A case of refractory systemic lupus erythematosus with monocytosis exhibiting somatic KRAS mutation

BACKGROUND: Systemic lupus erythematosus (SLE), an autoimmune disorder that damages various organ systems, is caused by a combination of genetic and environmental factors. Although germline mutations of several genes are known to cause juvenile SLE, most of the susceptibility genetic variants of adult SLE are common variants of the population, somatic mutations that cause or exacerbate SLE have not been reported. We hereby report a refractory SLE case with monocytosis accompanying somatic KRAS mutation that have been shown to cause lupus-like symptoms. CASE PRESENTATION: A 60-year-old female patient who had been diagnosed with SLE was admitted to our hospital. Although prednisolone and tacrolimus treatments had kept her thrombocytopenia and anti-DNA Ab level at bay for more than 4 years, a diagnosis of transverse myelitis was made when she became acutely ill with pleocytosis. Elevated cells (predominately monocytes), protein, IgG, and IL-6 levels were also found in the cerebrospinal fluid (CSF) of the patient. Standard pulse treatments of methylprednisolone, high-dose of prednisolone, and intravenous cyclophosphamide in combination with plasma exchange could not alleviate the refractory neural and autoimmune manifestation. Monocytosis of peripheral blood was also noted. Flow cytometric analysis revealed elevated ratio of CD14+CD16+ atypical monocytes, which excluded the possibility of chronic myelomonocytic leukemia. Lupus-like symptoms with monocytosis reminded us of Ras-associated autoimmune leukoproliferative disorder, and Sanger sequencing of KRAS and NRAS genes from the patients' peripheral blood mononuclear cells (PBMC), sorted CD3+ lymphocytes and CD14+ monocytes, and cerebrospinal fluid were performed. An activating KRAS somatic mutation was found in the patients' DNA at the time of encephalomyelitis diagnosis. CONCLUSION: Somatic mutations of some genes including KRAS may cause the refractoriness of SLE

A physiologically-based pharmacokinetic model of oseltamivir phosphate and its carboxylate metabolite for rats and humans

Oseltamivir phosphate (OP, Tamiflu®) is a widely used prodrug for the treatment of influenza viral infections. Orally administered OP is rapidly hydrolyzed by the carboxylesterases in animals to oseltamivir carboxylate (OC), a potent influenza virus neuraminidase inhibitor. The goals of this study were to develop and validate a physiologically-based pharmacokinetic (PBPK) model of OP/OC in rats and humans, and to predict the internal tissue doses for OP and OC in humans after receiving OP orally. To this end, a PBPK model of OP/OC was first developed in the rat, which was then scaled up to humans by replacing the physiological and biochemical parameters with human-specific values. The proposed PBPK model consisted of an OP and an OC sub-models each containing nine first-order, flow-limited tissue/organ compartments. OP metabolism to OC was assumed to carry out mainly by hepatic carboxylesterases although extra-hepatic metabolism also occurred especially in the plasma. The PBPK model was developed and validated by experimental data from our laboratories and from the literature. The proposed PBPK model accurately predicted the pharmacokinetic behavior of OP and OC in humans and rats after receiving a single or multiple doses of OP orally or an OC dose i.v. The PBPK model was used to predict the internal tissue doses of OP and OC in a hypothetical human after receiving the recommended dose of 75 mg/kg OP b.i.d. for 6 days. Steady-state OC concentrations in the plasma and major organs such as the lung and the brain were higher than the minimum in vitro IC50 reported for H1N1 influenza virus neuraminidase, confirming OP is an effective, anti-viral agent. OP side-effects in the gastrointestinal tract and brain of humans were explainable by the tissue doses found in these organs. The PBPK model provides a quantitative tool to evaluate the relationship between an externally applied dose of OP and the internal tissue doses in humans. As such the model can be used to adjust the dose regimens for adult patients in disease states e.g., renal failure and liver damage

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

RETINOIC ACID RECEPTOR ALPHA IN GERM CELLS IN IMPORTANT FOR MITOSIS OF SPERMATOGONIA, SPERMATOGONIAL DIFFERENTIATION AND MEIOSIS

Spermatogenesis is governed by vitamin A, as shown by vitamin A deficient (VAD) testes, which lack advanced germ cells. Vitamin A signaling is mediated by retinoid receptors. There are two families of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with alpha, beta and gamma subtypes. Retinoic acid receptor alpha (RARA), plays a significant role in the testis such that Rara-null males are infertile because of severe germ cell loss.Striking similarities of the testicular phenotypes are detected between Rara-null and VAD mice: severely degenerated testes, lack of germ cells, sloughing of mature spermatids, and infertility. To discern the molecular function of RARA in germ cells, Rara was conditionally deleted using stimulated by retinoic acid 8 (STRA8)-iCRE. With RARA function disabled in germ cells, morphological abnormalities detected in the testes included lack of germ cell organization, lack of lumen, sloughing cells, and vacuolization. Not surprisingly, germ-cell specific Rara conditional knockout mice (cKO) had a dramatic reduction in epididymal sperm number. Further analysis of cKO testes demonstrated decreased spermatogonial proliferation and differentiation, while meiotic defects such as reduced synapsis, synaptonemal fragmentation, and unrepaired double strand breaks were increased. Furthermore, functional spermatogonial transplantation assays pointed to the possibility that RARA regulates spermatogonial stem cell colonization and proliferation, as shown by the reduction of donor-derived spermatogenesis from the cKO donor germ cells. The lack of RARA in the testes clearly shows quantifiable deficiencies during spermatogonial proliferation, differentiation, and meiosis.Microarray gene expression studies of mRNAs from the enriched germ cells from wild type and cKO mice provided molecular evidence that RARA regulates spermatogonial differentiation at postnatal day 4 (P4) and meiosis at P8. Cell differentiation, cell adhesion, cell migration, and other pathways related to the early steps of spermatogonial differentiation were found to be functional categories significant in germ cells from P4. These were very distinct from synapsis, synaptonemal complex formation, and crossover formation related to meiosis, which were functional categories significant in germ cells from P8. In conjunction with phenotypic abnormalities, we provide gene expression evidence that RARA mediates retinoic acid function during spermatogonial proliferation, differentiation, and meiosis

RETINOIC ACID RECEPTOR ALPHA IN GERM CELLS IN IMPORTANT FOR MITOSIS OF SPERMATOGONIA, SPERMATOGONIAL DIFFERENTIATION AND MEIOSIS

Thesis (Ph.D.), School of Molecular Biosciences, Washington State UniversitySpermatogenesis is governed by vitamin A, as shown by vitamin A deficient (VAD) testes, which lack advanced germ cells. Vitamin A signaling is mediated by retinoid receptors. There are two families of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with alpha, beta and gamma subtypes. Retinoic acid receptor alpha (RARA), plays a significant role in the testis such that Rara-null males are infertile because of severe germ cell loss. Striking similarities of the testicular phenotypes are detected between Rara-null and VAD mice: severely degenerated testes, lack of germ cells, sloughing of mature spermatids, and infertility. To discern the molecular function of RARA in germ cells, Rara was conditionally deleted using stimulated by retinoic acid 8 (STRA8)-iCRE. With RARA function disabled in germ cells, morphological abnormalities detected in the testes included lack of germ cell organization, lack of lumen, sloughing cells, and vacuolization. Not surprisingly, germ-cell specific Rara conditional knockout mice (cKO) had a dramatic reduction in epididymal sperm number. Further analysis of cKO testes demonstrated decreased spermatogonial proliferation and differentiation, while meiotic defects such as reduced synapsis, synaptonemal fragmentation, and unrepaired double strand breaks were increased. Furthermore, functional spermatogonial transplantation assays pointed to the possibility that RARA regulates spermatogonial stem cell colonization and proliferation, as shown by the reduction of donor-derived spermatogenesis from the cKO donor germ cells. The lack of RARA in the testes clearly shows quantifiable deficiencies during spermatogonial proliferation, differentiation, and meiosis. Microarray gene expression studies of mRNAs from the enriched germ cells from wild type and cKO mice provided molecular evidence that RARA regulates spermatogonial differentiation at postnatal day 4 (P4) and meiosis at P8. Cell differentiation, cell adhesion, cell migration, and other pathways related to the early steps of spermatogonial differentiation were found to be functional categories significant in germ cells from P4. These were very distinct from synapsis, synaptonemal complex formation, and crossover formation related to meiosis, which were functional categories significant in germ cells from P8. In conjunction with phenotypic abnormalities, we provide gene expression evidence that RARA mediates retinoic acid function during spermatogonial proliferation, differentiation, and meiosis.School of Molecular Biosciences, Washington State Universit

Younger Adults Are More Likely to Increase Fruit and Vegetable Consumption and Decrease Sugar Intake with the Application of Dietary Monitoring

Establishing healthy eating habits is considered to be a sustainable strategy for health maintenance, and mobile applications (apps) are expected to be highly effective among the young-aged population for healthy eating promotion. The purpose of this study was to investigate the effectiveness of a dietary monitoring app on younger adults’ nutrition knowledge and their dietary habits. A controlled-experimental study was performed with one experimental group having a three-hour nutrition seminar and 12 weeks of dietary monitoring with the app, and one control group receiving a three-hour nutrition seminar. Behavioral feedback delivered by the app was evaluated in facilitating the transfer of nutritional knowledge to nutrition behavior. A total of 305 younger adults aged from 19 to 31 were recruited. Baseline and post-intervention nutrition knowledge and dietary behavior were collected. All mean scores of post-GNKQ-R increased from baseline for both the control and the experimental groups. The mean differences of sugar intake, dietary fiber intake, and vitamin C intake for the experimental group were significantly more than those for the control group (all p p < 0.001). For those younger adults with a relatively large body size, they were more likely to increase fruit consumption with the application of dietary monitoring