A unified censored normal regression model for qPCR differential gene expression analysis

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is considered as the gold standard for accurate, sensitive, and fast measurement of gene expression. Prior to downstream statistical analysis, RT-qPCR fluorescence amplification curves are summarized into one single value, the quantification cycle (Cq). When RT-qPCR does not reach the limit of detection, the Cq is labeled as undetermined . Current state of the art qPCR data analysis pipelines acknowledge the importance of normalization for removing non-biological sample to sample variation in the Cq values. However, their strategies for handling undetermined Cq values are very ad hoc. We show that popular methods for handling undetermined values can have a severe impact on the downstream differential expression analysis. They introduce a considerable bias and suffer from a lower precision. We propose a novel method that unites preprocessing and differential expression analysis in a single statistical model that provides a rigorous way for handling undetermined Cq values. We compare our method with existing approaches in a simulation study and on published microRNA and mRNA gene expression datasets. We show that our method outperforms traditional RT-qPCR differential expression analysis pipelines in the presence of undetermined values, both in terms of accuracy and precision

Ibrutinib does not have clinically relevant interactions with oral contraceptives or substrates of CYP3A and CYP2B6

Ibrutinib may inhibitintestinal CYP3A4 and induce CYP2B6 and/or CYP3A. Secondary to potential induction, ibrutinib may reduce the exposure and effectiveness of oral contraceptives (OCs). This phase I study evaluated the effect of ibrutinib on the pharmacokinetics of the CYP2B6 substrate bupropion, CYP3A substrate midazolam, and OCs ethinylestradiol (EE) and levonorgestrel (LN). Female patients (N = 22) with B-cell malignancies received single doses of EE/LN (30/150 μg) and bupropion/midazolam (75/2 mg) during a pretreatment phase on days 1 and 3, respectively (before starting ibrutinib on day 8), and again after ibrutinib 560 mg/day for ≥ 2 weeks. Intestinal CYP3A inhibition was assessed on day 8 (single-dose ibrutinib plus single-dose midazolam). Systemic induction was assessed at steady-state on days 22 (EE/LN plus ibrutinib) and 24 (bupropion/midazolam plus ibrutinib). The geometric mean ratios (GMRs; test/reference) for maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) were derived using linear mixed-effects models (90% confidence interval within 80%-125% indicated no interaction). On day 8, the GMR for midazolam exposure with ibrutinib coadministration was ≤ 20% lower than the reference, indicating lack of intestinal CYP3A4 inhibition. At ibrutinib steady-state, the Cmax and AUC of EE were 33% higher than the reference, which was not considered clinically relevant. No substantial changes were noted for LN, midazolam, or bupropion. No unexpected safety findings were observed. A single dose of ibrutinib did not inhibit intestinal CYP3A4, and repeated administration did not induce CYP3A4/2B6, as assessed using EE, LN, midazolam, and bupropion

Public Deposits In Biological Resource Centres Is An Essential Part Of Fair Science

F A I R s c i e n c e a i m s a t s h a r i n g s c i e n t i f i c o u t p u t i n s u c h a w a y a s t o m a x i m i z e t h e a c c e s s , r e u s e a n d i m p a c t o f r e s e a r c h . T h i s a l l o w s t r a n s p a r e n c y o f r e s u l t s , r e p r o d u c i b i l i t y o f e x p e r i m e n t s , c u m u l a t i v e r e s e a r c h , a n d a v o i d s a w a s t e o f r e s s o u r c e s ( 1 ) . A l t h o u g h t h e F A I R d a t a p r i n c i p l e i s b e c o m i n g a w e l l - k n o w n c o n c e p t , l e s s a t t e n t i o n i s g i v e n t o i t s a p p l i c a t i o n t o b i o l o g i c a l r e s o u r c e s . I n l i f e s c i e n c e s , p u b l i c m i c r o b i a l - a n d p l a s m i d c o l l e c t i o n s r e p r e s e n t a h i s t o r i c a l e x a m p l e o f F A I R s c i e n c e , t h a n k s t o t h e i r l o n g s t a n d i n g e x p e r i e n c e i n t h e p r e s e r v a t i o n a n d d i s t r i b u t i o n o f l i v i n g m i c r o b i a l s t r a i n s a n d ( g ) D N A f o r f u r t h e r s c i e n t i f i c i n v e s t i g a t i o n s o r d e v e l o p m e n t , w h i l e r e s p e c t i n g ( i n t e r ) n a t i o n a l l e g i s l a t i o n s a n d c a s e - s p e c i f i c r e s t r i c t i o n s a s d e f i n e d b y t h e c l i e n t s . T h e s e b i o l o g i c a l r e s o u r c e c e n t r e s ( B R C ) p r o v i d e w e l l - c h a r a c t e r i z e d , q u a l i t y - c o n t r o l l e d a n d a u t h e n t i c a t e d s t r a i n s , p l a s m i d s a n d a s s o c i a t e d d a t a ( 2 ) . T h e y a l s o s u p p o r t t h e b i o - i n d u s t r y , f o r w h i c h t h e d i v e r s i t y o f n a t u r a l l y o c c u r r i n g o r g e n e t i c a l l y e n g i n e e r e d m i c r o o r g a n i s m s a r e a n i n v a l u a b l e s o u r c e o f a p p l i c a t i o n s . T h e r e s p o n s i b i l i t y t o d e p o s i t t h e m i c r o o r g a n i s m s a n d g e n e t i c r e s o u r c e s i n p u b l i c B R C s i s s h a r e d b y r e s e a r c h e r s , f u n d i n g a g e n c i e s a n d p u b l i s h e r s ( 2 ) . L i f e s c i e n t i s t s n e e d t o b e c o m e m o r e a w a r e o f t h e i m p o r t a n c e o f s t r a i n a n d p l a s m i d c o n s e r v a t i o n a n d g r o w a c c u s t o m e d t o d e p o s i t t h e m d u r i n g t h e p u b l i c a t i o n p r o c e s s o r a t t h e e n d o f p r o j e c t s . G o v e r n m e n t a l f u n d i n g p o l i c i e s s h o u l d r e q u e s t i n t h e i r c o n t r a c t s t h e d e p o s i t o f b i o l o g i c a l m a t e r i a l s i s o l a t e d o r c o n s t r u c t e d d u r i n g f i n a n c e d p r o j e c t s . R e g a r d i n g p u b l i s h e r s , m o s t j o u r n a l s e n c o u r a g e a u t h o r s t o d e p o s i t t h e i r d a t a s e t s ( c o d e s , s e q u e n c e s , e t c ) i n p u b l i c r e p o s i t o r i e s b u t v e r y f e w s p e c i f i c a l l y r e q u i r e d e p o s i t o f b i o l o g i c a l m a t e r i a l a n d c u l t i v a t e d s t r a i n s i n p u b l i c c o l l e c t i o n s . E d i t o r s s h o u l d t h e r e f o r e i m p l e m e n t m e c h a n i s m s f o r a c t i v e a g r e e m e n t b y a u t h o r s t o d e p o s i t s t r a i n s a n d o t h e r g e n e t i c r e s o u r c e s w h e n s u b m i t t i n g a n a r t i c l e , o r t o j u s t i f y w h y i t w o u l d n o t b e p o s s i b l e . S u c h m e c h a n i s m s c o u l d f o l l o w T r a n s p a r e n c y a n d O p e n n e s s P r o m o t i o n g u i d e l i n e s ( 3 ) f o r j o u r n a l s t h a t i n c l u d e s t a n d a r d s f o r research materials.Belgian Consortium of Collections of Microorganism

Green infrastructure can promote plant functional connectivity in a grassland species around fragmented semi‐natural grasslands in NW‐Europe

Species may benefit from green infrastructure, i.e. the network of natural and anthropogenic habitat remnants in human-dominated landscapes, if it helps isolated populations in remaining habitat patches to be functionally connected. The importance of green infrastructure is therefore increasingly emphasized in conservation policy to counter biodiversity loss. However, there is limited evidence, particularly in plants, that green infrastructure promotes functional connectivity, i.e. supports the colonization of habitat patches across a landscape. We applied landscape genetics to test whether the green infrastructure supports structural and functional connectivity in the grassland perennial Galium verum, in 35 landscapes in Belgium, Germany and Sweden. We used multivariate genetic clustering techniques, nestedness analyses and conditional inference trees to examine landscape-scale patterns in genetic diversity and structure of plant populations in the green infrastructure surrounding semi-natural grasslands. Inferred functional connectivity explained genetic variation better than structural connectivity, yielding positive effects on genetic variation. The road verge network, a major structural component of the green infrastructure and its functional connectivity, most effectively explained genetic diversity and composition in G. verum. Galium verum ramets occupying the surrounding landscape proved to be genetic subsets of focal grassland populations, shaping a nested landscape population genetic structure with focal grasslands, particularly ancient ones, harbouring unique genetic diversity. This nested pattern weakened as road network density increased, suggesting road verge networks enable high landscape occupancy by increased habitat availability and facilitates gene flow into the surrounding landscape. Our study proposes that green infrastructure can promote functional connectivity, providing that a plant species can survive outside of core habitat patches. As this often excludes habitat specialist species, conservation practice and policy should primarily focus on ancient, managed semi-natural grasslands. These grasslands both harbour unique genetic diversity and act as primary gene and propagule sources for the surrounding landscape, highlighting their conservation value

Stable reference genes for the measurement of transcript abundance during larval caste development in the honeybee

Many genes are differentially regulated by caste development in the honeybee. Identifying and understanding these differences is key to discovering the mechanisms underlying this process. To identify these gene expression differences requires robust methods to measure transcript abundance. RT-qPCR is currently the gold standard to measure gene expression, but requires stable reference genes to compare gene expression changes. Such reference genes have not been established for honeybee caste development. Here, we identify and test potential reference genes that have stable expression throughout larval development between the two female castes. In this study, 15 candidate reference genes were examined to identify the most stable reference genes. Three algorithms (GeNorm, Bestkeeper and NormFinder) were used to rank the candidate reference genes based on their stability between the castes throughout larval development. Of these genes Ndufa8 (the orthologue of a component of complex one of the mitochondrial electron transport chain) and Pros54 (orthologous to a component of the 26S proteasome) were identified as being the most stable. When these two genes were used to normalise expression of two target genes (previously found to be differentially expressed between queen and worker larvae by microarray analysis) they were able to more accurately detect differential expression than two previously used reference genes (awd and RpL12). The identification of these novel reference genes will be of benefit to future studies of caste development in the honeybee

Stearoyl-CoA Desaturase-1 (SCD1) Augments Saturated Fatty Acid-Induced Lipid Accumulation and Inhibits Apoptosis in Cardiac Myocytes

Mismatch between the uptake and utilization of long-chain fatty acids in the myocardium leads to abnormally high intracellular fatty acid concentration, which ultimately induces myocardial dysfunction. Stearoyl-Coenzyme A desaturase-1 (SCD1) is a rate-limiting enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids. Previous studies have shown that SCD1-deficinent mice are protected from insulin resistance and diet-induced obesity; however, the role of SCD1 in the heart remains to be determined. We examined the expression of SCD1 in obese rat hearts induced by a sucrose-rich diet for 3 months. We also examined the effect of SCD1 on myocardial energy metabolism and apoptotic cell death in neonatal rat cardiac myocytes in the presence of SFAs. Here we showed that the expression of SCD1 increases 3.6-fold without measurable change in the expression of lipogenic genes in the heart of rats fed a high-sucrose diet. Forced SCD1 expression augmented palmitic acid-induced lipid accumulation, but attenuated excess fatty acid oxidation and restored reduced glucose oxidation. Of importance, SCD1 substantially inhibited SFA-induced caspase 3 activation, ceramide synthesis, diacylglycerol synthesis, apoptotic cell death, and mitochondrial reactive oxygen species (ROS) generation. Experiments using SCD1 siRNA confirmed these observations. Furthermore, we showed that exposure of cardiac myocytes to glucose and insulin induced SCD1 expression. Our results indicate that SCD1 is highly regulated by a metabolic syndrome component in the heart, and such induction of SCD1 serves to alleviate SFA-induced adverse fatty acid catabolism, and eventually to prevent SFAs-induced apoptosis

An open-label, multicenter, phase Ib study investigating the effect of apalutamide on ventricular repolarization in men with castration-resistant prostate cancer

Purpose: Phase Ib study evaluating the effect of apalutamide, at therapeutic exposure, on ventricular repolarization by applying time-matched pharmacokinetics and electrocardiography (ECG) in patients with castration-resistant prostate cancer. Safety of daily apalutamide was also assessed. Methods: Patients received 240 mg oral apalutamide daily. Time-matched ECGs were collected via continuous 12-lead Holter recording before apalutamide (Day − 1) and on Days 1 and 57 (Cycle 3 Day 1). Pharmacokinetics of apalutamide were assessed on Days 1 and 57 at matched time points of ECG collection. QT interval was corrected for heart rate using Fridericia correction (QTcF). The primary endpoint was the maximum mean change in QTcF (ΔQTcF) from baseline to Cycle 3 Day 1 (steady state). Secondary endpoints were the effect of apalutamide on other ECG parameters, pharmacokinetics of apalutamide and its active metabolite, relationship between plasma concentrations of apalutamide and QTcF, and safety. Results: Forty-five men were enrolled; 82% received treatment for ≥ 3 months. At steady state, the maximum ΔQTcF was 12.4 ms and the upper bound of its associated 90% CI was 16.0 ms. No clinically meaningful effects of apalutamide were reported for heart rate or other ECG parameters. A concentration-dependent increase in QTcF was observed for apalutamide. Most adverse events (AEs) (73%) were grade 1–2 in severity. No patients discontinued due to QTc prolongation or AEs. Conclusion: The effect of apalutamide on QTc prolongation was modest and does not produce a clinically meaningful effect on ventricular repolarization. The AE profile was consistent with other studies of apalutamide

The c-Met tyrosine kinase inhibitor JNJ-38877605 causes renal toxicity through species-specific insoluble metabolite formation

Purpose: The receptor tyrosine kinase c-Met plays an important role in tumorigenesis and is a novel target for anticancer treatment. This phase I, first-in-human trial, explored safety, pharmacokinetics, pharmacodynamics, and initial antitumor activity of JNJ-38877605, a potent and selective c-Met inhibitor. Experimental Design: We performed a phase I dose-escalation study according to the standard 3+3 design. Results: Even at subtherapeutic doses, mild though recurrent renal toxicity was observed in virtually all patients. Renal toxicity had not been observed in preclinical studies in rats and dogs. Additional preclinical studies pointed toward the rabbit as a suitable toxicology model, as the formation of the M10 metabolite of JNJ-38877605 specifically occurred in rabbits and humans. Additional toxicology studies in rabbits clearly demonstrated that JNJ-38877605 induced species-specific renal toxicity. Histopathological evaluation in rabbits revealed renal crystal formation with degenerative and inflammatory changes. Identification of the components of these renal crystals revealed M1/3 and M5/6 metabolites. Accordingly, it was found that humans and rabbits showed significantly increased systemic exposure to these metabolites relative to other species. These main culprit insoluble metabolites were generated by aldehyde oxidase activity. Alternative dosing schedules of JNJ-3877605 and concomitant probenecid administration in rabbits failed to prevent renal toxicity at dose levels that could be pharmacologically active. Conclusions: Combined clinical and correlative preclinical studies suggest that renal toxicity of JNJ-38877605 is caused by the formation of species-specific insoluble metabolites. These observations preclude further clinical development of JNJ-38877605