We present the results of a 5-month photometric time-series survey for stellar rotation periods combined with a 4-year radial-velocity survey for membership and binarity in the 220 Myr open cluster M34. We report surface rotation periods for 120 stars, 83 of which are kinematic and photometric late-type cluster members. A comparison to previous work serves to illustrate the importance of 1 WIYN Open Cluster Study XLV

Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected persons have identified numerous examples of HLA-associated mutations within or adjacent T cell epitopes, but the potential impact of most mutations on epitope production and presentation remains unclear. The multistep breakdown of proteins into epitopes includes trimming of N-extended peptides into epitopes by aminopeptidases before loading onto MHC-I molecules. Defining sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. Here we identified cytosolic aminopeptidases cleavage preferences in primary cells, its impact on HIV antigen degradation into epitopes in primary human cell extracts by mass spectrometry, and on epitope presentation to CTL. We observed a hierarchy of preferred amino acid cleavage by cytosolic aminopeptidases. We demonstrated that flanking mutations producing more or less cleavable motifs can increase or decrease epitope production and presentation by up to 14-fold. We found that the efficiency of epitope production correlates with cleavability of flanking residues. These in vitro findings were supported by in vivo population-level analyses of clinically-derived viral sequences from 1134 antiretroviral-naïve HIV-infected persons: HLA-associated mutations immune pressures drove the selection of residues that are less cleavable by aminopeptidases predominantly at N-flanking sites, leading to reduced epitope production and immune recognition. These results underscore an important and widespread role of antigen processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.

Dendritic cells (DCs), macrophages (MPs) and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous antigens preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum (ER) translocation, trimming and MHC-I presentation. Here we compared the capacity of DCs, MPs and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848 and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs and monocytes. Differences in antigen processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load.

Focal cartilage defects caused by joint injury have a limited capacity to self-repair and, if left untreated, can lead to the early onset of osteoarthritis. The current standard of care, microfracture surgery, induces an endogenous repair response, but typically results in poorly integrated fibrocartilage, rather than native hyaline cartilage. The objective of this study was to test the hypothesis that a self-assembling peptide hydrogel functionalized with the proanabolic growth factor heparin-binding insulin-like growth factor-1 (HB-IGF-1) may improve integration between native cartilage and neotissue when combined with a brief enzymatic pretreatment to the defect site. This enzymatic pretreatment releases proteoglycans from the walls of the surrounding native cartilage in a controlled manner and, thereby, creates space for newly synthesized repair tissue to anchor and integrate with adjacent host cartilage. We used an in vitro model in which a cylindrical annulus of native cartilage was pretreated with trypsin over a 2-min period and then filled with a chondrocyte-seeded [KLDL] 3 hydrogel functionalized with proanabolic HB-IGF-1 that had been premixed into the gel. This procedure was deemed to be clinically tractable in the context of ongoing parallel animal studies as a method to augment the microfracture procedure. The trypsin pretreatment depleted proteoglycan content of adjacent cartilage in a controlled manner without inducing cell death. The addition of HB-IGF-1 was found to stimulate matrix biosynthesis both in the surrounding cartilage and the chondrocyte-seeded KLD scaffold, and to enhance mechanical integration of neotissue into native matrix.

No abstract

We have determined the change in the number of proteins bound non-specifically to DNA as a function of applied force using force–extension measurements on tethered DNA. Using magnetic tweezers, single molecules of λ DNA were repeatedly stretched and relaxed in the absence and presence of 170 nM λ repressor protein (CI). CI binds to six specific sites of λ DNA with nanomolar affinity and also binds non-specifically with micromolar affinity. The force versus extension data were analyzed using a recently developed theoretical framework for quantitative determination of protein binding to the DNA. The results indicate that the non-specific binding of CI changes the force–extension relation significantly in comparison to that of naked DNA. The DNA tether used in our experiment would have about 640 bound repressors, if it was completely saturated with bound proteins. We find that as the pulling force on DNA is reduced from 4.81 to 0.13 pN, approximately 138 proteins bind to DNA, which is about 22% of the length of the tethered DNA. Our results show that 0.13 pN is not low enough to cause saturation of DNA by repressor and 4.81 pN is also not high enough to eliminate all the repressors bound to DNA. This demonstrates that the force–extension relation provides an effective approach for estimating the number of proteins bound non-specifically to a DNA molecule.

Tissue engineering approaches using growth factor-functionalized acellular scaffolds to support and guide repair driven by endogenous cells are thought to require a careful balance between cell recruitment and growth factor release kinetics. The objective of this study was to identify a growth factor combination that accelerates progenitor cell migration into self-assembling peptide hydrogels in the context of cartilage defect repair. A novel 3D gel-to-gel migration assay enabled quantification of the chemotactic impact of platelet-derived growth factor-BB (PDGF-BB), heparin-binding insulin-like growth factor-1 (HB-IGF-1), and transforming growth factorb1 (TGF-b1) on progenitor cells derived from subchondral bovine trabecular bone (bone-marrow progenitor cells, BM-PCs) encapsulated in the peptide hydrogel [KLDL] 3 . Only the combination of PDGF-BB and TGF-b1 stimulated significant migration of BM-PCs over a 4-day period, measured by confocal microscopy. Both PDGF-BB and TGF-b1 were slowly released from the gel, as measured using their 125

The objective of this study was to improve cartilage repair and integration using self‐assembling KLD hydrogel functionalized with platelet‐derived growth factor‐BB and heparin‐binding insulin‐like growth factor‐1 with associated enzymatic trypsin pre‐treatment of the native cartilage. Bilateral osteochondral defects were created at the central portion of the femoral trochlear groove of 48 skeletally mature, white New Zealand rabbits. One limb received a randomly assigned treatment and the contralateral limb served as the control. Treated defects were exposed to trypsin for 2 min and filled with self‐assembling KLD hydrogel only, or associated to growth factors. All control limbs received KLD hydrogel alone or received only trypsin but not hydrogel. Ninety days post‐defect creation, the rabbits were euthanized and magnetic resonance imaging, radiography, macroscopic evaluation, histology, and immunohistochemistry of the joint and repaired tissue were performed. Mixed model analyses of variance were utilized to assess the outcome parameters and individual comparisons were performed using Least Square Means procedure and differences with p‐value < 0.05 were considered significant. Trypsin enzymatic pre‐treatment improved cellular morphology, cluster formation and subchondral bone reconstitution. Platelet‐derived growth factor‐BB improved subchondral bone healing and basal integration. Heparin‐binding insulin‐like growth factor‐1 associated with platelet‐derived growth factor improved tissue and cell morphology. The authors conclude that self‐assembling KLD hydrogel functionalized with platelet‐derived growth factor and heparin‐binding insulin‐like growth factor‐1 with associated enzymatic pre‐treatment of the native cartilage with trypsin resulted in an improvement on the cartilage repair process. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2307–2315, 2019