Plasma temperature measurement with a silicon photomultiplier (SiPM).

The temperature of a nonneutral plasma confined in a Penning-Malmberg trap can be determined by slowly lowering one side of the trap's electrostatic axial confinement barrier; the temperature is inferred from the rate at which particles escape the trap as a function of the barrier height. In many experiments, the escaping particles are directed toward a microchannel plate, and the resulting amplified charge is collected on a phosphor screen. The screen is used for imaging the plasma but can also be used as a Faraday cup (FC) for a temperature measurement. The sensitivity limit is then set by microphonic noise enhanced by the screen's high-voltage bias. Alternately, a silicon photomultiplier (SiPM) can be employed to measure the charge via the light emitted from the phosphor screen. This decouples the signal from the microphonic noise and allows the temperature of colder and smaller plasmas to be measured than could be measured previously; this paper focuses on the advantages of a SiPM over a FC.

Discovery and development of next generation sGC stimulators with diverse multidimensional pharmacology and broad therapeutic potential.

Nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC), an enzyme that catalyzes the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine-3',5'-monophophate (cGMP), transduces many of the physiological effects of the gasotransmitter NO. Upon binding of NO to the prosthetic heme group of sGC, a conformational change occurs, resulting in enzymatic activation and increased production of cGMP. cGMP modulates several downstream cellular and physiological responses, including but not limited to vasodilation. Impairment of this signaling system and altered NO-cGMP homeostasis have been implicated in cardiovascular, pulmonary, renal, gastrointestinal, central nervous system, and hepatic pathologies. sGC stimulators, small molecule drugs that synergistically increase sGC enzyme activity with NO, have shown great potential to treat a variety of diseases via modulation of NO-sGC-cGMP signaling. Here, we give an overview of novel, orally available sGC stimulators that Ironwood Pharmaceuticals is developing. We outline the non-clinical and clinical studies, highlighting pharmacological and pharmacokinetic (PK) profiles, including pharmacodynamic (PD) effects, and efficacy in a variety of disease models.

A Case of Successful Thromboelastographic Guided Resuscitation after Postpartum Hemorrhage and Cardiac Arrest.

Amniotic fluid embolism (AFE) is an unusual cause of life threatening peri partum hemorrhage (PPH). AFE resuscitation is often associated with renal and respiratory insufficiency, and a coagulopathy similar to disseminated intravascular coagulation (DIC). Resuscitation requires immediate recognition and limited use of crystalloid. We present a case of PPH caused by AFE with resultant cardiac arrest, renal and respiratory failure, and DIC-like coagulopathy, whose successful resuscitation was guided by perfusionist-directed serial thromboelastography (TEG). Viscoelastic tests (VET)s, including the TEG and rotational thromboelastometry (ROTEM), may provide more individualized blood component therapy (BCT) in the treatment of severe PPH associated with AFE as has been previously noted with trauma resuscitation in the literature. However, VET's efficacy is often limited by a lack of standardization, quality assurance norms, and consistent operator proficiency. We suggest that there may be a role for perfusionsts adept at utilizing TEG in the optimization of BCT and adjunctive hemostatic agents in severely hemorrhagic patients. This patient's successful resuscitation demonstrates the importance of resuscitation guided by the perfusionist or other medical professionals with expertise in TEG guided resuscitation and how the administration of specific blood products and hemostatic agents guided by the TEG can help optimize patient outcomes in comparison to traditional 1:1:1 packed red blood cells (PRBC) /fresh frozen plasma (FFP) /platelets ratios given to severely hemorrhaging patients.

Tiered approaches to chromatographic bioanalytical method performance evaluation: recommendation for best practices and harmonization from the Global Bioanalysis Consortium harmonization team.

The A2 harmonization team, a part of the Global Bioanalysis Consortium (GBC), focused on defining possible tiers of chromatographic-based bioanalytical method performance. The need for developing bioanalytical methods suitable for the intended use is not a new proposal and is already referenced in regulatory guidance language. However, the practical implementation of approaches that differ from the well-established full validation requirements has proven challenging. Advances in technologies, the need to progress drug development more efficiently, and emerging new drug compound classes support the use of categorized tiers of bioanalytical methods. This paper incorporated the input from an international team of experienced bioanalysts to surmise the advantages and the challenges of tiered approaches and to provide recommendations on paths forward.

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice.

AIMS/HYPOTHESIS: Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signalling. Hepatic PTP1B deficiency, using the Alb-Cre promoter to drive Ptp1b deletion from birth in mice, improves glucose homeostasis, insulin sensitivity and lipid metabolism. The aim of this study was to investigate the therapeutic potential of decreasing liver PTP1B levels in obese and insulin-resistant adult mice. METHODS: Inducible Ptp1b liver-specific knockout mice were generated using SA-Cre-ER(T2) mice crossed with Ptp1b floxed (Ptp1b(fl/fl)) mice. Mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance. Tamoxifen was administered in the HFD to induce liver-specific deletion of Ptp1b (SA-Ptp1b(-/-) mice). Body weight, glucose homeostasis, lipid homeostasis, serum adipokines, insulin signalling and endoplasmic reticulum (ER) stress were examined. RESULTS: Despite no significant change in body weight relative to HFD-fed Ptp1b(fl/fl) control mice, HFD-fed SA-Ptp1b(-/-) mice exhibited a reversal of glucose intolerance as determined by improved glucose and pyruvate tolerance tests, decreased fed and fasting blood glucose and insulin levels, lower HOMA of insulin resistance, circulating leptin, serum and liver triacylglycerols, serum NEFA and decreased HFD-induced ER stress. This was associated with decreased glycogen synthase, eukaryotic translation initiation factor-2α kinase 3, eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase 2 phosphorylation, and decreased expression of Pepck. CONCLUSIONS/INTERPRETATION: Inducible liver-specific PTP1B knockdown reverses glucose intolerance and improves lipid homeostasis in HFD-fed obese and insulin-resistant adult mice. This suggests that knockdown of liver PTP1B in individuals who are already obese/insulin resistant may have relatively rapid, beneficial therapeutic effects.

Low energy prepulse for 10 Hz operation of a soft-x-ray laser.

The influence on Nickel-like Molybdenum soft-x-ray laser performance and stability of a low energy laser prepulse arriving prior to the main laser pumping pulses is experimentally investigated. A promising regime for 10 Hz operation has been observed. A four times increase in soft-x-ray laser operation time with a same target surface is demonstrated. This soft-x-ray laser operation mode corresponds to an optimum delay between the prepulse and the main pulses and to a prepulse energy greater than 20 mJ. We also show that this regime is not associated with a weaker degradation of the target or any reduced ablation rate. Therefore the role of preplasma density gradient in this effect is discussed.

Evidence for parent-of-origin effects on genetic variability of beef traits.

Imprinted genes are involved in many aspects of development in mammals, plants, and perhaps birds and may play a role in growth and carcass composition of slaughter animals. In the presence of genomic imprinting the expression and, consequently, the effect on the phenotype of maternal and paternal alleles are different. For genetic evaluation genomic imprinting can be accounted for by incorporating 2 additive genetic effects per animal; the first corresponds to a paternal and the second to a maternal expression pattern of imprinted genes. This model holds whatever the mode of imprinting may be: paternal or maternal, full or partial, or any combination thereof. A set of slaughter data from 65,233 German Simmental fattening bulls was analyzed with respect to the relative importance of the genetic imprinting variance. Besides slaughter weight, net daily BW gain, and killing out percentage, there were 22 other traits describing the carcass composition. The latter traits were evaluated by automatic video-imaging devices and were composed of weights of valuable cuts as well as fat and meatiness grade. The number of ancestors in the pedigree was 356,880. Genomic imprinting significantly contributed to the genetic variance of 10 traits, with estimated proportions between 8 and 25% of the total additive genetic variance. For 6 of these traits, the maternal contribution to the imprinting variance was larger than the paternal, whereas for all other traits the reverse was true. Fat grade only showed a paternal contribution to the imprinting variance. Estimates of animal model heritabilities of automatic video-imaging-recorded carcass traits ranged between 20 and 30%.

Expression of an Nkx3.1-CRE gene using ROSA26 reporter mice.

The genetic locus of Nkx3.1, an early murine marker of sclerotome and prostate development, was disrupted by a knock in of CRE recombinase via homologous recombination in embryonic stem cells. Cell fate mapping revealed previously unidentified cell lineages expanded from Nkx3.1-expressing cell populations and recapitulated reported Nkx3.1 expression patterns. In lineage trace experiments of E18.5 Nkx3.1-CRE; R26R embryos novel staining was observed in areas of the lungs, portions of the duodenum, and vertebral elements of the skeleton. beta-galactosidase activity measured in Nkx3.1-CRE; R26R and Nkx3.2-CRE; R26R embryos was observed in overlapping regions of the sclerotome but no apparent change in Nkx3.1 expression was seen in the Nkx3.2 mutants by in situ hybridization.

S100-mediated signal transduction in the nervous system and neurological diseases.

This article presents new information regarding the complement/level of S100 family members expressed in the brain and reviews the contribution of brain S100 family members to nervous system function and disease. A total of ten S100 family members are reported in the literature to be expressed in brain -S100A1, S100A2, S100A4, S100A5, S100A6, S100A10, S100A11, S100A13, S100B, and S100Z. Quantitative Northern blot analysis detected no S100A3, S100A8, S100A9 or S100A14 mRNA in mouse brain suggesting that these family members are not expressed in the brain. In addition, there was a 100-fold range in the mRNA levels for the six family members that were detected in mouse brain: S100A1/S100B levels were 5-fold higher than S100A6/S100A10 levels and 100-fold higher than S100A4/S100A13 levels. Five of these six family members (S1100A1, S100A6, S100A10, S100A13, and S100B) exhibited age-dependent increases in expression in adult mice that ranged from 5- to 20-fold. Although previous studies on S100 function in the nervous system have focused on S100B, other family members (S100A1, S100A3, S100A4, S100A5) have been implicated in neurological diseases. Like S100B, intra- and inter-cellular forms of these family members have been linked to cell growth, cell differentiation, and apoptotic pathways. Studies presented here demonstrate that ablation of S100A1 expression in PC12 cells results in increased resistance to Abeta peptide induced cell death, stabilization of intracellular [Ca2+] homeostasis, and reduced amyloid precursor protein expression. Altogether, these results confirm that S100-mediated signal transduction pathways play an important role in nervous system function/disease and implicate S100A1 in the neuronal cell dysfunction/death that occurs in Alzheimer's disease.

Inhibition of p53 transcriptional activity by the S100B calcium-binding protein.

The levels of S100 Ca(2+)-binding proteins correlate with the progression of certain tumors, but their role, if any, in carcinogenesis is still poorly understood. S100B protein associates with both the p53 oligomerization domain (residues 325-355) and the extreme C terminus of the tumor suppressor p53 (residues 367-392). Consequently, S100B inhibits p53 tetramer formation and p53 phosphorylation mediated by protein kinase C, on p53 C-terminal end. In this report, we show that the S100B protein decreases p53 DNA binding and transcriptional activity. The effect of S100B is reflected in vivo by a reduced accumulation of p53, p21, and MDM2 protein levels in co-transfection assays and in response to bleomycin. The S100B can still interact with p53 in the absence of p53 extreme C-terminal end and reduce the expression of p53 downstream effector genes. These data indicate that S100B does not require p53 extreme C-terminal end to inhibit p53 activity. Collectively, these findings imply that elevated levels of S100B in tumors such as astrocytomas and gliomas could inhibit p53 functions and contribute to cancer progression.

Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays.

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.

Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.

Determination of metrifonate enantiomers in blood and brain samples using liquid chromatography on a chiral stationary phase coupled to tandem mass spectrometry.

A novel enantioselective assay is described for the simultaneous determination of the metrifonate enantiomers BAY z 7216 and BAY z 7217 in extracts of whole blood samples obtained from rats, mice, rabbits and Beagle dogs as well as in rat brain tissue using liquid chromatography tandem mass spectrometry (LC/MS/MS) with thermally and pneumatically assisted electrospray ionization (TurboIonSpray(R)). Chromatographic separation is achieved on a chiral normal phase column with a mobile phase containing 0.25% water only. The total run time per sample is 11.0 min giving chromatographic base line separation of the enantiomers. Compared with previous methods this assay offers a higher sample throughput, excellent ruggedness and higher sensitivity. The limits of quantification for each enantiomer are 5.00 microg/L from 0.5 mL whole blood and 7.50 ng/g (ppb) using 0.333 g brain tissue, respectively. Similar assay specifications have been derived for the two enantiomers. The method has been validated for the analysis of blood samples from low and high dosed preclinical pharmacokinetic and toxicokinetic studies, corresponding to two analytical working ranges like e.g. 5.00 to 1000 microg/L and 200 to 40000 microg/L (0. 200 to 40.0 mg/L). For rat brain tissue the validated concentration range is 7.50 to 750 ng/g (ppb).

Development, validation and application of assays to quantify metrifonate and 2,2-dichlorovinyl dimethylphosphate in human body fluids.

Gas chromatographic procedures [GC with electron-capture detection (ECD) and GC-MS] for the quantitative analysis of metrifonate and its active metabolite 2,2-dichlorovinyl dimethylphosphate (DDVP) in human blood and urine were developed, validated, and applied to the analysis of clinical study samples. Analysis of metrifonate involved extraction of acidified blood with ethyl acetate followed by solid-phase clean-up of the organic extract. Acidified urine was extracted with dichloromethane and the residue of evaporated organic phase was reconstituted in toluene. ECD and diethyl analogue of metrifonate internal standard (I.S.) were used for quantitation of metrifonate. The metrifonate lower limit of quantitation (LOQ) was 10.0 microg/l. The DDVP metabolite was chromatographed separately after cyclohexane extraction of acidified blood and urine using d6-DDVP I.S. and MS detection. The LOQ of DDVP was 1 microg/l. Stability studies have confirmed that the matrix should be acidified prior to storage at -20 degrees C or -80 degrees C to inhibit chemical and enzymatic degradation of the analytes and to avoid overestimation of DDVP concentrations. Metrifonate was found to be stable in acidified human blood after 20 months of storage at -20 degrees C and after 23 months of storage at -80 degrees C. Under these conditions DDVP was found to be stable after 12 months of storage. Both assay procedures were cross-validated by different world-wide laboratories and found to be accurate and robust during analyses of clinical study samples.

Comparison of turbulent-flow chromatography with automated solid-phase extraction in 96-well plates and liquid-liquid extraction used as plasma sample preparation techniques for liquid chromatography-tandem mass spectrometry.

Turbulent flow chromatography (TFC) combined with the high selectivity and sensitivity of tandem mass spectrometry (MS-MS) is a new technique for the fast direct analysis of drugs from crude plasma. TFC in the 96-well plate format reduces significantly the time required for sample clean-up in the laboratory. For example, for 100 samples the workload for a technician is reduced from about 8 h by a manual liquid-liquid extraction (LLE) assay to about 1 h in the case of TFC. Sample clean-up and analysis are performed on-line on the same column. Similar chromatographic performance and validation results were achieved using HTLC Turbo-C18 columns (Cohesive Technologies) and Oasis HLB extraction columns (Waters). One 96-well plate with 96 plasma samples is analyzed within 5.25 h, corresponding to 3.3 min per sample. Compared to this LLE and analysis of 96 samples takes about 16 h. Two structurally different and highly protein bound compounds, drug A and drug B, were analyzed under identical TFC conditions and the assays were fully validated for the application to toxicokinetics studies (compliant with Good Laboratory Practices-GLP). The limit of quantitation was 1.00 microg/l and the linear working range covered three orders of magnitude for both drugs. In the case of drug A the quality of analysis by TFC was similar to the reference LLE assay and slightly better than automated solid-phase extraction in 96-well plates. The accuracy was -3.1 to 6.7% and the precision was 3.1 to 6.8% in the case of drug A determined for dog plasma by TFC-MS-MS. For drug B the accuracy was -3.7 to 3.5% and the precision was 1.6 to 5.4% for rat plasma, which is even slightly better than what was achieved with the validated protein precipitation assay.

Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice.

We compared the lambda cII/cI transgenic mutation assay described by Jakubczak et al. [(1996): Proc Natl Acad Sci USA 93:9073-9078] to the previously established Big Blue assay. Genomic DNA isolated from liver, spleen, and lung tissue of control or ethylnitrosourea (ENU)-treated Big Blue mice (100 mg/kg i.p., single dose) was packaged into phage (five animals, two packagings per DNA sample) which were simultaneously plated for lacI and cII/cI mutant frequency (MF) and titer. Mean MF of control animals was higher for cII/cI than lacI for all three tissues examined (spontaneous cII/cI MF divided by spontaneous lacI MF = 2.9, 3.1, and 1.7 for liver, spleen, and lung, respectively). The differences were statistically significant for liver and spleen, but not lung. The ENU-induced MF measured by subtracting control MFs from ENU-treated MFs was higher in the cII/cI assay than lacI (liver = 23.0 x 10(-5) for cII/cI vs. 15.1 x 10(-5) for lacI; spleen = 64.8 x 10(-5) for cII/cI vs. 36.1 x 10(-5) for lacI; lung = 17.1 x 10(-5) for cII/cI vs. 15.8 x 10(-5) for lacI). Fold increase over control values measured by dividing MF of ENU-treated animals by appropriate control values was higher for lacI than cII/cI (liver = 4.4-fold for lacI vs. 2.7 for cII/cI; spleen = 13.1-fold for lacI vs. 8.4 for cII/cI; and lung = 5.6-fold for lacI vs. 4.0 for cII/cI). Despite these differences, overall results were similar for the two mutational endpoints. These results suggest that the cII/cI assay may be an acceptable alternative to lacI where transgenic mutation studies are indicated.

Lack of response to multiple genotoxic agents at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys.

We tested the ability of a series of known genotoxic agents to cause mutations at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys as measured by the ability to form clones in the presence of 6-thioguanine. Ethylmethane sulfonate (EMS, 300 mg/kg i.p.), chloroethylmethane sulfonate (CI-EMS, 35 or 50 mg/kg i.p.), and the Pharmacia & Upjohn antitumor agents adozelesin (1.6, 4, 6, or 8 microg/kg i.v.) and CC-1065 (6 microg/kg i.v.) were all negative in the hprt mutation test. Results with cyclophosphamide (CP, 75 mg/kg i.v.) were equivocal. Adozelesin, CC-1065, and CI-EMS treatments increased the percentage of T-lymphocytes with chromosome aberrations, as well as inducing types of aberrations not seen in control cells. EMS and CP were not tested for chromosome aberrations. We have previously shown that treatment of monkeys with 77 mg/kg ENU substantially increased the hprt mutant frequency, with a lag time of approximately 77 days between treatment and peak MF values. The results of the present study suggest a low sensitivity of the hprt mutation assay to certain classes of genotoxic agents in cynomolgus monkeys.

Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice.

Big Blue mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor-intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073-9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C --> A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3' flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G --> A and G --> T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G --> A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations.

DNA sequence analysis of hprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment.

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T-cell mutant frequency (44.2 x 10(-6)) was still 26-fold higher than background (1.7 x 10(-6)), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR-amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C --> A:T and five A:T --> G:C transitions, one G:C --> C:G, eight A:T --> T:A, and six A:T --> C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU-induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence.