Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.     

Hepatic Insulin Resistance in Obese Non-Diabetic Subjects and in Type 2 Diabetic Patients

Objective: Obese non-diabetic patients are characterized by an extra-hepatic insulin resistance. Whether obese patients also have decreased hepatic insulin sensitivity remains controversial. Research Methods and Procedures: To estimate their hepatic insulin sensitivity, we measured the rate of exogenous insulin infusion required to maintain mildly elevated glycemia in obese patients with type 2 diabetes, obese non-diabetic patients, and lean control subjects during constant infusions of somatostatin and physiological low-glucagon replacement infusions. To account for differences in insulin concentrations among the three groups of subjects, an additional protocol was also performed in healthy lean subjects with higher insulin infusion rates and exogenous dextrose infusion. Results: The insulin infusion rate required to maintain glycemia at 8.5 mM was increased 4-fold in obese patients with type 2 diabetes and 1.5-fold in obese non-diabetic patients. The net endogenous glucose production (measured with 6,6-²H₂-glucose) and total glucose output (measured with 2-²H₁-glucose) were ∼30% lower in the patients than in the lean subjects. Net endogenous glucose production and total glucose output were both markedly increased in both groups of obese patients compared with lean control subjects during hyperinsulinemia. Discussion: Our data indicate that both obese non-diabetic and obese type 2 diabetic patients have a blunted suppressive action of insulin on glucose production, indicating hepatic and renal insulin resistance.     

A comparison of the multilevel MIMIC model to the multilevel regression and mixed ANOVA model for the estimation and testing of a cross-level interaction effect: A simulation study

When observing data on a patient-reported outcome measure in, for example, clinical trials, the variables observed are often correlated and intended to measure a latent variable. In addition, such data are also often characterized by a hierarchical structure, meaning that the outcome is repeatedly measured within patients. To analyze such data, it is important to use an appropriate statistical model, such as structural equation modeling (SEM). However, researchers may rely on simpler statistical models that are applied to an aggregated data structure. For example, correlated variables are combined into one sum score that approximates a latent variable. This may have implications when, for example, the sum score consists of indicators that relate differently to the latent variable being measured. This study compares three models that can be applied to analyze such data: the multilevel multiple indicators multiple causes (ML-MIMIC) model, a univariate multilevel model, and a mixed analysis of variance (ANOVA) model. The focus is on the estimation of a cross-level interaction effect that presents the difference over time on the patient-reported outcome between two treatment groups. The ML-MIMIC model is an SEM-type model that considers the relationship between the indicators and the latent variable in a multilevel setting, whereas the univariate multilevel and mixed ANOVA model rely on sum scores to approximate the latent variable. In addition, the mixed ANOVA model uses aggregated second-level means as outcome. This study showed that the ML-MIMIC model produced unbiased cross-level interaction effect estimates when the relationships between the indicators and the latent variable being measured varied across indicators. In contrast, under similar conditions, the univariate multilevel and mixed ANOVA model underestimated the cross-level interaction effect.     

Predictors for length of hospital stay in patients with community-acquired Pneumonia: Results from a Swiss Multicenter study

ABSTRACT: BACKGROUND: Length of hospital stay (LOS) in patients with community-acquired pneumonia (CAP) is variable and directly related to medical costs. Accurate estimation of LOS on admission and during follow-up may result in earlier and more efficient discharge strategies. METHODS: This is a prospective multicenter study including patients in emergency departments of 6 tertiary care hospitals in Switzerland between October 2006 and March 2008. Medical history, clinical data at presentation and health care insurance class were collected. We calculated univariate and multivariate cox regression models to assess the association of different characteristics with LOS. In a split sample analysis, we created two LOS prediction rules, first including only admission data, and second including also additional inpatient information. RESULTS: The mean LOS in the 875 included CAP patients was 9.8 days (95%CI 9.3-10.4). Older age, respiratory rate >20pm, nursing home residence, chronic pulmonary disease, diabetes, multilobar CAP and the pneumonia severity index class were independently associated with longer LOS in the admission prediction model. When also considering follow-up information, low albumin levels, ICU transfer and development of CAP-associated complications were additional independent risk factors for prolonged LOS. Both weighted clinical prediction rules based on these factors showed a high separation of patients in Kaplan Meier Curves (p logrank <0.001 and <0.001) and a good calibration when comparing predicted and observed results. CONCLUSIONS: Within this study we identified different baseline and follow-up characteristics to be strong and independent predictors for LOS. If validated in future studies, these factors may help to optimize discharge strategies and thus shorten LOS in CAP patients.     

Thermography imaging during static and controlled thermoregulation in complex regional pain syndrome type 1: diagnostic value and involvement of the central sympathetic system

Background  

Complex Regional Pain Syndrome type 1 (CRPS1) is a clinical diagnosis based on criteria describing symptoms of the disease.     

Intracolonial social distance, signaling modality and association choice in the greater sac-winged bat (Saccopteryx bilineata)

Understanding patterns and motivations for social spacing greatly illuminates the structure and internal dynamics of given groups or social systems. The highly social, polygynous greater sac-winged bat, Saccopteryx bilineata, represents an excellent model for social spacing analysis, since the choice of individual roost-sites within a day-roost constitutes an enduring, often repeated decision about relative proximity to conspecifics. Day-roost colonies consist of one or multiple harem groups, each with several females and a single male. Additionally, non-harem males without females may be present. A social-distance–time-budget metric revealed that harem males, females, and non-harem males differed significantly in their respective spatial associations while roosting. Harem males and females were most closely associated, with harem males located at the center of harems instead of at the borders. Non-harem males associated significantly closer with one another than with harem females that they were trying to access. The signaling modality mediating social interactions depended on the respective social distances between communicating bats. Our results suggest a concentric social organization based around harem males, which may select roost sites in closest possible proximity to females to enhance courtship signal strength. This constitutes an interesting deviation from the normative form of harem maintenance, patrolling borders, in mammals.     

Induced responses of nestling great tits reduce hen flea reproduction

The dynamics of host–parasite interactions depend to a large extent on the effect of host responses on parasite fitness. Exposure to parasites may induce behavioural or physiological responses in hosts that may reduce the subsequent survival or reproductive output of the parasite. Neonate hosts may further directly obtain immunologically active substances from their mother, for instance via milk in mammals or egg yolk in birds. However, the relative importance of maternally‐derived and self‐generated responses in inducing parasite resistance is poorly understood, especially in free‐living vertebrates. Here we investigate the complementary effect of experimentally induced maternal and neonate responses in great tit (Parus major) hosts on the reproductive success of their common ectoparasite, the hen flea (Ceratophyllus gallinae). In the laboratory we measured the number of eggs and larvae produced by individual flea females collected from host nests. In addition, the total number of larvae produced by an experimentally set number of flea females in the host's nestbox was assessed under field conditions. There was no indication of maternally‐transferred parasite resistance, since exposing the mother to fleas during the laying period did not affect the reproductive rate of fleas exploiting her offspring early or late in the nestling cycle. Independent of the maternal treatment, exposure of neonates to fleas early in the nestling period reduced the reproductive output of fleas late in the nestling cycle. The effect of the induced nestling response was seasonal, reducing flea reproduction in nests of early‐breeding hosts but not in nests of late‐breeding ones. Larvae production in the nestbox and in the laboratory was positively correlated, but under natural conditions the neonate response did not affect the size of the flea larvae population. Our results indicate induced responses as a means by which neonate avian hosts resist ectoparasites. Other factors, such as the environmental temperature and density‐dependent larval competition, may be more important in determining the size of the future parasite populations.