The structure and function of vertebrate fibroblast growth factor receptor 1

The vertebrate fibroblast growth factor receptor 1 (FGFR1) is alternatively spliced generating multiple splice variants that are differentially expressed during embryo development and in the adult body. The restricted expression patterns of FGFR1 isoforms, together with differential expression and binding of specific ligands, leads to activation of common FGFR1 signal transduction pathways, but may result in distinctively different biological responses as a result of differences in cellular context. FGFR1 isoforms are also present in the nucleus in complex with various fibroblast growth factors where they function to regulate transcription of target genes.Casper Groth and Michael Lardell

Mitochondrion to endoplasmic reticulum apposition length in zebrafish embryo spinal progenitors is unchanged in response to perturbations associated with Alzheimer’s disease

Published: June 21, 2017Mutations in the human genes PRESENILIN1 (PSEN1), PRESENILIN2 (PSEN2) and AMYLOID BETA A4 PRECURSOR PROTEIN (APP) have been identified in familial Alzheimer's disease (AD). The length of mitochondrion-endoplasmic reticulum (M-ER) appositions is increased in Psen1-/-/Psen2-/- double knockout murine embryonic fibroblasts and in fibroblasts from AD-affected individuals. Development of an easily accessible, genetically manipulable, in vivo system for studying M-ER appositions would be valuable so we attempted to manipulate M-ER apposition length in zebrafish (Danio rerio) embryos. We injected fertilized zebrafish eggs with antisense morpholino oligonucleotides (MOs) that inhibit expression of zebrafish familial AD gene orthologues psen1 and psen2. Furthermore, we treated zebrafish embryos with DAPT (a highly specific γ-secretase inhibitor) or with sodium azide (to mimic partially hypoxic conditions). We then analyzed M-ER apposition in an identified, presumably proliferative neural cell type using electron microscopy. Our analysis showed no significant differences in M-ER apposition lengths at 48 hours post fertilization (hpf) between psen1 & psen2 MO co-injected embryos, embryos treated with DAPT, or sodium azide, and control embryos. Instead, the distribution of M-ER apposition lengths into different length classes was close to identical. However, this indicates that it is feasible to reproducibly measure M-ER size distributions in zebrafish embryos. While our observations differ from those of murine and human studies, this may be due to differences in cellular differentiation and metabolic state, cell age, or species-specific responses. In particular, by focusing on a presumably proliferative embryonic cell type, we may have selected a cell heavily already reliant on anaerobic glycolysis and less responsive to factors affecting M-ER apposition. Future examination of more differentiated, more secretory cell types may reveal measurable responses of M-ER apposition to environmental and genetic manipulation.Morgan Newman, Lena Halter, Anne Lim, Michael Lardell

The Zebrafish equivalent of Alzheimer's disease-associated PRESENILIN Isoform PS2V regulates inflammatory and other responses to hypoxic stress

Dominant mutations in the PRESENILIN genes PSEN1 and PSEN2 cause familial Alzheimer's disease (fAD) that usually shows onset before 65 years of age. In contrast, genetic variation at the PSEN1 and PSEN2 loci does not appear to contribute to risk for the sporadic, late onset form of the disease (sAD), leading to doubts that these genes play a role in the majority of AD cases. However, a truncated isoform of PSEN2, PS2V, is upregulated in sAD brains and is induced by hypoxia and high cholesterol intake. PS2V can increase γ-secretase activity and suppress the unfolded protein response (UPR), but detailed analysis of its function has been hindered by lack of a suitable, genetically manipulable animal model since mice and rats lack this PRESENILIN isoform. We recently showed that zebrafish possess an isoform, PS1IV, that is cognate to human PS2V. Using an antisense morpholino oligonucleotide, we can block specifically the induction of PS1IV that normally occurs under hypoxia. Here, we exploit this ability to identify gene regulatory networks that are modulated by PS1IV. When PS1IV is absent under hypoxia-like conditions, we observe changes in expression of genes controlling inflammation (particularly sAD-associated IL1B and CCR5), vascular development, the UPR, protein synthesis, calcium homeostasis, catecholamine biosynthesis, TOR signaling, and cell proliferation. Our results imply an important role for PS2V in sAD as a component of a pathological mechanism that includes hypoxia/oxidative stress and support investigation of the role of PS2V in other diseases, including schizophrenia, when these are implicated in the pathology.Esmaeil Ebrahimie, Seyyed Hani Moussavi Nik, Morgan Newman, Mark Van Der Hoek and Michael Lardell

Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences

Objective: Repeat expansion of polyglutamine tracks leads to a group of inherited human neurodegenerative disorders. Studying such repetitive sequences is required to gain insight into the pathophysiology of these diseases. PCR-based manipulation of repetitive sequences, however, is challenging due to the absence of unique primer binding sites or the generation of non-specific products. Results: We have utilised the degeneracy of the genetic code to generate a polyglutamine sequence with low repeat similarity. This strategy allowed us to use conventional PCR to generate multiple constructs with approximately defined numbers of glutamine repeats. We then used these constructs to measure the in vivo variation in autophagic degradation activity related to the different numbers of glutamine repeats, providing an example of their applicability to study repeat expansion diseases. Our simple and easily generalised method of generating low repetition DNA sequences coding for uniform stretches of amino acid residues provides a strategy for generating particular lengths of polyglutamine tracts using standard PCR and cloning protocols.Dhanushika Ratnayake, Morgan Newman and Michael Lardell

Trueperella pyogenes endocarditis in a Swiss farmer: a case report and review of the literature.

BACKGROUND Trueperella pyogenes (T. pyogenes) is a bacterium that colonizes the skin and mucosal surfaces of various domestic and wild animals. It rarely leads to infections in humans, with only a few descriptions available in the literature. CASE PRESENTATION A 71-year-old Swiss farmer with a history of recurring basal cell carcinoma and metastasized pancreatic neuroendocrine tumor presented with signs of sepsis after a three-day history of general weakness, malaise and fever. Clinical and echocardiographic findings, as well as persistent bacteremia were consistent with mitral valve endocarditis caused by T. pyogenes. The patient's condition gradually improved under antibiotic treatment with piperacillin/tazobactam (empiric therapy of sepsis), and later penicillin G based on resistance testing. He was discharged after 13 days and continued outpatient antibiotic therapy with ceftriaxone, resulting in a total antibiotic treatment duration of six weeks. This is the first literature review of T. pyogenes endocarditis in humans. Among nine cases of T. pyogenes endocarditis, three patients had documented contact with farm animals and five had an underlying condition that compromised the immune system. While antibiotic resistance of T. pyogenes is an emerging concern, susceptibility to beta-lactam antibiotics seems to persist. The mortality of T. pyogenes endocarditis described in the literature was high, with 66% of patients not surviving the disease. CONCLUSIONS T. pyogenes is a rare causative organism of infectious endocarditis in humans and descriptions are mainly restricted to case reports. In our review of the literature, we found that both an impaired immune system and contact with farm animals might be risk factors. Growth of T. pyogenes in blood cultures is unlikely to be missed during routine analysis, as it shows marked beta-hemolysis on blood agar culture plates, which generally leads to further characterization of the bacteria. Susceptibility to penicillin, ceftriaxone, and macrolides seems to be retained and the reported mortality in the few patients with T. pyogenes endocarditis is high

Brain transcriptome analysis of a familial Alzheimer's disease-like mutation in the zebrafish presenilin 1 gene implies effects on energy production

To prevent or ameliorate Alzheimer's disease (AD) we must understand its molecular basis. AD develops over decades but detailed molecular analysis of AD brains is limited to postmortem tissue where the stresses initiating the disease may be obscured by compensatory responses and neurodegenerative processes. Rare, dominant mutations in a small number of genes, but particularly the gene PRESENILIN 1 (PSEN1), drive early onset of familial AD (EOfAD). Numerous transgenic models of AD have been constructed in mouse and other organisms, but transcriptomic analysis of these models has raised serious doubts regarding their representation of the disease state. Since we lack clarity regarding the molecular mechanism(s) underlying AD, we posit that the most valid approach is to model the human EOfAD genetic state as closely as possible. Therefore, we sought to analyse brains from zebrafish heterozygous for a single, EOfAD-like mutation in their PSEN1-orthologous gene, psen1. We previously introduced an EOfAD-like mutation (Q96_K97del) into the endogenous psen1 gene of zebrafish. Here, we analysed transcriptomes of young adult (6-month-old) entire brains from a family of heterozygous mutant and wild type sibling fish. Gene ontology (GO) analysis implies effects on mitochondria, particularly ATP synthesis, and on ATP-dependent processes including vacuolar acidification.Morgan Newman, Nhi Hin, Stephen Pederson and Michael Lardell

Identification and expression analysis of the zebrafish orthologues of the mammalian MAP1LC3 gene family

Autophagy is the principle pathway within cells involved in clearing damaged proteins and organelles. Therefore autophagy is necessary to maintain the turnover balance of peptides and homoeostasis. Autophagy occurs at basal levels under normal conditions but can be upregulated by chemical inducers or stress conditions. The zebrafish (Danio rerio) serves as a versatile tool to understand the functions of genes implicated in autophagy. We report the identification of the zebrafish orthologues of mammalian genes MAP1LC3A (map1lc3a) and MAP1LC3B (map1lc3b) by phylogenetic and conserved synteny analysis and we examine their expression during embryonic development. The zebrafish map1lc3a and map1lc3b genes both show maternally contributed transcripts in early embryogenesis. However, levels of map1lc3a transcript steadily increase until at least 120h post-fertilisation while the levels of map1lc3b show a more variable pattern across developmental time. We have also validated the LC3I ratio/LC3I immunoblot autophagy assay in the presence of chloroquine (a lysosomal proteolysis inhibitor). We found that the LC3II/LC3I ratio is significantly increased in the presence of sodium azide with chloroquine supporting that hypoxia induces autophagy in zebrafish. This was supported by our qPCR assay that showed increased map1lc3a transcript levels in the presence of sodium azide. In contrast, levels of map1lc3b transcripts were reduced in the presence of rapamycin but the decrease in the presence of sodium azide did not reach statistical significance. Our study supports the use of zebrafish for analysing the interplay between hypoxia, development and autophagy.Swamynathan Ganesan, Seyyed Hani Moussavi Nik, Morgan Newman, Michael Lardell

The comparison of methods for measuring oxidative stress in zebrafish brains

The zebrafish is a versatile model organism with the potential to contribute to our understanding of the molecular pathological mechanisms underlying Alzheimer's disease (AD). An early characteristic of AD brain pathology is lipid peroxidation resulting from oxidative stress. However, changes in lipid peroxidation have not yet been assessed in zebrafish brains, and an earlier attempt to observe changes in F₂-isoprostane levels in the brains of zebrafish exposed to hypoxia was unsuccessful. In this article, we examine the utility of various assays of lipid peroxidation and more general assays of intracellular oxidative stress to detect the changes in oxidative stress in the brains of adult zebrafish exposed to hypoxia or explanted into a sodium azide solution for chemical mimicry of hypoxia. Levels of F₂-isoprostanes and F₄-neuroprostanes were low and variable in zebrafish brains such that statistically significant changes due to hypoxia or chemical mimicry of hypoxia could not be observed. However, measurement of lipid hydroperoxides did reveal significant changes in lipid peroxidation under these conditions, while analyses of catalase gene expression and an assay based on 2',7'-dicholorofluorescein oxidation also revealed changes in oxidative stress levels.Seyyed Hani Moussavi Nik, Kevin Croft, Trevor A. Mori, and Michael Lardell

Utilización de la densidad de incidencia, en la valoración de faciores de riesgo de la infección nosocomial

ResumenEn la literatura se han propuesto las medidas de densidad de incidencia para la valoración de la infección nosocomial. A través de una cohorte retrospectiva de 843 pacientes se realiza una comparación entre la razón de densidades de incidencia (RDI) y el cociente de incidencias acumuladas o riesgo relativo (RR) en la cuantificación de factores de riesgo de la infección hospitalaria. Las variables que se analizaron fueron la intervención quirúrgica, su duración, el tipo de cirugía, la gravedad de la enfermedad subyacente y la edad. En todas las situaciones la RDI fue inferior al RR, al eliminar la prolongación de estancia producida por los distintos factores de riesgo. Ejemplos de ello fueron el que la RDI para la operación fue de 2,78, mientras que el RR ofreció valores de 6,46, o en la edad, donde los mayores de 60 años presentaron un RR significativo de 1,67, mientras que la RDI fue de 0,96. Esto sugiere que el empleo de la RDI facilitaría la comparabilidad de los resultados entre diferentes centros en la cuantificación de factores de riesgo para la infección nosocomial, aparte de cuantificar con más exactitud los distintos factores de riesgo, lo que tiene importancia a la hora de establecer las medidas de control.SummarySeveral authors have suggested that incidence density should be used in studying nosocomial infection. We assess several risk factors for hospital infection by two ratios, the incidence density ratio (IDR) and the relative risk (RR), in an historical cohort of 843 patients. The variables analyzed were: operation, its length, type of surgical wound, severity of underlying disease, and age. The IDR figures were always lower than those yielded by the RR. For example, the IDR for operated patients was 2.78, whereas RR yielded a figure of 6.46, or the IDR for patients >60 years old was 0.96, whereas the RR achieved a significant value of 1.67. This suggests that the use of IDR lo analyze risk factors for nosocomial infection improves comparability of results obtained in different hospital settings. Also, it may allow a more exact quantification of an effect. These facts influence implementation of nosocomial infection control measures