Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries

Abstract The potential public health impact of foodborne parasites transmitted via contaminated fresh produces indicates the necessity for robust and reliable laboratory methods for their detection and identification on this infection vehicle. Standardization of methods for detection of common FBP in fresh produce is to be expected and ensuring that the DNA extraction approach is most appropriate for the FBP of interest and for the matrix being analyzed is also important. Therefore, the aim of the present study was to compare the efficacy of two commercially available DNA extraction procedures, the UNEX-based method and DNeasy PowerSoil kit in the detection of three protozoan parasites, C. cayetanensis, C. parvum, and T. gondii, on contaminated berries. Oocysts of each parasite were spiked into the pellets of raspberry and blueberry washes. The spiked pellets were then randomly assigned to DNA extraction using either the PowerSoil or UNEX method, with DNA extraction with both methods performed by two independent analysts. The detection rate when berry washes were spiked with 20 oocysts of C. cayetanensis, T. gondii, and C. parvum was 95%, 85%, and 40%, respectively, when using the PowerSoil kit; whereas the equivalent results using the UNEX method were 55%, 60%, and 5%, respectively. In addition, significantly lower Cq values were achieved for each parasite in the samples spiked with 500 oocysts when the PowerSoil kit was used. Possible reasons for these results are discussed, and include the composition of both the beads and the buffers in each method

Treatment against helminths in Norwegian sheep: a questionnaire-based survey

A questionnaire was distributed to 5487 farmers throughout Norway in order to obtain information about management practices regarding helminth infections in sheep. In addition, the farmers' perceptions of helminths and anthelmintic efficacy were investigated. Most farmers (80%) treated prophylactically against nematodes, and 24% also used prophylactic treatment against Fasciola hepatica. Overall, few farmers (11%) used parasitological analysis as a tool to assess the timing of treatment, but rather based it on other factors such as previous experience (70%). In the surveyed sheep flocks, the use of benzimidazoles was reduced from 2018 (52%) to 2019 (47%) (p < 0.01), whereas the use of macrocyclic lactones increased from 2017 (23%) to 2019 (36%) (p < 0.001). Poor anthelmintic efficacy was suspected by 10% of the farmers, and 11% reported that helminths were an increasing problem in their flocks. The majority of farmers (72%) considered their veterinarian as the most important advisor for treatment of parasites, but reported a high level of uncertainty regarding which parasites were present in their flocks, with unknown status most frequently reported for Haemonchus contortus (71.5%). This is probably related to the fact that very few farmers (15%) regularly test their animals for parasites. The present study provides up-to-date information on treatment practices for helminths in Norwegian sheep flocks

Pre- and post-race intestinal microbiota in long-distance sled dogs and associations with performance

Although our understanding of the role of the gut microbiota in different diseases is improving, our knowledge regarding how the gut microbiota affects functioning in healthy individuals is still limited. Here, we hypothesize that the gut microbiota could be associated with sled dog endurance-race performance. We investigated the gut microbiota in 166 fecal samples from 96 Alaskan Huskies, representing 16 teams participating in the 2016 Femund Race (400 km) in Norway, relating the microbiota composition to performance and metadata derived from questionnaires. For 16S rRNA gene sequencing-derived compositional data, we found a strong negative association between Enterobacteriaceae (dysbiosis-associated) and Clostridium hiranonis (normobiosis-associated). The teams with the best performances showed both the lowest levels of dysbiosis-associated bacteria prior to the race and the lowest change (decrease) in these bacteria after the race. Taken together, our results support the hypothesis that normobiosis-associated bacteria are involved in resilience mechanisms, potentially preventing growth of Enterobacteriaceae during the race.publishedVersio

Meta-analysis of whole-exome sequencing data from two independent cohorts finds no evidence for rare variant enrichment in Parkinson disease associated loci

Parkinson disease (PD) is a complex neurodegenerative disorder influenced by both environmental and genetic factors. While genome wide association studies have identified several susceptibility loci, many causal variants and genes underlying these associations remain undetermined. Identifying these is essential in order to gain mechanistic insight and identify biological pathways that may be targeted therapeutically. We hypothesized that gene-based enrichment of rare mutations is likely to be found within susceptibility loci for PD and may help identify causal genes. Whole-exome sequencing data from two independent cohorts were analyzed in tandem and by meta-analysis and a third cohort genotyped using the NeuroX-array was used for replication analysis. We employed collapsing methods (burden and the sequence kernel association test) to detect gene-based enrichment of rare, protein-altering variation within established PD susceptibility loci. Our analyses showed trends for three genes (GALC, PARP9 and SEC23IP), but none of these survived multiple testing correction. Our findings provide no evidence of rare mutation enrichment in genes within PD-associated loci, in our datasets. While not excluding that rare mutations in these genes may influence the risk of idiopathic PD, our results suggest that, if such effects exist, much larger sequencing datasets will be required for their detection.publishedVersio

Common gene expression signatures in Parkinson’s disease are driven by changes in cell composition

The etiology of Parkinson’s disease is largely unknown. Genome-wide transcriptomic studies in bulk brain tissue have identified several molecular signatures associated with the disease. While these studies have the potential to shed light into the pathogenesis of Parkinson’s disease, they are also limited by two major confounders: RNA post-mortem degradation and heterogeneous cell type composition of bulk tissue samples. We performed RNA sequencing following ribosomal RNA depletion in the prefrontal cortex of 49 individuals from two independent case-control cohorts. Using cell type specific markers, we estimated the cell type composition for each sample and included this in our analysis models to compensate for the variation in cell type proportions. Ribosomal RNA depletion followed by capture by random primers resulted in substantially more even transcript coverage, compared to poly(A) capture, in post-mortem tissue. Moreover, we show that cell type composition is a major confounder of differential gene expression analysis in the Parkinson’s disease brain. Accounting for cell type proportions attenuated numerous transcriptomic signatures that have been previously associated with Parkinson’s disease, including vesicle trafficking, synaptic transmission, immune and mitochondrial function. Conversely, pathways related to endoplasmic reticulum, lipid oxidation and unfolded protein response were strengthened and surface as the top differential gene expression signatures in the Parkinson’s disease prefrontal cortex. Our results indicate that differential gene expression signatures in Parkinson’s disease bulk brain tissue are significantly confounded by underlying differences in cell type composition. Modeling cell type heterogeneity is crucial in order to unveil transcriptomic signatures that represent regulatory changes in the Parkinson’s disease brain and are, therefore, more likely to be associated with underlying disease mechanisms.publishedVersio

Meta-analysis of whole-exome sequencing data from two independent cohorts finds no evidence for rare variant enrichment in Parkinson disease associated loci

Parkinson disease (PD) is a complex neurodegenerative disorder influenced by both environmental and genetic factors. While genome wide association studies have identified several susceptibility loci, many causal variants and genes underlying these associations remain undetermined. Identifying these is essential in order to gain mechanistic insight and identify biological pathways that may be targeted therapeutically. We hypothesized that gene-based enrichment of rare mutations is likely to be found within susceptibility loci for PD and may help identify causal genes. Whole-exome sequencing data from two independent cohorts were analyzed in tandem and by meta-analysis and a third cohort genotyped using the NeuroX-array was used for replication analysis. We employed collapsing methods (burden and the sequence kernel association test) to detect gene-based enrichment of rare, protein-altering variation within established PD susceptibility loci. Our analyses showed trends for three genes (GALC, PARP9 and SEC23IP), but none of these survived multiple testing correction. Our findings provide no evidence of rare mutation enrichment in genes within PD-associated loci, in our datasets. While not excluding that rare mutations in these genes may influence the risk of idiopathic PD, our results suggest that, if such effects exist, much larger sequencing datasets will be required for their detection.publishedVersio

Parasite detection in food:Current status and future needs for validation

Background Many parasites (protozoa and helminths) can be transmitted through food and lead to infections with high morbidity, as well as disease outbreaks. Although the importance of foodborne parasites (FBP) is recognised by many sectors of the food industry, standardized analytical methods and validation procedures for testing food for FBP are lacking. Scope and approach:Current methods for detection of FBP, and their validation, are critically reviewed, focusing on priority FBP in Europe: the helminths Echinococcus multilocularis, Echinococcus granulosus, Taenia saginata, Trichinella spp., and Anisakidae, and the protozoa Toxoplasma gondii, Cryptosporidium spp., and Giardia duodenalis. Key findings and conclusions:Standard methods exist for detection of T. saginata in beef, and Trichinella spp. in meat (and are mandatory at meat inspection in Europe), Anisakidae in fish, and Cryptosporidium spp. and G. duodenalis in leafy green vegetables and berry fruits. For other FBP or foods, methods used in sample surveys have been described, but validation data are generally absent; limits of detection are not provided, ring trials have rarely been performed, and for most FBP quality control materials, proficiency schemes, and reference standards are lacking. The use of surrogate particles or organisms for method development or validation purposes needs to be carefully considered. Documented procedures for validation, such as ISO17468 and ISO16140-2:2016 that were established for bacteria, are mostly inappropriate for FBP. The development and application of standardized and validated detection methods would enhance understanding of the foodborne route of transmission, improve risk assessments, and help identify and verify critical control points.Peer Reviewe

The NADPARK study: A randomized phase I trial of nicotinamide riboside supplementation in Parkinson’s disease

We conducted a double-blinded phase I clinical trial to establish whether nicotinamide adenine dinucleotide (NAD) replenishment therapy, via oral intake of nicotinamide riboside (NR), is safe, augments cerebral NAD levels, and impacts cerebral metabolism in Parkinson’s disease (PD). Thirty newly diagnosed, treatment-naive patients received 1,000 mg NR or placebo for 30 days. NR treatment was well tolerated and led to a significant, but variable, increase in cerebral NAD levels—measured by 31phosphorous magnetic resonance spectroscopy—and related metabolites in the cerebrospinal fluid. NR recipients showing increased brain NAD levels exhibited altered cerebral metabolism, measured by 18fluoro-deoxyglucose positron emission tomography, and this was associated with mild clinical improvement. NR augmented the NAD metabolome and induced transcriptional upregulation of processes related to mitochondrial, lysosomal, and proteasomal function in blood cells and/or skeletal muscle. Furthermore, NR decreased the levels of inflammatory cytokines in serum and cerebrospinal fluid. Our findings nominate NR as a potential neuroprotective therapy for PD, warranting further investigation in larger trials.publishedVersio