The Evf2 Ultraconserved Enhancer lncRNA Functionally and Spatially Organizes Megabase Distant Genes in the Developing Forebrain.

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.

Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells.

BACKGROUND: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. RESULTS: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. CONCLUSIONS: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.

Positive selection in dNTPase SAMHD1 throughout mammalian evolution.

The vertebrate protein SAMHD1 is highly unusual in having roles in cellular metabolic regulation, antiviral restriction, and regulation of innate immunity. Its deoxynucleoside triphosphohydrolase activity regulates cellular dNTP concentration, reducing levels below those required by lentiviruses and other viruses to replicate. To counter this threat, some primate lentiviruses encode accessory proteins that bind SAMHD1 and induce its degradation; in turn, positive diversifying selection has been observed in regions bound by these lentiviral proteins, suggesting that primate SAMHD1 has coevolved to evade these countermeasures. Moreover, deleterious polymorphisms in human SAMHD1 are associated with autoimmune disease linked to uncontrolled DNA synthesis of endogenous retroelements. Little is known about how evolutionary pressures affect these different SAMHD1 functions. Here, we examine the deeper history of these interactions by testing whether evolutionary signatures in SAMHD1 extend to other mammalian groups and exploring the molecular basis of this coevolution. Using codon-based likelihood models, we find positive selection in SAMHD1 within each mammal lineage for which sequence data are available. We observe positive selection at sites clustered around T592, a residue that is phosphorylated to regulate SAMHD1 activity. We verify experimentally that mutations within this cluster affect catalytic rate and lentiviral restriction, suggesting that virus-host coevolution has required adaptations of enzymatic function. Thus, persistent positive selection may have involved the adaptation of SAMHD1 regulation to balance antiviral, metabolic, and innate immunity functions.

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers.

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.

The missing link: allostery and catalysis in the anti-viral protein SAMHD1.

Vertebrate protein SAMHD1 (sterile-α-motif and HD domain containing protein 1) regulates the cellular dNTP (2'-deoxynucleoside-5'-triphosphate) pool by catalysing the hydrolysis of dNTP into 2'-deoxynucleoside and triphosphate products. As an important regulator of cell proliferation and a key player in dNTP homeostasis, mutations to SAMHD1 are implicated in hypermutated cancers, and germline mutations are associated with Chronic Lymphocytic Leukaemia and the inflammatory disorder Aicardi-Goutières Syndrome. By limiting the supply of dNTPs for viral DNA synthesis, SAMHD1 also restricts the replication of several retroviruses, such as HIV-1, and some DNA viruses in dendritic and myeloid lineage cells and resting T-cells. SAMHD1 activity is regulated throughout the cell cycle, both at the level of protein expression and post-translationally, through phosphorylation. In addition, allosteric regulation further fine-tunes the catalytic activity of SAMHD1, with a nucleotide-activated homotetramer as the catalytically active form of the protein. In cells, GTP and dATP are the likely physiological activators of two adjacent allosteric sites, AL1 (GTP) and AL2 (dATP), that bridge monomer-monomer interfaces to stabilise the protein homotetramer. This review summarises the extensive X-ray crystallographic, biophysical and molecular dynamics experiments that have elucidated important features of allosteric regulation in SAMHD1. We present a comprehensive mechanism detailing the structural and protein dynamics components of the allosteric coupling between nucleotide-induced tetramerization and the catalysis of dNTP hydrolysis by SAMHD1.

Biological treatment of the knee with platelet-rich plasma or bone marrow aspirate concentrates.

- Knee pathologies including focal cartilage injuries, osteoarthritis (OA), and ligament injuries are common. The poor regeneration and healing potential of cartilage has led to the search for other treatment modalities with improved healing capacity. Furthermore, with an increasing elderly population that desires to remain active, the burden of knee pathologies is expected to increase. Increased sports participation and the desire to return to activities faster is also demanding more effective and minimally invasive treatment options. Thus, the use of biologic agents in the treatment of knee pathologies has emerged as a potential option. Despite the increasing use of biologic agents for knee pathology, there are conflicting results on the efficacy of these products. Furthermore, strong data supporting the optimal preparation methods and composition for widely used biologic agents, such as platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC), largely remain absent from the literature. This review presents the literature on the most commonly employed biologic agents for the different knee pathologies.

A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa.

CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA_TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA. We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins.

Biochemical characterization and comparison of two closely related active mariner transposases.

Most DNA transposons move from one genomic location to another by a cut-and-paste mechanism and are useful tools for genomic manipulations. Short inverted repeat (IR) DNA sequences marking each end of the transposon are recognized by a DNA transposase (encoded by the transposon itself). This enzyme cleaves the transposon ends and integrates them at a new genomic location. We report here a comparison of the biophysical and biochemical properties of two closely related and active mariner/Tc1 family DNA transposases: Mboumar-9 and Mos1. We compared the in vitro cleavage activities of the enzymes on their own IR sequences, as well as cross-recognition of their inverted repeat sequences. We found that, like Mos1, untagged recombinant Mboumar-9 transposase is a dimer and forms a stable complex with inverted repeat DNA in the presence of Mg(2+) ions. Mboumar-9 transposase cleaves its inverted repeat DNA in the manner observed for Mos1 transposase. There was minimal cross-recognition of IR sequences between Mos1 and Mboumar-9 transposases, despite these enzymes having 68% identical amino acid sequences. Transposases sharing common biophysical and biochemical properties, but retaining recognition specificity toward their own IR, are a promising platform for the design of chimeric transposases with predicted and improved sequence recognition.

Platform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis.

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2'-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes.

Exploring knotting mechanisms in protein folding.

One of the most striking topological features to be found in a protein is that of a distinct knot formed by the path of the polypeptide backbone. Such knotted structures represent some of the smallest "self-tying" knots observed in Nature. Proteins containing a knot deep within their structure add an extra complication to the already challenging protein-folding problem; it is not obvious how, during the process of folding, a substantial length of polypeptide chain manages to spontaneously thread itself through a loop. Here, we probe the folding mechanism of YibK, a homodimeric alpha/beta-knot protein containing a deep trefoil knot at its carboxy terminus. By analyzing the effect of mutations made in the knotted region of the protein we show that the native structure in this area remains undeveloped until very late in the folding reaction. Single-site destabilizing mutations made in the knot structure significantly affect only the folding kinetics of a late-forming intermediate and the slow dimerization step. Furthermore, we find evidence to suggest that the heterogeneity observed in the denatured state is not caused by isomerization of the single cis proline bond as previously thought, but instead could be a result of the knotting mechanism. These results allow us to propose a folding model for YibK where the threading of the polypeptide chain and the formation of native structure in the knotted region of the protein occur independently as successive events.

A quantitative serum biomarker of circulating collagen X effectively correlates with endochondral fracture healing.

Currently, there are no standardized methods for quantitatively measuring fracture repair. Physicians rely on subjective physical examinations and qualitative evaluation of radiographs to detect mineralized tissue. Since most fractures heal indirectly through a cartilage intermediate, these tools are limited in their diagnostic utility of early repair. Prior to converting to the bone, cartilage undergoes hypertrophic maturation, characterized by the deposition of a provisional collagen X matrix. The objective of this study was to characterize the kinetics of a novel collagen X biomarker relative to other biological measurements of fracture healing using a murine model of endochondral fracture repair in which a closed, mid-shaft tibia fracture was created using the classic drop-weight technique. Serum was collected 5 to 42 days post-fracture in male and female mice and compared to uninjured controls (n = 8-12). Collagen X in the serum was quantified using a recently validated ELISA-based bioassay ("Cxm")1 and compared to genetic and histological markers of fracture healing and inflammation. We found the Cxm biomarker reliably increased from baseline to a statistically unique peak 14 days post-fracture that then resolved to pre-fracture levels by 3 weeks following injury. The shape and timing of the Cxm curve followed the genetic and histological expression of collagen X but did not show a strong correlation with local inflammatory states. Assessment of fracture healing progress is crucial to making correct and timely clinical decisions for patients. This Cxm bioassay represents a minimally invasive, inexpensive technique that could provide reliable information on the biology of the fracture to significantly improve clinical care.

Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis.

SAMHD1 regulates cellular 2'-deoxynucleoside-5'-triphosphate (dNTP) homeostasis by catalysing the hydrolysis of dNTPs into 2'-deoxynucleosides and triphosphate. In CD4+ myeloid lineage and resting T-cells, SAMHD1 blocks HIV-1 and other viral infections by depletion of the dNTP pool to a level that cannot support replication. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome and hypermutated cancers. Furthermore, SAMHD1 sensitises cancer cells to nucleoside-analogue anti-cancer therapies and is linked with DNA repair and suppression of the interferon response to cytosolic nucleic acids. Nevertheless, despite its requirement in these processes, the fundamental mechanism of SAMHD1-catalysed dNTP hydrolysis remained unknown. Here, we present structural and enzymological data showing that SAMHD1 utilises an active site, bi-metallic iron-magnesium centre that positions a hydroxide nucleophile in-line with the Pα-O5' bond to catalyse phosphoester bond hydrolysis. This precise molecular mechanism for SAMHD1 catalysis, reveals how SAMHD1 down-regulates cellular dNTP and modulates the efficacy of nucleoside-based anti-cancer and anti-viral therapies.

Characterization of the chondrogenic and osteogenic potential of male and female human muscle-derived stem cells: Implication for stem cell therapy.

People of all backgrounds are susceptible to bone and cartilage damage, and these injuries can be debilitating. Current treatments for bone and cartilage injuries are less than optimal, and we are interested in developing new approaches to treat these diseases, specifically using human muscle-derived stem cells (hMDSCs). Our lab previously demonstrated that sex differences exist between male and female murine MDSCs; thus, this paper sought to investigate whether sex differences also exist in hMDSCs. In the present study, we characterized the chondrogenic and osteogenic sex differences of hMDSCs in vitro and in vivo. We performed in vitro osteogenic and chondrogenic differentiation using hMDSC pellet cultures. As demonstrated by microCT, histology, and immunohistochemistry, male hMDSCs were more chondrogenic and osteogenic than their female counterparts in vitro. No differences were observed based on the sex of hMDSCs in osteogenic and chondrogenic gene expression and cell surface markers. For our in vivo study, we transduced hMDSCs with lenti-BMP2/GFP and transplanted these cells into critical-sized calvarial defects in mice. MicroCT results revealed that male hMDSCs regenerated more bone at 2 weeks and demonstrated higher bone density at 4 and 6 weeks than female hMDSCs. Histology demonstrated that both male and female hMDSCs regenerated functional bone. Clinical relevance: These studies reinforce that stem cells isolated from male and female patients differ in function, and we should disclose the sex of cells used in future studies. Considering sex differences of hMDSCs may help to improve cell-based therapies for autologous cell treatment of bone and cartilage damage. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1339-1349, 2019.

Variations in Blood Supply From Proximal to Distal in the Ulnar Collateral Ligament of the Elbow: A Qualitative Descriptive Cadaveric Study.

BACKGROUND: The vascular supply of the ulnar collateral ligament (UCL) is unknown. Previous studies reported varying success in return-to-play rates after nonoperative management of partial UCL tears and suggested a varying healing capacity as possibly related to the location of the UCL injury. PURPOSE: To analyze the macroscopic vascular anatomy of the UCL of the elbow. STUDY DESIGN: Descriptive laboratory study. METHODS: Eighteen fresh-frozen male cadaveric elbows from 9 donors were sharply dissected 15 cm proximal to the medial epicondyle. Sixty milliliters of India ink was injected through the brachial artery of each elbow. Arms were then frozen at -10°C, radial side down, in 15° to 20° of elbow flexion. A band saw was used to section the frozen elbows into 5-mm coronal or sagittal sections. Sections were cleared for visualization with the modified Spalteholz technique. Images of the specimens were taken, and qualitative description of UCL vascularity was undertaken. RESULTS: The authors consistently found a dense blood supply to the proximal UCL, while the distal UCL was hypovascular. They also observed a possible osseous contribution to the proximal UCL from the medial epicondyle in addition to an artery from the flexor/pronator musculature that consistently appeared to provide vascularity to the proximal UCL. The degree of vascular penetration from proximal to distal in the UCL ranged from 39% to 68% of the overall UCL length, with a 49% mean length of vascular penetration of the UCL. CONCLUSION: This study found a difference in the vascular supply of the UCL. The proximal UCL was well vascularized, while the distal UCL was hypovascular. This difference in vascular supply may be a factor in the differential healing capacities of the UCL based on the location of injury. CLINICAL RELEVANCE: An improved understanding of the macroscopic vascular supply of the UCL may aid in the clinical management of partial UCL tears and suggests an indication for these treatments with respect to location of UCL injuries.

Quantitative and Qualitative Assessment of the Posterior Medial Meniscus Anatomy: Defining Meniscal Ramp Lesions.

BACKGROUND: Meniscal ramp lesions have been defined as a tear of the peripheral attachment of the posterior horn of the medial meniscus (PHMM) at the meniscocapsular junction or an injury to the meniscotibial attachment. Precise anatomic descriptions of these structures are limited in the current literature. PURPOSE: To quantitatively and qualitatively describe the PHMM and posteromedial capsule anatomy pertaining to the location of a meniscal ramp lesion with reference to surgically relevant landmarks. STUDY DESIGN: Descriptive laboratory study. METHODS: Fourteen male nonpaired fresh-frozen cadavers were used. The locations of the posteromedial meniscocapsular and meniscotibial attachments were identified. Measurements to surgically relevant landmarks were performed with a coordinate measuring system. To further analyze the posteromedial meniscocapsular and meniscotibial attachments, hematoxylin and eosin and alcian blue staining were conducted on a separate sample of 10 nonpaired specimens. RESULTS: The posterior meniscocapsular attachment had a mean ± SD length of 20.2 ± 6.0 mm and attached posteroinferiorly to the PHMM at a mean depth of 36.4% of the total posterior meniscal height. The posterior meniscotibial ligament attached on the PHMM 16.5 mm posterior and 7.7 mm medial to the center of the posterior medial meniscal root attachment. The meniscotibial ligament tibial attachment was 5.9 ± 1.3 mm inferior to the articular cartilage margin of the posterior medial tibial plateau. The posterior meniscocapsular attachment converged with the meniscotibial ligament at the most posterior point of the meniscocapsular junction in all specimens. Histological staining of the meniscocapsular and meniscotibial ligament PHMM attachments showed similar structure, cell density, and fiber directionality, with no qualitative difference in the makeup of their collagen matrices across all specimens. CONCLUSION: The anatomy of the area where a medial meniscal ramp tear occurs revealed that the 2 posterior meniscal attachments merged at a common attachment on the PHMM. Histological analysis validated a shared attachment point of the meniscocapsular and meniscotibial attachments of the PHMM. CLINICAL RELEVANCE: The findings of this study provide the anatomic foundation for an improved understanding of the meniscocapsular and meniscotibial attachments of the PHMM, which may help provide a more precise definition of a meniscal ramp lesion.

Evaluation of Endothelial and Vascular-Derived Progenitor Cell Populations in the Proximal and Distal UCL of the Elbow: A Comparative Study.

Background: Vascular-derived progenitor and endothelial cell populations (CD31, CD34, CD146) are capable of multipotent differentiation at the site of injured ligamentous tissue to aid in the intrinsic healing response. Proximal ulnar collateral ligament (UCL) tears have been reported to have better healing capability when compared with distal UCL tears. Purpose: To compare the vascular composition of the proximal and distal insertions of the anterior bundle of the UCL of the elbow via known markers of endothelial and vascular-derived progenitor cells (CD31, CD34, CD146). Study Design: Descriptive laboratory study. Methods: UCLs were harvested from 10 nonpaired fresh-frozen human cadaveric elbows and transected into proximal and distal portions. Endothelial and vascular-derived progenitor cell densities were assessed with 4 staining groups: CD31 (immunohistochemistry) and CD31/α-smooth muscle actin (α-SMA), CD34/α-SMA, and CD146/α-SMA (immunofluorescence). CD31 immunohistochemistry identified endothelial progenitor cells in the UCL. Later staining of the same slides with α-SMA demonstrated the relationship of progenitor cells to the surrounding vasculature. Fluorescent staining was quantified by calculating the proportion of positively stained nuclei versus the total number of nuclei in the proximal and distal UCL. Results: CD31+ cells were present in the proximal and distal sections of all 10 UCLs. Fluorescent staining revealed no significant differences in the ratio of CD31 to total nuclei between the distal (median, 36% [range, 23%-53%]) and proximal UCL (39% [22%-56%]) (P = .432, Wilcoxon signed-rank test). Similarly, no differences were seen between CD34 distal (39% [24%-64%]) and proximal regions (46% [28%-63%]) (P = .846, Wilcoxon signed-rank test) or CD146 distal (40% [12%-65%]) and proximal regions (40% [22%-51%]) (P ≥ .999, Wilcoxon signed-rank test). Conclusion: Analysis of UCL tissues demonstrated equal distributions of vascular endothelial and vascular-derived progenitor cell markers throughout the proximal and distal UCL. Unlike that of the medial collateral ligament of the knee, the microvascular composition of the proximal and distal UCL insertions was not different, suggesting a well-vascularized ligament throughout its course. Clinical Relevance: These findings investigate one of the possible contributors to UCL healing after injury, which may provide insight into operative and nonoperative management of UCL injuries in the future. This study also indicates that reasons other than differences in progenitor cell density alone may explain the clinical healing differences seen between proximal and distal UCL tears. A better understanding of the microvascular environment and associated blood supply is warranted to understand the healing capability of the UCL.

Structural basis for DNA 3'-end processing by human tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1.

Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration.

BACKGROUND: Human muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study. METHODS: We isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells' age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA). RESULTS: We found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1. CONCLUSION: hMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.

A bend, flip and trap mechanism for transposon integration.

Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.

Structural basis of Mos1 transposase inhibition by the anti-retroviral drug Raltegravir.

DNA transposases catalyze the movement of transposons around genomes by a cut-and-paste mechanism related to retroviral integration. Transposases and retroviral integrases share a common RNaseH-like domain with a catalytic DDE/D triad that coordinates the divalent cations required for DNA cleavage and integration. The anti-retroviral drugs Raltegravir and Elvitegravir inhibit integrases by displacing viral DNA ends from the catalytic metal ions. We demonstrate that Raltegravir, but not Elvitegravir, binds to Mos1 transposase in the presence of Mg(2+) or Mn(2+), without the requirement for transposon DNA, and inhibits transposon cleavage and DNA integration in biochemical assays. Crystal structures at 1.7 Å resolution show Raltegravir, in common with integrases, coordinating two Mg(2+) or Mn(2+) ions in the Mos1 active site. However, in the absence of transposon ends, the drug adopts an unusual, compact binding mode distinct from that observed in the active site of the prototype foamy virus integrase.