Characterization of a Novel Protease in Staphylococcus aureus

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms encoding the N-terminal extension of L27. Cleavage of L27 by Prp represents a new target for potential antibiotic therapy. In order to characterize this protease, Prp has been overexpressed and purified. Using an assay we have developed, based on cleavage of a fluorogenic peptide derived from the conserved L27 cleavage sequence, we have undertaken an analysis of the enzyme kinetics and substrate specificity for Prp cleavage and tested predictions made based on a structural model using active-site mutants

Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N-terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N-terminal extension also encode a sequence-specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un-cleavable N-terminal extension, or lacking the extension (pre-cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N-terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site-directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway

A Belmont Report for Animals?

Human and animal research both operate within established standards. In the United States, criticism of the human research environment and recorded abuses of human research subjects served as the impetus for the establishment of the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, and the resulting Belmont Report. The Belmont Report established key ethical principles to which human research should adhere: respect for autonomy, obligations to beneficence and justice, and special protections for vulnerable individuals and populations. While current guidelines appropriately aim to protect the individual interests of human participants in research, no similar, comprehensive, and principled effort has addressed the use of (nonhuman) animals in research. Although published policies regarding animal research provide relevant regulatory guidance, the lack of a fundamental effort to explore the ethical issues and principles that should guide decisions about the potential use of animals in research has led to unclear and disparate policies. Here, we explore how the ethical principles outlined in the Belmont Report could be applied consistently to animals. We describe how concepts such as respect for autonomy and obligations to beneficence and justice could be applied to animals, as well as how animals are entitled to special protections as a result of their vulnerability

Enhancement of the superconducting gap by nesting in CaKFe4As4 - a new high temperature superconductor

We use high resolution angle resolved photoemission spectroscopy and density functional theory with experimentally obtained crystal structure parameters to study the electronic properties of CaKFe4As4. In contrast to related CaFe2As2 compounds, CaKFe4As4 has high Tc of 35K at stochiometric composition. This presents unique opportunity to study properties of high temperature superconductivity of iron arsenic superconductors in absence of doping or substitution. The Fermi surface consists of three hole pockets at $\Gamma$ and two electron pockets at the $M$ point. We find that the values of the superconducting gap are nearly isotropic, but significantly different for each of the FS sheets. Most importantly we find that the overall momentum dependence of the gap magnitudes plotted across the entire Brillouin zone displays a strong deviation from the simple cos(kx)cos(ky) functional form of the gap function, proposed in the scenario of the Cooper-pairing driven by a short range antiferromagnetic exchange interaction. Instead, the maximum value of the gap is observed for FS sheets that are closest to the ideal nesting condition in contrast to the previous observations in some other ferropnictides. These results provide strong support for the multiband character of superconductivity in CaKFe4As4, in which Cooper pairing forms on the electron and the hole bands interacting via dominant interband repulsive interaction, enhanced by FS nesting}.Comment: 5 pages, 4 figure

A novel mode of capping protein-regulation by Twinfilin

Cellular actin assembly is controlled at the barbed ends of actin filaments, where capping protein (CP) limits polymerization. Twinfilin is a conserved in vivo binding partner of CP, yet the significance of this interaction has remained a mystery. Here, we discover that the C-terminal tail of Twinfilin harbors a CP-interacting (CPI) motif, identifying it as a novel CPI-motif protein. Twinfilin and the CPI-motif protein CARMIL have overlapping binding sites on CP. Further, Twinfilin binds competitively with CARMIL to CP, protecting CP from barbed-end displacement by CARMIL. Twinfilin also accelerates dissociation of the CP inhibitor V-1, restoring CP to an active capping state. Knockdowns of Twinfilin and CP each cause similar defects in cell morphology, and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity. Together, these observations define Twinfilin as the first \u27pro-capping\u27 ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle

Scholarly research productivity among otolaryngology residency graduates and its relationship to future academic achievement

Background: The Accreditation Council for Graduate Medical Education (ACGME) requires that all residencies participate in research. This growing emphasis on research during residency has made it increasingly important for medical students to gain familiarity with the basic principles of research before beginning residency training. Thus, our goal is to determine if an association exists between publication rates before, during, or after otolaryngology residency training and whether publication efforts may predict future academic achievement. If such an association exists, perhaps otolaryngology residency program directors and education policymakers could use it as a predictive tool to screen future applicants.Methods: In this cross-sectional analysis, we selected a random sample of 50 otolaryngology residency programs listed on Doximity. From these programs, we assembled a list of graduating residents from 2013, 2014, and 2015. Using SCOPUS, PubMed, and Google Scholar, a list of publications for each graduate was compiled and data were extracted in an independent, double-blinded fashion by two investigators.Results: Of the 50 randomly selected otolaryngology residency programs included in this analysis, 27 (54%) programs representing 207 residents were included. Before residency, graduates published a mean of 0.7 (SD=2.3) articles and a mean of 0.2 (SD=0.8) first author publications. During residency, graduates published a mean of 4.1 (SD=5.0) articles and a mean of 2.2 (SD=3.2) first author publications. After residency, graduates published a mean of 5.4 (SD= 9.6) articles and a mean of 1.8 (SD=2.8) first author publications. Residents who pursued a fellowship had more publications (t205=-5.5, p <.001) and more first author publications (t205=-5.3, p <.001) than residents who did not pursue fellowship training. Residents who chose careers in academic medicine had a higher number of mean total publications (t205=-7.2, p <.001) and first author publications (t205=-7.0, p <.001) than those in private practice.Conclusion: Otolaryngology residency graduates are actively involved in research opportunities throughout their medical training. Research productivity significantly correlated with future fellowship training, the pursuit of an academic career, and overall h-index. Residents who published more research were more likely to enter fellowship training and academic careers. Our results indicate that promoting greater physician involvement in the research process may strengthen confidence in the interpretation and application of research findings and ultimately lead to future academic success

PDGF Receptor-α Does Not Promote HCMV Entry into Epithelial and Endothelial Cells but Increased Quantities Stimulate Entry by an Abnormal Pathway

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128–131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα–transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128–131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128–131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types

Interest in Otolaryngology-Head and Neck Surgery residency: Can Google Trends be a predictive tool?

Google Trends, an online internet search tool, was used to evaluate the association between the U.S. senior applicant pool for Otolaryngology-Head and Neck Surgery (Oto-HNS) residency programs and internet search queries for Oto-HNS residency. Retrospective analysis was performed on the relative search interest for Oto-HNS residencies using Google Trends. Google Trends data was compiled and compared to the National Residency Match Program (NRMP) data. Analysis demonstrated a recent decrease in relative search volume interest in Oto-HNS residencies. The Google Trends analysis mirrored the data from the NRMP which reported a relative decrease in the number of medical school graduate applicants in the field of Oto-HNS. These results suggest that online search tools such as Google Trends may be a useful tool providing insight into the interests of medical school graduates in Oto-HNS residency