Numerical Model for the Determination of Erythrocyte Mechanical Properties and Wall Shear Stress in vivo From Intravital Microscopy.

The mechanical properties and deformability of Red Blood Cells (RBCs) are important determinants of blood rheology and microvascular hemodynamics. The objective of this study is to quantify the mechanical properties and wall shear stress experienced by the RBC membrane during capillary plug flow in vivo utilizing high speed video recording from intravital microscopy, biomechanical modeling, and computational methods. Capillaries were imaged in the rat cremaster muscle pre- and post-RBC transfusion of stored RBCs for 2-weeks. RBC membrane contours were extracted utilizing image processing and parametrized. RBC parameterizations were used to determine updated deformation gradient and Lagrangian Green strain tensors for each point along the parametrization and for each frame during plug flow. The updated Lagrangian Green strain and Displacement Gradient tensors were numerically fit to the Navier-Lame equations along the parameterized boundary to determined Lame's constants. Mechanical properties and wall shear stress were determined before and transfusion, were grouped in three populations of erythrocytes: native cells (NC) or circulating cells before transfusion, and two distinct population of cells after transfusion with stored cells (SC1 and SC2). The distinction, between the heterogeneous populations of cells present after the transfusion, SC1 and SC2, was obtained through principle component analysis (PCA) of the mechanical properties along the membrane. Cells with the first two principle components within 3 standard deviations of the mean, were labeled as SC1, and those with the first two principle components greater than 3 standard deviations from the mean were labeled as SC2. The calculated shear modulus average was 1.1±0.2, 0.90±0.15, and 12 ± 8 MPa for NC, SC1, and SC2, respectively. The calculated young's modulus average was 3.3±0.6, 2.6±0.4, and 32±20 MPa for NC, SC1, and SC2, respectively. o our knowledge, the methods presented here are the first estimation of the erythrocyte mechanical properties and shear stress in vivo during capillary plug flow. In summary, the methods introduced in this study may provide a new avenue of investigation of erythrocyte mechanics in the context of hematologic conditions that adversely affect erythrocyte mechanical properties

Telomere Length Shows No Association with BRCA1 and BRCA2 Mutation Status

This study aimed to determine whether telomere length (TL) is a marker of cancer risk or genetic status amongst two cohorts of BRCA1 and BRCA2 mutation carriers and controls. The first group was a prospective set of 665 male BRCA1/2 mutation carriers and controls (mean age 53 years), all healthy at time of enrolment and blood donation, 21 of whom have developed prostate cancer whilst on study. The second group consisted of 283 female BRCA1/2 mutation carriers and controls (mean age 48 years), half of whom had been diagnosed with breast cancer prior to enrolment. TL was quantified by qPCR from DNA extracted from peripheral blood lymphocytes. Weighted and unweighted Cox regressions and linear regression analyses were used to assess whether TL was associated with BRCA1/2 mutation status or cancer risk. We found no evidence for association between developing cancer or being a BRCA1 or BRCA2 mutation carrier and telomere length. It is the first study investigating TL in a cohort of genetically predisposed males and although TL and BRCA status was previously studied in females our results don't support the previous finding of association between hereditary breast cancer and shorter TL

Epigallocatechin-3-gallate, a prototypic chemopreventative agent for protection against cisplatin-based ototoxicity

AbstractCisplatin-induced ototoxicity is one of the major factors limiting cisplatin chemotherapy. Ototoxicity results from damage to outer hair cells (OHCs) and other regions of the cochlea. At the cellular level, cisplatin increases reactive oxygen species (ROS) leading to cochlear inflammation and apoptosis. Thus, ideal otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatin's chemotherapeutic efficacy. In this study, we show that epigallocatechin-3-gallate (EGCG) is a prototypic agent exhibiting these properties of an effect otoprotective agent. Rats administered oral EGCG demonstrate reduced cisplatin-induced hearing loss, reduced loss of OHCs in the basal region of the cochlea and reduced oxidative stress and apoptotic markers. EGCG also protected against the loss of ribbon synapses associated with inner hair cells and Na+/K+ ATPase α1 in the stria vascularis and spiral ligament. In vitro studies showed that EGCG reduced cisplatin-induced ROS generation and ERK1/2 and signal transducer and activator of transcription-1 (STAT1) activity, but preserved the activity of STAT3 and Bcl-xL. The increase in STAT3/STAT1 ratio appears critical for mediating its otoprotection. EGCG did not alter cisplatin-induced apoptosis of human-derived cancer cells or cisplatin antitumor efficacy in a xenograft tumor model in mice because of its inability to rescue the downregulation of STAT3 in these cells. These data suggest that EGCG is an ideal otoprotective agent for treating cisplatin-induced hearing loss without compromising its antitumor efficacy.</jats:p

Nucleic Acids — The Use of Nucleic Acid Testing in Molecular Diagnostics

In 1989 Roche entered into an agreement with Cetus to develop diagnostic applications for the novel technique polymerase chain reaction (PCR). A new area of molecular diagnostics began and genes and pathogen genomes have been used to diagnose disease since that point. Automated laboratory platforms were created to facilitate the workflow and allow for accurate and precise processing of patient blood samples in a highly streamlined manner. In this chapter the use of nucleic acids in molecular diagnostics will be described and their application to important human diseases. Examples are discussed with respect to which nucleic acid marker has provided strong clinical utility and impact to healthcare

An Overview of Prognosis Health Management Research at GRC for Gas Turbine Engine Structures With Special Emphasis on Deformation and Damage Modeling

Herein a general, multimechanism, physics-based viscoelastoplastic model is presented in the context of an integrated diagnosis and prognosis methodology which is proposed for structural health monitoring, with particular applicability to gas turbine engine structures. In this methodology, diagnostics and prognostics will be linked through state awareness variable(s). Key technologies which comprise the proposed integrated approach include 1) diagnostic/detection methodology, 2) prognosis/lifing methodology, 3) diagnostic/prognosis linkage, 4) experimental validation and 5) material data information management system. A specific prognosis lifing methodology, experimental characterization and validation and data information management are the focal point of current activities being pursued within this integrated approach. The prognostic lifing methodology is based on an advanced multi-mechanism viscoelastoplastic model which accounts for both stiffness and/or strength reduction damage variables. Methods to characterize both the reversible and irreversible portions of the model are discussed. Once the multiscale model is validated the intent is to link it to appropriate diagnostic methods to provide a full-featured structural health monitoring system

A homozygous genome‐edited Sept2‐EGFP fibroblast cell line

Septins are a conserved, essential family of GTPases that interact with actin, microtubules, and membranes and form scaffolds and diffusion barriers in cells. Several of the 13 known mammalian septins assemble into nonpolar, multimeric complexes that can further polymerize into filamentous structures. While some GFP‐coupled septins have been described, overexpression of GFP‐tagged septins often leads to artifacts in localization and function. To overcome this ubiquitous problem, we have here generated a genome‐edited rat fibroblast cell line expressing Septin 2 (Sept2) coupled to enhanced green fluorescent protein (EGFP) from both chromosomal loci. We characterize these cells by genomic polymerase chain reaction (PCR) for genomic integration, by western blot and reverse transcriptase‐PCR for expression, by immunofluorescence and immunoprecipitation for the colocalization of septins with one another and cellular structures and for complex formation of different septins. By live cell imaging, proliferation and migration assays we investigate proper function of septins in these cells. We find that EGFP is incorporated into both chromosomal loci and only EGFP‐coupled Sept2 is expressed in homozygous cells. We find that endogenous Sept2‐EGFP exhibits expression levels, localization and incorporation into cellular septin complexes similar to the wt in these cells. The expression level of other septins is not perturbed and cell division and cell migration proceed normally. We expect our cell line to be a useful tool for the cell biology of septins, especially for quantitative biology

A novel human pain insensitivity disorder caused by a point mutation in ZFHX2

Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-Type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs. awx326media1 5680039660001 The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.We thank the Medical Research Council (J.J.C., Career Development Award, G1100340), Wellcome Trust (200183/ Z/15/Z and 101054/Z/13/Z) and Arthritis Research UK (20200) for generous support and Shionogi for an academic research grant (165302). Thanks to the University of Siena for partially funding this research. J.T.B. is supported by a Research Fellowship from the Alzheimer�s Society. J.D.R. received funding from the Wellcome Trust through the London Pain Consortium and from Colciencias through a Francisco Jose de Caldas Scholarship (LASPAU, Harvard University). D.L.H.B. is a Wellcome senior clinical scientist (ref. no. 095698z/11/z and 202747/Z/16/Z) and member of the Wellcome Pain Consortium.Scopu

A study of the expression and cellular function of the human FAM111B gene

POIKTMP, a multi-systemic fibrosing disease, results from mutations in the human FAM111B gene. Studies have also suggested high expression of this gene in cancers. Despite rising interest in the pathological effects of FAM111B mutations and overexpression of FAM111B, knowledge of the physiological role of this gene remains limited. Therefore, this study sought out to provide insights into the cellular function of FAM111B and to investigate the pathological effect of the FAM111B Y621D mutation. First, bioinformatics studies coupled with quantitative PCR and Western blots analysis were employed to assess FAM111B gene and protein expression in cancerous and non-cancerous cell lines. Subsequently, FAM111B gene expression was downregulated and upregulated in the human fibrosarcoma (HT1080) cell line by RNA-interference mediated gene silencing and recombinant gene expression technologies. The effect of these FAM111B dysregulations was studied using cellbased functional assays: proliferation, apoptosis, migration, and invasion assays. Furthermore, the functional pathways and interacting proteins of the FAM111B protein was determined using mass spectroscopy proteomics. Finally, preliminary studies in a POIKTMP patient-derived fibroblasts were attempted to recapitulate the results obtained using the HT1080 cell line. The results from this study indicated that FAM111B gene and protein overexpression occurs in cancer cells. Second, the depletion of FAM111B suggests a decelerated rate of cell proliferation and migration (14%), and increased apoptosis (1.4-fold). Conversely, overexpression of FAM111B resulted in a marked reduction in apoptosis (3-fold) and increased cell migration by 27 %, howbeit, no evidence of increased proliferation. Furthermore, Y621D FAM111B mutant cells showed reduced expression of FAM111B, decreased apoptosis (1.1-fold), cellular invasion (24%), and indicates an increase in cell proliferation and migration (18 %). The proteomics data suggested wild-type FAM111B interacts with HSP7C, a molecular chaperone, which alongside BAG3 and BCL2 to minimise apoptosis. Similarly, Y621D's interaction with G3V3W4, a component of the 20S proteasome complex involved in the proteolytic degradation of damaged proteins, may suggest the rapid clearance of this mutant protein