Continuous and Long-Term Volume Measurements with a Commercial Coulter Counter

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter.National Institutes of Health (U.S.) (EUREKA Exceptional, Unconventional Research Enabling Knowledge Acceleration (R01GM085457))National Institutes of Health (U.S.) (contract R21CA137695)National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874

Current characterization methods for cellulose nanomaterials

International audienc

Characterizing friction for fiber reinforced composites manufacturing: Method development and effect of process parameters

In automated layup manufacturing processes of fiber-reinforced polymer composites, the quality of the manufactured part is strongly dependent on frictional behavior. Improper control of frictional forces can lead to defect formation. Frictional sliding rheometry tests provide an innovative methodology to accurately characterize the tool-ply friction of unidirectional (UD) prepreg employing unique annular plate geometries. The effect of processing parameters (temperature, velocity, and normal force) on the frictional response of a carbon fiber prepreg was studied. Moreover, utilizing custom designed plate geometries coupled with optically transparent fixtures allowed for in-situ quantification of the prepreg-rigid surface contact area along with simultaneous characterization of the process parameter-dependent frictional mechanisms. Our findings highlight the reduction in frictional forces with increasing temperature, attributed to the increased resin flowability, while increases in sliding rates resulted in a pronounced increase in the frictional forces. The effect of applied load on the frictional characteristics was more complicated due to contributions from both the adhesive and normal forces. Finally, the results were interpreted in light of the contact area measurements performed at different temperatures, normal force, and sliding rate

Nitric Oxide Down-Regulates Topoisomerase I and Induces Camptothecin Resistance in Human Breast MCF-7 Tumor Cells

<div><p>Camptothecin (CPT), a topoisomerase I poison, is an important drug for the treatment of solid tumors in the clinic. Nitric oxide (^·NO), a physiological signaling molecule, is involved in many cellular functions, including cell proliferation, survival and death. We have previously shown that ^·NO plays a significant role in the detoxification of etoposide (VP-16), a topoisomerase II poison <i>in vitro</i> and in human melanoma cells. ^·NO/^·NO-derived species are reported to modulate activity of several important cellular proteins. As topoisomerases contain a number of free sulfhydryl groups which may be targets of ^·NO/^·NO-derived species, we have investigated the roles of ^·NO/^·NO-derived species in the stability and activity of topo I. Here we show that ^·NO/^·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines. Importantly, ^·NO treatment induced a significant resistance to CPT only in MCF-7 cells. This resistance to CPT did not result from loss of topoisomerase I activity as there were no differences in topoisomerase I-induced DNA cleavage <i>in vitro</i> or in tumor cells, but resulted from the stabilization/induction of bcl2 protein. This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance. Because tumors <i>in vivo</i> are heterogeneous and contaminated by infiltrating macrophages, ^·NO-induced down-regulation of topoisomerase I protein combined with bcl2 protein stabilization could render certain tumors highly resistant to CPT and drugs derived from it in the clinic.</p></div

Markers of fertility in reproductive microbiomes of male and female endangered black-footed ferrets (Mustela nigripes)

Abstract Reproductive microbiomes contribute to reproductive health and success in humans. Yet data on reproductive microbiomes, and links to fertility, are absent for most animal species. Characterizing these links is pertinent to endangered species, such as black-footed ferrets (Mustela nigripes), whose populations show reproductive dysfunction and rely on ex-situ conservation husbandry. To understand microbial contributions to animal reproductive success, we used 16S rRNA amplicon sequencing to characterize male (prepuce) and female (vaginal) microbiomes of 59 black-footed ferrets at two ex-situ facilities and in the wild. We analyzed variation in microbiome structure according to markers of fertility such as numbers of viable and non-viable offspring (females) and sperm concentration (males). Ferret vaginal microbiomes showed lower inter-individual variation compared to prepuce microbiomes. In both sexes, wild ferrets harbored potential soil bacteria, perhaps reflecting their fossorial behavior and exposure to natural soil microbiomes. Vaginal microbiomes of ex-situ females that produced non-viable litters had greater phylogenetic diversity and distinct composition compared to other females. In males, sperm concentration correlated with varying abundances of bacterial taxa (e.g., Lactobacillus), mirroring results in humans and highlighting intriguing dynamics. Characterizing reproductive microbiomes across host species is foundational for understanding microbial biomarkers of reproductive success and for augmenting conservation husbandry