Statistical issues related to dietary intake as the response variable in intervention trials.

The focus of this paper is dietary intervention trials. We explore the statistical issues involved when the response variable, intake of a food or nutrient, is based on self-report data that are subject to inherent measurement error. There has been little work on handling error in this context. A particular feature of self-reported dietary intake data is that the error may be differential by intervention group. Measurement error methods require information on the nature of the errors in the self-report data. We assume that there is a calibration sub-study in which unbiased biomarker data are available. We outline methods for handling measurement error in this setting and use theory and simulations to investigate how self-report and biomarker data may be combined to estimate the intervention effect. Methods are illustrated using data from the Trial of Nonpharmacologic Intervention in the Elderly, in which the intervention was a sodium-lowering diet and the response was sodium intake. Simulations are used to investigate the methods under differential error, differing reliability of self-reports relative to biomarkers and different proportions of individuals in the calibration sub-study. When the reliability of self-report measurements is comparable with that of the biomarker, it is advantageous to use the self-report data in addition to the biomarker to estimate the intervention effect. If, however, the reliability of the self-report data is low compared with that in the biomarker, then, there is little to be gained by using the self-report data. Our findings have important implications for the design of dietary intervention trials. © 2016 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd

mTOR independent alteration in ULK1 Ser758 phosphorylation following chronic LRRK2 kinase inhibition

Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In this study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson's disease linked protein Leucine Rich Repeat Kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity

Biallelic interferon regulatory factor 8 mutation: A complex immunodeficiency syndrome with dendritic cell deficiency, monocytopenia, and immune dysregulation

Background: The homozygous K108E mutation of interferon regulatory factor 8 (IRF8) is reported to cause dendritic cell (DC) and monocyte deficiency. However, more widespread immune dysfunction is predicted from the multiple roles ascribed to IRF8 in immune cell development and function. Objective: We sought to describe the effect on hematopoiesis and immunity of the compound heterozygous R83C/R291Q mutation of IRF8, which is present in a patient with recurrent viral infection, granuloproliferation, and intracerebral calcification. Methods: Variant IRF8 alleles were identified by means of exome sequencing, and their function was tested by using reporter assays. The cellular phenotype was studied in detail by using flow cytometry, functional immunologic assay transcriptional profiling, and antigen receptor profiling. Results: Both mutations affected conserved residues, and R291Q is orthologous to R294, which is mutated in the BXH2 IRF8-deficient mouse. R83C showed reduced nuclear translocation, and neither mutant was able to regulate the Ets/IRF composite element or interferon-stimulated response element, whereas R291Q retained BATF/JUN interactions. DC deficiency and monocytopenia were observed in blood, dermis, and lung lavage fluid. Granulocytes were consistently increased, dysplastic, and hypofunctional. Natural killer cell development and maturation were arrested. TH1, TH17, and CD8+ memory T-cell differentiation was significantly reduced, and T cells did not express CXCR3. B-cell development was impaired, with fewer memory cells, reduced class-switching, and lower frequency and complexity of somatic hypermutation. Cell-specific gene expression was widely disturbed in interferon- and IRF8-regulated transcripts. Conclusions: This analysis defines the clinical features of human biallelic IRF8 deficiency, revealing a complex immunodeficiency syndrome caused by DC and monocyte deficiency combined with widespread immune dysregulation

Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs

Pathogenic Parkinson's disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation

LRRK2 is one of the most important genetic contributors to Parkinson's disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations

Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells

In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo

DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions