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The use of subatmospheric pressure to promote wound healing has increased in popularity during the last several years. The original studies on granulation tissue formation used a 125-mmHg vacuum. The use of alternative sources of subatmospheric pressure has led to many questions regarding efficacy or risk. In this report a swine model is used to quantify and compare the effects of low vacuum suction (25 mmHg) and high vacuum suction (500 mmHg) produced by various vacuum pumps and wall suction systems with the standard 125-mmHg vacuum. Additionally, the effects of an unregulated air leak in the sealing system were examined. All four wound treatments were examined on each of 4 pigs. Wounds were treated until one of the wounds had granulated to a level flush with the surrounding tissue. Wounds treated with the standard 125-mmHg vacuum had filled with granulation tissue by day 8. At this time wounds treated with 25 mmHg had filled 21.2% with new granulation tissue, and wounds treated with 500 mmHg had filled 5.9% with new tissue. Wounds treated with 125 mmHg with a hole in the sealing drape had increased in size 197% because of the debridement of necrotic tissue. In conclusion, wounds treated with a 125-mmHg vacuum exhibited a significant (p< 0.0001) increase in the rate of granulation tissue formation compared with treatment at 25 mmHg or 500 mmHg. The presence of an unregulated air leak in the sealing drape results in significant progression (p < 0.0001) of the wound secondary to dehydration and progressive necrosis.

Common genetic variants can have profound effects on cellular function, but studying these effects in primary human tissue samples and during development is challenging. Human induced pluripotent stem cell (iPSC) technology holds great promise for assessing these effects across a range of differentiation contexts. Here, we use an efficient multiplexing strategy to differentiate 215 iPS cell lines towards a midbrain neural fate, including dopaminergic neurons, and profile over 1 million cells sampled across three differentiation timepoints using single cell RNA sequencing. We find that the proportion of neuronal cells produced by each cell line is highly reproducible over different experimental batches, and identify robust molecular markers in pluripotent cells that predict line-to-line differences in cell fate. We identify expression quantitative trait loci (eQTL) that manifest at different stages of neuronal development, and in response to rotenone-induced oxidative stress. We find 1,284 eQTL that colocalise with a known risk locus for a neurological trait, 46% of which are not found in the GTEx catalogue. Our study illustrates how coupling single cell transcriptomics with long-term iPSC differentiation can profile mechanistic effects of human trait-associated genetic variants in otherwise inaccessible cell states.

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells, and this is thought to be important in chemoresistance. A method for the assessment of ALDH activity in viable cells recently has been developed and made commercially available in a kit format. In this study, we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Via multicolor flow cytometry of cord blood ALDH + cells, we have expanded on their phenotypic analysis. We then assessed the incidence, morphology, phenotype, and nonobese diabetic/ severe combined immunodeficiency engraftment ability of ALDH + cells from acute myeloid leukemia (AML) samples. AML samples had no ALDH + cells at all, an extremely rare nonmalignant stem/progenitor cell population, or a less rare, leukemic stem cell population. Hence, in addition to identifying nonmalignant stem cells within some AML samples, a high ALDH activity also identifies some patients' CD34 + / CD38 -leukemic stem cells. The incidence of normal or leukemic stem cells with an extremely high ALDH activity may have important implications for resistance to chemotherapy. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis. Stem Cells 2005;23:752-760

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) assay is the current model for assessment of human normal and leukemic stem cells. We explored why 51% of 59 acute myeloid leukemia (AML) patients were unable to initiate leukemia in NOD/SCID mice. Increasing the cell dose, using more permissive recipients, and alternative tissue sources did not cause AML engraftment in most previously nonengrafting AML samples. Homing of AML cells to the marrow was the same between engrafters and nonengrafters. FLT3 internal tandem duplication (ITD) and nucleophosmin mutations occurred at a similar frequency in engrafters and nonengrafters. The only variable that was related to engraftment ability was the karyotypically defined risk stratification of individual AML cases. Of interest, follow-up of younger patients with intermediate-risk AML revealed a significant difference in overall survival between NOD/SCID engrafting and nonengrafting AMLs. Hence, the ability of AML to engraft in the NOD/SCID assay seems to be an inherent property of AML cells, independent of homing, conditioning, or cell frequency/source, which is directly related to prognosis. Our results suggest an important difference between leukemic initiating cells between engrafting and nonengrafting AML cases that correlates with treatment response.

DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d.

role of mechanics in the ecophysiology of prokaryotic cells come to the forefront [6,[9][10][11][12][13][14][15][16][17], highlighting the governing biophysical principles that drive colony formation. arXiv:1703.04504v2 [cond-mat.soft]