Phlpp1 Expression in Osteoblasts Plays a Modest Role in Bone Homeostasis.

Prior work demonstrated that Phlpp1 deficiency alters limb length and bone mass, but the cell types involved and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within bone-forming osteoblasts, we crossed Phlpp1 floxed mice with mice harboring type 1 collagen (Col1a12.3kb)-Cre. Mineralization of bone marrow stromal cell cultures derived from Phlpp1 cKOCol1a1 was unchanged, but levels of inflammatory genes (eg, Ifng, Il6, Ccl8) and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratios were enhanced by either Phlpp1 ablation or chemical inhibition. Micro-computed tomography of the distal femur and L5 vertebral body of 12-week-old mice revealed no alteration in bone volume per total volume, but compromised femoral bone microarchitecture within Phlpp1 cKOCol1a1 conditional knockout females. Bone histomorphometry of the proximal tibia documented no changes in osteoblast or osteoclast number per bone surface but slight reductions in osteoclast surface per bone surface. Overall, our data show that deletion of Phlpp1 in type 1 collagen-expressing cells does not significantly alter attainment of peak bone mass of either males or females, but may enhance inflammatory gene expression and the ratio of RANKL/OPG. Future studies examining the role of Phlpp1 within models of advanced age, inflammation, or osteocytes, as well as functional redundancy with the related Phlpp2 isoform are warranted. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Ten simple rules for funding scientific open source software.

Scientific research increasingly relies on open source software (OSS). Funding OSS development requires intentional focus on issues of scholarly credit, unique forms of labor, maintenance, governance, and inclusive community-building. Such issues cut across different scientific disciplines that make them of interest to a variety of funders and institutions but may present challenges in understanding generalized needs. Here we present 10 simple rules for investing in scientific OSS and the teams who build and maintain it.

Image-based analysis of primary human neutrophil chemotaxis in an automated direct-viewing assay.

Multi-well assays based on the Boyden chamber have enabled highly parallel studies of chemotaxis-the directional migration of cells in response to molecular gradients-while direct-viewing approaches have allowed more detailed questions to be asked at low throughput. Boyden-based plates provide a count of cells that pass through a membrane, but no information about cell appearance. In contrast, direct-viewing devices enable the observation of cells during chemotaxis, which allows measurement of many parameters including area, shape, and location. Here we show automated chemotaxis and cell morphology assays in a 96-unit direct-viewing plate. Using only 12000 primary human neutrophils per datum, we measured dose-dependent stimulation and inhibition of chemotaxis and quantified the effects of inhibitors on cell area and elongation. With 60 parallel conditions we demonstrated 5-fold increase in throughput compared to previously reported direct-viewing approaches.

Phosphorylation-dependent sumoylation of estrogen-related receptor alpha1.

We previously showed that estrogen-related receptor alpha1 (ERRalpha1) can compete with estrogen receptor alpha (ERalpha) for binding to estrogen response elements (EREs), repressing transcription in the mammary carcinoma cell line MCF-7. Given that ERRalpha1 can function in the absence of ligands and exists as a phosphoprotein in vivo, we wished to determine sites of phosphorylation involved in regulating its transcriptional activity. Using a combination of electrophoretic mobility shift analysis, phospho-specific fluorescent dye staining, and site-directed mutagenesis, we identified two novel in vivo sites of phosphorylation in the A/B ligand-independent activation domain of ERRalpha1 at Ser19 and Ser22. Inhibition of phosphorylation at amino acid residue 22 did not have a significant effect on ERRalpha1's transcriptional activity. However, mutation of amino acid residue 19 from serine to alanine enhanced two-fold ERRalpha1's response to the coactivator GRIP-1. We also identified two sites of sumoylation at Lys14 and Lys403. We found that inhibition of sumoylation at Lys14 could enhance five-fold ERRalpha1's response to coactivator GRIP-1. Furthermore, phosphorylation of Ser19 enhanced the sumoylation at Lys14. Taken together, we conclude that phosphorylation at Ser19 and sumoylation at Lys14 within the A/B domain play roles in regulating ERRalpha1's transcriptional activities via affecting its response to coactivators.