We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the ...interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations-for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population.
The optoelectronic microrobot Zhang, Shuailong; Scott, Erica Y.; Singh, Jastaranpreet ...
Proceedings of the National Academy of Sciences - PNAS,
07/2019, Letnik:
116, Številka:
30
Journal Article
Recenzirano
Odprti dostop
Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to ...manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of “load,” “transport,” and “deliver,” which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell–cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond.
We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects ...single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.
Heart failure (HF) is commonly referred to as an epidemic, posing major clinical and public health challenges. Yet, contemporary data on its magnitude and implications are scarce.
To evaluate recent ...trends in HF incidence and outcomes overall and by preserved ejection fraction (HFpEF) or reduced ejection fraction (HFrEF).
Incidence rates of HF in Olmsted County, Minnesota (population, approximately 144,248), between January 1, 2000, and December 31, 2010, were assessed.
Patients identified with incident HF (n = 2762) (mean age, 76.4 years; 43.1% male) were followed up for all-cause and cause-specific hospitalizations (through December 2012) and death (through March 2014).
The age- and sex-adjusted incidence of HF declined substantially from 315.8 per 100,000 in 2000 to 219.3 per 100,000 in 2010 (annual percentage change, -4.6), equating to a rate reduction of 37.5% (95% CI, -29.6% to -44.4%) over the last decade. The incidence declined for both HF types but was greater (interaction P = .08) for HFrEF (-45.1%; 95% CI, -33.0% to -55.0%) than for HFpEF (-27.9%; 95% CI, -12.9% to -40.3%). Mortality was high (24.4% for age 60 years and 54.4% for age 80 years at 5 years of follow-up), frequently ascribed to noncardiovascular causes (54.3%), and did not decline over time. The risk of cardiovascular death was lower for HFpEF than for HFrEF (multivariable-adjusted hazard ratio, 0.79; 95% CI, 0.67-0.93), whereas the risk of noncardiovascular death was similar (1.07; 95% CI, 0.89-1.29). Hospitalizations were common (mean, 1.34; 95% CI, 1.25-1.44 per person-year), particularly among men, and did not differ between HFpEF and HFrEF. Most hospitalizations (63.0%) were due to noncardiovascular causes. Hospitalization rates for cardiovascular causes did not change over time, whereas those for noncardiovascular causes increased.
Over the last decade, the incidence of HF declined substantially, particularly for HFrEF, contrasting with no apparent change in mortality. Noncardiovascular conditions have an increasing role in hospitalizations and remain the most frequent cause of death. These results underscore the need to augment disease-centric management approaches with holistic strategies to reduce the population burden of HF.
Abstract
There is great interest in the development of micromotors which can convert energy to motion in sub-millimeter dimensions. Micromachines take the micromotor concept a step further, ...comprising complex systems in which multiple components work in concert to effectively realize complex mechanical tasks. Here we introduce light-driven micromotors and micromachines that rely on optoelectronic tweezers (OET). Using a circular micro-gear as a unit component, we demonstrate a range of new functionalities, including a touchless micro-feed-roller that allows the programming of precise three-dimensional particle trajectories, multi-component micro-gear trains that serve as torque- or velocity-amplifiers, and micro-rack-and-pinion systems that serve as microfluidic valves. These sophisticated systems suggest great potential for complex micromachines in the future, for application in microrobotics, micromanipulation, microfluidics, and beyond.
The choice of drugs for patients with status epilepticus that is refractory to treatment with benzodiazepines has not been thoroughly studied.
In a randomized, blinded, adaptive trial, we compared ...the efficacy and safety of three intravenous anticonvulsive agents - levetiracetam, fosphenytoin, and valproate - in children and adults with convulsive status epilepticus that was unresponsive to treatment with benzodiazepines. The primary outcome was absence of clinically evident seizures and improvement in the level of consciousness by 60 minutes after the start of drug infusion, without additional anticonvulsant medication. The posterior probabilities that each drug was the most or least effective were calculated. Safety outcomes included life-threatening hypotension or cardiac arrhythmia, endotracheal intubation, seizure recurrence, and death.
A total of 384 patients were enrolled and randomly assigned to receive levetiracetam (145 patients), fosphenytoin (118), or valproate (121). Reenrollment of patients with a second episode of status epilepticus accounted for 16 additional instances of randomization. In accordance with a prespecified stopping rule for futility of finding one drug to be superior or inferior, a planned interim analysis led to the trial being stopped. Of the enrolled patients, 10% were determined to have had psychogenic seizures. The primary outcome of cessation of status epilepticus and improvement in the level of consciousness at 60 minutes occurred in 68 patients assigned to levetiracetam (47%; 95% credible interval, 39 to 55), 53 patients assigned to fosphenytoin (45%; 95% credible interval, 36 to 54), and 56 patients assigned to valproate (46%; 95% credible interval, 38 to 55). The posterior probability that each drug was the most effective was 0.41, 0.24, and 0.35, respectively. Numerically more episodes of hypotension and intubation occurred in the fosphenytoin group and more deaths occurred in the levetiracetam group than in the other groups, but these differences were not significant.
In the context of benzodiazepine-refractory convulsive status epilepticus, the anticonvulsant drugs levetiracetam, fosphenytoin, and valproate each led to seizure cessation and improved alertness by 60 minutes in approximately half the patients, and the three drugs were associated with similar incidences of adverse events. (Funded by the National Institute of Neurological Disorders and Stroke; ESETT ClinicalTrials.gov number, NCT01960075.).
Background Anaphylaxis is a potentially life-threatening systemic allergic reaction. Objective We aimed to determine the incidence rate and causes of anaphylaxis during a 10-year period in Olmsted ...County, Minnesota. Methods Using the resources of the Rochester Epidemiology Project, a comprehensive records linkage system, we performed a population-based incidence study in Olmsted County, Minnesota, from 2001 through 2010. All cases with a diagnosis of anaphylactic shock and 20% of cases with related diagnoses were manually reviewed. The relationships of age group, sex, and year of anaphylaxis with incidence rates were assessed by fitting Poisson regression models. Results Six hundred thirty-one cases of anaphylaxis were identified. The median age was 31 years (interquartile range, 19-44 years). The overall age- and sex-adjusted incidence rate was 42 (95% CI, 38.7-45.3) per 100,000 person-years. There was a significant increase in the overall incidence of anaphylaxis during the study period, with an average increase of 4.3% per year ( P < .001). In addition, there was a 9.8% increase per year in the incidence rate of food-related anaphylaxis. Food-related anaphylaxis was most common in children aged 0 to 9 years, venom-related anaphylaxis was most common in those 20 to 39 years of age, and medication-related anaphylaxis was most common in those 30 to 39 years of age. Conclusion The overall incidence rate of anaphylaxis was 42 per 100,000 person-years from 2001-2010 in Olmsted County, Minnesota. The incidence of anaphylaxis increased over time, and several inciting triggers were uniquely associated with different age groups.
The early part of the last deglaciation is characterised by a ~40 ppm atmospheric CO2 rise occurring in two abrupt phases. The underlying mechanisms driving these increases remain a subject of ...intense debate. Here, we successfully reproduce changes in CO2, δ13C and Δ14C as recorded by paleo-records during Heinrich stadial 1 (HS1). We show that HS1 CO2 increase can be explained by enhanced Southern Ocean upwelling of carbon-rich Pacific deep and intermediate waters, resulting from intensified Southern Ocean convection and Southern Hemisphere (SH) westerlies. While enhanced Antarctic Bottom Water formation leads to a millennial CO2 outgassing, intensified SH westerlies induce a multi-decadal atmospheric CO2 rise. A strengthening of SH westerlies in a global eddy-permitting ocean model further supports a multi-decadal CO2 outgassing from the Southern Ocean. Our results highlight the crucial role of SH westerlies in the global climate and carbon cycle system with important implications for future climate projections.
Background Minimal clinically important differences (MCIDs) for the American Shoulder and Elbow Surgeons (ASES) score, the Simple Shoulder Test (SST), and a visual analog scale (VAS) measuring pain ...have not been previously described using an anchor-based method after shoulder arthroplasty. The purpose of this study was to determine the MCIDs for these measures after shoulder arthroplasty for glenohumeral arthritis or advanced rotator cuff disease. Methods Primary anatomic total shoulder arthroplasty (TSA), primary reverse TSA, or hemiarthroplasty was performed in 326 patients by 1 of 5 shoulder and elbow surgeons. The SST score, ASES score, and VAS pain score were collected preoperatively and at a minimum of 2 years postoperatively (mean, 3.5 years). The MCIDs were calculated for the ASES score, SST score, and VAS pain score using an anchor-based method. Results The MCIDs for the ASES score, SST score, and VAS pain score were 20.9 ( P < .001), 2.4 ( P < .0001), and 1.4 ( P = .0158), respectively. Duration of follow-up and type of arthroplasty (anatomic TSA vs reverse TSA) did not have a significant effect on the MCIDs ( P > .1) except shorter follow-up correlated with a larger MCID for the ASES score ( P = .0081). Younger age correlated with larger MCIDs for all scores ( P < .024). Female sex correlated with larger MCIDs for the VAS pain score ( P = .123) and ASES score ( P = .05). Conclusions Patients treated with a shoulder arthroplasty require a 1.4-point improvement in the VAS pain score, a 2.4-point improvement in the SST score, and a 21-point improvement in the ASES score to achieve a minimal clinical importance difference from the procedure.
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