Although cellular senescence drives multiple age-related co-morbidities through the senescence-associated secretory phenotype, in vivo senescent cell identification remains challenging. Here, we generate a gene set (SenMayo) and validate its enrichment in bone biopsies from two aged human cohorts. We further demonstrate reductions in SenMayo in bone following genetic clearance of senescent cells in mice and in adipose tissue from humans following pharmacological senescent cell clearance. We next use SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from human and murine bone marrow/bone scRNA-seq data. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Using this senescence panel, we are able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways. SenMayo also represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

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Cell development requires tight yet dynamic control of protein production. Here, we use parallel RNA and ribosome profiling to study translational regulatory dynamics during murine terminal erythropoiesis. Our results uncover pervasive translational control of protein synthesis, with widespread alternative translation initiation and termination, robust discrimination of long noncoding from micropeptide-encoding RNAs, and dynamic use of upstream open reading frames. Further, we identify hundreds of messenger RNAs (mRNAs) whose translation efficiency is dynamically controlled during erythropoiesis and that enrich for target sites of RNA-binding proteins that are specific to hematopoietic cells, thus unraveling potential regulators of erythroid translational programs. A major such program involves enhanced decoding of specific mRNAs that are depleted in terminally differentiating/enucleating cells with decreasing transcriptional capacity. We find that RBM38, an erythroid-specific RNA-binding protein previously implicated in splicing, interacts with the general translation initiation factor eIF4G and promotes translation of a subset of these irreplaceable mRNAs. Inhibition of RBM38 compromises translation in erythroblasts and impairs their maturation, highlighting a key function for this protein during erythropoiesis. These findings thus reveal critical roles for dynamic translational control in supporting specialized mammalian cell formation.

bPoint mutations with unclear molecular mechanisms are often associated with vancomycin resistance in Staphylococcus aureus. Here, we observed that the walK (G223D) mutation caused decreased expression of genes associated with cell wall metabolism, decreased autolytic activity, thickened cell walls, and reduced vancomycin susceptibility. A phosphorylation assay showed that WalK (G223D) exhibited reduced autophosphorylation, which led to reduced phosphorylation of WalR. An electrophoretic mobility shift assay indicated that WalK (G223D)-phosphorylated WalR had a reduced capacity to bind to the atlA promoter. (2), graRS (3, 4), and walKR (5, 6), were shown to contribute to the development of VISA (5, 7). However, the molecular mechanisms have been incompletely defined (8). WalK is a sensor kinase of the WalKR two-component regulatory system (9-11), and walK mutations across the spectrum of the domains that contribute to two-component regulatory function have been found in many clinical VISA strains isolated from various countries and laboratory-derived VISA (5, 6). Nevertheless, the same mutations are not found in both clinical and laboratoryderived VISA strains. Thus, a key question is whether the mechanism of laboratory-derived VISA strains is analogous to that of clinical VISA strains. WThe laboratory-derived VISA strain, designated SV-1, was selected by serial passage of the susceptible S. aureus strain MW2 through progressively increasing concentrations of vancomycin according to a previously described protocol (5). To identify the genetic changes that confer vancomycin resistance, whole-genome sequencing of wild-type MW2 and SV-1 was completed at the National Center for Gene Research using the paired-end sequencing of Solexa. Five mutations were identified (Table 1) and further confirmed by PCR and sequencing (see the supplemental material). Among the mutations, a single-nucleotide polymorphism within walK (conferring the G223D amino acid change) in SV-1 was also found in the clinical VISA strain JKD6008 (6), supporting the validity of our approach in identifying clinically relevant resistance mechanisms and suggesting that this point mutation probably plays an important role in reduced vancomycin susceptibilities in clinical S. aureus isolates.To determine the effect of the walK mutation, allelic replacement was performed to generate the walK mutant using the vector pBTs, which was derived from pBT2 and pKOR1 (12, 13). To construct pBTs, the segment containing antisense secY, which can inhibit colony formation on agar plates, was cloned into pBT2, and the fragment containing the walK point mutation was cloned into pBTs (see the supplemental material). Antibiotic susceptibilities of the walK mutant were evaluated by determining the MICs of vancomycin and daptomycin using

Cellular senescence is a plausible and potentially tractable mechanism of aging. Molecular hallmarks of senescent cells, including the expression of cyclin-dependent kinase inhibitors (CDKIs), DNA damage-response proteins, and mediators and components of the senescence-associated secretory phenotype (SASP), increase in multiple tissues with advancing age and chronic disease (Tuttle et al., 2020). Indeed, in mice, targeted elimination of senescent cells by genetic approaches and candidate senotherapeutic drugs restores tissue health and attenuates the progression of numerous age-related

Cellular senescence is a plausible mediator of inflammation-related tissue dysfunction. In the aged brain, senescent cell identities and the mechanisms by which they exert adverse influence are unclear. Here we used high-dimensional molecular profiling, coupled with mechanistic experiments, to study the properties of senescent cells in the aged mouse brain. We show that senescence and inflammatory expression profiles increase with age and are brain region- and sex-specific. p16-positive myeloid cells exhibiting senescent and disease-associated activation signatures, including upregulation of chemoattractant factors, accumulate in the aged mouse brain. Senescent brain myeloid cells promote peripheral immune cell chemotaxis in vitro. Activated resident and infiltrating immune cells increase in the aged brain and are partially restored to youthful levels through p16-positive senescent cell clearance in female p16-InkAttac mice, which is associated with preservation of cognitive function. Our study reveals dynamic remodeling of the brain immune cell landscape in aging and suggests senescent cell targeting as a strategy to counter inflammatory changes and cognitive decline.

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