PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites

Next-generation sequencing (NGS) studies have identified large numbers of genetic variants that are predicted to alter miRNA-mRNA interactions. We developed a novel high-throughput bioassay, PASSPORT-seq, that can functionally test in parallel 100s of these variants in miRNA binding sites (mirSNPs). The results are highly reproducible across both technical and biological replicates. The utility of the bioassay was demonstrated by testing 100 mirSNPs in HEK293, HepG2, and HeLa cells. The results of several of the variants were validated in all three cell lines using traditional individual luciferase assays. Fifty-five mirSNPs were functional in at least one of three cell lines (FDR ≤ 0.05); 11, 36, and 27 of them were functional in HEK293, HepG2, and HeLa cells, respectively. Only four of the variants were functional in all three cell lines, which demonstrates the cell-type specific effects of mirSNPs and the importance of testing the mirSNPs in multiple cell lines. Using PASSPORT-seq, we functionally tested 111 variants in the 3' UTR of 17 pharmacogenes that are predicted to alter miRNA regulation. Thirty-three of the variants tested were functional in at least one cell line

Pharmacogenetics and Practice: Tailoring Prescribing for Safety and Effectiveness

The promise of pharmacogenomics testing, to find the right medication at the right dose for the right patient at the right time, sits at the heart of precision medicine. Identifying genetic variants that contribute to inter-patient variability in drug disposition and effect allows clinicians to select a more appropriate medication for a patient’s condition by limiting adverse drug events and maximizing beneficial effects. However, as pharmacogenomics is increasingly integrated into prevention-based healthcare, a major obstacle to effective implementation of pharmacogenomics testing is the lack of adequate knowledge of healthcare providers on interpretation of these test results

Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes

Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1–3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observed in situ in human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism

Overexpression of a Prefoldin β subunit gene reduces biomass recalcitrance in the bioenergy crop Populus.

Prefoldin (PFD) is a group II chaperonin that is ubiquitously present in the eukaryotic kingdom. Six subunits (PFD1-6) form a jellyfish-like heterohexameric PFD complex and function in protein folding and cytoskeleton organization. However, little is known about its function in plant cell wall-related processes. Here, we report the functional characterization of a PFD gene from Populus deltoides, designated as PdPFD2.2. There are two copies of PFD2 in Populus, and PdPFD2.2 was ubiquitously expressed with high transcript abundance in the cambial region. PdPFD2.2 can physically interact with DELLA protein RGA1_8g, and its subcellular localization is affected by the interaction. In P. deltoides transgenic plants overexpressing PdPFD2.2, the lignin syringyl/guaiacyl ratio was increased, but cellulose content and crystallinity index were unchanged. In addition, the total released sugar (glucose and xylose) amounts were increased by 7.6% and 6.1%, respectively, in two transgenic lines. Transcriptomic and metabolomic analyses revealed that secondary metabolic pathways, including lignin and flavonoid biosynthesis, were affected by overexpressing PdPFD2.2. A total of eight hub transcription factors (TFs) were identified based on TF binding sites of differentially expressed genes in Populus transgenic plants overexpressing PdPFD2.2. In addition, several known cell wall-related TFs, such as MYB3, MYB4, MYB7, TT8 and XND1, were affected by overexpression of PdPFD2.2. These results suggest that overexpression of PdPFD2.2 can reduce biomass recalcitrance and PdPFD2.2 is a promising target for genetic engineering to improve feedstock characteristics to enhance biofuel conversion and reduce the cost of lignocellulosic biofuel production

Assessment of lymphatic filariasis prior to re-starting mass drug administration campaigns in coastal Kenya.

BACKGROUND: Lymphatic filariasis (LF) is a debilitating disease associated with extensive disfigurement and is one of a diverse group of diseases referred to as neglected tropical diseases (NTDs) which mainly occur among the poorest populations. In line with global recommendations to eliminate LF, Kenya launched its LF elimination programme in 2002 with the aim to implement annual mass drug administration (MDA) in order to interrupt LF transmission. However, the programme faced financial and administrative challenges over the years such that sustained annual MDA was not possible. Recently, there has been renewed interest to eliminate LF and the Kenyan Ministry of Health, through support from World Health Organization (WHO), restarted annual MDA in 2015. The objective of this study was to evaluate the current status of LF infection in the endemic coastal region of Kenya before MDA campaigns were restarted. RESULTS: Ten sentinel sites in Kwale, Kilifi, Tana River, Lamu, and Taita-Taveta counties in coastal Kenya were selected for participation in a cross-sectional survey of LF infection prevalence. At least 300 individuals in each sentinel village were sampled through random house-to-house visits. During the day, the point-of-care immunochromatographic test (ICT) was used to detect the presence of Wuchereria bancrofti circulating filarial antigen in finger prick blood samples collected from residents of the selected sentinel villages. Those individuals who tested positive with the ICT test were requested to provide a night-time blood sample for microfilariae (MF) examination. The overall prevalence of filarial antigenaemia was 1.3% (95% CI: 0.9-1.8%). Ndau Island in Lamu County had the highest prevalence (6.3%; 95% CI: 4.1-9.7%), whereas sites in Kilifi and Kwale counties had prevalences?<?1.7%. Mean microfilarial density was also higher in Ndau Island (234 MF/ml) compared to sentinel sites in Kwale and Kilifi counties (< 25 MF/ml). No LF infection was detected in Tana River and Taita-Taveta counties. Overall, more than 88% of the study participants reported to have used a bed net the previous night. CONCLUSIONS: Prevalence of LF infection is generally very low in coastal Kenya, but there remain areas that require further rounds of MDA if the disease is to be eliminated as a public health problem in line with the ongoing global elimination efforts. However, areas where there was no evidence of LF transmission should be considered for WHO-recommended transmission assessment surveys in view of stopping MDA

Implementation of a Renal Precision Medicine Program: Clinician Attitudes and Acceptance

A precision health initiative was implemented across a multi-hospital health system, wherein a panel of genetic variants was tested and utilized in the clinical care of chronic kidney disease (CKD) patients. Pharmacogenomic predictors of antihypertensive response and genomic predictors of CKD were provided to clinicians caring for nephrology patients. To assess clinician knowledge, attitudes, and willingness to act on genetic testing results, a Likert-scale survey was sent to and self-administered by these nephrology providers (N = 76). Most respondents agreed that utilizing pharmacogenomic-guided antihypertensive prescribing is valuable (4.0 ± 0.7 on a scale of 1 to 5, where 5 indicates strong agreement). However, the respondents also expressed reluctance to use genetic testing for CKD risk stratification due to a perceived lack of supporting evidence (3.2 ± 0.9). Exploratory sub-group analyses associated this reluctance with negative responses to both knowledge and attitude discipline questions, thus suggesting reduced exposure to and comfort with genetic information. Given the evolving nature of genomic implementation in clinical care, further education is warranted to help overcome these perception barriers

Genotype-Guided Hydralazine Therapy

Background: Despite its approval in 1953, hydralazine hydrochloride continues to be used in the management of resistant hypertension, a condition frequently managed by nephrologists and other clinicians. Hydralazine hydrochloride undergoes metabolism by the N-acetyltransferase 2 (NAT2) enzyme. NAT2 is highly polymorphic as approximately 50% of the general population are slow acetylators. In this review, we first evaluate the link between NAT2 genotype and phenotype. We then assess the evidence available for genotype-guided therapy of hydralazine, specifically addressing associations of NAT2 acetylator status with hydralazine pharmacokinetics, antihypertensive efficacy, and toxicity. Summary: There is a critical need to use hydralazine in some patients with resistant hypertension. Available evidence supports a significant link between genotype and NAT2 enzyme activity as 29 studies were identified with an overall concordance between genotype and phenotype of 92%. The literature also supports an association between acetylator status and hydralazine concentration, as fourteen of fifteen identified studies revealed significant relationships with a consistent direction of effect. Although fewer studies are available to directly link acetylator status with hydralazine antihypertensive efficacy, the evidence from this smaller set of studies is significant in 7 of 9 studies identified. Finally, 5 studies were identified which support the association of acetylator status with hydralazine-induced lupus. Clinicians should maintain vigilance when prescribing maximum doses of hydralazine. Key Messages: NAT2 slow acetylator status predicts increased hydralazine levels, which may lead to increased efficacy and adverse effects. Caution should be exercised in slow acetylators with total daily hydralazine doses of 200 mg or more. Fast acetylators are at risk for inefficacy at lower doses of hydralazine. With appropriate guidance on the usage of NAT2 genotype, clinicians can adopt a personalized approach to hydralazine dosing and prescription, enabling more efficient and safe treatment of resistant hypertension

Toward a chemical reanalysis in a coupled chemistry-climate model: an evaluation of MOPITT CO assimilation and its impact on tropospheric composition

We examine in detail a 1 year global reanalysis of carbon monoxide (CO) that is based on joint assimilation of conventional meteorological observations and Measurement of Pollution in The Troposphere (MOPITT) multispectral CO retrievals in the Community Earth System Model (CESM). Our focus is to assess the impact to the chemical system when CO distribution is constrained in a coupled full chemistry-climate model like CESM. To do this, we first evaluate the joint reanalysis (MOPITT Reanalysis) against four sets of independent observations and compare its performance against a reanalysis with no MOPITT assimilation (Control Run). We then investigate the CO burden and chemical response with the aid of tagged sectoral CO tracers. We estimate the total tropospheric CO burden in 2002 (from ensemble mean and spread) to be 371 ± 12% Tg for MOPITT Reanalysis and 291 ± 9% Tg for Control Run. Our multispecies analysis of this difference suggests that (a) direct emissions of CO and hydrocarbons are too low in the inventory used in this study and (b) chemical oxidation, transport, and deposition processes are not accurately and consistently represented in the model. Increases in CO led to net reduction of OH and subsequent longer lifetime of CH4 (Control Run: 8.7 years versus MOPITT Reanalysis: 9.3 years). Yet at the same time, this increase led to 5-10% enhancement of Northern Hemisphere O3 and overall photochemical activity via HOx recycling. Such nonlinear effects further complicate the attribution to uncertainties in direct emissions alone. This has implications to chemistry-climate modeling and inversion studies of longer-lived species

Characteristics and Predictive Value of Blood Transcriptome Signature in Males with Autism Spectrum Disorders

Autism Spectrum Disorders (ASD) is a spectrum of highly heritable neurodevelopmental disorders in which known mutations contribute to disease risk in 20% of cases. Here, we report the results of the largest blood transcriptome study to date that aims to identify differences in 170 ASD cases and 115 age/sex-matched controls and to evaluate the utility of gene expression profiling as a tool to aid in the diagnosis of ASD. The differentially expressed genes were enriched for the neurotrophin signaling, long-term potentiation/depression, and notch signaling pathways. We developed a 55-gene prediction model, using a cross-validation strategy, on a sample cohort of 66 male ASD cases and 33 age-matched male controls (P1). Subsequently, 104 ASD cases and 82 controls were recruited and used as a validation set (P2). This 55-gene expression signature achieved 68% classification accuracy with the validation cohort (area under the receiver operating characteristic curve (AUC): 0.70 [95% confidence interval [CI]: 0.62–0.77]). Not surprisingly, our prediction model that was built and trained with male samples performed well for males (AUC 0.73, 95% CI 0.65–0.82), but not for female samples (AUC 0.51, 95% CI 0.36–0.67). The 55-gene signature also performed robustly when the prediction model was trained with P2 male samples to classify P1 samples (AUC 0.69, 95% CI 0.58–0.80). Our result suggests that the use of blood expression profiling for ASD detection may be feasible. Further study is required to determine the age at which such a test should be deployed, and what genetic characteristics of ASD can be identified