CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery.

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Safety and exposure of once-daily ritonavir-boosted atazanavir in HIV-infected pregnant women.

OBJECTIVE: There are limited antiretroviral options for use in the treatment of HIV infection during pregnancy. The purpose of this study was to assess the safety, efficacy and appropriate dosing regimen for ritonavir (RTV)-boosted atazanavir in HIV-1-infected pregnant women. METHODS: In this nonrandomized, open-label study, HIV-infected pregnant women were dosed with either 300/100 mg (n=20) or 400/100 mg (n=21) atazanavir/RTV once-daily (qd) in combination with zidovudine (300 mg) and lamivudine (150 mg) twice daily in the third trimester. Pharmacokinetic parameters [maximum observed plasma concentration (C(max) ), trough observed plasma concentration 24 hour post dose (C(min) ) and area under concentration-time curve in one dosing interval (AUC(τ) )] were determined and compared with historical values (300/100 mg atazanavir/RTV) for HIV-infected nonpregnant adults (n=23). RESULTS: At or before delivery, all mothers achieved HIV RNA <50 HIV-1 RNA copies/mL and all infants were HIV DNA negative at 6 months of age. The third trimester AUC(τ) for atazanavir/RTV 300/100 mg was 21% lower than historical data, but the C(min) values were comparable. The C(min) value for atazanavir/RTV 400/100 mg was 39% higher than the C(min) for atazanavir/RTV 300/100 mg in historical controls, but the AUC(τ) values were comparable. Twice as many patients in the 400/100 mg group (62%) had an increase in total bilirubin (>2.5 times the upper limit of normal) as in the 300/100 mg group (30%). Atazanavir (ATV) was well tolerated with no unanticipated adverse events. CONCLUSIONS: In this study, use of atazanavir/RTV 300/100 mg qd produced C(min) comparable to historical data in nonpregnant HIV-infected adults. When used in combination with zidovudine/lamivudine, it suppressed HIV RNA in all mothers and prevented mother-to-child transmission of HIV-1 infection. During pregnancy, the pharmacokinetics, safety and efficacy demonstrated that a dose adjustment is not required for ATV.

Limits on the asymmetry parameters for two- and three-photon photofragmentation.

The known constraint, -1

Effect of rifampin on steady-state pharmacokinetics of atazanavir with ritonavir in healthy volunteers.

Mycobacterium tuberculosis is a concern in patients with human immunodeficiency virus (HIV) infection. Rifampin (RIF), an agent used against M. tuberculosis, is contraindicated with most HIV protease inhibitors. Atazanavir (ATV) has clinical efficacy comparable to a standard of care regimen in naive patients and, when dosed with low-dose ritonavir (RTV), also in treatment-experienced patients. We evaluated here the safety and pharmacokinetics of ATV, resulting from three regimens of ATV, RTV, and RIF in 71 healthy subjects. The pharmacokinetics for ATV and RTV were assessed after 6 and 10 days of dosing with ATV 400 mg (n = 53) and with ATV-RTV at 300 and 100 mg (ATV/RTV 300/100; n = 52), respectively. Steady-state pharmacokinetics for ATV, RTV, RIF, and desacetyl-rifampin (des-RIF) were measured after 10 days of dosing of ATV/RTV/RIF 300/100/600 (n = 17), ATV/RTV/RIF 300/200/600 (n = 17), or ATV/RTV/RIF 400/200/600 (n = 14). An RIF 600-alone arm was enrolled as a control group (n = 18). With ATV/RTV/RIF 400/200/600, ATV area under the concentration-time curve values were comparable, but the C(min) values were lower relative to ATV 400 alone. ATV exposures were substantially reduced for the other RIF-containing regimens relative to ATV 400 alone and for all regimens relative to ATV/RTV 300/100 alone. RIF and des-RIF exposures were 1.6- to 2.5-fold higher than with RIF 600 alone. The incidence of grade 3/4 alanine aminotransferase/aspartate aminotransferase values was limited to 1 subject each in both the ATV/RTV/RIF 300/200/600 and the ATV/RTV/RIF 400/200/600 treatments. Coadministration of ATV with RIF was safe and generally well tolerated. Since ATV exposures were reduced in all regimens, ATV and RIF should not be coadministered at the dosing regimens studied.

Quantum level structures at a Fermi resonance with angular momentum: classical periodic orbits, catastrophe maps and quantum monodromy.

The Xiao-Kellman catastrophe map, for the classification of classical periodic orbits of the standard 2:1 Fermi resonance Hamiltonian is extended to species with finite vibrational angular momentum. The influence of the classical periodic orbit structure on different organizations of the quantum mechanical eigenvalues, in the four regions of the map, is strikingly demonstrated. The quantum eigenvalue lattices in angular momentum and energy space show dislocations attributable to a topological effect, termed quantum monodromy. Analogues with quantum monodromy in quasi-linear molecules and LiCN/LiNC isomerisation are demonstrated.

Quantum initial value representation simulation of water trimer far infrared absorption spectrum.

We extend the technique of quantum propagation on a grid of trajectory guided coupled coherent states to simulate experimental absorption spectra. The approach involves calculating the thermally averaged dipole moment autocorrelation function by means of quantum propagation in imaginary time. The method is tested on simulation of the far infrared spectrum of water trimer based on a three-dimensional model potential. Results are in good agreement with experiment and with other calculations.

Cell-cell interactions influence resistance and survival of Salmonella serotype Typhimurium to environmental stress.

AIMS: The aim of this work was to study the effects of prolonged nutrient stress on survival, cell interactions and resistance to inimical processes in Salmonella serotype Typhimurium. METHODS AND RESULTS: Salmonella Typhimurium cells were subjected to prolonged incubation in the stationary phase of growth and the properties of starved cells (old) were investigated with reference to those of exponentially-growing cells (young). Competition experiments between old and young cells revealed cell-cell interactions that influenced stationary phase survival and response of the bacterium to heat stress. During prolonged incubation of cells, cycles of resistance and sensitivity to heat stress were identified. Competition experiments between old and young cells revealed that the resistance of young cells to heat increased to levels more like those of stationary phase cells than growing cells. The presence of old cells influenced the phenotype of young cells, possibly by means of cell-cell interactions. There was no evidence for the involvement of any extracellularly-produced factors in this phenomenon, but a requirement that the old competitor cells be viable could be demonstrated. CONCLUSIONS: It is proposed that the complex interactions within stationary phase cultures of Salm. Typhimurium may be due to cycles of mutation in concert with an as yet undefined interaction between old cells and growing ones. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence for active and diverse responses to nutrient stress within populations of Salm. Typhimurium that promote survival and that may be important for the success of this bacterium as a pathogen.

Refractory coronary artery spasm with superimposed thrombosis: successful treatment with Palmaz-Schatz stent.

Prinzmetal variant angina due to epicardial coronary artery spasm is a disease usually treated with drug therapy with successful results. A case of variant angina, refractory to conventional pharmacological treatment, and complicated by coronary artery thrombosis, was treated with percutaneous transluminal coronary angioplasty and stenting with good immediate and late clinical results.

[The early angiographic follow-up of myocardial revascularization in a minithoracotomy: the results of the first 100 consecutive cases]. / Il controllo angiografico precoce della rivascolarizzazione miocardica in minitoracotomia: risultati dei primi cento casi consecutivi.

The use of the left internal thoracic artery anastomized to the left anterior descending coronary artery via a small left thoracotomy to revascularize the anterior wall of the left ventricle has gained wide acceptance since its introduction into clinical practice a few years ago. A mandatory, postoperative angiographic control was suggested in order to check the surgical results of this new method of revascularization. We herein analyze the results of the in-hospital angiographic control of a series of 100 consecutive patients who underwent minimally invasive coronary artery bypass. In all 100 patients the thoracic graft, the anastomosis and the target vessel were patent, with no anomalies in 90 subjects. In 4 patients, a sharp angulation of the thoracic artery in the last third before the anastomosis to the native vessel was observed; in 3 subjects, the arterial graft had been anastomized to a diseased tract of the target vessel and in 3 cases a significant stenosis of the target vessel beyond the anastomosis was documented; in 2 cases the persistence of a thoracic artery branch was discovered. Since 1) neither in-hospital total occlusion of the thoracic graft to the left anterior descending coronary artery via a small thoracotomy was documented nor a significant incidence of major anomalies was observed; 2) the anomalies documented seem to be clinically negligible and may regress in the midterm postoperative period; 3) Doppler flow analysis is able to detect not only the patency but also the presence of significant stenosis in the arterial graft; the in-hospital angiographic control of this surgical technique should be limited to patients with abnormal ultrasonic data or with reappearance of myocardial ischemia in the anterior wall of the left ventricle, thus not reducing the advantages in terms of speed and cost-control of this type of myocardial revascularization.

Neonatal sex hormones have 'organizational' effects on the hypothalamic-pituitary-adrenal axis of male rats.

Sex hormones have activational effects on the hypothalamic-pituitary-adrenal (HPA) axis in adulthood: For example, corticosterone release is influenced by gonadal status. These experiments investigated whether sex hormones have organizational effects on the HPA axis of male rats: Do sex hormones have relatively permanent effects on its development? In adults, both neonatal (neoGDX) and adult gonadectomy (adult GDX) resulted in elevated corticosterone (CORT) levels in response to stress compared to intact rats. Five days of testosterone propionate (TP) replacement was not as effective at attenuating CORT levels in neoGDX rats as in adult GDX rats. Neonatal GDX elevated corticosterone binding globulin (CBG) levels, whereas adult GDX was without effect. In Experiment 2 the effects of neonatal gonadectomy and neonatal treatment with either TP, estradiol benzoate (EB), or oil vehicle was examined. Despite 14 days of hormone replacement, neoGDX showed elevated CORT levels in response to stress compared to all other groups. A single neonatal dose of TP or EB in neoGDX rats eliminated the increased responsiveness. Neonatal TP and EB were without effect in sham-operated rats. Plasma CBG levels were elevated in neoGDX groups regardless of neonatal hormone treatment. Corticosteroid receptor binding levels were examined in various brain areas and the pituitary in two groups most different in their androgen experience: NeoGDX and shams that did not receive treatments as adults. NeoGDX had lower levels of glucocorticoid receptor, and higher levels of mineralocorticoid receptor binding in the pituitary. No other receptor differences were found. These experiments suggest that neonatal sex hormones influence the sensitivity of the HPA axis to sex hormones in adulthood and, thus, that they have organizational effects in addition to activational effects on HPA function.

Assessment of left internal mammary artery grafts using dipyridamole Doppler echocardiography.

Color Doppler echocardiography of the left mammary artery was combined with dipyridamole testing in order to assess the presence of significant (>70%) graft stenosis in 87 patients with a mammary artery graft to the left anterior descending coronary artery presenting with chest pain. Occluded grafts are detected by absent diastolic flow velocities at baseline, whereas the response of the diastolic flow velocity to dipyridamole distinguishes patients with critical versus noncritical stenosis of a patent graft.

Cellular basis for improved left ventricular pump function after digoxin therapy in experimental left ventricular failure.

OBJECTIVES: The present study examined left ventricular (LV) and myocyte contractile performance and electrophysiologic variables after long-term digoxin treatment in a model of LV failure. BACKGROUND: A fundamental therapeutic agent for patients with chronic LV dysfunction is the cardiac glycoside digoxin. However, whether digoxin has direct effects on myocyte contractile function and electrophysiologic properties in the setting of chronic LV dysfunction remains unexplored. METHODS: Left ventricular and isolated myocyte function and electrophysiologic variables were examined in five control dogs, five dogs after the development of long-term rapid pacing (rapid pacing, 220 beats/min, 4 weeks) and five dogs with rapid pacing given digoxin (0.25 mg/day) during the pacing period (rapid pacing and digoxin). RESULTS: Left ventricular ejection fraction decreased in the dogs with rapid pacing compared with that in control dogs (30 +/- 2% vs. 68 +/- 3%, p < 0.05) and was higher with digoxin than that in the rapid pacing group (38 +/- 3%, p = 0.038). Left ventricular end-diastolic volume increased in the rapid pacing group compared with the control group (84 +/- 6 ml vs. 59 +/- 7 ml, p < 0.05) and remained increased with digoxin (79 +/- 6 ml). Isolated myocyte shortening velocity decreased in the rapid pacing group compared with the control group (37 +/- 1 microns/s vs. 59 +/- 1 microns/s, p < 0.05) and increased with digoxin compared with rapid pacing (46 +/- 1 microns/s, p < 0.05). Action potential maximal upstroke velocity was diminished in the rapid pacing group compared with the control group (135 +/- 6 V/s vs. 163 +/- 9 V/s, p < 0.05) and increased with digoxin compared with rapid pacing (155 +/- 12 V/s, p < 0.05). Action potential duration increased in the rapid pacing group compared with the control group (247 +/- 10 vs. 216 +/- 6 ms, p < 0.05) and decreased with digoxin compared with rapid pacing (219 +/- 12 ms, p < 0.05). CONCLUSIONS: In this model of rapid pacing-induced LV failure, digoxin treatment improved LV pump function, enhanced isolated myocyte contractile performance and normalized myocyte action potential characteristics. This study provides unique evidence to suggest that the cellular basis for improved LV pump function with digoxin treatment in the setting of LV failure has a direct and beneficial effect on myocyte contractile function and electrophysiologic measures.

Prompt and safe femoral hemostasis with a collagen device after intracoronary implantation of Palmaz-Schatz stents.

We report the safety and efficacy of sealing the femoral puncture site with percutaneously applied collagen after Palmaz-Schatz stent implantation in 100 consecutive patients. Patients were anticoagulated with continuous heparin infusion, overlapping oral anticoagulants, and antiplatelet therapy by dextran, aspirin, and dipyridamole. At the time of sheath removal and collagen application, the mean activated partial thromboplastin time and prothrombin time values expressed as international normalized ratio were 3.2 +/- 2.1 and 1.6 +/- 0.7, respectively. The hemostasis time ranged from 1 to 8 minutes (mean 2.18 +/- 2.08 minutes). Only two (2%) patients had major puncture-site bleeding (not seal related in one case) that required surgery and blood transfusions. Small (< 6 cm) and medium (6 to 10 cm) hematomas observed in 12 (12%) and 2 (2%) patients, respectively, resolved spontaneously without sequelae. Local infection developed in 2 (2%) patients, who were successfully treated with antibiotics without clinical consequences. Subacute stent thrombosis was observed in only 1 (1%) patient. Repeat catheterization through the same femoral artery was performed at 6-month follow-up in 55 patients without difficulty or vascular complications. These findings suggest that percutaneous collagen application after coronary stenting is a secure method of achieving prompt and effective femoral hemostasis with a low incidence of major vascular bleeding complications despite intense anticoagulation. Stable hemostasis may allow continued full-dose anticoagulation, reducing the risk of stent subacute thrombosis.

LV and myocyte structure and function after early recovery from tachycardia-induced cardiomyopathy.

Left ventricular (LV) function and mass were measured in six conscious dogs at weekly intervals during the progression of tachycardia-induced dilated cardiomyopathy (DCM) and during a 1-mo recovery period from DCM (post-DCM). LV end-diastolic volume and LV wall stress increased and LV ejection fraction decreased with each week of pacing. Despite the increased LV wall stress, LV mass did not change during the progression of tachycardia DCM. One week post-DCM resulted in an improved LV ejection fraction and normalization of neurohormonal profiles. However, 1 wk post-DCM was accompanied by a 26% increase in LV mass and persistent LV chamber dilation. Isolated myocyte function was examined and compared with that in six normal control dogs. Myocyte percent and myocyte velocity of shortening were 19 and 32% lower, respectively, in the post-DCM group compared with controls. Thus termination of the tachycardia subsequent to the development of DCM resulted in persistent LV chamber dilation and abnormalities in myocyte contractile function. The improved LV pump function with early recovery from tachycardia-induced DCM was mediated by LV hypertrophy and a subsequent reduction in LV wall stress rather than a normalization of LV geometry and myocyte contractile function.

Quantitative evaluation of AFO use with myelomeningocele children.

Although the sample size is small, results are very positive for the use of AFO's with the myelomeningocele child. The increase in pertinent gait parameters in conjunction with a decrease in excess hip, knee, and ankle flexion demonstrate the benefits gained in using AFO's to assist the myelomeningocele child in ambulation. Further, the decrease in excess muscle activation time and co-contraction facilitate a decrease in energy expenditure, thus allowing for more ambulation the limited results from this study provide a basis from which further research may be gleaned. The use of quantitative measures in evaluating the benefits of AFO's provides objective data from which future design and application recommendations may be made.

Antigenic stimulation and the occurrence of multiple myeloma.

To evaluate whether potent or prolonged stimulation of the immune system increases the risk of multiple myeloma, the authors compared 698 myeloma cases which occurred between July 1, 1977 and June 30, 1981 in four geographic areas of the United States with 1,683 demographically similar controls from the same areas. Cases and controls were interviewed about past exposures which may have involved antigenic challenge. Although few positive associations emerged, those most consistent with the immune stimulation hypothesis were modest associations between myeloma and a history of rheumatic fever (relative risk (RR) = 1.74, 95% confidence interval (CI) = 1.09-2.77) and between myeloma and urinary tract infection (RR = 1.30, 95% CI = 1.00-1.69, when self-respondent cases were compared with controls). Little association was found between the risk of myeloma and the number of past viral illnesses, number of bacterial illnesses, or number of allergy desensitization injections. Myeloma risk was found to be inversely related to the number of diseases against which a subject reported having been immunized, perhaps because of differences in socioeconomic status between cases and controls. These findings provide little support for the immune system stimulation hypothesis of myeloma etiology, but because of the limitations of interview techniques for assessing antigen exposure, further studies using laboratory methods may be warranted.

Bladder cancer, drinking water source, and tap water consumption: a case-control study.

Data from a population-based case-control interview study of incident bladder cancer in 10 areas of the United States were used to estimate relative risks among white men (2,116 cases, 3,892 controls) and women (689 cases, 1,366 controls) according to beverage intake level and type of water source. Individual year-by-year profiles of water source and treatment were developed by linking lifetime residential information with historical water utility data from an ancillary survey. Risk of bladder cancer increased with intake level of beverages made with tap water. The odds ratio (OR) for the highest vs. lowest quintile of tap water consumption was 1.43 [95% confidence interval (CI) = 1.23, 1.67; chi 2 for trend = 26.3, P less than .001]. The risk gradient with intake was restricted to persons with at least a 40-year exposure to chlorinated surface water and was not found among long-term users of nonchlorinated ground water. The ORs for the highest vs. lowest quintiles of tap water intake were 1.7 and 2.0, respectively, among subjects with 40-59 and greater than or equal to 60 years' exposure. Duration of exposure to chlorinated surface water was associated with bladder cancer risk among women and nonsmokers of both sexes. Among non-smoking respondents with tap water consumption above the population median, the OR increased with exposure duration to a level of 3.1 (CI = 1.3, 7.3; chi 2 for trend = 6.3, P = .01) for greater than or equal to 60 years of residence at places served by chlorinated surface water (vs. non-chlorinated ground water users). These results extend findings of earlier epidemiologic studies and are consistent with environmental chemistry and toxicologic data demonstrating the presence of genotoxic by-products of chlorine disinfection in treated surface waters.