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Complex I has an essential role in ATP production by coupling electron transfer from NADH to quinone with translocation of protons across the inner mitochondrial membrane. Isolated complex I deficiency is a frequent cause of mitochondrial inherited diseases. Complex I has also been implicated in cancer, ageing, and neurodegenerative conditions. Until recently, the understanding of complex I deficiency on the molecular level was limited due to the lack of high-resolution structures of the enzyme. However, due to developments in single particle cryoelectron microscopy (cryo-EM), recent studies have reported nearly atomic resolution maps and models of mitochondrial complex I. These structures significantly add to our understanding of complex I mechanism and assembly. The disease-causing mutations are discussed here in their structural context.

HIV-1 Env, a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding displaces V1V2 loops from Env's apex, allowing coreceptor binding and opening Env to enable gp41-mediated fusion. We present 3.54 angstrom and 4.06 angstrom cryoelectron microscopy structures of partially open soluble native-like Env trimers (SOSIPs) bound to CD4. One structure, a complex with a coreceptor-mimicking antibody that binds both CD4 and gp120, stabilizes the displaced V1V2 and reveals its structure. Comparing partially and fully open Envs with closed Envs shows that gp41 re-arrangements are independent of the CD4-induced rearrangements that result in V1V2 displacement and formation of a 4-stranded bridging sheet. These findings suggest ordered conformational changes before coreceptor binding: (1) gp120 opening inducing side-chain rearrangements and a compact gp41 central helix conformation, and (2) 4-stranded bridging-sheet formation and V1V2 displacement. These analyses illuminate potential receptor-induced Env changes and inform design of therapeutics disrupting viral entry.

Hypoglycemia can alter arousal and negatively impact mood. This study tests the hypothesis that acute drops in glucose metabolism cause an aversive state mediated by monoamine activity. In experiment 1, male Sprague-Dawley rats were either food deprived (FD) or pre-fed (PF) and tested on conditioned place avoidance (CPA; biased place conditioning design; 3 pairings drug/vehicle, each 30 min-long) induced by the glucose antimetabolite 2-deoxy-d-glucose (2-DG; 0, 300 or 500 mg/kg, SC). Locomotion and blood glucose were also assessed. Experiment 2 examined whether clonidine (noradrenergic alpha 2 agonist, 0, 10 or 40 mu g/kg, SC) or bupropion (monoamine reuptake blocker, 0, 10 or 30 mg/kg, SC) could alter CPA induced by 500 mg/kg 2-DG. In experiment 3, blood corticosterone (CORT) was measured in response to 500 mg/kg 2-DG, alone or in combination with 40 mu g/kg clonidine or 30 mg/kg bupropion. Finally, experiment 4 controlled for possible place conditioning induced by 10 or 40 mu g/kg clonidine, or 10 or 30 mg/kg bupropion injected without 2-DG. It was found that 2-DG increased blood glucose and produced a robust CPA. The feeding status of the animals modulated these effects, including CORT levels. Both clonidine and bupropion attenuated the effects of 2-DG on CPA and CORT, but only bupropion reversed suppression of locomotion. Taken together, these results in rats suggest that impaired glucose metabolism can negatively impact arousal and mood via effects on HPA and monoamine systems.

The epithelial barrier of the gastrointestinal tract is home to numerous intraepithelial T cells (IETs). IETs are functionally adapted to the mucosal environment and are among the first adaptive immune cells to encounter microbial and dietary antigens. They possess hallmark features of tissue-resident T cells: they are long-lived nonmigratory cells capable of rapidly responding to antigen challenges independent of T cell recruitment from the periphery. Gut-resident T cells have been implicated in the relapsing and remitting course and persisting low-grade inflammation of chronic gastrointestinal diseases, including IBD and coeliac disease. So far, most data IETs have been derived from experimental animal models; however, IETs and the environmental makeup differ between mice and humans. With advances in techniques, the number of human studies has grown exponentially in the past 5 years. Here, we review the literature on the involvement of human IETs in gut homeostasis and inflammation, and how these cells are influenced by the microbiota and dietary antigens. Finally, targeting of IETs in therapeutic interventions is discussed. Broad insight into the function and role of human IETs in gut homeostasis and inflammation is essential to identify future diagnostic, prognostic and therapeutic strategies.

Fibrolamellar carcinomas are characterized by activation of protein kinase A, a kinase composed of catalytic and regulatory subunits. PRKACA encodes a catalytic subunit of protein kinase A, and almost all fibrolamellar carcinomas have a heterozygous 400-kb deletion that leads to the fusion of DNAJB1 and PRKACA. The resulting DNAJB1-PRKACA fusion transcript is believed to activate protein kinase A by dysregulation of the catalytic portion of the protein. In contrast, PRKAR1A encodes one of the regulatory subunits of protein kinase A. We hypothesized that loss of function of this regulatory unit could also lead to protein kinase A activation and thus to fibrolamellar carcinoma. Because PRKAR1A mutations underlie the Carney complex, we searched for liver tumors in individuals with the Carney complex. We identified 3 individuals with fibrolamellar carcinomas and a personal history of the Carney complex. All three tumors displayed the typical morphology of fibrolamellar carcinoma and were positive for arginase, cytokeratin 7, and cluster of differentiation 68. Fluorescence in situ hybridization was negative for PRKACA rearrangements. However, PRKAR1A sequencing identified pathogenic mutations in two of two cases with successful sequencing. In addition, all three cases were negative for PRKAR1A protein expression, consistent with inactivation of this key regulatory unit of protein kinase A. We also identified one additional fibrolamellar carcinoma in an individual without a documented history of the Carney complex who was negative for PRKACA rearrangements but had loss of PRKAR1A protein expression as well as PRKAR1A mutations. Conclusion: Fibrolamellar carcinoma can be part of the Carney complex; in this setting, fibrolamellar carcinomas have inactivating PRKAR1A mutations instead of the DNAJB1-PRKACA fusion gene found in sporadic fibrolamellar carcinomas, providing an alternate means for activation of protein kinase A. (Hepatology 2017).

Prior studies demonstrate that astrotactin (ASTN1) provides a neuronal receptor for glial-guided CNS migration. Here we report that ASTN1 binds N-cadherin (CDH2) and that the ASTN1: CDH2 interaction supports cell-cell adhesion. To test the function of ASTN1: CDH2 binding in glial-guided neuronal migration, we generated a conditional loss of Cdh2 in cerebellar granule cells and in glia. Granule cell migration was slowed in cerebellar slice cultures after a conditional loss of neuronal Cdh2, and more severe migration defects occurred after a conditional loss of glial Cdh2. Expression in granule cells of a mutant form of ASTN1 that does not bind CDH2 also slowed migration. Moreover, in vitro chimeras of granule cells and glia showed impaired neuron-glia attachment in the absence of glial, but not neuronal, Cdh2. Thus, cis and trans bindings of ASTN1 to neuronal and glial CDH2 form an asymmetric neuron-glial bridge complex that promotes glial-guided neuronal migration.

Background: Inherited CARD9 deficiency constitutes a primary immunodeficiency predisposing uniquely to chronic and invasive fungal infections. Certain mutations are shown to negatively impact CARD9 protein expression and/or NF-kappa B activation, but the underlying biochemical mechanism remains to be fully understood. Objectives: To investigate a possible founder origin of a known CARD9 R70W mutation in five families of Turkish origin. To explore the biochemical mechanism of immunodeficiency by R70W CARD9. Methods: We performed haplotype analysis using microsatellite markers and SNPs. We designed a model system exploiting a gain-of-function (GOF) CARD9 L213L1 mutant that triggers constitutive NE-kappa B activation, analogous to an oncogenic CARD11 mutant, to study NF-KB signaling and signalosome formation. We performed reporter assays, immunoprecipitation and confocal imaging on HEK cells overexpressing different CARD9 variants. Results: We identified a common haplotype, thus providing evidence for a common Turkish founder. CARD9 R70W failed to activate NF-kappa B and abrogated NF-kappa B activation by WT CARD9 and by GOF CARD9. Notably, R70W CARD9 also exerted negative effects on NF-kappa B activation by CARD10, CARD11, and CARD14. Consistent with the NF-kappa B results, the R70W mutation prevented GOF CARD9 to pull down the signalosome partner proteins BCL10 and MALT1. This reflected into drastic reduction of BCL10 filamentous assemblies in a cellular context. Indeed, structural analysis revealed that position R70 in CARD9 maps at the putative interface between successive CARD domains in CARD9 filaments. Conclusions: The R70W mutation in CARD9 prevents NF-kappa B activation by inhibiting productive interactions with downstream BCL10 and MALT1, necessary for assembly of the filamentous CARD9-BCL10-MALT1 signalosome.

The ichthyoses are rare skin disorders with immune and barrier aberrations. Identifying blood phenotypes may advance targeted therapeutics. We aimed to compare frequencies of skin homing/cutaneous lymphocyte antigen (+) versus systemic/cutaneous lymphocyte antigen (-) "polar" CD4(+)/CD8(+) and activated T-cell subsets in ichthyosis versus atopic dermatitis, psoriasis, and control blood, with appropriate clinical correlations. Flow cytometry was used to measure IFN-gamma, IL-13, IL-9, IL-17, and IL-22 cytokines in CD4(+)/CD8(+) T cells, with inducible co-stimulator molecule and HLA-DR defining mid-and long-term T-cell activation, respectively. We compared peripheral blood from 47 patients with ichthyosis (congenital ichthyosiform erythroderma, lamellar ichthyosis, epidermolytic ichthyosis, and Netherton syndrome) with 43 patients with atopic dermatitis and 24 patients with psoriasis and 59 age-matched controls. Clinical measures included the ichthyosis severity score, with subsets for erythema and scaling, trans-epidermal water loss, and pruritus. All ichthyoses had excessive inducible co-stimulator molecule activation (P<0.001), particularly epidermolytic ichthyosis. Significantly elevated IL-17-(P<0.05) and IL-22-producing (P<0.01) T cells characterized ichthyoses, mainly Netherton syndrome and congenital ichthyosiform erythroderma (P<0.05). Increased T helper 2/cytotoxic T cell 2/T helper 9 (P<0.05) and similar IFN-gamma frequencies (P>0.1) versus controls were also noted. IL-17/IL-22-producing cells clustered with clinical measures, whereas IFN-gamma clustered with age. Our data show peripheral blood IL-17/IL-22 activation across the ichthyoses, correlating with clinical measures. Targeted therapies should dissect the relative contribution of polar cytokines to disease pathogenesis.

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and estab- lish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.

Differentiation of embryonic stem cells in vitro is an important tool in dissecting and understanding the mechanisms that govern early embryologic development. In recent years, there has been considerable progress in creating organoids that model gastrulation, neurulation or organogenesis. However, one of the key challenges is reproducibility. Geometrically confining stem cell colonies considerably improves reproducibility and provides quantitative control over differentiation and tissue shape. Here, we review recent advances in controlling the two-dimensional or three-dimensional organization of cells and the effect on differentiation phenotypes. Improved methods of geometrical control will allow for an even more detailed understanding of the mechanisms underlying embryologic development and will eventually pave the way for the highly reproducible generation of specific tissue types.