Symptoms of infection, such as fever, anorexia and lethargy, are ubiquitous among vertebrates. Rather than nonspeci® c manifestations of illness, these responses are organized, adaptive strategies that are often critical to host survival. During times of energetic shortage such as winter, however, it may be detrimental for individuals to prolong energetically demanding symptoms such as fever. Individuals may adjust their immune responses prior to winter by using day length to anticipate energetically-demanding conditions. If the expression of sickness behaviours is constrained by energy availability, then cytokine production, fever, and anorexia should be attenuated in infected Siberian hamsters housed under simulated winter photoperiods. We housed hamsters in either long (14 L : 10 D) or short (10 L : 14 D) day lengths and assessed cytokines, anorexia and fever following injections of lipopolysaccharide (LPS). Short days attenuated the response to lipopolysaccharide, by decreasing the production of interleukin (IL)-6 and IL-1b , and diminishing the duration of fever and anorexia. Short-day exposure in hamsters also decreased the ingestion of dietary iron, a nutrient vital to bacterial replication. Taken together, short day lengths attenuated the symptoms of infection, presumably to optimize energy expenditure and survival outcome.

Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

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Interleukin-1 (IL-1) has been implicated as a critical mediator of neuroimmune communication. In the brain, the functional receptor for IL-1, type 1 IL-1 receptor (IL-1R1), is localized primarily to the endothelial cells. In this study, we created an endothelial-specific IL-1R1 knockdown model to test the role of endothelial IL-1R1 in mediating the effects of IL-1. Neuronal activation in the hypothalamus was measured by c-fos expression in the paraventricular nucleus and the ventromedial preoptic area. In addition, two specific sickness symptoms, febrile response and reduction of locomotor activity, were studied. Intracerebroventricular injection of IL-1 induced leukocyte infiltration into the CNS, activation of hypothalamic neurons, fever, and reduced locomotor activity in normal mice. Endothelialspecific knockdown of IL-1R1 abrogated all these responses. Intraperitoneal injection of IL-1 also induced neuronal activation in the hypothalamus, fever, and reduced locomotor activity, without inducing leukocyte infiltration into the brain. Endothelial-specific knockdown of IL-1R1 suppressed intraperitoneal IL-1-induced fever, but not the induction of c-fos in hypothalamus. When IL-1 was given intravenously, endothelial knockdown of IL-1R1 abolished intravenous IL-1-induced CNS activation and the two monitored sickness symptoms. In addition, endothelial-specific knockdown of IL-1R1 blocked the induction of cyclooxygenase-2 expression induced by all three routes of IL-1 administration. These results show that the effects of intravenous and intracerebroventricular IL-1 are mediated by endothelial IL-1R1, whereas the effects of intraperitoneal IL-1 are partially dependent on endothelial IL-1R1.

Disruption of neuronal Ca2ϩ homeostasis contributes to neurodegenerative diseases through mechanisms that are not fully understood. A polymorphism in CALHM1, a recently described ion channel that regulates intracellular Ca 2ϩ levels, is a possible risk factor for late-onset Alzheimer's disease. Since there are six potentially redundant CALHM family members in humans, the physiological and pathophysiological consequences of CALHM1 function in vivo remain unclear. The nematode Caenorhabditis elegans expresses a single CALHM1 homolog, CLHM-1. Here we find that CLHM-1 is expressed at the plasma membrane of sensory neurons and muscles. Like human CALHM1, C. elegans CLHM-1 is a Ca 2ϩ -permeable ion channel regulated by voltage and extracellular Ca 2ϩ. Loss of clhm-1 in the body-wall muscles disrupts locomotory kinematics and biomechanics, demonstrating that CLHM-1 has a physiologically significant role in vivo. The motility defects observed in clhm-1 mutant animals can be rescued by muscle-specific expression of either C. elegans CLHM-1 or human CALHM1, suggesting that the function of these proteins is conserved in vivo. Overexpression of either C. elegans CLHM-1 or human CALHM1 in neurons is toxic, causing degeneration through a necrotic-like mechanism that is partially Ca 2ϩ dependent. Our data show that CLHM-1 is a functionally conserved ion channel that plays an important but potentially toxic role in excitable cell function.

Acute myeloid leukemia (AML) is a heterogeneous malignancy. Despite the advances in past decades, the clinical outcomes of AML patients remain poor. Leukemia stem cells (LSCs) is the major cause of the recurrence of AML even after aggressive treatment making, promoting development of LSC-targeted agents is an urgent clinical need. Although the antitumor activity of disulfiram (DS), an approved anti-alcoholism drug, has been demonstrated in multiple types of tumors including hematological malignancies such as AML, it remains unknown whether this agent would also be able to target cancer stem cells like LSCs. Here, we report the in vitro and in vivo activity of DS in combination with copper (Cu) against CD34+/CD38+ leukemia stem-like cells sorted from KG1α and Kasumi-1 AML cell lines, as well as primary CD34+ AML samples. DS plus Cu (DS/Cu) displayed marked inhibition of proliferation, induction of apoptosis, and suppression of colony formation in cultured AML cells while sparing the normal counterparts. DS/Cu also significantly inhibited the growth of human CD34+/CD38+ leukemic cell-derived xenografts in NOD/SCID mice. Mechanistically, DS/Cu-induced cytotoxicity was closely associated with activation of the stress-related ROS-JNK pathway as well as simultaneous inactivation of the pro-survival Nrf2 and nuclear factor-κB pathways. In summary, our findings indicate that DS/Cu selectively targets leukemia stem-like cells both in vitro and in vivo, thus suggesting a promising LSC-targeted activity of this repurposed agent for treatment of relapsed and refractory AML.

The Hippo pathway plays a critical role in cell growth and tumorigenesis. The activity of TEA domain transcription factor 4 (TEAD4) determines the output of Hippo signaling; however, the regulation and function of TEAD4 has not been explored extensively. Here, we identified glucocorticoids (GC) as novel activators of TEAD4. GC treatment facilitated glucocorticoid receptor (GR)-dependent nuclear accumulation and transcriptional activation of TEAD4. TEAD4 positively correlated with GR expression in human breast cancer, and high expression of TEAD4 predicted poor survival of patients with breast cancer. Mechanistically, GC activation promoted GR interaction with TEAD4, forming a complex that was recruited to the TEAD4 promoter to boost its own expression. Functionally, the activation of TEAD4 by GC promoted breast cancer stem cells maintenance, cell survival, metastasis, and chemoresistance both in vitro and in vivo. Pharmacologic inhibition of TEAD4 inhibited GC-induced breast cancer chemoresistance. In conclusion, our study reveals a novel regulation and functional role of TEAD4 in breast cancer and proposes a potential new strategy for breast cancer therapy. Significance: This study provides new insight into the role of glucocorticoid signaling in breast cancer, with potential for clinical translation.

Objective. While the effects of biomechanical signals in the form of joint movement and exercise are known to be beneficial to inflamed joints, limited information is available regarding the intracellular mechanisms of their actions. This study was undertaken to examine the intracellular mechanisms by which biomechanical signals suppress proinflammatory gene induction by the interleukin-1-␤ (IL-1␤)-induced NF-B signaling cascade in articular chondrocytes.Methods. Primary rat articular chondrocytes were exposed to biomechanical signals in the form of cyclic tensile strain, and the effects on the NF-B signaling cascade were examined by Western blot analysis, real-time polymerase chain reaction, and immunofluorescence.Results. Cyclic tensile strain rapidly inhibited the IL-1␤-induced nuclear translocation of NF-B, but not its IL-1␤-induced phosphorylation at serine 276 and serine 536, which are necessary for its transactivation and transcriptional efficacy, respectively. Examination of upstream events revealed that cyclic tensile strain also inhibited the cytoplasmic protein degradation of IB␤ and IB␣, as well as repressed their gene transcription. Additionally, cyclic tensile strain induced a rapid nuclear translocation of IB␣ to potentially prevent NF-B binding to DNA. Furthermore, the inhibition of IL-1␤-induced degradation of IB by cyclic tensile strain was mediated by down-regulation of IB kinase activity.Conclusion. These results indicate that the signals generated by cyclic tensile strain act at multiple sites within the NF-B signaling cascade to inhibit IL-1␤-induced proinflammatory gene induction. Taken together, these findings provide insight into how biomechanical signals regulate and reduce inflammation, and underscore their potential in enhancing the ability of chondrocytes to curb inflammation in diseased joints.