Proteomic characterization of 1,000 human and murine neutrophils freshly isolated from blood and sites of sterile inflammation.

Neutrophils are indispensable for defense against pathogens. Injured tissue-infiltrated neutrophils can establish a niche of chronic inflammation and promote degeneration. Studies investigated transcriptome of single-infiltrated neutrophils which could misinterpret molecular states of these post mitotic cells. However, neutrophil proteome characterization has been challenging due to low harvests from affected tissues. Here, we present a workflow to obtain proteome of 1,000 murine and human tissue-infiltrated neutrophils. We generated spectral libraries containing ∼6,200 mouse and ∼5,300 human proteins from circulating neutrophils. 4,800 mouse and 3,400 human proteins were recovered from 1,000 cells with 102-108 copies/cell. Neutrophils from stroke-affected mouse brains adapted to the glucose-deprived environment with increased mitochondrial activity and ROS-production while cells invading inflamed human oral cavities increased phagocytosis and granule release. We provide an extensive protein repository for resting human and mouse neutrophils, identify proteins lost in low input samples, thus enabling the proteomic characterization of limited tissue infiltrated neutrophils.

Clinorotation inhibits myotube formation by fluid motion, not by simulated microgravity.

To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat - a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments.

Optimization of Heat Accumulation during Femtosecond Laser Drilling Borehole Matrices by Using a Simplex Algorithm.

We report on an optimization study of percussion drilling thin metal sheets employing a high repetition rate, high power femtosecond laser with respect to the resulting heat accumulation. A specified simplex algorithm was employed to optimize the spatial drilling sequence, whereas a simplified thermal simulation using COMSOL was validated by comparing its results to the temperature measurements using an infrared camera. Optimization for drilling borehole matrices was aspired with respect to the generated temperature across the processed specimen, while the drilling strategy was altered in its spatial drilling sequence and by using multi-spot approaches generated by a spatial light modulator. As a result, we found that an optimization strategy based on limited consecutive holes in a Moore neighborhood led to reduced temperatures and the shortest process times.