Neuronal activity regulates Matrin 3 abundance and function in a calcium-dependent manner through calpain-mediated cleavage and calmodulin binding.

RNA-binding protein (RBP) dysfunction is a fundamental hallmark of amyotrophic lateral sclerosis (ALS) and related neuromuscular disorders. Abnormal neuronal excitability is also a conserved feature in ALS patients and disease models, yet little is known about how activity-dependent processes regulate RBP levels and functions. Mutations in the gene encoding the RBP Matrin 3 (MATR3) cause familial disease, and MATR3 pathology has also been observed in sporadic ALS, suggesting a key role for MATR3 in disease pathogenesis. Here, we show that glutamatergic activity drives MATR3 degradation through an NMDA receptor-, Ca2+-, and calpain-dependent mechanism. The most common pathogenic MATR3 mutation renders it resistant to calpain degradation, suggesting a link between activity-dependent MATR3 regulation and disease. We also demonstrate that Ca2+ regulates MATR3 through a nondegradative process involving the binding of Ca2+/calmodulin to MATR3 and inhibition of its RNA-binding ability. These findings indicate that neuronal activity impacts both the abundance and function of MATR3, underscoring the effect of activity on RBPs and providing a foundation for further study of Ca2+-coupled regulation of RBPs implicated in ALS and related neurological diseases.

Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks.

The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis.

Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the gastrointestinal tract. The cytokine TNF-alpha (TNF-α) plays a pivotal role in mediating this inflammatory response. RNA interference (RNAi) holds great promise for the specific and selective silencing of aberrantly expressed genes, such as TNF-α in IBD. The aim of this study was to investigate the efficacy of an amphiphilic cationic cyclodextrin (CD) vector for effective TNF-α siRNA delivery to macrophage cells and to mice with induced acute-colitis. The stability of CD.siRNA was examined by gel electrophoresis in biorelevant media reflecting colonic fluids. RAW264.7 cells were transfected with CD.TNF-α siRNA, stimulated with lipopolysaccharide (LPS) and TNF-α and IL-6 responses were measured by PCR and ELISA. Female C57BL/6 mice were exposed to dextran sodium sulphate (DSS) and treated by intrarectal administration with either CD.siRNA TNF-α or a control solution. In vitro, siRNA in CD nanocomplexes remained intact and stable in both fed and fasted simulated colonic fluids. RAW264.7 cells transfected with CD.TNF-α siRNA and stimulated with LPS displayed a significant reduction in both gene and protein levels of TNF-α and IL-6. CD.TNF-α siRNA-treated mice revealed a mild amelioration in clinical signs of colitis, but significant reductions in total colon weight and colonic mRNA expression of TNF-α and IL-6 compared to DSS-control mice were detected. This data indicates the clinical potential of a local CD-based TNF-α siRNA delivery system for the treatment of IBD.

Natural killer cells protect mice from DSS-induced colitis by regulating neutrophil function via the NKG2A receptor.

Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and infection defense but their role in chronic inflammatory disorders such as inflammatory bowel disease is less clear. Here, we investigated the role of NK cells in dextran sodium sulfate (DSS)-induced colitis in mice. Depletion of NK cells impairs the survival of mice with colitis and is linked with dramatic increases in colonic damage, leukocyte infiltration, and pro-inflammatory profiles. Mice depleted of NK cells had increased numbers of neutrophils in colons and mesenteric lymph nodes, compared with control mice, in addition to acquiring a hyper-activation status. In vitro and in vivo studies demonstrate that NK cells downregulate pro-inflammatory functions of activated neutrophils, including reactive oxygen species and cytokine production, by direct cell-to-cell contact involving the NK cell-inhibitory receptor NKG2A. Our results indicate an immunoregulatory mechanism of action of NK cells attenuating DSS-induced colitis neutrophil-mediated inflammation and tissue injury via NKG2A-dependent mechanisms.

Evidence for two independent associations with type 1 diabetes at the 12q13 locus.

Genome-wide association studies have identified associations between type 1 diabetes and single-nucleotide polymorphisms (SNPs) at chromosome 12q13, surrounding the gene ERBB3. Our objective was to fine map this region to further localize causative variants. Re-sequencing identified more than 100 putative SNPs in an 80-kb region at 12q13. By genotyping 42 SNPs, spanning ∼214 kb, in 382 affected sibling pair type 1 diabetes families, we were able to genotype or tag 67 common SNPs (MAF≥0.05) identified from HapMap CEU data and CEU data from the 1000 Genomes Project, plus additional rare coding variants identified from our re-sequencing efforts. In all, 15 SNPs provided nominal evidence for association (P≤0.05), with type 1 diabetes. The most significant associations were observed with rs2271189 (P=4.22 × 10(-5)), located in exon 27 of the ERBB3 gene, and an intergenic SNP rs11171747 (P=1.70 × 10(-4)). Follow-up genotyping of these SNPs in 2740 multiplex type 1 diabetes families validated these findings. After analyzing variants spanning more than 200 kb, we have replicated associations from previous GWAS and provide evidence for novel associations with type 1 diabetes. The associations across this region could be entirely accounted for by two common SNPs, rs2271189 and rs11171747.

Use of bioluminescence imaging to track neutrophil migration and its inhibition in experimental colitis.

Inflammatory bowel disease (IBD) is associated with neutrophil infiltration into the mucosa and crypt abscesses. The chemokine interleukin (IL)-8 [murine homologues (KC) and macrophage inflammatory protein (MIP)-2] and its receptor CXCR2 are required for neutrophil recruitment; thus, blocking this engagement is a potential therapeutic strategy. In the present study, we developed a preclinical model of neutrophil migration suitable for investigating the biology of and testing new drugs that target neutrophil trafficking. Peritoneal exudate neutrophils from transgenic ß-actin-luciferase mice were isolated 12h after intraperitoneal injection with thioglycollate, and were assessed phenotypically and functionally. Exudate cells were injected intravenously into recipients with dextran sodium sulphate (DSS)-induced colitis followed by bioluminescence imaging of whole-body and ex vivo organs at 2, 4 and 16-22h post-transfer. Anti-KC antibody or an isotype control were administered at 20 µg/mouse 1h before transfer, followed by whole-body and organ imaging 4h post-transfer. The peritoneal exudate consisted of 80% neutrophils, 39% of which were CXCR2(+) . In vitro migration towards KC was inhibited by anti-KC. Ex vivo bioluminescent imaging showed that neutrophil trafficking into the colon of DSS recipients was inhibited by anti-KC 4h post-cell transfer. In conclusion, this study describes a new approach for investigating neutrophil trafficking that can be used in preclinical studies to evaluate potential inhibitors of neutrophil recruitment.

Role of the toxic neutrophil count in the early diagnosis of Kawasaki disease.

OBJECTIVE: To determine whether the toxic neutrophil count (TNC), defined as the sum of the number of peripheral blood neutrophils with vacuoles plus the number with toxic granulations per 100 neutrophils examined, can be used as an aid to early diagnosis of Kawasaki disease (KD). METHODS: Prospective evaluation at a tertiary care pediatric hospital of 56 acutely febrile children with at least one other clinical criterion for KD. Clinical characteristics and TNC were compared for 3 groups of patients: those with (1) definite KD, (2) probable KD, and (3) unlikely KD. The sensitivity and specificity of the TNC at various cutoff points was determined. RESULTS: We evaluated 56 patients (Group 1, N=27; Group 2, N=4; Group 3, N=25). Mean TNC (TNC/mm3) were higher in the patients with definite KD compared to patients with unlikely KD (38 vs 24; p=0.037). At a TNC cutoff of > or =70, the test had a specificity of 100%, but a sensitivity of only 18%. The likelihood ratio (the number of times more likely this TNC result is to be found in KD versus non-KD subjects) was 8.89. At a cutoff of > or =10, the test had a high sensitivity of 92% and specificity of 38%. CONCLUSION: No laboratory test replaced the need for careful clinical evaluation in cases of suspected KD. The TNC may be a useful adjunct to the clinical assessment of children with KD, particularly at the extremes of measurement.

Innovations in case flow analysis: using midlier variance analysis.

This article presents the use of midlier and case flow analysis during a retrospective chart review to study the costs of hospital length of stay. The results showed that considerable savings can be realized by using these methods for one group of patients with the same diagnosis. We were able to redefine and track patients during their pre-, peri-, and postoperative period and estimated the time and cost opportunities for work flow improvement. We concluded that considerable savings are possible by extending the analysis to patients in other DRG groups.

Influencing physician behavior with CQI: a case study.

Health care reform will require unprecedented levels of cooperation among physicians, health care administrators, and other providers in order to ensure high-quality, affordable care for all. At the University of Massachusetts Medical Center, CQI techniques helped engage physicians in an effort to substantially alter ordering patterns to cut costs and achieve quality goals.

Value of degenerative change in neutrophils as a diagnostic test for Kawasaki syndrome.

STUDY OBJECTIVE: To determine whether the presence of vacuoles and toxic granulation in neutrophils can be used as a diagnostic test to help differentiate children with Kawasaki syndrome from those with clinically similar illnesses. DESIGN: Peripheral blood smears of 23 patients with Kawasaki syndrome, 23 disease control patients, and 23 hematology laboratory control subjects were examined in random order by technicians unaware of either the diagnosis or the previously recorded laboratory results. SETTING: Tertiary care children's hospital in Ottawa, Canada. PATIENTS: All 23 consecutive patients with Kawasaki syndrome satisfied established criteria for the diagnosis. Disease control patients were selected from the hospital registry of patients with other illnesses frequently considered as part of the differential diagnosis for Kawasaki syndrome. MEASUREMENTS AND MAIN RESULTS: Compared with disease control patients, patients with Kawasaki syndrome had a higher percentage of neutrophils with vacuoles (mean +/- SEM, 31% +/- 5% vs 14% +/- 3%; p = 0.006) and toxic granulation (mean +/- SEM, 43% +/- 7% vs 14% +/- 4%; p less than 0.001). If the sum of the number of neutrophils with vacuoles and the number with toxic granulation (per 100 mature neutrophils examined) was at least 70, this "toxic neutrophil" test had a specificity of 0.96 and a likelihood ratio of a positive test result of 12. CONCLUSIONS: Degenerative change in neutrophils is common in the early stages of Kawasaki syndrome. The toxic neutrophil test is potentially a helpful adjunct to the clinical examination, particularly in the case of infants and other patients with subtle manifestations who might otherwise be at risk for delayed diagnosis.

Kinetics of xanthan production when NH3-N limits biomass synthesis and glucose limits polysaccharide synthesis.

The bacterium Xanthomonas campestris, which synthesizes the commercially important polysaccharide xanthan, was grown aseptically in 1.2 L fermenters using semicontinuous cell culture technique (d' = 0.0035 h-1). The effects of carbon-substrate concentration on xanthan production were investigated at three initial glucose concentrations (Go = 15, 20, 25 g/L). Cell biomass synthesis was nitrogen-limited by use of a chemically defined medium that contained NH3-N as the sole nitrogen source at a concentration where it was exhausted before glucose. A linear relationship between biomass synthesis and NH3-N depletion was observed. This relationship remained valid only until NH3-N exhaustion, after which biomass concentration slowly rose another 20 percent before declining. Another linear relationship was found between xanthan synthesis and glucose uptake. This relationship was unaffected by the disappearance of NH3-N and held through glucose exhaustion. The quasi-stoichiometric yield coefficients obtained for each linear relationship were not affected by G0-. Biomass synthesis kinetics showed no variation with G0 before NH3-N exhaustion; afterwards, cell biomass decline was delayed by increasing G0. Xanthan synthesis kinetics displayed no detectable response to depletion of NH3-N and plateauing of biomass concentration; however, there was a marked slow down in the net rate of xanthan synthesis and a drop in xanthan yield after cell biomass decline became noticeable.

A semicontinuous culture model that links cell growth to extracellular nutrient concentration.

During semicontinuous culture, a sample of fixed volume is removed at regular time intervals to make measurements and/or harvest culture components, and an equal volume of fresh medium is immediately added to the culture, thereby instantaneously enhancing nutrient concentrations and diluting cell concentration. The resulting cell concentration versus time curve (i.e., the actual cell growth curve) has a saw-toothed appearance because of the periodic dilution of cell concentration. The observed cell concentrations correspond to the peaks of the saw-toothed curve. Cell growth rates are estimated from the locus of observed cell concentrations (i.e., from the apparent growth curve obtained by connecting the peaks of the saw-toothed curve). The sole preexisting model (Fencl's mode) for estimating cell growth rate is valid only when the cells are growing exponentially at a constant rate between samplings. This model has limited validity: despite the periodic enhancement of nutrient concentration, cell growth between samplings eventually causes nutrient depletion, and the cells cease to grow exponentially. Failure to recognize the limits of validity for Fencl' model has resulted in many erroneous applications of the model and, consequently, many incorrect estimates of cell growth rates. To provide a means for correctly estimating cell growth rates, Fencl's exponential model was extended, and a new model that describes the effects of nutrient depletion on cell growth in semi-continuous culture was obtained. The new model shows that exhaustion of a single growth-limiting nutrient in semicontinuous culture causes the locus of cell concentrations observed at time intervals of Deltat to follow a logistic growth curve. The actual cell growth rate was shown to equal the apparent logistic growth rate plus the effective dilution rate -Deltat(-1) In (1 - f), where f is the ratio of sample volume to total culture volume. Moreover, the model predicts that both the apparent logistic growth rate and the apparent steady-state cell concentration should rise linearly with the concentration of growth-limiting nutrient in the input medium, but fall linearly with increases in the effective dilution rate. The new logistic model for nutrient-limited cell growth in semicontinuous culture was successfully tested using published data for Asterionella formosa, Cyclotella meneghiniana, Daucus carota, and strain L mouse cells.

Quality assurance programs cope with today's demands.

Pathologists face new demands resulting from malpractice litigation, health regulations, increased competition, and heightened consumer activism. Yet despite the concerns about quality and cost that beset the field, the pathologist's mission remains the same: the provision of quality care. Against the backdrop of current pressures, the authors describe the benefits and organization of a quality assurance program to help achieve this goal.

Risk management enhances quality care and protection from liability.

Two externally imposed economic pressures that grip the health care industry are on a collision course: tightening reimbursement and exploding costs of malpractice litigation and insurance. Physicians practice defensive medicine to cope with the latter, but regulatory agencies are no longer willing to pay for this habit. Pathologists must be aware of the laboratory's liability exposure, which is likely to increase in the future. Hospitals and physicians are turning to risk management programs to help limit potential losses and monitor performance.

Survival after high blood alcohol levels. Association with first-order elimination kinetics.

A patient survived acute alcohol intoxication with an unprecedented blood alcohol level of 1,500 mg/dL. Treatment included peritoneal dialysis and intravenously administered fructose. Alcohol elimination followed first-order kinetics, in which the elimination rate is proportional to concentration, rather than zero order in which the metabolic rate is independent of concentration. The report provides further evidence for first-order elimination kinetics with high blood alcohol levels and for an adaptive increase in alcohol metabolism with long-term alcohol consumption.