Potently neutralizing and protective human antibodies against SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.

Does Surgical Approach Affect Dislocation Rate After Total Hip Arthroplasty in Patients Who Have Prior Lumbar Spinal Fusion? A Retrospective Analysis of 16,223 Cases.

BACKGROUND: Lumbar spinal fusion (LSF) is a risk factor for dislocation following total hip arthroplasty (THA). The effect of the surgical approach on this association has not been investigated. This study examined the association between the surgical approach and dislocation following THA in patients who had prior LSF. METHODS: We retrospectively reviewed 16,223 primary elective THAs at our institution from June 2011 to September 2022. Patients who had LSF prior to THA were identified using International Classification of Diseases (ICD) codes. Patients were stratified by LSF history, surgical approach, and intraoperative robot or navigation use to compare dislocation rates. There were 8,962 (55.2%) posterior, 5,971 (36.8%) anterior, and 1,290 (8.0%) laterally based THAs. Prior LSF was identified in 323 patients (2.0%). Binary logistic regressions were used to assess the association of patient factors with dislocation risk. RESULTS: There were 177 dislocations identified in total (1.1%). In nonadjusted analyses, the dislocation rate was significantly higher following the posterior approach among all patients (P = .003). Prior LSF was associated with a significantly higher dislocation rate in all patients (P < .001) and within the posterior (P < .001), but not the anterior approach (P = .514) subgroups. Multivariate regressions demonstrated anterior (OR [odds ratio] = 0.64, 95% CI [confidence interval] 0.45 to 0.91, P = .013), and laterally based (OR = 0.42, 95% CI 0.18 to 0.96, P = .039) approaches were associated with decreased dislocation risk, whereas prior LSF (OR = 4.28, 95% CI 2.38 to 7.69, P < .001) was associated with increased dislocation risk. Intraoperative technology utilization was not significantly associated with dislocation in the multivariate regressions (OR = 0.72, 95% CI 0.49 to 1.06, P = .095). CONCLUSIONS: The current study confirmed that LSF is a significant risk factor for dislocation following THA; however, anterior and laterally based approaches may mitigate dislocation risk in this population. In multivariate analyses, including surgical approach, LSF, and several perioperative variables, intraoperative technology utilization was not found to be significantly associated with dislocation risk.

Cell population growth kinetics in the presence of stochastic heterogeneity of cell phenotype.

Recent studies at individual cell resolution have revealed phenotypic heterogeneity in nominally clonal tumor cell populations. The heterogeneity affects cell growth behaviors, which can result in departure from the idealized uniform exponential growth of the cell population. Here we measured the stochastic time courses of growth of an ensemble of populations of HL60 leukemia cells in cultures, starting with distinct initial cell numbers to capture a departure from the uniform exponential growth model for the initial growth ("take-off"). Despite being derived from the same cell clone, we observed significant variations in the early growth patterns of individual cultures with statistically significant differences in growth dynamics, which could be explained by the presence of inter-converting subpopulations with different growth rates, and which could last for many generations. Based on the hypothesis of existence of multiple subpopulations, we developed a branching process model that was consistent with the experimental observations.

Explicit effort may not influence perceptuomotor decision-making.

Many decisions that humans make are enacted by the action system. For example, humans use reach-to-grasp movements when making perceptuomotor decisions between and obtaining fruits of varying quality from a pile. Recent work suggests that the characteristics of each action alternative may influence the decision itself-there may be a bias away from making perceptuomotor alternatives associated with high effort when participants are unaware of the effort differences between responses. The present study examined if perceptuomotor decisions were influenced by explicit reaching effort differences. Neurotypical human participants were presented with random dot motion stimuli in which most dots moved in random directions and varying percentages of remaining dots moved coherently left- or rightward. Participants reported leftward motion judgements by performing leftward (or left hand) reaching movements and rightward motion judgements by performing rightward (or right hand) reaching movements. A resistance band was affixed to participants' wrists and to the table in different configurations. The configurations allowed for one movement/motion direction judgement to always require stretching of the band and, therefore, require relatively more effort. Across a set of experiments, the response context (i.e. selecting directions within a limb or selecting between limbs) and the effort difference between responses were manipulated. Overall, no experiment revealed a bias away from the perceptuomotor decision associated with high effort. Based on these results, it is concluded that, in this biomechanical context, explicit effort may not influence perceptuomotor decision-making and may point to a contextual influence of action effort on perceptuomotor decision-making.

Discovery of Marburg virus neutralizing antibodies from virus-naïve human antibody repertoires using large-scale structural predictions.

Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. We computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a full-length native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.

Human Monoclonal Antibodies to Escherichia coli Outer Membrane Protein A Porin Domain Cause Aggregation but Do Not Alter In Vivo Bacterial Burdens in a Murine Sepsis Model.

Escherichia coli is one of the most frequent human pathogens, increasingly exhibits antimicrobial resistance, and has complex interactions with the host immune system. E. coli exposure or infection can result in the generation of antibodies specific for outer membrane protein A (OmpA), a multifunctional porin. We identified four OmpA-specific naturally occurring antibodies from healthy human donor B cells and assessed their interactions with E. coli and OmpA. These antibodies are highly specific for OmpA, exhibiting no cross-reactivity to a strain lacking ompA and retaining binding to both laboratory and clinical isolates of E. coli in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays. One monoclonal antibody (Mab), designated ECOL-11, is specific for the extracellular N-terminal porin domain of OmpA and induces growth phase-specific bacterial aggregation. This aggregation is not induced by the fragment antigen binding (Fab) form of the MAb, suggesting the importance of bivalency for this aggregating activity. ECOL-11 decreases adhesion and phagocytosis of E. coli by RAW 264.7 macrophage-like cells, possibly by inhibiting the adhesion functions of OmpA. Despite this in vitro phenotype, organ E. coli burdens were not altered by antibody prophylaxis in a murine model of lethal E. coli septic shock. Our findings support the importance of OmpA at the host-pathogen interface and begin to explore the implications and utility of E. coli-specific antibodies in human hosts.

Tourniquet Use Is Associated With Reduced Blood Loss and Fewer Reoperations in Aseptic Revision Total Knee Arthroplasty.

BACKGROUND: Although tourniquet use in primary total knee arthroplasty (TKA) has been widely studied, the outcomes associated with tourniquet use in revision TKA (rTKA) remains relatively unexplored. This study investigates surgical outcomes and patient satisfaction in association with tourniquet use during aseptic rTKA. METHODS: We retrospectively reviewed all patients who underwent rTKA for aseptic causes at our institution from 2011 to 2020. Patients were separated into 2 cohorts based on tourniquet inflation during the procedure. Outcomes of interest included estimated blood loss, change in hemoglobin, surgical time, length of stay, reoperation rate, and Knee Injury and Osteoarthritis Outcome Score for Joint Replacement (KOOS, JR) scores. RESULTS: Of the 1212 patients included, 1007 (83%) underwent aseptic rTKA with the use of a tourniquet and 205 (17%) without the use of a tourniquet. The mean tourniquet inflation time was 93.0 minutes (standard deviation 33.3 minutes). Blood loss was significantly less for patients in the tourniquet cohort as measured through estimated blood loss (224.1 vs 325.1 mL, P < .001) and change in preoperative to postoperative hemoglobin (1.75 vs 2.04 g/dL, P < .001). There were no statistical differences in surgical time (P = .267) and length of stay (P = .206) between the 2 groups. The reoperation rate was significantly greater for patients who did not have a tourniquet utilized (20.5% vs 15.0%, P = .038). Delta improvement in KOOS, JR scores from baseline to 3 months postoperatively did not statistically differ between the 2 cohorts (P = .560). CONCLUSION: Although delta improvements in KOOS, JR scores were similar for both cohorts, patients who did not have a tourniquet inflated during aseptic rTKA had increased blood loss and were more likely to undergo subsequent reoperation compared to patients who did. LEVEL III EVIDENCE: Retrospective Cohort Study.

Presence of back pain prior total knee arthroplasty and its effects on short-term patient-reported outcome measures.

PURPOSE: Back pain may both decrease patient satisfaction after TKA and confound outcome assessment in satisfied patients. Our primary objective was to determine whether preoperative back pain is associated with differences in postoperative patient-reported outcome measures (PROMs). METHODS: We retrospectively reviewed 234 primary TKA patients who completed PROMs preoperatively and 12 weeks postoperatively, which included a back pain questionnaire, the Knee Injury and Osteoarthritis Outcome Score for Joint Replacement (KOOS JR) and the Forgotten Joint Score-12 (FJS-12). Cohorts were defined based on the severity of preoperative back pain (none, mild, moderate and severe) and compared. Demographics were compared using ANOVA and Chi-square analysis. Univariate ANCOVA analysis was utilized to compare PROMs while accounting for significant demographic differences. RESULTS: Both preoperative KOOS JR scores (none: 47.90, mild: 47.61, moderate: 44.61 and severe: 38.70; p = 0.013) and 12-week postoperative KOOS JR scores (none: 61.24, mild: 64.94, moderate: 57.48 and severe: 57.01; p = 0.012) had a statistically significant inverse relationship with regard to the intensity of preoperative back pain. Although FJS-12 scores at the 12-week postoperative period trended lower with increasing levels of preoperative back pain (p = 0.362), it did not reach statistical significance. Patients who reported severe back pain preoperatively achieved the largest delta improvement from baseline compared to those with lesser pain intensity (p = 0.003). Patients who had a 2-grade improvement in their back pain achieved significantly higher KOOS JR scores 12 weeks postoperatively compared to patients with either 1-grade or no improvement (63.53 vs. 55.98; p = 0.042). Both preoperative (47.99 vs. 41.11; p = 0.003) and 12-week postoperative (64.06 vs. 55.73; p < 0.001) KOOS JR scores were statistically higher for those who reported mild or no back pain pre-and postoperatively than those who reported moderate or severe back pain pre-and postoperatively. CONCLUSION: Knee pain and back pain both exert negative effects on outcome instruments designed to measure pain and function. Although mean improvement from pre- to postoperative KOOS JR scores for patients with severe pre-existing back pain was higher than their counterparts, this statistical difference is likely not clinically significant. This implies that all patients may experience similar benefits from TKA despite the presence or absence of back pain. Attempts to measure TKA outcomes using PROMs should seek to control for lumbago and other sources of body pain. Level of Evidence IIIRetrospective Cohort Study.

Improved exercise capacity in cyclophilin-D knockout mice associated with enhanced oxygen utilization efficiency and augmented glucose uptake via AMPK-TBC1D1 signaling nexus.

We previously reported in HEK 293T cells that silencing the mitochondrial peptidyl prolyl isomerase cyclophilin-D (Cyp-D) reduces Vo2. We now report that in vivo Cyp-D ablation using constitutive Cyp-D knockout (KO) mice also reduces Vo2 both at rest (∼15%) and during treadmill exercise (∼12%). Yet, despite Vo2 reduction, these Cyp-D KO mice ran longer (1071 ± 77 vs. 785 ± 79 m; P = 0.002), for longer time (43 ± 3 vs. 34 ± 3 min; P = 0.004), and at higher speed (34 ± 1 vs. 29 ± 1 m/s; P ≤ 0.001), resulting in increased work (87 ± 6 vs. 58 ± 6 J; P ≤ 0.001). There were parallel reductions in carbon dioxide production, but of lesser magnitude, yielding a 2.3% increase in the respiratory exchange ratio consistent with increased glucose utilization as respiratory substrate. In addition, primary skeletal muscle cells of Cyp-D KO mice subjected to electrical stimulation exhibited higher glucose uptake (4.4 ± 0.55 vs. 2.6 ± 0.04 pmol/mg/min; P ≤ 0.001) with enhanced AMPK activation (0.58 ± 0.06 vs. 0.38 ± 0.03 pAMPK/ß-tubulin ratio; P ≤ 0.01) and TBC1 (Tre-2/USP6, BUB2, Cdc16) domain family, member 1 (TBC1D1) inactivation. Likewise, pharmacological activation of AMPK also increased glucose uptake (3.2 ± 0.3 vs. 2.3 ± 0.2 pmol/mg/min; P ≤ 0.001). Moreover, lactate and ATP levels were increased in these cells. Taken together, Cyp-D ablation triggered an adaptive response resulting in increased exercise capacity despite less oxygen utilization associated with increased glucose uptake and utilization involving AMPK-TBC1D1 signaling nexus.-Radhakrishnan, J., Baetiong, A., Kaufman, H., Huynh, M., Leschinsky, A., Fresquez, A., White, C., DiMario, J. X., Gazmuri, R. J. Improved exercise capacity in cyclophilin-D knockout mice associated with enhanced oxygen utilization efficiency and augmented glucose uptake via AMPK-TBC1D1 signaling nexus.

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells.

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or "tipping point" at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations.

Molecular pathology of adverse local tissue reaction caused by metal-on-metal implants defined by RNA-seq.

Total hip arthroplasty (THA) alleviates hip pain and improves joint function. Current implant design permits long-term survivorship of THAs, but certain metal-on-metal (MoM) articulations can portend catastrophic failure due to adverse local tissue reactions (ALTR). Here, we identified biological and molecular differences between periacetabular synovial tissues of patients with MoM THA failure undergoing revision THA compared to patients undergoing primary THA for routine osteoarthritis (OA). Analysis of tissue biopsies by RNA-sequencing (RNA-seq) revealed that MoM patient samples exhibit significantly increased expression of immune response genes but decreased expression of genes related to extracellular matrix (ECM) remodeling. Thus, interplay between local tissue inflammation and ECM degradation may account for the pathology and compromised clinical outcomes in select patients with MoM implants. We conclude that adverse responses of host tissues to implant materials result in transcriptomic modifications in patients with MoM implants that permit consideration of strategies that could mitigate ECM damage.

KLF10 Gene Expression Modulates Fibrosis in Dystrophic Skeletal Muscle.

Dystrophic skeletal muscle is characterized by fibrotic accumulation of extracellular matrix components that compromise muscle structure, function, and capacity for regeneration. Tissue fibrosis is often initiated and sustained through transforming growth factor-ß (TGF-ß) signaling, and Krüppel-like factor 10 (KLF10) is an immediate early gene that is transcriptionally activated in response to TGF-ß signaling. It encodes a transcriptional regulator that mediates the effects of TGF-ß signaling in a variety of cell types. This report presents results of investigation of the effects of loss of KLF10 gene expression in wild-type and dystrophic (mdx) skeletal muscle. On the basis of RT-PCR, Western blot, and histological analyses of mouse tibialis anterior and diaphragm muscles, collagen type I (Col1a1) and fibronectin gene expression and protein deposition were increased in KLF10-/- mice, contributing to increased fibrosis. KLF10-/- mice displayed increased expression of genes encoding SMAD2, SMAD3, and SMAD7, particularly in diaphragm muscle. SMAD4 gene expression was unchanged. Expression of the extracellular matrix remodeling genes, MMP2 and TIMP1, was also increased in KLF10-deficient mouse muscle. Histological analyses and assays of hydroxyproline content indicated that the loss of KLF10 increased fibrosis. Dystrophic KLF10-null mice also had reduced grip strength. The effects of loss of KLF10 gene expression were most pronounced in dystrophic diaphragm muscle, suggesting that KLF10 moderates the fibrotic effects of TGF-ß signaling in chronically damaged regenerating muscle.

Molecular pathology of total knee arthroplasty instability defined by RNA-seq.

Total knee arthroplasty (TKA) is a durable and reliable procedure to alleviate pain and improve joint function. However, failures related to flexion instability sometimes occur. The goal of this study was to define biological differences between tissues from patients with and without flexion instability of the knee after TKA. Human knee joint capsule tissues were collected at the time of primary or revision TKAs and analyzed by RT-qPCR and RNA-seq, revealing novel patterns of differential gene expression between the two groups. Interestingly, genes related to collagen production and extracellular matrix (ECM) degradation were higher in samples from patients with flexion instability. Partitioned clustering analyses further emphasized differential gene expression patterns between sample types that may help guide clinical interpretations of this complication. Future efforts to disentangle the effects of physical and biological (e.g., transcriptomic modifications) risk factors will aid in further characterizing and avoiding flexion instability after TKA.

C/EBPα represses slow myosin heavy chain 2 gene expression in developing avian myotubes.

BACKGROUND: The CCAAT/enhancer binding proteins (C/EBP) comprise a family of transcription factors that regulate many cellular processes. Little is known of their function during embryonic and fetal myogenesis. Slow myosin heavy chain 2 (MyHC2) is a marker of the slow avian skeletal muscle fiber type, and slow MyHC2 gene regulation involves molecular pathways that lead to muscle fiber type diversification. METHODS: The biological effects of C/EBPα and C/EBPß expression were analyzed by use of a general C/EBP activity reporter and by slow MyHC2 promoter-reporter constructs transfected into specific myogenic cell lineages. The effects of C/EBPα and C/EBPß expression were also analyzed by immunocytochemical detection of slow MyHC2. C/EBPα interaction with the slow MyHC2 promoter was assessed by electromobility shift assays. RESULTS: C/EBPα and C/EBPß are present in embryonic fast and fast/slow avian myogenic lineages. Overexpression of C/EBPα cDNA repressed slow MyHC2 promoter activity in embryonic myotubes and in both electrically stimulated fetal myotubes. Deletion analysis of the slow MyHC2 promoter-luciferase reporter demonstrated that the transcriptional repression mediated by C/EBPα occurs within the first 222bp upstream from exon 1 of the slow MyHC2 gene. Electromobility shift assays determined that C/EBPα can bind to a non-canonical C/EBP site within the slow MyHC2 gene, and mutation of this site reduced transcriptional repression of the slow MyHC2 gene. CONCLUSION: C/EBPα, but not C/EBPß, represses slow MyHC2 promoter activity via a non-canonical C/EBP binding element. GENERAL SIGNIFICANCE: Members of the C/EBP family of transcription factors differentially regulate genes indicative of distinct muscle fiber types.

Muscle fiber type specific activation of the slow myosin heavy chain 2 promoter by a non-canonical E-box.

Different mechanisms control skeletal muscle fiber type gene expression at specific times in vertebrate development. Embryonic myogenesis leading to formation of primary muscle fibers in avian species is largely directed by myoblast cell commitment to the formation of diverse fiber types. In contrast, development of different secondary fiber types during fetal myogenesis is partly determined by neural influences. In both primary and secondary chicken muscle fibers, differential expression of the slow myosin heavy chain 2 (MyHC2) gene distinguishes fast from fast/slow muscle fibers. This study focused on the transcriptional regulation of the slow MyHC2 gene in primary myotubes formed from distinct fast/slow and fast myogenic cell lineages. Promoter deletion analyses identified a discrete 86 bp promoter segment that conferred fiber type, lineage-specific gene expression in fast/slow versus fast myoblast derived primary myotubes. Sequence analysis and promoter activity assays determined that this segment contains two functional cis-regulatory elements. One element is a non-canonical E-box, and electromobility shift assays demonstrated that both cis-elements interacted with the E-protein, E47. The results indicate that primary muscle fiber type specific expression of the slow MyHC2 gene is controlled by a novel mechanism involving a transcriptional complex that includes E47 at a non-canonical E-box.

Repression of myoblast proliferation and fibroblast growth factor receptor 1 promoter activity by KLF10 protein.

BACKGROUND: FGFR1 gene expression regulates myoblast proliferation and differentiation, and its expression is controlled by Krüppel-like transcription factors. RESULTS: KLF10 interacts with the FGFR1 promoter, repressing its activity and cell proliferation. CONCLUSION: KLF10 represses FGFR1 promoter activity and thereby myoblast proliferation. SIGNIFICANCE: A model of transcriptional control of chicken FGFR1 gene regulation during myogenesis is presented. Skeletal muscle development is controlled by regulation of myoblast proliferation and differentiation into muscle fibers. Growth factors such as fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation and differentiation in numerous tissues, including skeletal muscle. Transcriptional regulation of FGFR1 gene expression is developmentally regulated by the Sp1 transcription factor, a member of the Krüppel-like factor (KLF) family of transcriptional regulators. Here, we show that another KLF transcription factor, KLF10, also regulates myoblast proliferation and FGFR1 promoter activity. Expression of KLF10 reduced myoblast proliferation by 86%. KLF10 expression also significantly reduced FGFR1 promoter activity in myoblasts and Sp1-mediated FGFR1 promoter activity in Drosophila SL2 cells. Southwestern blot, electromobility shift, and chromatin immunoprecipitation assays demonstrated that KLF10 bound to the proximal Sp factor binding site of the FGFR1 promoter and reduced Sp1 complex formation with the FGFR1 promoter at that site. These results indicate that KLF10 is an effective repressor of myoblast proliferation and represses FGFR1 promoter activity in these cells via an Sp1 binding site.

Structural studies of several clinically important oncology drugs in complex with human serum albumin.

BACKGROUND: Serum albumin is a major pharmacokinetic effector of drugs. To gain further insight into albumin binding chemistry, the crystal structures of six oncology agents were determined in complex with human serum albumin at resolutions of 2.8 to 2.0Å: camptothecin, 9-amino-camptothecin, etoposide, teniposide, bicalutamide and idarubicin. METHODS: Protein crystal growth and low temperature X-ray crystallography RESULTS: These large, complex drugs are all bound within the subdomain IB binding region which can be described as a hydrophobic groove formed by α-helices h7, h8 and h9 covered by the extended polypeptide L1. L1 creates a binding cavity with two access sites, one between loop L1 and α-helices h7 and h8 (distal site: IBd) and the other between L1 and α-helix h9 (proximal site: IBp). Camptothecin (2.4Å) and 9 amino camptothecin (2.0Å) are clearly bound as the open lactone form (IBp). Idarubicin (2.8Å) binds in a DNA like dimer complex via an intermolecular π stacking arrangement in IBd. Bicalutamide (2.4Å) is bound in a folded intramolecular π stacking arrangement between two aromatic rings in IBd similar to idarubicin. Teniposide (2.7Å) and etoposide (2.7Å), despite small chemical differences, are bound in two distinctly different sites at or near IB. Teniposide is internalized via primarily hydrophobic interactions and spans through both openings (IBp-d). Etoposide is bound between the exterior of IB and IIA and exhibits an extensive hydrogen bonding network. CONCLUSIONS: Subdomain IB is a major binding site for complex heterocyclic molecules. GENERAL SIGNIFICANCE: The structures have important implications for drug design and development. This article is part of a Special Issue entitled Serum Albumin.

Understanding gene circuits at cell-fate branch points for rational cell reprogramming.

Cell-type reprogramming, the artificial induction of a switch of cell lineage and developmental stage, holds great promise for regenerative medicine. However, how does the metazoan body itself 'program' the various cell lineages in the first place? Knowledge of how multipotent cells make cell-fate decisions and commit to a particular lineage is crucial for a rational reprogramming strategy and to avoid trial-and-error approaches in choosing the appropriate set of transcription factors to use. In the past few years, a general principle has emerged in which small gene circuits of cross-inhibition and self-activation govern the decision at branch points of cell development. A formal theoretical treatment of such circuits that deal with their dynamics on the 'epigenetic landscape' could offer some guidance to find the optimal way of cell reprogramming.

Catheter-directed thrombolysis for severe pulmonary embolism in pediatric patients.

BACKGROUND: Catheter-directed thrombolytic (CDT) therapies for severe pulmonary embolism (PE) have been shown to be effective and safe when compared with systemic thrombolysis in adults. Pediatric studies assessing efficacy and safety of CDT for PE are lacking. Hence, our aim was to review CDT as a therapy for pediatric PE. METHODS: We retrospectively reviewed charts of patients aged <18 years, who underwent CDT for main or major branch pulmonary artery occlusion associated with hypotension or right ventricular dysfunction secondary to PE during a 3-year period, in our tertiary care academic Pediatric Intensive Care Unit. RESULTS: Six CDT interventions were performed on 5 patients with PE (median age: 16.5 years). All patients presented with chest pain and dyspnea. The predisposing factors for thrombogenesis differed in all patients, and all had multiple risk factors. Five of six procedures (83%) were accompanied by ultrasound agitation with EKOS endowave infusion system (ultrasound-accelerated CDT [UCDT]), whereas 1 had CDT without ultrasound agitation. Complete resolution of PE occurred in 4 instances (67%) at 24 hr, whereas in 2 cases (33%), there was partial resolution. One patient with complete resolution underwent another successful UCDT after 4 months for recurrence. Clinical parameters (heart rate, respiratory rate, blood pressure, and oxygen saturations) and echocardiographic findings improved after treatment in all the patients. Median duration of hospital stay was 9 days with no mortality and treatment-related complications. All patients were discharged with long-term anticoagulation. CONCLUSIONS: Our case series is the first that describes CDT/UCDT as an effective and safe therapy for pediatric patients with severe PE. CDT is known to accelerate fibrinolysis via focused delivery of thrombolytic agent to the thrombus site. For carefully selected patients, CDT/UCDT provides a useful treatment option for severe PE irrespective of the etiology, predisposing conditions, and associated comorbidities.

Genome-wide expression analysis and EMX2 gene expression in embryonic myoblasts committed to diverse skeletal muscle fiber type fates.

BACKGROUND: Primary skeletal muscle fibers form during embryonic development and are characterized as fast or slow fibers based on contractile protein gene expression. Different avian primary muscle fiber types arise from myoblast lineages committed to formation of diverse fiber types. To understand the basis of embryonic muscle fiber type diversity and the distinct myoblast lineages that generate this diversity, gene expression analyses were conducted on differentiated muscle fiber types and their respective myoblast precursor lineages. RESULTS: Embryonic fast muscle fibers preferentially expressed 718 genes, and embryonic fast/slow muscle fibers differentially expressed 799 genes. Fast and fast/slow myoblast lineages displayed appreciable diversity in their gene expression profiles, indicating diversity of precursor myoblasts. Several genes, including the transcriptional regulator EMX2, were differentially expressed in both fast/slow myoblasts and muscle fibers vs. fast myoblasts and muscle fibers. EMX2 was localized to nuclei of fast/slow myoblasts and muscle fibers and was not detected in fast lineage cells. Furthermore, EMX2 overexpression and knockdown studies indicated that EMX2 is a positive transcriptional regulator of the slow myosin heavy chain 2 (MyHC2) gene promoter activity in fast/slow muscle fibers. CONCLUSIONS: These results indicate the presence of distinct molecular signatures that characterize diverse embryonic myoblast lineages before differentiation.