Activity

It is often desirable to evaluate the ability of cells to move in an unrestricted manner in multiple directions without chemical gradients. By combining the standard radial migration assay with injection-molded gaskets and a rigid fixture, we have developed a highly reliable and sensitive method for observing and measuring radial cell migration. This method is adapted for use on high-throughput automated imaging systems. The use of injection-molded gaskets enables low-cost replacement of cell-wetted components. Moreover, the design enables secondary placement of attractants and co-cultures. This device and its enhanced throughput permit the use of therapeutic screening to evaluate phenotypic responses, for example, cancer cell migration response due to drugs or chemical signals. This approach is orthogonal to other 2D cell migration applications, such as scratch wound assays, although here we offer a noninvasive, enhanced-throughput device, which currently is not commercially available but is easily constructed. The proposed device is a systematic, reliable, rapid application to monitor phenotypic responses to chemotherapeutic screens, genetic alterations (e.g., RNAi and CRISPR), supplemental regimens, and other approaches, offering a reliable methodology to survey unbiased and noninvasive cell migration.CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.Mitochondria and peroxisomes are highly dynamic, multifunctional organelles. Both perform key roles for cellular physiology and homoeostasis by mediating bioenergetics, biosynthesis, and/or signalling. To support cellular function, they must be properly distributed, of proper size, and be able to interact with other organelles. Accumulating evidence suggests that the small atypical GTPase Miro provides a central signalling node to coordinate mitochondrial as well as peroxisomal dynamics. In this review, I summarize our current understanding of Miro-dependent functions and molecular mechanisms underlying the proper distribution, size and function of mitochondria and peroxisomes.This article describes the cross-cultural adaption and psychometric testing of the Family Nursing Practice Scale (FNPS) German version. The FNPS aims to examine self-reported family nursing practice skills and reciprocity in the nurse-family relationship. Using a cross-sectional design, 583 acute and critical care nurses were invited to complete the FNPS German version. Exploratory factor analysis was used to assess the structural validity. Internal consistency was determined using Cronbach’s alpha. BLU-222 ic50 A total of 317 nurses returned a completed online questionnaire. Principal axis factor analysis suggests a one-factor solution in which all 10 items are retained, accounting for 36% of the variance. Cronbach’s alpha was .84. In contrast to the original version, our findings indicate the unidimensionality of the construct. The FNPS German version appears to be a valid and reliable scale to assess nurses’ perception of their family nursing practice proficiency. Further testing is needed to confirm the unidimensionality and to establish test-retest reliability.Autophagy summarizes evolutionarily conserved, intracellular degradation processes targeting cytoplasmic material for lysosomal degradation. These encompass constitutive processes as well as stress responses, which are often found dysregulated in diseases. Autophagy pathways help in the clearance of damaged organelles, protein aggregates and macromolecules, mediating their recycling and maintaining cellular homeostasis. Protein-protein interaction networks contribute to autophagosome biogenesis, substrate loading, vesicular trafficking and fusion, protein translocations across membranes and degradation in lysosomes. Hypothesis-free proteomic approaches tremendously helped in the functional characterization of protein-protein interactions to uncover molecular mechanisms regulating autophagy. In this review, we elaborate on the importance of understanding protein-protein-interactions of varying affinities and on the strengths of mass spectrometry-based proteomic approaches to study these, generating new mechanistic insights into autophagy regulation. We discuss in detail affinity purification approaches and recent developments in proximity labeling coupled to mass spectrometry, which uncovered molecular principles of autophagy mechanisms. Abbreviations AMPK AMP-activated protein kinase; AP-MS affinity purification-mass spectrometry; APEX2 ascorbate peroxidase-2; ATG autophagy related; BioID proximity-dependent biotin identification; ER endoplasmic reticulum; GFP green fluorescent protein; iTRAQ isobaric tag for relative and absolute quantification; MS mass spectrometry; PCA protein-fragment complementation assay; PL-MS proximity labeling-mass spectrometry; PtdIns3P phosphatidylinositol-3-phosphate; PTM posttranslational modification; PUP-IT pupylation-based interaction tagging; RFP red fluorescent protein; SILAC stable isotope labeling by amino acids in cell culture; TAP tandem affinity purification; TMT tandem mass tag.