (BodyGuardian) was used for monitoring heart rate (HR) and respiratory rate (RR), via single-lead electrocardiogram (ECG) recordings, together with physical activity. We measured data collection rates, compared device readouts with conventional measures, and monitored changes in HR measures during the amphetamine challenge. Completeness of data collection was good for the Actiwatch (96%) and lower for the BodyGuardian (80%). A good correlation was observed between device and in-clinic measures for HR (r = 0.99; P < 0.001), but was poor for RR (r = 0.39; P = 0.004). Manual reviews of selected ECG strips corresponding to HR measures below, within, and above the normal range were consistent with BodyGuardian measurements. The BodyGuardian device detected clear HR responses after amphetamine administration while subjects were physically active, whereas conventional measures collected at predefined timepoints while subjects were resting and supine did not. Wearable digital technology shows promise for monitoring human subjects for physiologic changes and pharmacologic responses, although fit-for-purpose evaluation and validation continues to be important prior to the wider deployment of these devices.

Evaluation of Wearable Digital Devices in a Phase I Clinical Trial.

We assessed the performance of two US Food and Drug Administration (FDA) 510(k)-cleared wearable digital devices and the operational feasibility of deploying them to augment data collection in a 10-day residential phase I clinical trial. The Phillips Actiwatch Spectrum Pro (Actiwatch) was used to assess mobility and sleep, and the Vitalconnect HealthPatch MD (HealthPatch) was used for monitoring heart rate (HR), respiratory rate (RR), and surface skin temperature (ST). We measured data collection rates, compared device readouts with anticipated readings and conventional in-clinic measures, investigated data limitations, and assessed user acceptability. Six of nine study participants consented; completeness of data collection was adequate (> 90% for four of six subjects). A good correlation was observed between the HealthPatch device derived and in-clinic measures for HR (Pearson r = 0.71; P = 2.2e-16) but this was poor for RR (r = 0.08; P = 0.44) and ST (r = 0.14; P = 0.14). Manual review of electrocardiogram strips recorded during reported episodes of tachycardia > 180 beats/min showed that these were artefacts. The HealthPatch was judged to be not fit-for-purpose because of artefacts and the need for time-consuming manual review. The Actiwatch device was suitable for monitoring mobility, collecting derived sleep data, and facilitating the interpretation of vital sign data. These results suggest the need for fit-for-purpose evaluation of wearable devices prior to their deployment in drug development studies.

Modifications of Callender's Classification of Uveal Melanoma at the Armed Forces Institute of Pathology.

One hundred well-documented cases of uveal melanoma accessioned at the Armed Forces Institute of Pathology before 1970 were reviewed and reclassified to identify changes made in the Callender classification. We compared the new classification with the original classification to determine the effect of the changes on the prediction of outcome for the patient after enucleation. Staff pathologists had originally classified 52 of the 100 cases as spindle-cell type melanoma. Only 31 of the 100 cases were reclassified as spindle-cell types (two spindle-cell nevi and 29 spindle-cell melanomas). Tumors classified as mixed-cell type were further subdivided into groups based on the percentage and size of the epithelioid cells. Tumors formerly classified as spindle-cell type that contained small or rare epithelioid cells were reclassified as mixed-cell type. This improved the prediction of outcome for the patient. We found that nucleolar size and pleomorphism are important variables that should be considered in the classification of uveal melanomas.

Powering ex vivo tissue models in microfluidic systems.

This Frontiers review analyzes the rapidly growing microfluidic strategies that have been employed in attempts to create physio relevant 'organ-on-chip' models using primary tissue removed from a body (human or animal). Tissue harvested immediately from an organism, and cultured under artificial conditions is referred to as ex vivo tissue. The use of primary (organotypic) tissue offers unique benefits over traditional cell culture experiments, and microfluidic technology can be used to further exploit these advantages. Defining the utility of particular models, determining necessary constituents for acceptable modeling of in vivo physiology, and describing the role of microfluidic systems in tissue modeling processes is paramount to the future of organotypic models ex vivo. Virtually all tissues within the body are characterized by a large diversity of cellular composition, morphology, and blood supply (e.g., nutrient needs including oxygen). Microfluidic technology can provide a means to help maintain tissue in more physiologically relevant environments, for tissue relevant time-frames (e.g., matching the natural rates of cell turnover), and at in vivo oxygen tensions that can be controlled within modern microfluidic culture systems. Models for ex vivo tissues continue to emerge and grow in efficacy as mimics of in vivo physiology. This review addresses developments in microfluidic devices for the study of tissues ex vivo that can serve as an important bridge to translational value.

Prevalence of radiographic findings in individuals with chronic low back pain screened for a randomized controlled trial: secondary analysis and clinical implications.

OBJECTIVE: The purpose of this study is to measure the prevalence of graded disc degeneration, spondylolisthesis, transitional segmentation, and the distribution of sacral slope in patients 21 to 65 years of age with chronic low back pain (CLBP). METHODS: This retrospective study analyzed 247 digital lumbar radiographic series obtained during a randomized controlled trial of chiropractic patients with CLBP. Chronic low back pain was defined as pain in the low back lasting 12 weeks or longer. Radiographic findings of disc degeneration, spondylolisthesis, and lumbosacral transitional segmentation were graded by 2 authors using established classification criteria. Sacral slope was measured with a digital tool contained within imaging software. RESULTS: Lumbosacral transitional segments graded I to IV (Castellvi classification) were present in 14% of cases. Lumbar disc degeneration was most prevalent at L3-4 (49%), followed by L4-5 (42%), L2-3 (41%), L5-S1 (37%), and L1-2 (29%). Isthmic spondylolisthesis was present in 5% of cases, with L5 the most common location. Degenerative spondylolisthesis demonstrated a prevalence of 18%, most commonly occurring at L4. The prevalence of degenerative spondylolisthesis was 51% for women aged 50 to 59 years and 24% for men in the same age range. CONCLUSIONS: Moderate-severe disc degeneration, multilevel disc narrowing, and degenerative spondylolisthesis were common in individuals with CLBP with age more than 40 years. Isthmic spondylolisthesis was not more prevalent than what has been reported in other populations. Transitional segmentation was identified in a minority of participants, with some of these exhibiting accessory joints or fusion. Mean sacral slope in individuals with CLBP was not substantially different from mean slopes reported in other populations.

Volume-outcome relationship in revision hip replacement - Results from a low volume hospital.

INTRODUCTION: Mortality and morbidity are both increased during revision hip surgery. Higher hospital procedure volumes have been associated with lower rates of mortality and/or complications according to some reports - the "practice makes perfect" hypothesis. AIM: The aim of the study was to test "practice makes perfect; hypothesis with regards to revision hip surgery at our low volume hospital. METHODS: This is a retrospective study of all the patients who underwent revision hip arthroplasty under the care of the senior author between February 2002 and January 2006. Data was collected about the 30-day and one-year mortality, post-operative complications like deep vein thrombosis (DVT), pulmonary embolism (PE), superficial or deep wound infections, dislocations, and the Oxford hip score. RESULTS: The rate of revision hip surgery carried out in our hospital was 6.25 per year. There was no 30-day mortality, stroke within 3 months, dislocations within one year, re-admission within one month, one-year mortality and deep infections within one year. The final outcome after revision hip surgery, based on Oxford questionnaire, showed that 72% had an excellent outcome and 8% had poor outcome. CONCLUSION: Volume and outcome relationship may not contribute towards the final outcome when individual surgeons and hospitals are considered. Good general hospital care can greatly affect the health outcome for a particular procedure. Strategies aimed at improving the general hospital care may benefit the patients as much as volume based regionalization.

Diagnostic radiology in the tropics: technical considerations.

An estimated two thirds of the world's population is currently without access to diagnostic radiology services, and most of them live in resource-limited tropical regions with harsh environments. Most patients are diagnosed and treated in poorly equipped government-funded hospitals and clinics that have insufficiently trained staff and are barely operational. Any available imaging equipment is likely to be functioning suboptimally and be poorly maintained. The root of the problem is usually a lack of know-how and a quality culture, combined with insufficient basic equipment and infrastructure. Radiological imaging is an essential aspect of primary care and used in the critical diagnosis and management of trauma, tuberculosis, pneumonia, acquired immunodeficiency syndrome, cancer, and other respiratory and abdominal diseases. Considerations such as quality management and infrastructure, personnel, equipment, and radiation protection and safety are important to ensure the proper functioning and rational use of a diagnostic radiology facility in the tropics.

An evaluation of the experience of integrated GP training placements in the Kent, Surrey and Sussex Deanery.

Innovative/integrated training posts (ITPs) are an increasingly established feature of vocational training for GPStRs. They aim to extend exposure to primary care in the early years of specialty training. To date, there has been little evaluation of the post and no studies investigating how ITPs are variously structured. This research of an ITP scheme operated by the Kent, Surrey and Sussex (KSS) Deanery is the first comprehensive study of ITPs across a whole deanery. The registrars on integrated placement, their GP trainers and hospital/community-based education supervisors took part in the study reported here. Participants reported integrated training to be an effective model to develop GPStRs. Early and extended exposure and orientation to primary care were seen as key benefits. The secondary placement component provided relevant specialist training applicable to primary care. ITPs provided GPStRs with relevant learning to support the requirements of the GP curriculum. Problems identified by previous research such as integration within teams were not reported as an issue in this study. Continuity issues were, however, highlighted. Reporting on the different ITP models utilised within the Deanery, we find that GPStRs with greater exposure to primary care perceive the programme as being more effective than those with less exposure.

Impacts of off-road vehicles (ORVs) on macrobenthic assemblages on sandy beaches.

Sandy beaches are the prime sites for human recreation and underpin many coastal economies and developments. In many coastal areas worldwide, beach recreation relies on the use of off-road vehicles (ORVs) driven on the shore. Yet, the use of ORVs is not universally embraced due to social conflicts with other beach user groups and putative environmental consequences of vehicle traffic on sandy shores. Such ecological impacts of ORVs are, however, poorly understood for endobenthic invertebrates of the intertidal zone seawards of the dunes. Consequently, this study quantified the degree to which assemblages of intertidal beach invertebrates are affected by traffic. The study design comprised a series of temporally replicated spatial contrasts between two reference sites (no ORVs) and two beaches with heavy ORV traffic (in excess of 250,000 vehicles per year) located in SouthEast Queensland, Australia. Macrobenthic assemblages on ORV-impacted beaches had significantly fewer species at substantially reduced densities, resulting in marked shifts in community composition and structure. These shifts were particularly strong on the middle and upper shore where vehicle traffic was concentrated. Strong effects of ORVs were detectable in all seasons, but increased towards the summer months as a result of heavier traffic volumes. This study provides clear evidence that ORVs can have substantial impacts on sandy beach invertebrates that are manifested throughout the whole community. Demonstrating such an ecological impact caused by a single type of human use poses a formidable challenge to management, which needs to develop multi-faceted approaches to balance environmental, social, cultural, and economic arguments in the use of sandy shores, including management of "beach traffic."

Diagnosis of limited ophthalmic Wegener granulomatosis: distinctive pathologic features with ANCA test confirmation.

PURPOSE: To describe the clinical and histopathologic finding of very limited ophthalmic Wegener granulomatosis (WG). METHODS: Thirteen patients with scleritis, orbitopathy, episcleritis, and panuveitis were studied. They presented without evidence of lung or kidney disease, though eight had sinus involvement. We reviewed the biopsies for histopathologic findings consistent with WG, and tested for antineutrophil cytoplasmic antibodies antineutrophil cytoplasmic antibody (ANCA). RESULTS: WG was suggested by granulomatous foci, collagen necrosis, neutrophils/nuclear dust, plasma cells and infiltrating eosinophils. Granular degeneration of the interstitial collagen; mummification of the collagen with disappearance of fibroblastic nuclei; and a polymorphous infiltrate exhibiting plasma cells, lymphocytes, neutrophils, and eosinophils within the epithelioid granulomas should suggest the diagnosis. ANCA test results supported the diagnosis of WG in all cases. CONCLUSION: The described histologic characteristics are highly suggestive of WG. These findings along with clinical or laboratory findings, allow the diagnosis of very limited ophthalmic WG in the absence of systemic involvement.

Seasonal variation in pigeon body weight and delayed matching-to-sample performance.

The weights of 5 pigeons with free access to food, monitored over 3 calendar years in the laboratory, were found to fluctuate with season. All pigeons were at their heaviest in the winter and were lightest in the summer. Five different pigeons performed a standard delayed matching-to-sample task for 44 weeks from January to November. Their weights were held at 85% of their summer free-feeding weights, making their predicted deprivation level higher in the winter relative to predicted winter free-feeding weights. Slopes of forgetting functions fit to weekly response totals for each pigeon were shallower in winter, showing an improvement in accuracy with longer delays. Thus, delayed matching-to-sample performance may have been affected by the practice of maintaining the pigeons at a constant body weight throughout the calendar year.

Development of an evidence-based application and rubric for evaluating applicants' qualifications for promotion to professor.

OBJECTIVE: Every promotion committee is challenged by the need to make value judgments on the quantity and quality of peer work. Decisions based upon subjective assessments may not do justice to the applicant's or the institution's needs. The purpose of this article is to (1) describe the process a college promotion committee used to increase the objectivity brought to this activity, (2) present the tools developed that facilitated the collection and evaluation of faculty work, and (3) describe their usage in a promotion cycle. METHODS: The Professor Promotion Committee met weekly for 6 months engaging in lengthy and comprehensive discourse to capture the breadth of scholastic and service activities normally engaged in by faculty. RESULTS: The committee's work culminated in the development of 4 electronic applications soliciting specific evidence aligned with faculty work and 1 scoring rubric tied directly to the e-applications. More than 55 activities were identified, divided into 4 levels of accomplishment using quantitative and qualitative criteria and weighted according to their centrality to faculty work and relative importance to the institution. Each activity was assigned to one of the following categories: teaching/academic support, scholarship/research, service, and professional development. A consensus score based upon the evidence was used to generate promotion discussions. CONCLUSIONS: The committee believes the online application aids applicants in recognizing the breadth and depth of promotable work. It provides them the opportunity to structure their work in ways that enhance their chances for promotion. The evidence-based rubric helps to reduce subjectivity in the evaluation process.

Bilateral diffuse melanocytic proliferation associated with ovarian carcinoma and metastatic malignant amelanotic melanoma.

PURPOSE: To report a case of metastatic malignant amelanotic melanoma to the skin from a patient diagnosed with bilateral diffuse uveal melanocytic proliferation (BDUMP). This dermatological finding is a unique phenomenon associated with BDUMP. DESIGN: Retrospective case report. METHODS: We studied the case of a 66-year-old Caucasian woman with gradual onset of blurred vision in her right eye followed by her left eye. She had previously been diagnosed with ovarian carcinoma, and findings of funduscopic examinations were consistent with BDUMP. Metastatic examination revealed no evidence of liver involvement. Clinical and histopathological examinations of both enucleated eyes were consistent with BDUMP. RESULTS: The hematoxylin and eosin, S-100, and HMB-45 stains were consistent with metastatic malignant amelanotic melanoma to the skin. CONCLUSIONS: Although believed to have a low potential for metastasis, patients should be monitored and evaluated regularly to detect any new lesions not associated with their primary inciting carcinoma.

Correlation of comparative genomic hybridization results of 100 archival uveal melanomas with patient survival.

Comparative genomic hybridization (CGH) was used to elucidate DNA sequence copy number imbalances in 100 archival formalin-fixed, paraffin-embedded (FFPE) uveal melanoma cases. Of these 100 cases, 51 were from patients who survived >or=9 years post diagnosis without evidence of metastasis; the remaining 49 patients died from metastatic disease. Viable probe was generated from 82 of the 100 cases, allowing correlation of CGH findings with survival for all but 18 cases. Copy number imbalances revealed by CGH were tested for univariate prognostic significance. The most powerful predictor of a poor prognosis was gain of 18q11.2, which was subsequently compared with other significant chromosomal regions, as well as histologic and clinical factors, in a multivariate analysis. There was also evidence of differential X chromosome involvement in the survival correlations between male and female cases, which may be of significance to prognosis. This large-scale CGH analysis of archival material is intended to direct further gene-specific study of malignancy in uveal melanoma.

Molecular cytogenetic evaluation of 10 uveal melanoma cell lines.

Uveal melanoma is the most common intraocular tumor in adults and often results in unilateral blindness and/or death. Previous cytogenetic characterizations of this tumor consistently revealed chromosomal abnormalities involving chromosomes 3, 6, and 8; reports of other abnormalities vary in frequency. We defined cytogenetic abnormalities of this tumor using complementary in situ hybridization techniques on 10 uveal melanoma cell lines. Synthesis of comparative genomic hybridization (CGH) and spectral karyotyping (SKY) results revealed that chromosomal rearrangement is involved in DNA sequence copy number abnormalities throughout the genome, but monosomy 3 was not found. Monosomy 3 is thought to be a significant prognostic indicator, so its absence was investigated further. Fluorescence in situ hybridization (FISH) for chromosome 3 revealed approximately 1 centromere signal per cell, but probes for 3p and 3q revealed multiple telomere signals per cell, suggesting chromosomal rearrangement without whole-chromosome loss. Based on combined CGH, SKY, and FISH data, we propose that chromosome 3 is more frequently involved in chromosomal rearrangements than whole-chromosome loss in uveal melanoma. Future approaches should be designed to confirm and enhance the resolution of regions of imbalance in primary tumors. Once identified, conserved chromosomal alterations that contribute to uveal melanoma may reveal the underlying aspects of uveal melanoma onset, metastasis and resistance to current treatment modalities.