Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by alpha(2)-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RT-PCR, and confocal microscopy to test whether 5-HT(1A), SST(1), and/or SST(2) receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific 5-HT(1A) receptor antagonist WAY 100135 (1 microM) reduced the amplitude of IPSPs, an effect that persisted in the presence of the alpha(2)-adrenoceptor antagonist idazoxan (2 microM), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific SSTR(2) antagonist CYN 154806 (100 nM) and a specific SSTR(1) antagonist SRA 880 (3 microM) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for 5-HT(1A), SST(1), and SST(2) receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that 5-HT(1A), SST(1), and SST(2) receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes.

Immunostimulatory agents provide a new category of anti-microbial agents that activate the host's innate immune system allowing control of viral and/or bacterial infections. The TLR-2 agonist PEG-Pam2Cys has been shown to mediate potent anti-viral activity against influenza viruses when administered prophylactically (1). Here, we demonstrate that the treatment of mice with PEG-Pam2Cys does not compromise their ability to generate adaptive immune responses following subsequent challenge with influenza virus. The antibody induced in mice pre-treated with Pam2Cys possessed hemagglutination-inhibiting activities and the CD8(+) T-cell responses that were elicited provided protection against heterologous viral challenge. In the absence of an effective influenza vaccine, an agent that provides immediate protection against the virus and does not compromise the induction of influenza-specific immunity on exposure to infectious virus provides an opportunity for population immunity to be achieved through natural exposure to virus.

Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.

Huntington's disease arises from CAG codon-repeat expansions in the Htt gene, which leads to a Htt gene product with an expanded polyglutamine (polyQ) sequence. The length of the polyQ expansion correlates with an increased tendency to form aggregates and clustering into micrometer-plus sized inclusion bodies in neurons and other cell types. Yet after nearly 20 years since the genetic basis for HD was identified, our knowledge of how polyQ-expanded Htt fragment aggregation relates to disease mechanisms remains fragmentary and controversial. Challenges remain in defining the aggregation process at the molecular level and how this process is influenced by, or influences cellular activities. Insight is further confounded by the term "aggregation" being used to describe a composite of distinct processes that may have opposing consequences to cell health and survival. This review discusses these issues in light of a historic summary of Htt aggregation in the cellular milieu and the intrinsic attributes of polyQ-expanded Htt that lead to aggregation. Finally, discussion centers on strategies forward to improve our knowledge for how aggregation relates to cellular dysfunction.

A photoreactive analog of chloroquine, N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl)-4- azi do-2- hydroxybenzamide (referred to as ASA-Q), has been synthesized and shown to mimic the action of chloroquine in possessing substantial antimalarial activity against a chloroquine-sensitive strain of Plasmodium falciparum. As for chloroquine, ASA-Q is less effective at killing drug-resistant strains of malaria, and the resistance can be modulated using the reagent verapamil. ASA-Q has been radiolabeled with Na125I and used as a photoaffinity probe for labeling chloroquine-binding proteins in malaria-infected erythrocytes. Two proteins have been identified with apparent molecular masses of 42 and 33 kDa in both chloroquine-sensitive and chloroquine-resistant strains of malaria. Photoaffinity labeling of the two proteins by iodo-ASA-Q was competitively inhibited by an excess of unlabeled chloroquine. The structurally related antimalarials amodiaquine and quinine also inhibited labeling of the two proteins, while verapamil and doxycycline had no effect. We suggest that the two labeled proteins are the macromolecular targets of chloroquine action in malaria parasites.

Interleukin (IL)-10-producing B cells (B10 cells) have emerged as important regulatory elements with immunosuppressive roles. Chronic lymphocytic leukemia (CLL) B cells also secrete IL-10 and share features of B10 cells, suggesting a possible contribution of CLL B cells to immunosuppression in CLL patients. Factors controlling the emergence of B10 cells are not known. B-cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) is critical for B-cell maturation and survival, and is implicated in the development and progression of CLL. We sought to investigate the role of BAFF in the emergence of IL-10-producing regulatory B cells in healthy donors and CLL patients. Here, we report that BAFF signaling promotes IL-10 production by CLL B cells in a mouse model of CLL and in CLL patients. Moreover, BAFF-mediated IL-10 production by normal and CLL B cells is mediated via its receptor transmembrane activator and cyclophilin ligand interactor. Our work uncovered a major targetable pathway important for the generation of regulatory B cells that is detrimental to immunity in CLL.

Estrogen may be involved in psychosis by an interaction with central dopaminergic activity. Aromatase knockout mice are unable to produce estrogen and have been shown to display altered behavioural responses and effects of the dopamine releaser, amphetamine. This study investigates the effect of gonadal status on amphetamine-induced c-fos expression in the brains of female aromatase knockout and wildtype mice. Six groups of mice were treated intraperitoneally with saline or 5mg/kg amphetamine. Fos immunoreactivity was assessed in the cingulate cortex, caudate putamen and nucleus accumbens. Aromatase knockout mice showed markedly reduced amphetamine-induced Fos immunoreactivity compared to wildtype mice. However, the amphetamine response was restored in aromatase-knockout mice after ovariectomy, which reduced this effect in wildtype controls. Estrogen supplementation reversed the effect of ovariectomy in wildtype mice but had no additional significant effect in aromatase-knockout mice. These results indicate that mechanisms involved in amphetamine-induced c-fos expression are altered in aromatase knockout mice and that the primary hormone involved in this effect is not estrogen, but may be another factor released from the ovaries, such as an androgen. These results provide new insight into the effect of gonadal hormones on amphetamine induced c-fos expression in this mouse model of estrogen deficiency. These results could be important for our understanding of the role of sex steroid hormones in psychosis.

The pathogenesis of severe dengue remains unclear, particularly the mechanisms underlying the plasma leakage that results in hypovolaemic shock in a small proportion of individuals. Maximal leakage occurs several days after peak viraemia implicating immunological pathways. Skin is a highly vascular organ and also an important site of immune reactions with a high density of dendritic cells (DCs), macrophages and T cells. We obtained skin biopsies and contemporaneous blood samples from patients within 24 hours of onset of dengue shock syndrome (DSS), and from healthy controls. We analyzed cell subsets by flow cytometry, and soluble mediators and antibodies by ELISA; the percentage of migratory CD1a+ dermal DCs was significantly decreased in the DSS patients, and skin CD8+ T cells were activated, but there was no accumulation of dengue-specific antibodies. Inflammatory monocytic cells were not observed infiltrating the skin of DSS cases on whole-mount histology, although CD14dim cells disappeared from blood.

This is the frst detailed study of the relation between cesarean birth and child cognitive development. We measure diferences in child cognitive performance at 4 to 9 years of age between cesarean-born and vaginally-born children (n=3,666) participating in the Longitudinal Study of Australian Children (LSAC). LSAC is a nationally representative birth cohort surveyed biennially. Using multivariate regression, we control for a large range of confounders related to perinatal risk factors and the socioeconomic advantage associated with cesarean-born children. Across several measures, we fnd that cesarean-born children perform signifcantly below vaginally-born children, by up to a tenth of a standard deviation in national numeracy test scores at age 8–9. Estimates from a low-risk sub-sample and lower-bound analysis suggest that the relation is not spuriously related to unobserved confounding. Lower rates of breastfeeding and adverse child and maternal health outcomes that are associated with cesarean birth are found to explain less than a third of the cognitive gap, which points to the importance of other mechanisms such as disturbed gut microbiota. The fndings underline the need for a precautionary approach in responding to requests for a planned cesarean when there are no apparent elevated risks from vaginal birth.

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion.

Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites.