14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins.

Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.

Discovery of Potent Inhibitors of 11ß-Hydroxysteroid Dehydrogenase Type 1 Using a Novel Growth-Based Protocol of in Silico Screening and Optimization in CONTOUR.

With the continuous progress in ultralarge virtual libraries which are readily accessible, it is of great interest to explore this large chemical space for hit identification and lead optimization using reliable structure-based approaches. In this work, a novel growth-based screening protocol has been designed and implemented in the structure-based design platform CONTOUR. The protocol was used to screen the ZINC database in silico and optimize hits to discover 11ß-HSD1 inhibitors. In contrast to molecular docking, the virtual screening process makes significant improvements in computational efficiency without losing chemical equities through partitioning 1.8 million ZINC compounds into fragments, docking fragments to form key hydrogen bonds with anchor residues, reorganizing molecules into molecular fragment trees using matched fragments and common substructures, and then regrowing molecules with the help of developed intelligent growth features inside the protein binding site to find hits. The growth-base screening approach is validated by the high hit rate. A total of 50 compounds have been selected for testing; of these, 15 hits having diverse scaffolds are found to inhibit 11ß-HSD1 with IC50 values of less than 1 µM in a biochemical enzyme assay. The best hit which exhibits an enzyme IC50 of 33 nM is further developed to a novel series of bicyclic 11ß-HSD1 inhibitors with the best inhibition of enzyme IC50 of 3.1 nM. The final lead candidate exhibits IC50 values of 7.2 and 21 nM in enzyme and adipocyte assays, respectively, displayed greater than 1000-fold of selectivity over 11ß-HSD2 and two other related hydroxysteroid dehydrogenases, and can serve as good starting points for further optimization to develop clinical candidates.

Angiogenesis selectively requires the p110alpha isoform of PI3K to control endothelial cell migration.

Phosphoinositide 3-kinases (PI3Ks) signal downstream of multiple cell-surface receptor types. Class IA PI3K isoforms couple to tyrosine kinases and consist of a p110 catalytic subunit (p110alpha, p110beta or p110delta), constitutively bound to one of five distinct p85 regulatory subunits. PI3Ks have been implicated in angiogenesis, but little is known about potential selectivity among the PI3K isoforms and their mechanism of action in endothelial cells during angiogenesis in vivo. Here we show that only p110alpha activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110alpha led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110alpha exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110alpha activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110beta in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1alpha, whereas p110delta is expressed at low level and contributes only minimally to PI3K activity in endothelial cells. These results provide the first in vivo evidence for p110-isoform selectivity in endothelial PI3K signalling during angiogenesis.

Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo.

The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes. PrfA-dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand-induced allostery. Here, we report the identification of a solvent-accessible cavity within the PrfA N-terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide-anchoring motif and has its entrance on the opposite side of the ß-barrel. Site-directed mutations in this pocket impaired intracellular PrfA-dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation-deficient PrfA mutations. Neither PrfA(allo) substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell-to-cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues.

Crosstalk between reticular adherens junctions and platelet endothelial cell adhesion molecule-1 regulates endothelial barrier function.

OBJECTIVE: Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium. METHODS AND RESULTS: In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins ß-catenin and p120-catenin. ß-catenin binds to α-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-α increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-α. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-α-induced alterations of barrier function, including increased ß-catenin phosphorylation, without altering the actomyosin cytoskeleton. CONCLUSIONS: We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-α.

Listeria InlB Expedites Vacuole Escape and Intracellular Proliferation by Promoting Rab7 Recruitment via Vps34.

Rapid phagosomal escape mediated by listeriolysin O (LLO) is a prerequisite for Listeria monocytogenes intracellular replication and pathogenesis. Escape takes place within minutes after internalization from vacuoles that are negative to the early endosomal Rab5 GTPase and positive to the late endosomal Rab7. Using mutant analysis, we found that the listerial invasin InlB was required for optimal intracellular proliferation of L. monocytogenes. Starting from this observation, we determined in HeLa cells that InlB promotes early phagosomal escape and efficient Rab7 acquisition by the Listeria-containing vacuole (LCV). Recruitment of the class III phosphoinositide 3-kinase (PI3K) Vps34 to the LCV and accumulation of its lipid product, phosphatidylinositol 3-phosphate (PI3P), two key endosomal maturation mediators, were also dependent on InlB. Small interfering RNA (siRNA) knockdown experiments showed that Vps34 was required for Rab7 recruitment and early (LLO-mediated) escape and supported InlB-dependent intracellular proliferation. Together, our data indicate that InlB accelerates LCV conversion into an escape-favorable Rab7 late phagosome via subversion of class III PI3K/Vps34 signaling. Our findings uncover a new function for the InlB invasin in Listeria pathogenesis as an intracellular proliferation-promoting virulence factor. IMPORTANCE Avoidance of lysosomal killing by manipulation of the endosomal compartment is a virulence mechanism assumed to be largely restricted to intravacuolar intracellular pathogens. Our findings are important because they show that cytosolic pathogens like L. monocytogenes, which rapidly escape the phagosome after internalization, can also extensively subvert endocytic trafficking as part of their survival strategy. They also clarify that, instead of delaying phagosome maturation (to allow time for LLO-dependent disruption, as currently thought), via InlB L. monocytogenes appears to facilitate the rapid conversion of the phagocytic vacuole into an escape-conducive late phagosome. Our data highlight the multifunctionality of bacterial virulence factors. At the cell surface, the InlB invasin induces receptor-mediated phagocytosis via class I PI3K activation, whereas after internalization it exploits class III PI3K (Vsp34) to promote intracellular survival. Systematically elucidating the mechanisms by which Listeria interferes with PI3K signaling all along the endocytic pathway may lead to novel anti-infective therapies.

Adherens junctions connect stress fibres between adjacent endothelial cells.

BACKGROUND: Endothelial cell-cell junctions maintain endothelial integrity and regulate vascular morphogenesis and homeostasis. Cell-cell junctions are usually depicted with a linear morphology along the boundaries between adjacent cells and in contact with cortical F-actin. However, in the endothelium, cell-cell junctions are highly dynamic and morphologically heterogeneous. RESULTS: We report that endothelial cell-cell junctions can attach to the ends of stress fibres instead of to cortical F-actin, forming structures that we name discontinuous adherens junctions (AJ). Discontinuous AJ are highly dynamic and are increased in response to tumour necrosis factor (TNF)-alpha, correlating with the appearance of stress fibres. We show that vascular endothelial (VE)-cadherin/beta-catenin/alpha-catenin complexes in discontinuous AJ are linked to stress fibres. Moreover, discontinuous AJ connect stress fibres from adjacent cells independently of focal adhesions, of which there are very few in confluent endothelial cells, even in TNF-alpha-stimulated cells. RNAi-mediated knockdown of VE-cadherin, but not zonula occludens-1, reduces the linkage of stress fibres to cell-cell junctions, increases focal adhesions, and dramatically alters the distribution of these actin cables in confluent endothelial cells. CONCLUSIONS: Our results indicate that stress fibres from neighbouring cells are physically connected through discontinuous AJ, and that stress fibres can be stabilized by AJ-associated multi-protein complexes distinct from focal adhesions.

60-year-old man • chronic cough • history of GERD & dyslipidemia • throat tickle • Dx?

► Chronic cough ► History of GERD & dyslipidemia ► Throat tickle.

Attitudes toward osteopathic medicine scale: development and psychometrics.

OBJECTIVE: To develop a valid and reliable instrument for measuring attitudes toward osteopathic medicine. METHODS: Participants included 5,669 first-year students from 33 U.S. colleges of osteopathic medicine, who completed an online survey at the beginning of the 2019-2020 academic year. Using data from the nationwide Project in Osteopathic Medical Education and Empathy, we developed a 13-item instrument: Attitudes Toward Osteopathic Medicine Scale (ATOMS) and demonstrated the validity and reliability of its scores. The social desirability response bias was controlled in statistical analyses. RESULTS: The corrected item-total score correlations were all positive and statistically significant, and the effect sizes of item discrimination indices were large. Cronbach's coefficient alpha reliability was 0.83. Construct validity, corroborating face and content validity of the ATOMS, was supported by three components, emerged from factor analysis: "Perspectives on Osteopathic Medicine," "Osteopathic Diagnosis and Treatment," and "Holistic-Integrative Care." Correlations between ATOMS scores and scores of cognitive empathy, emotional empathy; orientation toward interprofessional collaboration; lifelong learning; and burnout were statistically significant in the expected direction, providing validity evidence for the ATOMS. Using the method of contrasted groups, significant differences in the ATOMS scores were found by gender, ethnicity, academic background, and career interest in the expected direction, supporting the validity of the ATOMS scores. National norms were developed to assess individual scores alongside national percentile ranks. CONCLUSIONS: The ATOMS, developed in a nationwide study, supported by strong psychometric evidence for measuring orientation toward osteopathic medicine, has implications for the assessment of osteopathic medical education, patient outcomes, and admission decisions.

Deciphering interplay between Salmonella invasion effectors.

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a 'signaling hub' during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cisbinary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen-host interaction.

Phosphoinositide 3-kinases in cell migration.

Cell migration is essential for many biological processes in animals and is a complex highly co-ordinated process that involves cell polarization, actin-driven protrusion and formation and turnover of cell adhesions. The PI3K (phosphoinositide 3-kinase) family of lipid kinases regulate cell migration in many different cell types, both through direct binding of proteins to their lipid products and indirectly through crosstalk with other pathways, such as Rho GTPase signalling. Emerging evidence suggests that the involvement of PI3Ks at different stages of migration varies even within one cell type, and is dependent on the combination of external stimuli, as well as on the signalling status of the cell. In addition, it appears that different PI3K isoforms have distinct roles in cell polarization and migration. This review describes how PI3K signalling is regulated by pro-migratory stimuli, and the diverse ways in which PI3K-mediated signal transduction contributes to different aspects of cell migration.

Topical Imiquimod Induces Severe Weakness and Myalgias After Three Applications: A Case Report.

Imiquimod can be used to treat superficial basal cell carcinoma, actinic keratosis, genital warts, and other skin conditions. The adverse events associated with this topical agent commonly include application site irritation, primarily erythema, as well as headache, myalgia, and fatigue. There are usually minimal systemic symptoms. We report the case of a patient who used topical imiquimod 5% cream on nine basal cell carcinoma lesions daily for three days and developed severe muscle weakness and the inability to walk. He fell twice, went to the emergency department, and was given 125mg injection of methylprednisolone. The imiquimod was then discontinued and he recovered almost back to baseline in 48 hours. We hypothesize the patient's reaction to the imiquimod was due to an immune etiology, potentially involving TLR7 and NF-κB as precipitators of this myopathy. Overall, this report demonstrates a potential severe and rapid adverse reaction to topical imiquimod administration not previously reported in the literature.

Navigating the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study: practical outcomes and implications for depression treatment in primary care.

The Sequenced Treatment Alternatives to Relieve Depression (STAR( *)D) study addressed many clinically relevant issues on treatment of depressed outpatients. The study used an equipoise-stratified randomization scheme that enhanced the real-world expediency of treatment options studied. Because patients who had significant comorbid medical and psychiatric problems were included, and care was provided in the outpatient primary care setting as well as in outpatient psychiatric centers, findings are relevant to primary care physicians. The use of measurement-based treatment protocols promotes objectivity in a realm of often subjective clinical decision making. Although STAR( *)D was unable to provide specific treatment comparisons for patients at all study levels, it succeeded in defining the prevalence of treatment-resistant depression and is a model for further practical clinical outcomes studies.

Different PI 3-kinase inhibitors have distinct effects on endothelial permeability and leukocyte transmigration.

Endothelial cells play a central role in inflammatory responses, mediating leukocyte and solute traffic from blood vessels into the tissue, and are therefore key targets for anti-inflammatory therapies. Phosphoinositide 3-kinases (PI3Ks) are important signal transducers in inflammation and cancer, however there are 8 different PI3K catalytic isoforms, several of which have been shown to play distinct roles in cellular responses. Isoform-selective inhibitors have recently been described, but their effects on endothelial cell responses have not been compared. Here we compare the effects of the pan-PI3K inhibitor wortmannin with that of four more isoform-selective inhibitors, PI-103, TGX-221, AS604850 and IC87114, on endothelial cells stimulated with the pro-inflammatory cytokine TNFα. We find that PI-103 and wortmannin are most effective at reducing both endothelial permeability and leukocyte transendothelial migration (TEM), which correlates with a decrease in both the activity of the tyrosine kinase Pyk2 and its association with VE-cadherin. PI-103-related compounds are therefore likely to be good candidates for treating chronic inflammatory responses involving TNFα.

Quantification of transendothelial migration using three-dimensional confocal microscopy.

Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using cultured cells, many model systems are unable to provide detailed visual information of cell morphologies and distribution of proteins such as junctional markers, as well as quantitative data on the rate of TEM. Improvements in imaging techniques have made microscopy-based assays an invaluable tool for studying this type of detailed cell movement in physiological processes. In this chapter, we describe a confocal microscopy-based method that can be used to assess TEM of both leukocytes and cancer cells across endothelial barriers in response to a chemotactic gradient, as well as providing information on their migration into a subendothelial extracellular matrix, designed to mimic that found in vivo.

Regulation of Listeria virulence: PrfA master and commander.

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection. These bacteria live as soil saprotrophs on decaying plant matter but also as intracellular parasites, using the cell cytosol as a replication niche. PrfA, a regulatory protein, integrates a number of environmental cues that signal the transition between these two contrasting lifestyles, activating a set of key virulence factors during host infection. While a number of details concerning the general mode of action of this virulence master switch have been elucidated, others remain unsolved. Recent work has revealed additional mechanisms that contribute to L. monocytogenes virulence modulation, often via cross-talk with PrfA, or by regulating new genes involved in host colonization.