Lysine methylation signaling in skeletal muscle biology: from myogenesis to clinical insights.

Lysine methylation signaling is well studied for its key roles in the regulation of transcription states through modifications on histone proteins. While histone lysine methylation has been extensively studied, recent discoveries of lysine methylation on thousands of non-histone proteins has broadened our appreciation for this small chemical modification in the regulation of protein function. In this review, we highlight the significance of histone and non-histone lysine methylation signaling in skeletal muscle biology, spanning development, maintenance, regeneration, and disease progression. Furthermore, we discuss potential future implications for its roles in skeletal muscle biology as well as clinical applications for the treatment of skeletal muscle-related diseases.

Global lysine methylome profiling using systematically characterized affinity reagents.

Lysine methylation modulates the function of histone and non-histone proteins, and the enzymes that add or remove lysine methylation-lysine methyltransferases (KMTs) and lysine demethylases (KDMs), respectively-are frequently mutated and dysregulated in human diseases. Identification of lysine methylation sites proteome-wide has been a critical barrier to identifying the non-histone substrates of KMTs and KDMs and for studying functions of non-histone lysine methylation. Detection of lysine methylation by mass spectrometry (MS) typically relies on the enrichment of methylated peptides by pan-methyllysine antibodies. In this study, we use peptide microarrays to show that pan-methyllysine antibodies have sequence bias, and we evaluate how the differential selectivity of these reagents impacts the detection of methylated peptides in MS-based workflows. We discovered that most commercially available pan-Kme antibodies have an in vitro sequence bias, and multiple enrichment approaches provide the most comprehensive coverage of the lysine methylome. Overall, global lysine methylation proteomics with multiple characterized pan-methyllysine antibodies resulted in the detection of 5089 lysine methylation sites on 2751 proteins from two human cell lines, nearly doubling the number of reported lysine methylation sites in the human proteome.

Going Online in Pandemic Time: A DivulgaMicro Workshop Experience.

DivulgaMicro is a Brazilian science communication and outreach project run by three young female scientists. In 2020, due to the COVID-19 pandemic, we promoted the first virtual edition of the DivulgaMicro Workshop. Here, we describe adaptations implemented to deliver the virtual edition and its assessment by the participants. We offered a 3-day workshop addressing manuscript writing and publishing, poster design and oral presentations, and strategies for effectively communicating with the lay public on Days 1, 2, and 3, respectively. Each daily session was divided into short lectures and active learning through interactive and cooperative activities. We applied pre- and postsession surveys daily to assess participants' learning, with an opinion questionnaire at the end of the workshop. We selected 50 of 221 applicants, but only 38 of those selected participated in the workshop. Correct answers were significantly higher (p < 0.01) in the postsession surveys each day. Most of the 34 participants who answered the opinion questionnaire (97%) would recommend the workshop to their colleagues. Participants stated that the workshop content was transmitted in a clear and straightforward manner, and they considered the online format suitable for knowledge acquisition. Participants were satisfied with the organization, dynamics, and interactivity. Topics addressed on Day 3 (61.8%) and Day 1 (23.5%) were most liked and were considered most useful in the participants' careers. We believe that the overall success of the workshop is due to the combination of short lectures with active-learning activities, the use of virtual platforms that enabled effective communication between participants and instructors, and the support of our collaborators.

A Read/Write Mechanism Connects p300 Bromodomain Function to H2A.Z Acetylation.

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active regulatory regions associated with gene expression. Although the Tip60 complex is proposed to acetylate H2A.Z, functional studies suggest additional enzymes are involved. Here, we show that p300 acetylates H2A.Z at multiple lysines. In contrast, we found that although Tip60 does not efficiently acetylate H2A.Z in vitro, genetic inhibition of Tip60 reduces H2A.Zac in cells. Importantly, we found that interaction between the p300-bromodomain and H4 acetylation (H4ac) enhances p300-driven H2A.Zac. Indeed, H2A.Zac and H4ac show high genomic overlap, especially at active promoters. We also reveal unique chromatin features and transcriptional states at enhancers correlating with co-occurrence or exclusivity of H4ac and H2A.Zac. We propose that differential H4 and H2A.Z acetylation signatures can also define the enhancer state. In conclusion, we show both Tip60 and p300 contribute to H2A.Zac and reveal molecular mechanisms of writer/reader crosstalk between H2A.Z and H4 acetylation through p300.

A T Cell View of the Bone Marrow.

The majority of T cells present in the bone marrow (BM) represent an activated/memory phenotype and most of these, if not all, are circulating T cells. Their lodging in the BM keeps them activated, turning the BM microenvironment into a "memory reservoir." This article will focus on how T cell activation in the BM results in both direct and indirect effects on the hematopoiesis. The hematopoietic stem cell niche will be presented, with its main components and organization, along with the role played by T lymphocytes in basal and pathologic conditions and their effect on the bone remodeling process. Also discussed herein will be how "normal" bone mass peak is achieved only in the presence of an intact adaptive immune system, with T and B cells playing critical roles in this process. Our main hypothesis is that the partnership between T cells and cells of the BM microenvironment orchestrates numerous processes regulating immunity, hematopoiesis, and bone remodeling.

Platelet-Rich Plasma Obtained with Different Anticoagulants and Their Effect on Platelet Numbers and Mesenchymal Stromal Cells Behavior In Vitro.

There are promising results in the use of platelet-rich plasma (PRP) for musculoskeletal tissue repair. However, the variability in the methodology for its obtaining may cause different and opposing findings in the literature. Particularly, the choice of the anticoagulant is the first definition to be made. In this work, blood was collected with sodium citrate (SC), ethylenediaminetetraacetic acid (EDTA), or anticoagulant citrate dextrose (ACD) solution A, as anticoagulants, prior to PRP obtaining. Hematological analysis and growth factors release quantification were performed, and the effects on mesenchymal stromal cell (MSC) culture, such as cytotoxicity and cell proliferation (evaluated by MTT method) and gene expression, were evaluated. The use of EDTA resulted in higher platelet yield in whole blood; however, it induced an increase in the mean platelet volume (MPV) following the blood centrifugation steps for PRP obtaining. The use of SC and ACD resulted in higher induction of MSC proliferation. On the other hand, PRP obtained in SC presented the higher platelet recovery after the blood first centrifugation step and a minimal change in MSC gene expression. Therefore, we suggest the use of SC as the anticoagulant for PRP obtaining.

Platelet-rich plasma releasate differently stimulates cellular commitment toward the chondrogenic lineage according to concentration.

Platelet-rich plasma has been used to treat articular cartilage defects, with the expectations of anabolic and anti-inflammatory effects. However, its role on cellular chondrogenic or fibrogenic commitment is still a controversy. Herein, the role of platelet-rich plasma releasate, the product obtained following platelet-rich plasma activation, on cellular commitment toward the chondrogenic lineage was evaluated in vitro. Human nasoseptal chondrogenic cells and human bone marrow mesenchymal stromal cells were used as cell types already committed to the chondrogenic lineage and undifferentiated cells, respectively, as different concentrations of platelet-rich plasma releasate were tested in comparison to commonly used fetal bovine serum. Low concentration of platelet-rich plasma releasate (2.5%) presented similar effects on cellular growth compared to 10% fetal bovine serum, for both cell types. In a three-dimensional culture system, platelet-rich plasma releasate alone did not induce full nasoseptal chondrogenic cells cartilage-like pellet formation. Nonetheless, platelet-rich plasma releasate played a significant role on cell commitment as high-passage nasoseptal chondrogenic cells only originated cartilage-like pellets when expanded in the presence of platelet-rich plasma releasate rather than fetal bovine serum. Histological analyses and measurements of pellet area demonstrated that even low concentrations of platelet-rich plasma releasate were enough to prevent nasoseptal chondrogenic cells from losing their chondrogenic potential due to in vitro expansion thereby promoting their recommitment. Low concentration of platelet-rich plasma releasate supplemented in chondrogenic medium also increased the chondrogenic potential of mesenchymal stromal cells seeded on collagen-hyaluronic acid scaffolds, as observed by an increase in chondrogenic-related gene expression, sulfated glycosaminoglycan production, and compressive modulus following in vitro culture. On the contrary, higher concentration of platelet-rich plasma releasate (10%) hampered some of these features. In conclusion, platelet-rich plasma releasate was able to prevent cellular chondrogenic capacity loss, inducing regain of their phenotype, and modulate cell commitment. Our data support the hypothesis of platelet-rich plasma chondrogenic potential, allowing fetal bovine serum substitution for platelet-rich plasma releasate at specific concentrations in culture medium when chondrogenic commitment is desired on specific cell types and moments of culture.

The bone marrow endosteal niche: how far from the surface?

Hematopoietic stem cells (HSC) self-renewal takes place in the same microenvironment in which massive hematopoietic progenitor proliferation, commitment, and differentiation will occur. This is only made possible if the bone marrow microenvironment comprises different specific niches, composed by different stromal cells that work in harmony to regulate all the steps of the hematopoiesis cascade. Histological and functional assays indicated that HSC and multipotent progenitors preferentially colonize the endosteal and subendosteal regions, in close association with the bone surface. Conversely, committed progenitors and differentiated cells are distributed in the central and perisinusoidal regions, respectively. Over the last decade, many investigative teams sought to define which cell types regulate the HSC niche, how they are organized, and to what extent they interface with each other. System dynamics requires different stromal cells to operate distinct functions over similar HSC pools rather than a single stromal cell type controlling everything. Therefore, our focus herein is to depict the players in the endosteal and subendosteal regions, named the endosteal niche, a necessary step to better understand the interactions of the HSC within the niche and to identify potential targets to manipulate and/or modulate normal and malignant HSC behavior.

Human bone marrow mesenchymal progenitors: perspectives on an optimized in vitro manipulation.

When it comes to regenerative medicine, mesenchymal stem cells (MSCs) are considered one of the most promising cell types for use in many cell therapies and bioengineering protocols. The International Society of Cellular Therapy recommended minimal criteria for defining multipotential MSC is based on adhesion and multipotency in vitro, and the presence or absence of select surface markers. Though these criteria help minimize discrepancies and allow some comparisons of data generated in different laboratories, the conditions in which cells are isolated and expanded are often not considered. Herein, we propose and recommend a few procedures to be followed to facilitate the establishment of quality control standards when working with mesenchymal progenitors isolation and expansion. Following these procedures, the classic Colony-Forming Unit-Fibroblast (CFU-f) assay is revisited and three major topics are considered to define conditions and to assist on protocol optimization and data interpretation. We envision that the creation of a guideline will help in the identification and isolation of long-term stem cells and short-term progenitors to better explore their regenerative potential for multiple therapeutic purposes.