A Video Bioinformatics Method to Quantify Cell Spreading and Its Application to Cells Treated with Rho-Associated Protein Kinase and Blebbistatin

Commercial software is available for performing video bioinformatics analysis on cultured cells. Such software is convenient and can often be used to create suitable protocols for quantitative analysis of video data with relatively little background in image processing. This chapter demonstrates that CL-Quant software, a commercial program produced by DRVision, can be used to automatically analyze cell spreading in time-lapse videos of human embryonic stem cells (hESC). Two cell spreading protocols were developed and tested. One was professionally created by engineers at DRVision and adapted to this project. The other was created by an undergraduate student with 1 month of experience using CL-Quant. Both protocols successfully segmented small spreading colonies of hESC, and, in general, were in good agreement with the ground truth which was measured using ImageJ. Overall the professional protocol performed better segmentation, while the user-generated protocol demonstrated that someone who had relatively little background with CL-Quant can successfully create protocols. The protocols were applied to hESC that had been treated with ROCK inhibitors or blebbistatin, which tend to cause rapid attachment and spreading of hESC colonies. All treatments enabled hESC to attach rapidly. Cells treated with the ROCK inhibitors or blebbistatin spread more than controls and often looked stressed. The use of the spreading analysis protocol can provide a very rapid method to evaluate the cytotoxicity of chemical treatment and reveal effects on the cytoskeleton of the cell. While hESC are presented in this chapter, other cell types could also be used in conjunction with the spreading protocol

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Characterizing the Pathways That Initiate and Stop Dynamic Blebbing in Human Embryonic Stem Cells

Dynamic blebs are membrane protrusions on the surface of healthy cells that function in cell division and migration. The purpose of this dissertation was to discover better methods to control dynamic blebbing by identifying pathways that initiate and inhibit blebbing in human embryonic stem cells (hESC). Live cell imaging experiments demonstrated that dynamic and apoptotic blebbing were morphologically and temporally distinct during passaging of hESC. Dynamic blebs retracted faster than apoptotic blebs and had an intact cytoskeleton. Dynamic blebbing was prolonged by depolymerization of microtubules and stopped by drugs that disrupted actin microfilaments or inhibited myosin II. Plating on laminin-521 or Matrigel overcoated with laminin-111 efficiently stopped blebbing by activating an integrin-focal adhesion kinase pathway. To identify the pathway that initiates dynamic blebbing, we tested the hypothesis that the P2X7 calcium channel is activated by ATP released during passaging. Immunocytochemistry and PCR showed that the P2X7 receptor is expressed in hESC, but not in cells starting differentiation. P2X7 inhibitors and siRNA decreased dynamic blebbing. Extracellular ATP concentration increased during passaging. While apyrase, which degrades ATP, reduced the percentage of blebbing cells, addition of ATP to the culture medium prolonged blebbing. When Ca2+ was chelated by either EGTA or BAPTA, dynamic blebbing was inhibited. Rac activation was associated with decreased blebbing and cell attachment, while blebbing was activated though the ROCK pathway. These data support the idea that dynamic blebbing in hESC is initiated by extracellular ATP binding to P2X7, allowing Ca2+ influx which in turn initiates dynamic blebbing via the ROCK- myosin II pathway. Decreased extracellular ATP is accompanied by activation of Rac, cessation of blebbing, and attachment. These results introduce better ways to control dynamic blebbing and improve cell survival during passaging. The use of P2X7 inhibitors and laminin substrates enables attachment and improves single hESC plating efficiency, which will facilitate quantitative work in drug discovery and toxicology. These results also introduce: (1) P2X7 as a cell surface marker for pluripotency; (2) hESC as an excellent model for studying dynamic blebbing and (3) the possibility of using hESC, which are similar to epiblast cells, to study cell movements during gastrulation

The P2X7 receptor is an upstream regulator of dynamic blebbing and a pluripotency marker in human embryonic stem cells

New methods are needed to reduce dynamic blebbing which inhibits cell attachment and survival during passaging of pluripotent stem cells. We tested the hypothesis that activation of the P2X7 receptor by extracellular ATP during passaging initiates dynamic blebbing. The P2X7 receptor was present in human embryonic stem cells (hESC), but not in differentiating cells. Extracellular ATP concentrations were 14× higher in medium during passaging. Addition of ATP to culture medium prolonged dynamic blebbing and inhibited attachment. Inhibition of P2X7 by specific drugs or by siRNA significantly reduced dynamic blebbing and improved cell attachment. When cells were incubated in calcium chelators (EGTA or BAPTA), blebbing decreased and attachment improved. Calcium influx was observed using Fura-4 when ATP was added to culture medium and inhibited in the presence of the P2X7 inhibitor. Over-expressing activated Rac in hESC reduced blebbing and promoted cell attachment, while a Rac inhibitor prolonged blebbing and reduced attachment. These data identify a pathway involving P2X7 that initiates and prolongs dynamic blebbing during hESC passaging. This pathway provides new insight into factors that increase dynamic blebbing and identifies new targets, such as P2X7, that can be used to improve the culture of cells with therapeutic potential