ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front–back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front–back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines.     

Structural,Biochemical, and Biophysical Characterization of Idelalisib Binding to Phosphoinositide 3-Kinase δ

Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.     

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.     

Plasmodium berghei histamine‐releasing factor favours liver‐stage development via inhibition of IL‐6 production and associates with a severe outcome of disease

Plasmodium spp., which causes malaria, produces a histamine‐releasing factor (HRF), an orthologue of mammalian HRF. Histamine‐releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre‐erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito‐injected parasite form into the erythrocyte‐infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL‐6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti‐IL‐6 antibodies or infection in IL‐6‐deficient mice and parasite HRF is sufficient to decrease IL‐6 synthesis, indicating a direct role of parasite HRF in reducing host IL‐6. The target cells modulated by HRF for IL‐6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down‐regulate a cytokine with anti‐parasite activity. Our data also highlight the link between a prolonged transition from liver to blood‐stage infection and reduced incidence of experimental cerebral malaria.     

Relationship Between Depression and Lower Urinary Tract Symptoms Secondary to Benign Prostatic Hyperplasia

This article provides an overview of current data on the relationship between depression and lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH), with a focus on pathophysiology and patient management implications. Review of the literature indicated a clear relationship between LUTS secondary to BPH and depression. It is unknown whether this relationship is bidirectional or unidirectional. Depression is associated with the impact of LUTS on quality of life in men with BPH. Research suggests that depression alters the experience of LUTS in this population. Medical and surgical treatments for BPH may impact quality of life and, therefore, depression. Results conflict on the exact nature of the relationship examined, and on the extent to which the relationship may be attributed to physiological factors such as inflammation. Practicing clinicians should consider using a brief self-administered scale to assess for depression in patients with BPH. There is a clear need for additional research to decisively determine the nature of the relationship between LUTS secondary to BPH and depression, as well as the extent to which change in either condition may be affected by the other.Key words: Benign prostatic hyperplasia, Depression, Lower urinary tract symptomsThe prevalence of benign prostatic hyperplasia (BPH) increases with age.¹ Approximately half of men over age 40 are diagnosed with BPH. Of these men, approximately 50% will develop significant and bothersome lower urinary tract symptoms (LUTS) secondary to BPH, which increase in prevalence between ages 40 and 80 years. LUTS secondary to BPH is associated with decreased quality of life and may include urgency/frequency, incontinence, and nocturia. Symptom severity is impacted by the degree of prostatic enlargement, which is highly variable.¹Depression is another common condition that severely and negatively impacts quality of life, with an estimated lifetime prevalence of 16.5% according to the National Institute of Mental Health.² Depression plays a role in the pathogenesis of a number of chronic diseases, including inflammatory bowel disease, arthritis, asthma, and diabetes³; a relationship has also been identified between depression and urologic diagnoses such as incontinence.⁴ Symptoms of BPH are associated with decreased quality of life and depression, and the literature strongly suggests that there may also be a pathophysiologic relationship between BPH and depression^5,6; in addition, depressive symptoms are also associated with treatments for BPH.^7–9Research has suggested that psychiatric parameters such as depression may have a putative role in the development of LUTS secondary to BPH.⁶ Furthermore, depression may pose an impediment to effective treatment for these patients. Improved understanding of the relationship between BPH and depression could lead to improved management. This area of research is important because clinical depression is associated with a significant increase in mortality, and early detection, intervention, and treatment of clinically relevant depressive symptoms are key factors in patient care.¹⁰Fewer studies have focused on the relationship between depressive symptoms or depressive disorders and BPH, or the nature and direction of this relationship. Thus, a systematic review of the relationship between depression and BPH is needed. We provide a comprehensive summary of contemporary published reports on LUTS secondary to BPH and depression to improve understanding of the relationship between these two conditions and provide a framework for future investigation.     

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells

Migratory front-back polarity emerges from the cooperative effect of myosin IIA (MIIA) and IIB (MIIB) on adhesive signaling. We demonstrate here that, during polarization, MIIA and MIIB coordinately promote localized actomyosin bundling, which generates large, stable adhesions that do not signal to Rac and thereby form the cell rear. MIIA formed dynamic actomyosin proto-bundles that mark the cell rear during spreading; it also bound to actin filament bundles associated with initial adhesion maturation in protrusions. Subsequent incorporation of MIIB stabilized the adhesions and actomyosin filaments with which it associated and formed a stable, extended rear. These adhesions did not turn over and no longer signal to Rac. Microtubules fine-tuned the polarity by positioning the front opposite the MIIA/MIIB-specified rear. Decreased Rac signaling in the vicinity of the MIIA/MIIB-stabilized proto-bundles and adhesions was accompanied by the loss of Rac guanine nucleotide exchange factor (GEFs), like βPIX and DOCK180, and by inhibited phosphorylation of key residues on adhesion proteins that recruit and activate Rac GEFs. These observations lead to a model for front-back polarity through local GEF depletion.     

Segmental Motions, Not a Two-State Concerted Switch, Underlie Allostery in CheY

The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using (15)N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg(2+) enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the μs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism.     

Normal phase and reverse phase HPLC-UV-MS analysis of process impurities for rapamycin analog ABT-578: application to active pharmaceutical ingredient process development

ABT-578, an active pharmaceutical ingredient (API), is a semi-synthetic tetrazole derivative of the fermented polyene macrolide rapamycin. Reverse phase (RP)-HPLC-UV-MS and normal phase (NP)-HPLC-UV-MS methods employing an LC/MSD trap with electrospray ionization (ESI) have been developed to track and map all significant impurities from the synthetic process. Trace-level tracking of key impurities occurring at various process points was achieved using complimentary methodologies, including a stability indicating reverse phase HPLC method capable of separating at least 25 starting materials and process-related impurities from the API (YMC-Pack Phenyl column, UV-MS, 210 nm) and a targeted reverse phase HPLC method capable of separating very polar compounds from crude reaction mixtures (Phenomenex Synergi Polar RP column, UV, 265 nm). In addition, a normal phase HPLC method condition with post-column modifier infusion is described for the separation of epimeric impurities, and analysis of aqueous-sensitive reactive species (YMC-Pack SIL column, UV-MS, 278 nm). Process control strategies were established with these combinations of analytical technologies for impurities analyses to enable a rich understanding of the ABT-578 process.     

Influence of genetic variation in the fungal endophyte of a grass on an herbivore and its parasitoid

Neotyphodium coenophialum (Glenn, Bacon, Price & Hanlin) (Ascomycota: Clavicipitaceae) is an endophytic fungus that lives symbiotically within grasses and produces alkaloids that can help protect its hosts from some insect pests. We used laboratory‐based experiments to investigate whether fungal genotype influences an herbivore and its parasitoid. We tested whether variation in novel isolates, plus a control lacking fungal infection, affected preference by fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae: Amphipyrini), and growth and survival of Euplectrus comstockii Howard (Hymenoptera: Eulophidae: Euplectrini), a parasitoid of fall armyworm. Caterpillars preferred leaf blades in choice experiments from uninfected tall fescue [Lolium arundinaceum (Schreb.) Darbysh., cultivar Jesup (Poaceae)] and tended to avoid blades from plants containing fungal isolates AR502, AR542, or the most common strain from pastures in Georgia, USA, in tall fescue. However, caterpillars fed as much on leaf blades from plants containing isolate AR502 as from those lacking infection. Parasitoid pupal mass was not influenced by fungal isolate, yet fungal isolate did influence parasitoid survival. Survival was higher than expected for parasitoids reared from hosts fed plants lacking fungal infection, but was lower than expected for those reared from hosts fed plants infected with the common strain or AR542 isolates. In contrast, parasitoids reared from hosts fed plants infected with isolate AR502 did not experience higher mortality than expected by chance. Our results show that N. coenophialum can modify bottom‐up trophic cascades through direct effects on herbivores, as well as indirect effects on a natural enemy of the herbivores and that the fungus may influence the tritrophic interaction in ways that counterbalance herbivore protection provided by the symbiont. Our work also shows that these effects are influenced by fungal genotype. As attempts are made to produce forage cultivars with strains of fungal endophyte that lack negative influences on livestock, it will be prudent for investigators to assess the multi‐trophic effects of these novel associations within agroecosystems.