Disentangling astroglial physiology with a realistic cell model in silico

Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K+ and generate Ca2+ signals that release molecular regulators of neural circuitry. The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K+ channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K+ hotspots. We show how intracellular Ca2+ buffers affect Ca2+ waves and why the classical Ca2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca2+ imaging

A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

BACKGROUND: TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. METHODS: In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. RESULTS: In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity. CONCLUSIONS: A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures

New challenges for BRCA testing:a view from the diagnostic laboratory

Increased demand for BRCA testing is placing pressures on diagnostic laboratories to raise their mutation screening capacity and handle the challenges associated with classifying BRCA sequence variants for clinical significance, for example interpretation of pathogenic mutations or variants of unknown significance, accurate determination of large genomic rearrangements and detection of somatic mutations in DNA extracted from formalin-fixed, paraffin-embedded tumour samples. Many diagnostic laboratories are adopting next-generation sequencing (NGS) technology to increase their screening capacity and reduce processing time and unit costs. However, migration to NGS introduces complexities arising from choice of components of the BRCA testing workflow, such as NGS platform, enrichment method and bioinformatics analysis process. An efficient, cost-effective accurate mutation detection strategy and a standardised, systematic approach to the reporting of BRCA test results is imperative for diagnostic laboratories. This review covers the challenges of BRCA testing from the perspective of a diagnostics laboratory

CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes

Characterising chromosome rearrangements: recent technical advances in molecular cytogenetics

Genomic rearrangements can result in losses, amplifications, translocations and inversions of DNA fragments thereby modifying genome architecture, and potentially having clinical consequences. Many genomic disorders caused by structural variation have initially been uncovered by early cytogenetic methods. The last decade has seen significant progression in molecular cytogenetic techniques, allowing rapid and precise detection of structural rearrangements on a whole-genome scale. The high resolution attainable with these recently developed techniques has also uncovered the role of structural variants in normal genetic variation alongside single-nucleotide polymorphisms (SNPs). We describe how array-based comparative genomic hybridisation, SNP arrays, array painting and next-generation sequencing analytical methods (read depth, read pair and split read) allow the extensive characterisation of chromosome rearrangements in human genomes

Satisfaction of search awareness in trauma radiology: Malignant renal lesion on a trauma thoracolumbar spine CT

Fast-paced trauma imaging can result in misses relating to the nonosseous structures included in the field of view. We report a case of a Bosniak type III renal cyst, later found to be clear cell renal cell carcinoma, incidentally noted on post-traumatic CT of the thoracic and lumbar spine. This case includes a discussion of the circumstances which could result in a radiologist missing this finding, the idea of satisfaction of search, the importance of maintaining a thorough search pattern, and the management and communication of incidental findings