Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

BLProt: prediction of bioluminescent proteins based on support vector machine and relieff feature selection

<p>Abstract</p> <p>Background</p> <p>Bioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances. Identification of bioluminescent proteins is more challenging due to their poor similarity in sequence. So far, no specific method has been reported to identify bioluminescent proteins from primary sequence.</p> <p>Results</p> <p>In this paper, we propose a novel predictive method that uses a Support Vector Machine (SVM) and physicochemical properties to predict bioluminescent proteins. BLProt was trained using a dataset consisting of 300 bioluminescent proteins and 300 non-bioluminescent proteins, and evaluated by an independent set of 141 bioluminescent proteins and 18202 non-bioluminescent proteins. To identify the most prominent features, we carried out feature selection with three different filter approaches, ReliefF, infogain, and mRMR. We selected five different feature subsets by decreasing the number of features, and the performance of each feature subset was evaluated.</p> <p>Conclusion</p> <p>BLProt achieves 80% accuracy from training (5 fold cross-validations) and 80.06% accuracy from testing. The performance of BLProt was compared with BLAST and HMM. High prediction accuracy and successful prediction of hypothetical proteins suggests that BLProt can be a useful approach to identify bioluminescent proteins from sequence information, irrespective of their sequence similarity. The BLProt software is available at <url>http://www.inb.uni-luebeck.de/tools-demos/bioluminescent%20protein/BLProt</url></p

Contribution of a Common Variant in the Promoter of the 1-α-Hydroxylase Gene (CYP27B1) to Fracture Risk in the Elderly

CYP27B1 encodes mitochondrial 1α-hydroxylase, which converts 25-hydroxyvitamin D to its active 1,25-dihydroxylated metabolite. We tested the hypothesis that common variants in the CYP27B1 promoter are associated with fracture risk. The study was designed as a population-based genetic association study, which involved 153 men and 596 women aged 65–101 years, who had been followed for 2.2 years (range 0.1–5.5) between 1999 and 2006. During the follow-up period, the incidence of fragility fractures was ascertained. Bone ultrasound attenuation (BUA) was measured in all individuals, as were serum 25-hydroxyvitamin D and PTH concentrations; 86% subjects had vitamin D insufficiency. Genotypes were determined for the –1260C>A (rs10877012) and +2838T>C (rs4646536) CYP27B1 polymorphisms. A reporter gene assay was used to assess functional expression of the –1260C>A CYP27B1 variants. The association between genotypes and fracture risk was analyzed by Cox’s proportional hazards model. We found that genotypic distribution of CYP27B1 –1260 and CYP27B1 +2838 polymorphisms was consistent with the Hardy-Weinberg equilibrium law. The two polymorphisms were in high linkage disequilibrium, with D′ = 0.96 and r2 = 0.94. Each C allele of the CYP27B1 –1260 polymorphism was associated with increased risk of fracture (hazard ratio = 1.34, 95% CI 1.03–1.73), after adjustment for age, sex, number of falls, and BUA. In transient transfection studies, a reporter gene downstream of the –1260(A)-containing promoter was more highly expressed than that containing the C allele. These data suggest that a common but functional variation within the CYP27B1 promoter gene is associated with fracture risk in the elderly

Towards plant-odor-related olfactory neuroethology in Drosophila

Drosophila melanogaster is today one of the three foremost models in olfactory research, paralleled only by the mouse and the nematode. In the last years, immense progress has been achieved by combining neurogenetic tools with neurophysiology, anatomy, chemistry, and behavioral assays. One of the most important tasks for a fruit fly is to find a substrate for eating and laying eggs. To perform this task the fly is dependent on olfactory cues emitted by suitable substrates as e.g. decaying fruit. In addition, in this area, considerable progress has been made during the last years, and more and more natural and behaviorally active ligands have been identified. The future challenge is to tie the progress in different fields together to give us a better understanding of how a fly really behaves. Not in a test tube, but in nature. Here, we review our present state of knowledge regarding Drosophila plant-odor-related olfactory neuroethology to provide a basis for new progress

Hedonic Taste in Drosophila Revealed by Olfactory Receptors Expressed in Taste Neurons

Taste and olfaction are each tuned to a unique set of chemicals in the outside world, and their corresponding sensory spaces are mapped in different areas in the brain. This dichotomy matches categories of receptors detecting molecules either in the gaseous or in the liquid phase in terrestrial animals. However, in Drosophila olfactory and gustatory neurons express receptors which belong to the same family of 7-transmembrane domain proteins. Striking overlaps exist in their sequence structure and in their expression pattern, suggesting that there might be some functional commonalities between them. In this work, we tested the assumption that Drosophila olfactory receptor proteins are compatible with taste neurons by ectopically expressing an olfactory receptor (OR22a and OR83b) for which ligands are known. Using electrophysiological recordings, we show that the transformed taste neurons are excited by odor ligands as by their cognate tastants. The wiring of these neurons to the brain seems unchanged and no additional connections to the antennal lobe were detected. The odor ligands detected by the olfactory receptor acquire a new hedonic value, inducing appetitive or aversive behaviors depending on the categories of taste neurons in which they are expressed i.e. sugar- or bitter-sensing cells expressing either Gr5a or Gr66a receptors. Taste neurons expressing ectopic olfactory receptors can sense odors at close range either in the aerial phase or by contact, in a lipophilic phase. The responses of the transformed taste neurons to the odorant are similar to those obtained with tastants. The hedonic value attributed to tastants is directly linked to the taste neurons in which their receptors are expressed

ANCA-associated vasculitis.

The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) are a group of disorders involving severe, systemic, small-vessel vasculitis and are characterized by the development of autoantibodies to the neutrophil proteins leukocyte proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA). The three AAV subgroups, namely granulomatosis with polyangiitis (GPA), microscopic polyangiitis and eosinophilic GPA (EGPA), are defined according to clinical features. However, genetic and other clinical findings suggest that these clinical syndromes may be better classified as PR3-positive AAV (PR3-AAV), MPO-positive AAV (MPO-AAV) and, for EGPA, by the presence or absence of ANCA (ANCA+ or ANCA-, respectively). Although any tissue can be involved in AAV, the upper and lower respiratory tract and kidneys are most commonly and severely affected. AAVs have a complex and unique pathogenesis, with evidence for a loss of tolerance to neutrophil proteins, which leads to ANCA-mediated neutrophil activation, recruitment and injury, with effector T cells also involved. Without therapy, prognosis is poor but treatments, typically immunosuppressants, have improved survival, albeit with considerable morbidity from glucocorticoids and other immunosuppressive medications. Current challenges include improving the measures of disease activity and risk of relapse, uncertainty about optimal therapy duration and a need for targeted therapies with fewer adverse effects. Meeting these challenges requires a more detailed knowledge of the fundamental biology of AAV as well as cooperative international research and clinical trials with meaningful input from patients