Analytical gradients and derivative couplings for dynamically weighted complete active space self-consistent field.

We recently introduced a Dynamically Weighted Complete Active Space Self-Consistent Field (DW-CASSCF) electronic structure for excited-state dynamics. In this Communication, we reformulate analytical gradients at this level of theory using a Lagrangian approach, thereby reducing the required number of coupled-perturbed CASSCF calculations to one per state gradient. In addition, we derive and implement derivative couplings at the DW-CASSCF level for the first time. We demonstrate the new formulation of DW-CASSCF gradients by optimizing a conical intersection for the p-hydroxybenzylidene-imidazolinone anion, the green fluorescent protein chromophore, to shed light on its observed radiationless decay dynamics in the ultraviolet region.

An outbreak of Escherichia coli O157:H7 infections following a dairy education school field trip in Washington state, 2015.

On 27 April 2015, Washington health authorities identified Escherichia coli O157:H7 infections associated with dairy education school field trips held in a barn 20-24 April. Investigation objectives were to determine the magnitude of the outbreak, identify the source of infection, prevent secondary illness transmission and develop recommendations to prevent future outbreaks. Case-finding, hypothesis generating interviews, environmental site visits and a case-control study were conducted. Parents and children were interviewed regarding event activities. Odds ratios (OR) and 95% confidence intervals (CI) were computed. Environmental testing was conducted in the barn; isolates were compared to patient isolates using pulsed-field gel electrophoresis (PFGE). Sixty people were ill, 11 (18%) were hospitalised and six (10%) developed haemolytic uremic syndrome. Ill people ranged in age from <1 year to 47 years (median: 7), and 20 (33%) were female. Twenty-seven case-patients and 88 controls were enrolled in the case-control study. Among first-grade students, handwashing (i.e. soap and water, or hand sanitiser) before lunch was protective (adjusted OR 0.13; 95% CI 0.02-0.88, P = 0.04). Barn samples yielded E. coli O157:H7 with PFGE patterns indistinguishable from patient isolates. This investigation provided epidemiological, laboratory and environmental evidence for a large outbreak of E. coli O157:H7 infections from exposure to a contaminated barn. The investigation highlights the often overlooked risk of infection through exposure to animal environments as well as the importance of handwashing for disease prevention. Increased education and encouragement of infection prevention measures, such as handwashing, can prevent illness.

Determination of ß-N-methylamino-L-alanine, N-(2-aminoethyl)glycine, and 2,4-diaminobutyric acid in Food Products Containing Cyanobacteria by Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry: Single-Laboratory Validation.

A single-laboratory validation study was completed for the determination of ß-N-methylamino-L-alanine (BMAA), N-(2-aminoethyl)glycine (AEG), and 2,4-diaminobutyric acid (DAB) in bulk natural health product supplements purchased from a health food store in Canada. BMAA and its isomers were extracted with acid hydrolysis to free analytes from protein association. Acid was removed with the residue evaporated to dryness and reconstituted with derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Fluor). Chromatographic separation and detection were achieved using RP ultra-performance LC coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode. Data from biological samples were evaluated for precision and accuracy across different days to ensure repeatability. Accuracy was assessed by spike recovery of biological samples using varying amino acid concentrations, with an average recovery across all samples of 108.6%. The analytical range was found to be 764-0.746 ng/mL prior to derivatization, thereby providing a linear range compatible with potentially widely varying analyte concentrations in commercial health food products. Both the U. S. Food and Drug Administration (FDA) and U. S. Pharmacopeia definitions were evaluated for determining method limits, with the FDA approach found to be most suitable having an LOD of 0.187 ng/mL and LLOQ of 0.746 ng/mL. BMAA in the collected specimens was detected at concentrations lower than 1 µg/g, while AEG and DAB were found at concentrations as high as 100 µg/g. Finding these analytes, even at low concentrations, has potential public health significance and suggests a need to screen such products prior to distribution. The method described provides a rapid, accurate, and precise method to facilitate that screening process.

The natural non-protein amino acid N-ß-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

N-ß-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

Communication: Smoothing out excited-state dynamics: analytical gradients for dynamically weighted complete active space self-consistent field.

State averaged complete active space self-consistent field (SA-CASSCF) is a workhorse for determining the excited-state electronic structure of molecules, particularly for states with multireference character; however, the method suffers from known issues that have prevented its wider adoption. One issue is the presence of discontinuities in potential energy surfaces when a state that is not included in the state averaging crosses with one that is. In this communication I introduce a new dynamical weight with spline (DWS) scheme that mimics SA-CASSCF while removing energy discontinuities due to unweighted state crossings. In addition, analytical gradients for DWS-CASSCF (and other dynamically weighted schemes) are derived for the first time, enabling energy-conserving excited-state ab initio molecular dynamics in instances where SA-CASSCF fails.

Reactivity of ß-methylamino-L-alanine in complex sample matrixes complicating detection and quantification by mass spectrometry.

ß-methylamino-l-alanine (BMAA) is a naturally occurring nonprotein amino acid originally discovered in cycad seeds and traditional foods of the Chamorro people of Guam. Recent research has implicated BMAA as a potential factor in neurodegenerative disease and described the production of BMAA in cyanobacteria, but conflicting results have complicated the interpretation of data. We hypothesized that the reactivity of BMAA with metal ions in the sample matrix and the formation of metal adducts in electrospray ionization mass spectrometry (MS) analysis confound results. Dilute solutions of TCA, MgCl(2), NaCl, CuCl(2), ZnCl(2) (0.01 M), or artificial ocean water (Instant Ocean, 3.5 g/L) reduced the signal attributable to the BMAA M + H(+) peak by 78-99.7%. The degree of adduct formation was significantly affected by MS settings such as induction voltage. A number of the detected ion peaks in BMAA standards were consistent with the formation of metal-BMAA complexes in addition to the adduct formation. A standard of Zn(BMAA)(2) was synthesized, and the effects of sample preparation, derivatization, column chromatography, pH, and interactions with serine were determined. Together, these data demonstrate that sample matrix, formation of adducts, and mass spectrometry settings complicate analysis of BMAA, that analysis by detection of the parent ion and daughter ion fragmentation patterns are highly susceptible to false negative findings, and that failure to detect BMAA cannot be considered proof of absence of the compound.

Diagnostic performance of PCA3 to detect prostate cancer in men with increased prostate specific antigen: a prospective study of 1,962 cases.

PURPOSE: The detection of prostate cancer relies primarily on abnormal digital rectal examination or increased serum prostate specific antigen concentration. However, low positive predictive values result in many men with increased prostate specific antigen and/or suspicious digital rectal examination having a negative biopsy. We investigated the value of the PCA3 (prostate cancer gene 3) urine test in predicting the likelihood of diagnosis of cancer before biopsy. MATERIALS AND METHODS: We performed a prospective, community based clinical trial to evaluate PCA3 score before any biopsy. This trial was conducted at 50 urology practices in the United States. Samples were obtained from 1,962 men with increased serum prostate specific antigen (greater than 2.5 ng/ml) and/or abnormal digital rectal examination before transrectal prostate needle biopsy. Study samples (urinary PCA3 and biopsies) were processed and analyzed by a central laboratory. Sensitivity-specificity analyses were conducted. RESULTS: A total of 1,913 urine samples (97.5%) were adequate for PCA3 testing. Of 802 cases diagnosed with prostate cancer 222 had high grade prostatic intraepithelial neoplasia or atypical small acinar proliferation and were suspicious for cancer, whereas 889 cases were benign. The traditional PCA3 cutoff of 35 reduced the number of false-positives from 1,089 to 249, a 77.1% reduction. However, false-negatives (missed cancers) increased significantly from 17 to 413, an increase of more than 2,300%. Lowering the PCA3 cutoff to 10 reduced the number of false-positives 35.4% and false-negatives only increased 5.6%. CONCLUSIONS: Urinary PCA3 testing in conjunction with prostate specific antigen has the potential to significantly decrease the number of unnecessary prostate biopsies.

Ultrafast internal conversion in ethylene. II. Mechanisms and pathways for quenching and hydrogen elimination.

Through a combined experimental and theoretical approach, we study the nonadiabatic dynamics of the prototypical ethylene (C(2)H(4)) molecule upon π → π(∗) excitation with 161 nm light. Using a novel experimental apparatus, we combine femtosecond pulses of vacuum ultraviolet and extreme ultraviolet (XUV) radiation with variable delay to perform time resolved photo-ion fragment spectroscopy. In this second part of a two part series, the XUV (17 eV < hν < 23 eV) probe pulses are sufficiently energetic to break the C-C bond in photoionization, or to photoionize the dissociation products of the vibrationally hot ground state. The experimental data is directly compared to excited state ab initio molecular dynamics simulations explicitly accounting for the probe step. Enhancements of the CH(2)(+) and CH(3)(+) photo-ion fragment yields, corresponding to molecules photoionized in ethylene (CH(2)CH(2)) and ethylidene (CH(3)CH) like geometries are observed within 100 fs after π → π(∗) excitation. Quantitative agreement between theory and experiment on the relative CH(2)(+) and CH(3)(+) yields provides experimental confirmation of the theoretical prediction of two distinct conical intersections and their branching ratio [H. Tao, B. G. Levine, and T. J. Martinez, J. Phys. Chem. A. 113, 13656 (2009)]. Evidence for fast, non-statistical, elimination of H(2) molecules and H atoms is observed in the time resolved H(2)(+) and H(+) signals.

"Virtual unwrapping" of a mummified hand.

The purpose of this work is to demonstrate the feasibility of medical virtual reality technologies in the investigation of a mummified hand. The Ulster Museum obtained the mummy hand, which originated from Thebes, without any identifying information. The mummified hand was investigated using conventional X-ray and 3D multi-slice Computed Tomography (CT). Imaging revealed a range of fractures of the wrist, metacarpals and phalanges whilst 3D CT demonstrated internal structures using volume rendering. The absence of any features of bone healing at the fracture sites would imply that they occurred just prior to death or in the mummified state possibly during excavation. Conventional X-ray imaging indicated that the hand, although small, was likely to have originated from an adult. Medical imaging and virtual reality display will enable us to produce a rapid prototyped model using fused deposition technology. Therefore, further paleopathological research can be performed on the replica without the need to handle the original specimen.

Cyanobacteria and cyanotoxins are present in drinking water impoundments and groundwater wells in desert environments.

Desert environments and drylands experience a drastic scarcity of water resources. To alleviate dependence on freshwater for drinking water needs, countries have invested in infrastructure development of desalination plants. Collectively, the countries of the Arabian Gulf produce 45% of the world's desalinated water, which is stored in dams, mega-reservoirs and secondary house water tanks to secure drinking water beyond daily needs. Improper storage practices of drinking water in impoundments concomitant with increased temperatures and light penetration may promote the growth of cyanobacteria and accumulation of cyanotoxins. To shed light on this previously unexplored research area in desert environments, we examined drinking and irrigation water of urban and rural environments to determine whether cyanobacteria and cyanotoxins are present, and what are the storage and transportation practices as well as the environmental parameters that best predict their presence. Cyanobacteria were present in 80% of the urban and 33% of the rural water impoundments. Neurotoxins BMAA, DAB and anatoxin-a(S) were not detected in any of the water samples, although they have been found to accumulate in the desert soils, which suggests a bioaccumulation potential if they are leached into the aquifer. A toxic BMAA isomer, AEG, was found in 91.7% of rural but none of the urban water samples and correlated with water-truck transportation, light exposure and chloride ions. The hepatotoxic cyanotoxin microcystin-LR was present in the majority of all sampled impoundments, surpassing the WHO provisional guideline of 1 µg/l in 30% of the urban water tanks. Finally, we discuss possible management strategies to improve storage and transportation practices in order to minimize exposure to cyanobacteria and cyanotoxins, and actions to promote sustainable use of limited water resources.

Effects of catecholamines on the coronary circulation in the Langendorff-type transplanted dog heart.

Intracoronary administration of isoprenaline, adrenaline, and the noradrenaline in the Langendorff-type transplanted dog heart transiently decreased coronary blood flow measured from the inflow vessel; flow then increased. The reduction in coronary flow coincided with increased myocardial contractility and was prevented by keeping the left ventricle empty. Propranolol prevented changes in flow and contractility. It is concluded that positive inotropic changes result in the ejection of accumulated Thesbesian flow from the ventricle and affect coronary flow measurements and that the interpretation of flow changes requires a steady state.

Liquid chromatography and mass spectrometry for the analysis of N-ß-methylamino-L-alanine with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

Numerous studies in the past decade have identified N-ß-methylamino-L-alanine (BMAA) as a putative environmental neurotoxin. Produced by cyanobacteria and accumulated at different levels of the trophic system, BMAA has been detected in the brain tissue of human patients that died from progressive neurodegenerative disease. Research into the mechanism of neurotoxicity has been hampered by conflicting results and disagreement in the literature over analytical methods used for quantification and detection. While several research approaches have been tested, the use of the derivatizing reagent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate is presented here as an effective and selective means for the analysis of BMAA and two co-occurring biological isomers, DAB and AEG, by liquid chromatography and tandem mass spectrometry.

The serotonin transporter in the midbrain of suicide victims with major depression.

BACKGROUND: The involvement of serotonin in depression and suicide has been proposed, because major depression is successfully treated by medications that specifically block the serotonin transporter, and there is evidence for a decrease in serotonin transporters in major depression and suicide. The midbrain dorsal raphe nucleus (DR) has been implicated as a site for diminished serotonergic activity in that suicide victims with major depression have a significant increase in serotonin-1A autoreceptors in the DR. METHODS: [(3)H]Paroxetine was used to label the serotonin transporter in the subnuclei of the DR at several rostral-to-caudal levels of the midbrain in ten pairs of suicide victims with major depression and age-matched psychiatrically normal control subjects. RESULTS: There was a significant increase in serotonin transporters in the entire DR progressing from rostral-to-caudal levels in both normal control subjects and suicide victims with major depression. At comparable rostral-to-caudal levels, there were no significant differences in [(3)H]paroxetine binding between depressed suicide victims and normal control subjects in either the entire DR or its constituent subnuclei. CONCLUSIONS: The pathophysiology of serotonin mechanisms in suicide victims with major depression does not appear to involve alterations in the binding of [(3)H]paroxetine to the serotonin transporter in the midbrain DR.

Potentiation of vasoconstrictor responses by 3- and 4-aminopyridine.

1. In concentrations that are known to reduce potassium conductance in many excitable membranes, 3 and 4-aminopyridine (3-AP, 4-AP) potentiate vasoconstrictor responses of the isolated ear artery of the rabbit to noradrenaline and histamine. 2. 3- and 4-AP have no effect on the responses of potassium-depolarized arteries to noradrenaline, histamine or calcium. 3. The results suggest that the aminopyridines have no direct effect on the contractile machinery or on pharmacomechanical coupling, but cause potentiation by influencing electrical events at the cell membrane. 4. 4-AP causes a greater potentiation of the response te electrical stimulation than of the response to noradrenaline. This suggests that the aminopyridines may also cause an increase in the amount of noradrenaline released in response to sympathetic nerve stimulation.

Modification of cardiovascular responses to histamine by dithiothreitol.

1 Histamine produced dose-dependent contractile responses on both isolated perfused ear arteries and aortic strips of the rabbit. These responses were blocked by mepyramine and potentiated by both metiamide and dithiothreitol. 2 In the presence of maximum potentiation by metiamide, dithiothreitol still potentiated the contractile response to histamine of both preparations. 3 In the presence of mepyramine, histamine produced dose-dependent reductions in the contractile response to noradrenaline. This vasodilator action of histamine was abolished by metiamide but was unaffected by dithiothreitol. 4 The vasodilator action of histamine on the human isolated perfused temporal artery and the positive inotropic effect of histamine on the isolated spontaneously beating atria of the rabbit were blocked by metiamide but unaffected by dithiothreitol. 5 It is concluded that the rabbit aorta, like the ear artery, contains both H1 and H2 histamine receptors and that dithiothreitol potentiates cardiovascular responses mediated by H1-receptors but not by H2-receptors.

Histamine H2-receptors in the human peripheral circulation.

Histamine (10 mug/min for 3 min) infused into the brachial artery caused an increase in forearm blood flow which was reduced by mepyramine (25 mg). This effect was most marked in the first minute of the infusion. Metiamide (25 mg) had no effect on the dilatation during the infusion but caused a quicker return of flow to the resting level. The response was abolished when both drugs were given in combination. It is concluded that the response is initiated mainly by stimulation of H1-receptors and maintained by H1- and H2-receptors; continued activity of H2-receptors may account for the slow return of flow to the pre-infusional level.

Suppression of tumorigenicity of rat liver epithelial tumor cell lines by a putative human 11p11.2-p12 liver tumor suppressor locus.

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of a rat liver tumor cell line (termed GN6TF) which contains well defined chromosomal aberrations involving rat chromosomes 1, 4, 7, and 10. In the present study, we investigated the potential of this human 11p11.2-p12 liver tumor suppressor locus to suppress the tumorigenic potential of two other rat liver tumor cell lines (GN3TG and GP10TA) following microcell-mediated introduction of human chromosome 11. These tumor cell lines are aneuploid and contain chromosomal abnormalities that are similar to the GN6TF tumor line. The tumorigenic potential and other phenotypic characteristics of GN3TG-11neo and GP10TA-11neo microcell hybrid (MCH) cell lines were variable, and dependent upon the status of the introduced human chromosome 11. MCH cell lines that retained the region of 11p11. 2-p12 delineated by microsatellite markers D11S1385 and D11S903 exhibited suppression of tumorigenicity in vivo (decrease in tumorigenicity and/or elongation of latency), whereas, the tumorigenic potential of one MCH line that lacked markers in this region of human 11p11.2-p12, but retained flanking markers, was not changed from that of the parental tumor cell line. The chromosomal interval between microsatellite markers D11S1385 and D11S903 encompasses the previously localized minimal liver tumor suppressor region, suggesting that a common locus is responsible for tumor suppression among the rat liver tumor cell lines examined. The results of the present study have verified the presence of a liver tumor suppressor locus within human 11p11.2-p12, and have identified a substantial number of microsatellite markers that are closely linked to this tumor suppressor region. These chromosomal markers will facilitate positional cloning of candidate genes from this region, and may prove useful for determining the involvement of this locus in the pathogenesis of human liver cancer.

Fibrin glue application through the flexible fiberoptic bronchoscope: closure of bronchopleural fistulas.

Closure of bronchopleural fistulas can be accomplished by applying fibrin glue through a flexible fiberoptic bronchoscope. The advantages of this method include the avoidance of general anesthesia and thoracotomy and the excellent extended access to the bronchial tree provided by the flexible bronchoscope.

Measurement of capillary pressure in humans using a venous occlusion method.

The venous occlusion technique was used to measure capillary pressure in the forearm and foot of man over a wide range of venous pressures. In six recumbent subjects venous pressure (Pv) in the forearm (mean +/- SE) was 9.3 +/- 1.4 mmHg and the venous occlusion estimate of capillary pressure (Pc) was 17.0 +/- 1.6 mmHg, whereas in another six subjects Pv in the foot was 17.1 +/- 1.2 mmHg and Pc was 23.4 +/- 2.5 mmHg. Venous pressure in the limbs was increased either by changes in posture or by venous congestion with a sphygmomanometer cuff. On standing Pv in the foot increased to 95.2 +/- 1.5 mmHg and Pc rose to 112.8 +/- 3.1 mmHg. The relationship established between venous pressure and capillary pressure in the forearm is Pc = 1.16 Pv + 8.1, whereas in the foot the relationship is Pc = 1.2 Pv + 1.6. The magnitude and duration of the changes in capillary pressure were also recorded during reactive hyperemia. The venous occlusion method of measuring capillary pressure is simple and easily applied to studies in humans.

A sterile, delayed-closure technique for placement of a left ventricular assist device.

A simplified technique of left ventricular assist device placement using Silastic sheeting attached to the presternal fascia and skin is described. This technique allows viewing of cardiac action and prevents cardiac compression by sternal closure. Removal of the assist device is simple because the sternum does not have to be reopened.