Restriction endonucleases can be used to study B-Z junctions in supercoiled DNA.

Plasmids containing (C-G)n inserts have been used to study the inhibition of cleavage by restriction endonucleases due to Z-DNA formation in negatively supercoiled plasmids. The enzyme BssHII, which recognizes G-C-G-C-G-C, is strongly inhibited when the insert forms Z-DNA. The BamHI recognition sequence (G-G-A-T-C-C) was placed in four different positions near the B-Z junction and the inhibition of BamHI cleavage was determined as a function of negative superhelical density. Formation of Z-DNA in the (C-G)n insert inhibited cleavage by BamHI when its recognition sequence was located immediately adjacent to the insert or four base pairs away from it. However, no inhibition was found when the BamHI recognition site was eight base pairs away. These experiments help to define the limits of the structural perturbation associated with the B-Z junction.     

Pregnant mice are not primed but can be primed to fetal alloantigens.

In this report we present evidence gathered from various stages of murine pregnancy that indicates that pregnant females have no demonstrable immune effector function directed against their semiallogeneic fetuses. Specifically, by using in vitro assays we found that cytotoxic lymphocytes were not present in pregnant mice, and pregnant mice challenged with radiolabeled paternal strain leukemia cells showed no evidence of immune elimination. Nonetheless, immunity measurable both in vitro and in vivo was readily induced by priming with the paternal strain cells. No harm to the fetus was observed in primed mothers. These results cast doubt on the relevance of mechanisms proposed that involve systemic active suppression during pregnancy. The results are compatible with the hypothesis that the placenta acts as a barrier, preventing fetal cells from priming the mother.     

Cytomegalovirus antigenemia assay or PCR can be used to monitor ganciclovir treatment in bone marrow transplant recipients.

The antigenemia assay, polymerase chain reaction (PCR) and rapid culture technique on buffy coat cells (DEAFF test) were used to monitor 37 cytomegalovirus (CMV) seropositive bone marrow transplant (BMT) recipients for active CMV infection during the first 3 months after BMT. The antigen assay and PCR demonstrated a comparable sensitivity for the detection of CMV in blood: discordant results were only obtained in the early or late phase of infection when the viral load was low. The antigen assay was more sensitive than the DEAFF test. Only 12 out of 40 antigen-positive samples yielded a positive result with DEAFF test, whereas viremia without antigenemia was never found. The discordance between these two tests increased further during antiviral therapy with ganciclovir. A correlation was observed between the duration of antigenemia during treatment and the recurrence of systemic CMV reactivation. Ten out of 11 patients with antigen-positive leukocytes present for more than 1 week after starting the treatment subsequently exhibited a relapse of active infection, whereas only three out of nine patients who resolved their antigenemia within 1 week did so. In conclusion, the antigen assay and PCR are useful techniques for detection of CMV infection in BMT patients. Test results obtained during therapy give reliable information regarding the viral load and the possibility of recurrence of antigenemia, and can be taken into account when prolonged administration of ganciclovir is considered.     

The activated clotting time (ACT) can be used to monitor enoxaparin and dalteparin after intravenous administration

BACKGROUND: The use of low-molecular weight heparin (LMWH) during percutaneous coronary intervention (PCI) has been limited by the presumed inability to monitor its anticoagulant effect using bedside assays. OBJECTIVES: This study was designed to compare the dose-response of enoxaparin, dalteparin and unfractionated heparin (UFH) on the activated clotting time (ACT), and to determine whether the ACT or aPTT can be used to monitor intravenous (IV) low molecular weight heparin (LMWH). METHODS: A total of 130 patients undergoing cardiac catheterization were assigned to intravenous enoxaparin 0.5 mg/kg, dalteparin 50 international units/kg or UFH 50 units/kg. Of the 130 patients, 46 (35%) underwent PCI, all of whom received a glycoprotein (GP) IIb/IIIa inhibitor. We measured ACT, activated partial thromboplastin time (aPTT) and plasma anti-Xa levels after serial sampling. RESULTS: Both enoxaparin and dalteparin induced a significant rise in the ACT and aPTT, with an ACT dose-response approximately one-half the magnitude of that obtained using UFH. The time course of changes in the ACT and aPTT after administration of enoxaparin and dalteparin was virtually identical, with a return to baseline at approximately 2 hours. The enoxaparin and dalteparin-treated patients successfully underwent PCI with no major hemorrhagic complications. CONCLUSIONS: The ACT is equally sensitive to IV enoxaparin and dalteparin. These data support an ACT-guided strategy for intravenously administered LMWH during PCI. Additional studies with larger patient populations may be indicated to determine the ideal target ACT for LMWH in PCI.     

The activated clotting time can be used to monitor the low molecular weight heparin dalteparin after intravenous administration

OBJECTIVES: This study was designed to compare the dose response of dalteparin versus unfractionated heparin (UFH) on the activated clotting time (ACT), and to determine whether the ACT can be used to monitor intravenous (IV) dalteparin during percutaneous coronary intervention (PCI). BACKGROUND: The use of low molecular weight heparin (LMWH) during PCI has been limited by the presumed inability to monitor its anticoagulant effect using bedside assays. METHODS: This study was performed in three phases. In vitro, ACTs were measured on volunteer (n = 10) blood samples spiked with increasing concentrations of dalteparin or UFH. To extend these observations in vivo, ACTs were then measured in patients (n = 15) who were sequentially treated with IV dalteparin and then UFH. Finally, a larger monitoring study was undertaken involving patients (n = 110) who received dalteparin 60 or 80 international U (IU)/kg alone or followed by abciximab. We measured ACT (Hemochron), activated partial thromboplastin time (aPTT), plasma anti-Xa and anti-IIa levels, tissue factor pathway inhibitor (TFPI) concentration, and plasma dalteparin concentration. RESULTS: Dalteparin induced a significant rise in the ACT with a smaller degree of variance as compared to UFH. Five min after administration of IV dalteparin 80 IU/kg the ACT increased from 125 s (122 s, 129 s) to 184 s (176 s, 191 s) (p < 0.001). The aPTT, anti-Xa and anti-IIa activities, and TFPI concentration also demonstrated significant increases following IV dalteparin. CONCLUSIONS: The ACT and aPTT are sensitive to IV dalteparin at clinically relevant doses. These data suggest that the ACT may be useful in monitoring the anticoagulant effect of intravenously administered dalteparin during PCI.     

Value of circulating cell-free DNA in diagnosis of hepatocelluar carcinoma

AIM:To investigate the value of combined detection of circulating cell-free DNA(cfDNA),a-fetal protein(AFP) and a L-fucosidase(AFU) for diagnosis of hepatocellular carcinoma(HCC).METHODS:Serum samples from 39 HCC patients and 45 normal controls were collected.Branched DNA(bDNA) was used to detect the level of cfDNA,and a receiver operating characteristic curve was employed to evaluate the diagnostic sensitivity,specificity,accuracy,positive predictive value,negative predictive value,positive likelihood ratio,negative likelihood ratio and Youden index,and to assess the diagnostic efficiency and their correlations with the clinicopathological features.AFP and AFU were detected by chemiluminescence and colorimetry,respectively.The significance of combined detection of the three biomarkers was discussed.RESULTS:cfDNA level was increased in 22 of the 39 HCC samples and in 2 of the 45 normal controls.cfDNA level in HCC samples was significantly higher than that in normal controls(P < 0.05).There were significant differences in sex and extra-and intrahepatic metastasis(P < 0.05).There was no significant correlation between cfDNA,AFP and AFU in the detection of HCC.The sensitivity of combined detection of cfDNA with one marker(AFP or AFU) and cfDNA with two markers(AFP and AFU) was 71.8%,87.2% and 89.7% vs 56.4%,53.8% and 66.7% for cfDNA,AFP and AFU used alone,respectively,the difference being statistically significant(P < 0.05).CONCLUSION:Quantitative analysis of cfDNA is sensitive and feasible,and the combined detection of cfDNA with AFP or AFU or both could improve the diagnostic sensitivity for HCC.     

Economic games can be used to promote cooperation in the field

We present experimental evidence of the impact of playing a game on real-life cooperation. The game was framed as a pest-management activity, the effectiveness of which depends on the decisions of others. Playing the game changes behavior in the field, increasing the participation in all collective activities directed at reducing pest pressure. The economic impact of those activities is important, leading to losses that are ∼20% lower than in the control group. Increased cooperation reflects changes in the understanding of others’ willingness to cooperate, not changes in the understanding of underlying technological interdependencies.

Cooperation with nonkin is a distinctive aspect of human life (1), maintained by norms of conditional behavior (2), where norms have the accepted definition of shared views of how individuals ought to behave in a given situation (3). Promoting and sustaining cooperation in a context where it hitherto did not exist may require the elimination of inaccurate beliefs about others’ actions and beliefs, i.e., overcoming pluralistic ignorance (4).A variety of approaches have been used to create belief shocks that may lead to changes in behavioral norms (5–7). Changing the law (8) may provide an indication of the acceptability of some practices and foster changes in behavior (9) or perceptions (10). Similarly, “edutainment ” through radio shows (11), soap operas (12), or movies (13) can provide new role models that motivate the adoption of behaviors that were privately desired, but perceived as publicly shunned. At a local scale, information about what others do or value has been shown to promote environmental conservation (14), reduce alcohol consumption among teenagers (15), or increase female labor participation (16).In this paper, we evaluate the feasibility of using a framed game as an approach to promote cooperation. Similar to social scientists (17) or students in a classroom (18), participants in these games may learn about others’ behavior in conditions that were hitherto-unobserved, including about others’ willingness to cooperate (thereby reducing pluralistic ignorance), without having to incur the costs of promoting it themselves. This possibility was first noticed by ref. 19, and, building on that suggestion, recent work has used economic games as a teaching tool about the potential benefits of cooperation, usually in contexts where such problems are relatively new, such as the overexploitation of aquifers due to expanded access to water pumps (20–22). We build on this work in two ways: by experimentally evaluating the impact of playing a game on behavior outside the laboratory (improving on earlier evidence that mostly relies on a before–after comparison) and by quantifying the relative importance of reducing ignorance about others’ willingness to cooperate.The game was framed as a way of reducing rodent damage to rice. In Asia, such damage has important impacts on food security (23), which are particularly noticeable during outbreaks, some of which have even been followed by famine and civil unrest (24). Given the long history of association between rodents and humans (25, 26), the importance and persistence of such large losses is usually traced to rodents’ reproductive behavior, in particular, high litter size, short gestation period, and early sexual maturity (27, 28). In the absence of coordinated control (either by limiting rodents’ access to food and/or culling rodents), rodents quickly multiply, rendering any individual effort almost useless. This is certainly the case of the black rat (Rattus rattus), one of the main rodent pest species in the world (29), including in northern Lao People’s Democratic Republic (PDR), the setting of our analysis (30). The challenge, then, is how to coordinate multiple farmers into practicing rodent control over an extended period of time, when there are incentives to free-ride on others’ efforts to reduce pest pressure (27, 28), a problem common to the management of other pests and invasive species (31, 32).The game simulates this coordination problem in a simplified way. Players have to decide whether to contribute to reduce pest pressure by deciding how much time to allocate to the production of a private good (rice), knowing that the payoff (shown in 28)]. Still focusing on symmetric strategies, the social optimum can also be achieved with higher contributions (own contribution = {2, 3}), although these strategies do not correspond to a Nash equilibrium. Details about the rules of the game, as well as a discussion of players’ behavior, are presented in Methods.Table 1.Time allocation and payoff table (excerpt)

Methotrexate should not be used for patients with end-stage kidney disease

Methotrexate is a widely used disease-modifying anti-rheumatic drug. Its effectiveness has been proven in placebo-controlled trials and in comparison with other disease-modifying anti-rheumatic drugs. The pharmacokinetics of methotrexate are highly variable and unpredictable. In patients with normal renal function, the recommended dose in rheumatoid arthritis ranges between 7.5 and 15 mg/week, but in recent years, even dosages up to 25 mg weekly are used. Toxicity includes myelosuppression, gastrointestinal adverse effects, hepatotoxicity and pneumonitis. Renal impairment and age are considered major risk factors for developing methotrexate toxicity, but studies show conflicting results. Whether methotrexate can be administered to patients with end-stage kidney disease has not been formally tested. The present case illustrates the severe side effects of low-dose methotrexate treatment in a patient with end-stage kidney disease. Seven other cases have reported similar and even more severe and irreversible consequences after low-dose regimen. In view of these side effects we strongly recommend to monitor toxicity rigorously in patients with stage 3 or stage 4 kidney disease and not to use methotrexate in patients with stage 5 kidney disease.     

Prolonged duration of response to infliximab in early but not late pediatric Crohn's disease

OBJECTIVES: Tumor necrosis factor-alpha plays a central role in chronic intestinal inflammation of Crohn's disease. Targeting this cytokine with the chimeric monoclonal antibody infliximab has emerged as an effective form of therapy in adult Crohn's disease patients. We sought to determine whether infliximab treatment would benefit pediatric patients with medically refractory Crohn's disease. We also assessed the duration of response, comparing children with early disease to children with long-standing (late) Crohn's disease. METHODS: Fifteen consecutive children (mean age 12.8 +/- 3.2 yr) with medically refractory Crohn's disease were enrolled in a prospective, open-label trial of a single, 5-mg/kg infliximab intravenous infusion. Medically refractory disease was defined as an inability to taper steroids, lack of response to immunomodulator therapy over 4 months, and active disease as measured by the Pediatric Crohn's Disease Activity Index (PCDAI). Primary endpoints included measurements of disease activity (PCDAI), steroid use, and duration of clinical response. RESULTS: In all, 14/15 children (94%) improved after infliximab infusion, with a significant decrease of both PCDAI and daily steroid use by 4 wk. Ten patients (67%) achieved complete remission by 10 wk. Among the 14 patients who responded, three of six children (50%) with early disease maintained clinical response through the 12-month trial period, compared to none of eight children with late disease. There were no serious complications associated with the use of infliximab in any of the patients. CONCLUSIONS: Infliximab is safe and effective in the short-term treatment of medically refractory pediatric Crohn's disease. More importantly, there is a remarkably prolonged duration of response after infliximab therapy in children with early compared to late Crohn's disease.     

PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for Pompe disease

Screening of blood films for the presence of periodic acid-Schiff (PAS)-positive lymphocyte vacuoles is sometimes used to support the diagnosis of Pompe disease, but the actual diagnostic value is still unknown. We collected peripheral blood films from 65 untreated Pompe patients and 51 controls. Lymphocyte vacuolization was quantified using three methods: percentage vacuolated lymphocytes, percentage PAS-positive lymphocytes, and a PAS score depending on staining intensity. Diagnostic accuracy of the tests was assessed using receiver operating characteristic (ROC) curves. All three methods fully discerned classic infantile patients from controls. The mean values of patients with milder forms of Pompe disease were significantly higher than those of controls, but full separation was not obtained. The area under the ROC curve was 0.98 for the percentage vacuolated lymphocytes (optimal cutoff value 3; sensitivity 91%, specificity 96%) and 0.99 for the percentage PAS-positive lymphocytes and PAS score (optimal cutoff value 9; sensitivity 100%, specificity 98%). Our data indicate that PAS-stained blood films can be used as a reliable screening tool to support a diagnosis of Pompe disease. The percentage of PAS-positive lymphocytes is convenient for use in clinical practice but should always be interpreted in combination with other clinical and laboratory parameters.     

A nasal cannula can be used to treat obstructive sleep apnea

RATIONALE: Obstructive sleep apnea syndrome is due to upper airway obstruction and is associated with increased morbidity. Although continuous positive airway pressure efficaciously treats obstructive apneas and hypopneas, treatment is impeded by low adherence rates. OBJECTIVES: To assess the efficacy on obstructive sleep apnea of a minimally intrusive method for delivering warm and humidified air through an open nasal cannula. METHODS: Eleven subjects (age, 49.7+/-5.0 yr; body mass index, 30.5+/-4.3 kg/m2), with obstructive apnea-hypopnea syndrome ranging from mild to severe (5 to 60 events/h), were administered warm and humidified air at 20 L/minute through an open nasal cannula. MEASUREMENTS AND MAIN RESULTS: Measurements were based on standard sleep-disordered breathing and arousal indices. In a subset of patients pharyngeal pressure and ventilation were assessed to determine the mechanism of action of treatment with nasal insufflation. Treatment with nasal insufflation reduced the mean apnea-hypopnea index from 28+/-5 to 10+/-3 events per hour (p<0.01), and reduced the respiratory arousal index from 18+/-2 to 8+/-2 events per hour (p<0.01). Treatment with nasal insufflation reduced the apnea-hypopnea index to fewer than 10 events per hour in 8 of 11 subjects, and to fewer than 5 events per hour in 4 subjects. The mechanism of action appears to be through an increase in end-expiratory pharyngeal pressure, which alleviated upper airway obstruction and improved ventilation. CONCLUSIONS: Our findings demonstrate clinical proof of concept that a nasal cannula for insufflating high airflows can be used to treat a diverse group of patients with obstructive sleep apnea.     

Left atrial overload can be used to estimate mitral regurgitant volume

This study was conducted to assess the accuracy of the estimated mitral regurgitant volume using both the left atrial filling volume and the systolic component of pulmonary vein flow expressed as the percent of its total. Since mitral regurgitation fills the left atrial chamber, the variation in atrial volume during ventricular systole has been proposed as a means to evaluate the severity of regurgitation. Although the correlation with invasive grading of mitral regurgitation is good, there is an unacceptable overlap among grades caused by the absence of information concerning pulmonary vein flow, which enters the left atrium while regurgitation occurs. The Doppler regurgitant volume, or Dp-RVol (mitral stroke volume minus aortic stroke volume) was quantified in 74 patients with any degree and etiology of mitral regurgitation. Atrial volumes were measured from the four-chamber apical view (biplane area-length method). The systolic time-velocity integral of pulmonary vein flow was expressed as the percent of the total (PVs%) (systolic-diastolic) time-velocity integral. These parameters were subjected to multivariate analysis and a regression equation was obtained. The equation was subsequently applied to a group of 31 patients without mitral regurgitation, as evaluated by color Doppler or continuous-wave Doppler and to the overall population (105 patients) in order to estimate the mitral regurgitant volume. In 74 patients with mitral regurgitation, the Doppler regurgitant volume was univariately correlated with the left atrial filling volume (r= 0.74; p<0.0001) and the systolic pulmonary vein velocity integral expressed as the percent of the total (r=0.67; p<0.0001). In multiple regression analysis, the combination of atrial filling and the pulmonary vein velocity integral provided the more accurate estimation of the regurgitant volume (R2=0.84; standard error of the estimate [SEE], 13.9 mL; p<0.0001; Dp-RVol equals 7.84+[1.08*left atrial filling volume] 2 [0.839*PVs%]). In 31 patients with no mitral regurgitation detected by color Doppler or continuous wave Doppler the estimated regurgitant volume was 4.3±6.6 mL. In the overall population the estimated regurgitant volume and the Doppler regurgitant volume correlated well with each other (R2=0.85; SEE, 11.5 mL; p<0.0001). The equation was 100% sensitive and 98% specific in detecting a regurgitant volume higher than 55 mL. The combination of the atrial filling volume and the systolic pulmonary vein time-velocity integral expressed as the percent of the total allows reliable estimation of the regurgitant volume in patients with mitral regurgitation. (c)2001 CHF, Inc.