Automated cell tracking using StarDist and TrackMate [version 1; peer review: awaiting peer review]

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images

Automated cell tracking using StarDist and TrackMate

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images. </div

Cerebellar Globular Cells Receive Monoaminergic Excitation and Monosynaptic Inhibition from Purkinje Cells

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control

Within-season changes in habitat use of forest-dwelling boreal bats

Bats utilize forests as roosting sites and feeding areas. However, it has not been documented how bats utilize these habitats in the boreal zone with methods afforded by recent technological advances. Forest structure and management practices can create a variety of three-dimensional habitats for organisms capable of flight, such as bats. Here, we study the presence of boreal bats in a forest forming a mosaic of different age classes, dominant tree species, canopy cover, soil fertility, and other environmental variables, throughout their active season in the summer using passive ultrasound detectors. Our results indicate a preference for mature forest by Eptesicus nilssonii and a pooled set of Myotis bats. Both groups of bats also showed temporal changes in their habitat use regarding forest age. In June and July, both groups occurred more often in mature than young forests, but from August onwards, the difference in occurrence became less evident in Myotis and disappeared completely in E. nilssonii. In addition, E. nilssonii was more often present in forests with low canopy cover, and its occurrence shifted from coniferous forests to deciduous forests during the season. The results reflect the within-season dynamics of bat communities and their ability to utilize different types of forest as environmental conditions change. Yet, the results most importantly emphasize the importance of mature forests to bat diversity and the need to conserve such environments in the boreal zone

A turbulent decade for NSAIDs: update on current concepts of classification, epidemiology, comparative efficacy, and toxicity

Non-steroidal anti-inflammatory drugs (NSAIDs) represent a diverse class of drugs and are among the most commonly used analgesics for arthritic pain worldwide, though long-term use is associated with a spectrum of adverse effects. The introduction of cyclooxygenase-2-selective NSAIDs early in the last decade offered an alternative to traditional NSAIDs with similar efficacy and improved gastrointestinal tolerability; however, emerging concerns about cardiovascular safety resulted in the withdrawal of two agents (rofecoxib and valdecoxib) in the mid-2000s and, subsequently, in an overall reduction in NSAID use. It is now understood that all NSAIDs are associated with some varying degree of gastrointestinal and cardiovascular risk. Guidelines still recommend their use, but little is known of how patients use these agents. While strategies and guidelines aimed at reducing NSAID-associated complications exist, there is a need for evidence-based algorithms combining cardiovascular and gastrointestinal factors that can be used to aid treatment decisions at an individual patient level

Patients with Endoscopically Visible Polypoid Adenomatous Lesions Within the Extent of Ulcerative Colitis Have an Increased Risk of Colorectal Cancer Despite Endoscopic Resection.

OBJECTIVES: Ulcerative colitis (UC) is associated with an increased risk of colorectal cancer (CRC). Few studies have looked at long-term outcomes of endoscopically visible adenomatous lesions removed by endoscopic resection in these patients. We aimed to assess the risk of developing CRC in UC patients with adenomatous lesions that develop within the segment of colitis compared to the remainder of an ulcerative colitis cohort. METHODS: We identified patients with a confirmed histological diagnosis of UC from 1991 to 2004 and noted outcomes till June 2011. The Kaplan-Meier method was used to estimate cumulative probability of subsequent CRC. Factors associated with risk of CRC were assessed in a Cox proportional hazards model. RESULTS: Twenty-nine of 301 patients with UC had adenomatous lesions noted within the segment of colitis. The crude incidence rate of developing colon cancer in patients with UC was 2.45 (95 % CI 1.06-4.83) per 1000 PYD and in those with UC and polypoid adenomas within the extent of inflammation was 11.07 (95 % CI 3.59-25.83) per 1000 PYD. Adjusted hazards ratio of developing CRC on follow-up in UC patients with polypoid dysplastic adenomatous lesions within the extent of inflammation was 4.0 (95 % CI 1.3-12.4). CONCLUSIONS: The risk of developing CRC is significantly higher in UC patients with polypoid adenomatous lesions, within the extent of inflammation, despite endoscopic resection. Patients and physicians should take the increased risk into consideration during follow-up of these patients

Effect of hosts on competition among clones and evidence of differential selection between pathogenic and saprophytic phases in experimental populations of the wheat pathogen Phaeosphaeria nodorum

<p>Abstract</p> <p>Background</p> <p>Monoculture, multi-cropping and wider use of highly resistant cultivars have been proposed as mechanisms to explain the elevated rate of evolution of plant pathogens in agricultural ecosystems. We used a mark-release-recapture experiment with the wheat pathogen <it>Phaeosphaeria nodorum </it>to evaluate the impact of two of these mechanisms on the evolution of a pathogen population. Nine <it>P. nodorum </it>isolates marked with ten microsatellite markers and one minisatellite were released onto five replicated host populations to initiate epidemics of Stagonospora nodorum leaf blotch. The experiment was carried out over two consecutive host growing seasons and two pathogen collections were made during each season.</p> <p>Results</p> <p>A total of 637 pathogen isolates matching the marked inoculants were recovered from inoculated plots over two years. Genetic diversity in the host populations affected the evolution of the corresponding <it>P. nodorum </it>populations. In the cultivar mixture the relative frequencies of inoculants did not change over the course of the experiment and the pathogen exhibited a low variation in selection coefficients.</p> <p>Conclusions</p> <p>Our results support the hypothesis that increasing genetic heterogeneity in host populations may retard the rate of evolution in associated pathogen populations. Our experiment also provides indirect evidence of fitness costs associated with host specialization in <it>P. nodorum </it>as indicated by differential selection during the pathogenic and saprophytic phases.</p

The Extended Learning Curve for Laparoscopic Fundoplication: A Cohort Analysis Of 400 Consecutive Cases

Many studies have looked at the learning curve associated with laparoscopic Nissen fundoplication (LNF) in a given institution. This study looks at the learning curve of a single surgeon with a large cohort of patients over a 10-year period. Prospective data were collected on 400 patients undergoing laparoscopic fundoplication for over 10 years. The patients were grouped consecutively into cohorts of 50 patients. The operating time, the length of postoperative hospital stay, the conversion rate to open operation, the postoperative dilatation rate, and the reoperation rate were analyzed. Results showed that the mean length of operative time decreased from 143 min in the first 50 patients to 86 min in the last 50 patients. The mean postoperative length of hospital stay decreased from 3.7 days initially to 1.2 days latterly. There was a 14% conversion to open operation rate in the first cohort compared with a 2% rate in the last cohort. Fourteen percent of patients required reoperation in the first cohort and 6% in the last cohort. Sixteen percent required postoperative dilatation in the first cohort. None of the last 150 patients required dilatation. In conclusion, laparoscopic fundoplication is a safe and effective operation for patients with gastroesophageal reflux disease. New techniques and better instrumentation were introduced in the early era of LNF. The learning curve, however, continues well beyond the first 20 patients

Cost-effectiveness of six strategies for Helicobacter pylori diagnosis and management in uninvestigated dyspepsia assuming a high resource intensity practice pattern

<p>Abstract</p> <p>Background</p> <p>Initial assessment of dyspepsia often includes noninvasive testing for <it>Helicobacter pylori </it>infection. Commercially available tests vary widely in cost and accuracy. Although there is extensive literature on the cost-effectiveness of <it>H. pylori </it>treatment, there is little information comparing the cost-effectiveness of various currently used, noninvasive testing strategies.</p> <p>Methods</p> <p>A Markov simulation was used to calculate cost per symptom-free year and cost per correct diagnosis. Uncertainty in outcomes was estimated using probabilistic sensitivity analysis.</p> <p>Results</p> <p>Under the baseline assumptions, cost per symptom-free year was $122 for empiric proton pump inhibitor (PPI) trial, and costs for the noninvasive test strategies ranged from$123 (stool antigen) to $129 (IgG/IgA combined serology). Confidence intervals had significant overlap.</p> <p>Conclusions</p> <p>Under our assumptions for how testing for <it>H. pylori </it>infection is employed in United States medical practice, the available noninvasive tests all have similar cost-effectiveness between one another as well as with empiric PPI trial.</p