Genetic forms of neurohypophyseal diabetes insipidus

In the majority of cases, hereditary neurohypophyseal diabetes insipidus (DI) is a monogenic disorder caused by mutations in the AVP gene. Dominant transmission is by far the most common form. In these patients, symptoms develop gradually at various ages during childhood, progressing with complete penetrance to polyuria and polydipsia that is usually severe. In autosomal dominant neurohypophyseal DI (ADNDI), the mutant prohormone is folding deficient and consequently retained in the ER, where it forms amyloid-like fibrillar aggregates. Degradation by proteasomes occurs, but their clearance capacity appears to be insufficient. Postmortem studies in affected individuals suggest a neurodegenerative process confined to vasopressinergic neurons. Other forms of genetic neurohypophyseal DI include the very rare autosomal recessive type, also caused by mutations in the AVP gene, and complex multiorgan disorders, such as Wolfram syndrome. In all individuals where a congenital form of DI is suspected, including nephrogenic types, genetic analysis should be performed

Limited Correlation of Shotgun Metagenomics Following Host Depletion and Routine Diagnostics for Viruses and Bacteria in Low Concentrated Surrogate and Clinical Samples

The etiologic cause of encephalitis, meningitis or meningo-encephalitis is unknown in up to 70% of cases. Clinical shotgun metagenomics combined with host depletion is a promising technique to identify infectious etiologies of central nervous system (CNS) infections. We developed a straightforward eukaryotic host nucleic acid depletion method that preserves intact viruses and bacteria for subsequent shotgun metagenomics screening of clinical samples, focusing on cerebrospinal fluid (CSF). A surrogate CSF sample for a CNS infection paradigm was used to evaluate the proposed depletion method consisting of selective host cell lysis, followed by enzymatic degradation of the liberated genomic DNA for final depletion with paramagnetic beads. Extractives were subjected to reverse transcription, followed by whole genome amplification and next generation sequencing. The effectiveness of the host depletion method was demonstrated in surrogate CSF samples spiked with three 1:100 dilutions of Influenza A H3N2 virus (qPCR Ct-values 20.7, 28.8, >42/negative). Compared to the native samples, host depletion increased the amount of the virus subtype reads by factor 7127 and 132, respectively, while in the qPCR negative sample zero vs. 31 (1.4E-4 %) virus subtype reads were detected (native vs. depleted). The workflow was applied to thirteen CSF samples of patients with meningo-/encephalitis (two bacterial, eleven viral etiologies), a serum of an Andes virus infection and a nose swab of a common cold patient. Unlike surrogate samples, host depletion of the thirteen human CSF samples and the nose swab did not result in more reads indicating presence of damaged pathogens due to, e.g., host immune response. Nevertheless, previously diagnosed pathogens in the human CSF samples (six viruses, two bacteria), the serum, and the nose swab (Human rhinovirus A31) were detected in the depleted and/or the native samples. Unbiased evaluation of the taxonomic profiles supported the diagnosed pathogen in two native CSF samples and the native and depleted serum and nose swab, while detecting various contaminations that interfered with pathogen identification at low concentration levels. In summary, damaged pathogens and contaminations complicated analysis and interpretation of clinical shotgun metagenomics data. Still, proper consideration of these issues may enable future application of metagenomics for clinical diagnostics

Susceptibility to Mycobacterium ulcerans Disease (Buruli ulcer) Is Associated with IFNG and iNOS Gene Polymorphisms

Buruli ulcer (BU) is a chronic necrotizing disease of the skin and subcutaneous fat tissue. The causative agent, Mycobacterium ulcerans, produces mycolactone, a macrolide toxin, which causes apoptosis of mammalian cells. Only a small proportion of individuals exposed to M. ulcerans develop clinical disease, as surrounding macrophages may control the infection by bacterial killing at an early stage, while mycolactone concentration is still low. Otherwise, bacterial multiplication leads to in higher concentrations of mycolactone, with formation of necrotizing lesions that are no more accessible to immune cells. By typing a cohort of 96 Ghanaian BU patients and 384 endemic controls without BU, we show an association between BU and single nucleotide polymorphisms (SNPs) in iNOS (rs9282799) and IFNG (rs2069705). Both polymorphisms influence promoter activity in vitro. A previously reported SNP in SLC11A1 (NRAMP, rs17235409) tended to be associated with BU. Altogether, these data reflect the importance of IFNG signaling in early defense against M. ulcerans infection

Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting

Unlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the TGN by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the ER. Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule sorting. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for sorting into secretory granules by self-aggregation

Role of protein aggregation and degradation in autosomal dominant neurohypophyseal diabetes insipidus

This review focuses on the cellular and molecular aspects underlying familial neurohypophyseal diabetes insipidus (DI), a rare disorder that is usually transmitted in an autosomal-dominant fashion. The disease, manifesting in infancy or early childhood and gradually progressing in severity, is caused by fully penetrant heterozygous mutations in the gene encoding prepro-vasopressin-neurophysin II, the precursor of the antidiuretic hormone arginine vasopressin (AVP). Post mortem studies in affected adults have shown cell degeneration in vasopressinergic hypothalamic nuclei. Studies in cells expressing pathogenic mutants and knock-in rodent models have shown that the mutant precursors are folding incompetent and fail to exit the endoplasmic reticulum (ER), as occurs normally with proteins that have entered the regulated secretory pathway. A portion of these mutants is eliminated via ER-associated degradation (ERAD) by proteasomes after retrotranslocation to the cytosol. Another portion forms large disulfide-linked fibrillar aggregates within the ER, in which wild-type precursor is trapped. Aggregation capacity is independently conferred by two domains of the prohormone, namely the AVP moiety and the C-terminal glycopeptide (copeptin). The same domains are also required for packaging into dense-core secretory granules and regulated secretion, suggesting a disturbed balance between the physiological self-aggregation at the trans-Golgi network and avoiding premature aggregate formation at the ER in the disease. The critical role of ERAD in maintaining physiological water balance has been underscored by experiments in mice expressing wild-type AVP but lacking critical components of the ERAD machinery. These animals also develop DI and show amyloid-like aggregates in the ER lumen. Thus, the capacity of the ERAD is exceeded in autosomal dominant DI, which can be viewed as a neurodegenerative disorder associated with the formation of amyloid ER aggregates. While DI symptoms develop prior to detectable cell death in transgenic DI mice, the eventual loss of vasopressinergic neurons is accompanied by autophagy, but the mechanism leading to cell degeneration in autosomal dominant neurohypophyseal DI still remains unknown

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells.

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway

Limited Correlation of Shotgun Metagenomics Following Host Depletion and Routine Diagnostics for Viruses and Bacteria in Low Concentrated Surrogate and Clinical Samples.

The etiologic cause of encephalitis, meningitis or meningo-encephalitis is unknown in up to 70% of cases. Clinical shotgun metagenomics combined with host depletion is a promising technique to identify infectious etiologies of central nervous system (CNS) infections. We developed a straightforward eukaryotic host nucleic acid depletion method that preserves intact viruses and bacteria for subsequent shotgun metagenomics screening of clinical samples, focusing on cerebrospinal fluid (CSF). A surrogate CSF sample for a CNS infection paradigm was used to evaluate the proposed depletion method consisting of selective host cell lysis, followed by enzymatic degradation of the liberated genomic DNA for final depletion with paramagnetic beads. Extractives were subjected to reverse transcription, followed by whole genome amplification and next generation sequencing. The effectiveness of the host depletion method was demonstrated in surrogate CSF samples spiked with three 1:100 dilutions of Influenza A H3N2 virus (qPCR Ct-values 20.7, 28.8, >42/negative). Compared to the native samples, host depletion increased the amount of the virus subtype reads by factor 7127 and 132, respectively, while in the qPCR negative sample zero vs. 31 (1.4E-4 %) virus subtype reads were detected (native vs. depleted). The workflow was applied to thirteen CSF samples of patients with meningo-/encephalitis (two bacterial, eleven viral etiologies), a serum of an Andes virus infection and a nose swab of a common cold patient. Unlike surrogate samples, host depletion of the thirteen human CSF samples and the nose swab did not result in more reads indicating presence of damaged pathogens due to, e.g., host immune response. Nevertheless, previously diagnosed pathogens in the human CSF samples (six viruses, two bacteria), the serum, and the nose swab (Human rhinovirus A31) were detected in the depleted and/or the native samples. Unbiased evaluation of the taxonomic profiles supported the diagnosed pathogen in two native CSF samples and the native and depleted serum and nose swab, while detecting various contaminations that interfered with pathogen identification at low concentration levels. In summary, damaged pathogens and contaminations complicated analysis and interpretation of clinical shotgun metagenomics data. Still, proper consideration of these issues may enable future application of metagenomics for clinical diagnostics

Fysiska åtgärder på Borås gator för barns säkerhet som gående och cyklister : analys av åtgärdernas effekter

Godkänd; 2005; 20080919 (andbra

Additional file 1: of Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting

Figure S1.. Localization of wild-type provasopressin and the ∆E47 DI mutant in transfected HN10 cells. Immunofluorescence localization of wild-type (wt) provasopressin and the DI mutant precursor ∆E47 expressed in HN10 cells 48 h after transfection. Cells were costained with antibodies against provasopressin (V; blue), KDEL as an ER marker (red), and CgA as a cargo of secretory granules (green). Wild-type provasopressin is detected in granules in the neuronal extension together with CgA (filled arrowheads), whereas ∆E47 in a majority of expressing cells is found in aggregates in the ER of the cell body costained with anti-KDEL (open arrowheads). Bar: 10 μm. (TIF 1759 kb