Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image ...segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.
The International Association for Hospice and Palliative Care developed a consensus-based definition of palliative care (PC) that focuses on the relief of serious health-related suffering, a concept ...put forward by the Lancet Commission Global Access to Palliative Care and Pain Relief.
The main objective of this article is to present the research behind the new definition.
The three-phased consensus process involved health care workers from countries in all income levels. In Phase 1, 38 PC experts evaluated the components of the World Health Organization definition and suggested new/revised ones. In Phase 2, 412 International Association for Hospice and Palliative Care members in 88 countries expressed their level of agreement with the suggested components. In Phase 3, using results from Phase 2, the expert panel developed the definition.
The consensus-based definition is as follows: Palliative care is the active holistic care of individuals across all ages with serious health-related suffering due to severe illness and especially of those near the end of life. It aims to improve the quality of life of patients, their families and their caregivers. The definition includes a number of bullet points with additional details as well as recommendations for governments to reduce barriers to PC.
Participants had significantly different perceptions and interpretations of PC. The greatest challenge faced by the core group was trying to find a middle ground between those who think that PC is the relief of all suffering and those who believe that PC describes the care of those with a very limited remaining life span.
Immune checkpoint inhibitors (ICI) produce durable antitumor responses but provoke autoimmune toxicities, including uncommon but potentially devastating neurologic toxicities. The clinical features, ...including the spectrum, timing, and outcomes, of ICI-induced neurologic toxicities are not well characterized.
We performed disproportionality analysis using Vigibase, the World Health Organization pharmacovigilance database, comparing neurologic adverse event (AE) reporting in patients receiving ICIs vs. the full database. Neurologic AEs were classified by group queries using Medical Dictionary for Regulatory Activities, between database inception to September 28, 2018. Associations between ICIs and neurologic AEs were assessed using reporting odds ratios (ROR) and information component (IC). IC compares observed and expected values to find associations between drugs and AEs using disproportionate Bayesian reporting; IC
(lower end of the IC 95% credibility interval) > 0 is considered statistically significant.
Among the full database, 18,518,994 AEs were reported, including 48,653 with ICIs. ICIs were associated with higher incidence of myasthenia gravis (0.47% of ICI reports vs. 0.04% of the full database, ROR 16.5 95% CI 14.5-18.9; IC
3.31), encephalitis (0.51% vs. 0.05%, ROR 10.4 95% CI 9.2-11.8; IC
3.15), peripheral neuropathy (1.16% vs. 0.67%, IC
0.68), and meningitis (0.15% vs. 0.06%, ROR 3.1 95% CI 2.5-3.9; IC
1.01). Myasthenia gravis and encephalitis were associated with anti-PD-1 whereas other neurologic AEs were associated with anti-CTLA-4. Myasthenia gravis was characterized by high fatality rates (~ 20%), early onset (median 29 days), and frequent concurrent myocarditis and myositis; whereas other neurologic AEs had lower fatality rates (6-12%), later onset (median 61-80 days), and were non-overlapping.
ICIs produce a spectrum of distinct classes of neurologic AEs that can cause significant morbidity and mortality and tend to occur early and with class-specific associations.
Neuronal polarity and spatial rearrangement of neuronal processes are central to the development of all mature nervous systems. Recent studies have highlighted the dynamic expression of ...Collapsin-Response-Mediator Proteins (CRMPs) in neuronal dendritic/axonal compartments, described their interaction with cytoskeleton proteins, identified their ability to activate L- and N-type voltage-gated calcium channels (VGCCs) and delineated their crucial role as signaling molecules essential for neuron differentiation and neural network development and maintenance. In addition, evidence obtained from genome-wide/genetic linkage/proteomic/translational approaches revealed that CRMP expression is altered in human pathologies including mental (schizophrenia and mood disorders) and neurological (Alzheimer's, prion encephalopathy, epilepsy and others) disorders. Changes in CRMPs levels have been observed after psychotropic treatments, and disrupting CRMP2 binding to calcium channels blocked neuropathic pain. These observations, altogether with those obtained from genetically modified mice targeting individual CRMPs and RNA interference approaches, pave the way for considering CRMPs as potential early disease markers and modulation of their activity as therapeutic strategy for disorders associated with neurite abnormalities.
The natural four-letter genetic alphabet comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has ...emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.
Gene therapy for motor neuron diseases requires efficient gene delivery to motor neurons (MNs) throughout the spinal cord and brainstem. The present study compared adeno-associated viral (AAV) vector ...serotypes 1, 6, 8, and 9 for spinal cord delivery in adult mice, by the intraparenchymal or intrathecal route of administration. Whereas intraparenchymal injections resulted in local transduction of the lumbar segment of the spinal cord, intrathecal injections led to a broader distribution, transducing cells along the sacral, lumbar, and lower thoracic spinal cord. Overall, AAV6 and AAV9 performed better than the other serotypes. Dramatic differences in cell-specific expression patterns could be observed when constructs bearing the chicken β-actin (Cba) versus cytomegalovirus (CMV) promoter were compared. In summary, intrathecal delivery of AAV6 or AAV9 vectors containing the CMV promoter yielded the strongest levels of biodistribution and MN transduction in the spinal cord.
BackgroundImmune checkpoint inhibitors (ICIs) have transformed treatment for melanoma, but identifying reliable biomarkers of response and effective modifiable lifestyle factors has been challenging. ...Obesity has been correlated with improved responses to ICI, although the association of body composition measures (muscle, fat, etc) with outcomes remains unknown.MethodsWe performed body composition analysis using Slice-o-matic software on pretreatment CT scans to quantify skeletal muscle index (SMI=skeletal muscle area/height2), skeletal muscle density (SMD), skeletal muscle gauge (SMG=SMI × SMD), and total adipose tissue index (TATI=subcutaneous adipose tissue area + visceral adipose tissue area/height2) of each patient at the third lumbar vertebrae. We then correlated these measures to response, progression-free survival (PFS), overall survival (OS), and toxicity.ResultsAmong 287 patients treated with ICI, body mass index was not associated with clinical benefit or toxicity. In univariable analyses, patients with sarcopenic obesity had inferior PFS (HR 1.4, p=0.04). On multivariable analyses, high TATI was associated with inferior PFS (HR 1.7, p=0.04), which was particularly strong in women (HR 2.1, p=0.03). Patients with intermediate TATI and high SMG had the best outcomes, whereas those with low SMG/high TATI had inferior PFS and OS (p=0.02 for both PFS and OS).ConclusionsBody composition analysis identified several features that correlated with improved clinical outcomes, although the associations were modest. As with other studies, we identified sex-specific associations that warrant further study.
BACKGROUNDCurrent metrics for TB transmission include TB notifications, disease mortality, and prevalence surveys. These metrics are helpful to national TB programs to assess the burden of disease, ...but they do not directly measure incident infection in the community. METHODSTo estimate incidence of
infection in Kampala, Uganda, we performed a prospective cohort study between 2014 and 2017 which enrolled of 1,275 adult residents without signs of tuberculous infection (tuberculin skin test TST <5 mm and no signs of TB disease) and followed them for conversion of TST at 1 year. RESULTSDuring follow-up, 194 participants converted the TST and 158 converted by one year. The incidence density of TST conversion was 13.2 conversions/100 person-year (95% CI 11.6-15.1), which corresponds to an annual cumulative incidence of tuberculous infection of 12.4% (95% CI 10.7-14.3). Cumulative incidence was greater among older participants and among men. Among participants who reported prior exposure to TB cases, the cumulative risk was highest among those reporting exposure during follow-up. CONCLUSIONSThe high annual incidence of infection suggests that residents of Kampala have adequate contact for infection with undetected, infectious cases of TB as they go about their daily lives. .
Despite intense research into the multifaceted etiology of neurodegenerative diseases (ND), they remain incurable. Here we provide a brief overview of several major ND and explore novel therapeutic ...approaches. Although the cause (s) of ND are not fully understood, the accumulation of misfolded/aggregated proteins in the brain is a common pathological feature. This aggregation may initiate disruption of Ca
signaling, which is an early pathological event leading to altered dendritic structure, neuronal dysfunction, and cell death. Presently, ND gene therapies remain unidimensional, elusive, and limited to modifying one pathological feature while ignoring others. Considering the complexity of signaling cascades in ND, we discuss emerging therapeutic concepts and suggest that deciphering the molecular mechanisms involved in dendritic pathology may broaden the phenotypic spectrum of ND treatment. An innovative multiplexed gene transfer strategy that employs silencing and/or over-expressing multiple effectors could preserve vulnerable neurons before they are lost. Such therapeutic approaches may extend brain health span and ameliorate burdensome chronic disease states.
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