The cloning and characterisation of the chicken tyrosinase-related protein gene family

Very little is known about the molecular and genetic mechanisms controlling pigmentation within the bird kingdom. The aim therefore, of this study was to contribute towards the understanding of the genetic regulation of avian pigmentation by the cloning and characterisation of the chicken Tyrosinase-related protein (TRP) gene family. To accomplish this goal, neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. Using RNA extracted from these pigmented melanocyte cultures, a novel embryonic chick cDNA library was constructed. Screening of this library for chicken equivalents of the mammalian TRP gene family yielded more than 200 cDNA clones. After sequencing, three of these clones, 88.3, pcTRP- 1.6 and pcTRP -2. 10, were found to encode chicken Tyrosinase (Tyr), Tyrosinase-related protein-1 (Tyrp1) and Tyrosinase-related protein-2 (Tyrp2), respectively. In addition, a chicken Microphthalmia (Mi) cDNA clone (M156) was isolated using a mouse Mi cDNA probe. Comparative analyses revealed that chicken Tyr, Tyrp1 and Tyrp2 share approximately 68%, 72% and 70% amino acid sequence identity with their vertebrate orthologues. Northern blot hybridisation analysis demonstrated that the chicken TRPs are expressed in RNA from cultured retinal pigment epithelial (RPE) cells as well as in whole eye RNA. The major transcript sizes for the chicken Tyr, Tyrp1 and Tyrp2 genes are 2.5 kb, 2.3 kb and 3.5 kb, respectively. In situ hybridisation studies confirmed that both chicken Tyr and Tyrp2 genes are expressed in a pigment cell-specific fashion with signals detected in both the skin and RPE of chick embryos. Genomic Southern blot hybridisation analyses strongly suggested that all three chicken TRP genes contain several introns that are likely to be conserved within the vertebrate TRP gene family. Furthermore, the chick Tyr, Tyrp1 and Tyrp2 genes were found to span approximately 5-19 kb, 5-11 kb and 15-30 kb, respectively of the chicken genome. Comparisons between a black and white chick breed at the Tyr and Tyrp1 loci revealed no gross rearrangements at either of these loci. However, 1-2 kb alterations were observed between the same breeds at the Tyrp2 locus. The nature and significance of this alteration is not known. The cloning of the chicken Tyr, Tyrp1 and Tyrp2 cDNAs constitutes the first molecular cloning and characterisation of any avian TRP gene family. Taken together therefore, this study contributes towards the further understanding of the molecular mechanisms regulating pigmentation as well as the evolution of gene families

Forkhead box F2 Regulation of Platelet-Derived Growth Factor and myocardin/Serum Response Factor Signaling is Essential for Intestinal Development

Alterations in the forkhead box F2 gene expression have been reported in numerous pathologies, and Foxf2−/− mice are perinatal lethal with multiple malformations; however, molecular mechanisms pertaining to Foxf2 signaling are severely lacking. In this study, Foxf2 requirements in murine smooth muscle cells were examined using a conditional knock-out approach. We generated novel Foxf2-floxed mice, which we bred to smMHC-Cre-eGFP mice to generate a mouse line with Foxf2 deleted specifically from smooth muscle. These mice exhibited growth retardation due to reduced intestinal length as well as inflammation and remodeling of the small intestine. Colons of Tg(smMHC-Cre-eGFP+/−);Foxf2−/− mice had expansion of the myenteric nerve plexus and increased proliferation of smooth muscle cells leading to thickening of the longitudinal smooth muscle layer. Foxf2 deficiency in colonic smooth muscle was associated with increased expression of Foxf1, PDGFa, PDGFb, PDGF receptor α, and myocardin. FOXF2 bound to promoter regions of these genes indicating direct transcriptional regulation. Foxf2 repressed Foxf1 promoter activity in co-transfection experiments. We also show that knockdown of Foxf2 in colonic smooth muscle cells in vitro and in transgenic mice increased myocardin/serum response factor signaling and increased expression of contractile proteins. Foxf2 attenuated myocardin/serum response factor signaling in smooth muscle cells through direct binding to the N-terminal region of myocardin. Our results indicate that Foxf2 signaling in smooth muscle cells is essential for intestinal development and serum response factor signaling

Microplastic Concentrations in Orange City Wastewater

Microplastics are increasingly polluting both terrestrial and aquatic ecosystems. Researchers have found that wastewater treatment plants are an entry point for microplastics into surface waters, and we wondered how effective our local wastewater treatment plant is in removing microplastics from wastewater, given that it was not engineered for the removal of microplastics. We sampled effluent water from the new wastewater treatment plant (WWTP) in Orange City, Iowa, to determine the amount of microplastics released from the plant into Orange City Slough. We found concentrations of 498 pieces of microplastic/cubic meter (0.498/L) in the effluent water, which is similar to published values for secondary wastewater treatment plants. Given that microplastics have been found in human blood and have been shown to cause various health effects in humans and other animals, we propose that WWTPs be engineered to prevent the release of microplastics into surface waters

The Effect of Cold Stratification on the Germination of Grassland Seeds

One factor that must be considered when reconstructing a prairie is how the prairie seeds being planted need to be prepared for germination. The probability of successful germination of these seeds is dependent on many factors, including exposure to cold temperatures for a prolonged period of time. To explore this idea, we collected seeds from 13 species of forbs and grasses, both native and non-native, and stored them at various temperatures for several weeks. We predicted that the germination of the seeds of native grassland species would be enhanced by cold stratification, while non-natives (especially forbs common to flower gardens) could be negatively affected by cold stratification, especially if they are native to an area with less-extreme winters. We also predicted that seeds stored at -80°C (a temperature much lower than they would experience in nature) would respond negatively to the treatment and be unable to germinate. We found that four species (Sow Thistle, Penstemon, Queen Anne’s Lace, and Yellow Foxtail) were significantly affected by the seeds being chilled or frozen, with non-native sow thistle and yellow foxtail responding negatively to being frozen and native foxglove penstemon germinating best at -80°C

Invertebrate Pitfall Surveys at Glacial Hills Preserve and Buena Vista County Park

Invertebrates are integral members of the ecosystems they inhabit. However, they are often overlooked and understudied. We performed two pitfall surveys in Buena Vista County in September 2021 to study the diversity and abundance of invertebrate species in the presence and absence of two invasive species: Eastern Redcedar (Juniperus virginiana) and Garlic Mustard (Alliaria petiola). We collected pitfall samples and brought them back to the lab for identification. We found no significant differences in invertebrate abundance between cedar and non-cedar samples, nor between mustard and non-mustard samples. We propose further research focusing on seasonal changes, as these invasives may have more impacts at certain times of year than others

Preprocessing and Quality Control Strategies for Illumina DASL Assay-Based Brain Gene Expression Studies with Semi-Degraded Samples

Available statistical preprocessing or quality control analysis tools for gene expression microarray datasets are known to greatly affect downstream data analysis, especially when degraded samples, unique tissue samples, or novel expression assays are used. It is therefore important to assess the validity and impact of the assumptions built in to preprocessing schemes for a dataset. We developed and assessed a data preprocessing strategy for use with the Illumina DASL-based gene expression assay with partially degraded postmortem prefrontal cortex samples. The samples were obtained from individuals with autism as part of an investigation of the pathogenic factors contributing to autism. Using statistical analysis methods and metrics such as those associated with multivariate distance matrix regression and mean inter-array correlation, we developed a DASL-based assay gene expression preprocessing pipeline to accommodate and detect problems with microarray-based gene expression values obtained with degraded brain samples. Key steps in the pipeline included outlier exclusion, data transformation and normalization, and batch effect and covariate corrections. Our goal was to produce a clean dataset for subsequent downstream differential expression analysis. We ultimately settled on available transformation and normalization algorithms in the R/Bioconductor package lumi based on an assessment of their use in various combinations. A log2-transformed, quantile-normalized, and batch and seizure-corrected procedure was likely the most appropriate for our data. We empirically tested different components of our proposed preprocessing strategy and believe that our results suggest that a preprocessing strategy that effectively identifies outliers, normalizes the data, and corrects for batch effects can be applied to all studies, even those pursued with degraded samples

Gene expression profiling of human whole blood samples with the Illumina WG-DASL assay

<p>Abstract</p> <p>Background</p> <p>Microarray-based gene expression analysis of peripheral whole blood is a common strategy in the development of clinically relevant biomarker panels for a variety of human diseases. However, the results of such an analysis are often plagued by decreased sensitivity and reliability due to the effects of relatively high levels of globin mRNA in whole blood. Globin reduction assays have been shown to overcome such effects, but they require large amounts of total RNA and may induce distinct gene expression profiles. The Illumina whole genome DASL assay can detect gene expression levels using partially degraded RNA samples and has the potential to detect rare transcripts present in highly heterogeneous whole blood samples without the need for globin reduction. We assessed the utility of the whole genome DASL assay in an analysis of peripheral whole blood gene expression profiles.</p> <p>Results</p> <p>We find that gene expression detection is significantly increased with the use of whole genome DASL compared to the standard IVT-based direct hybridization. Additionally, globin-probe negative whole genome DASL did not exhibit significant improvements over globin-probe positive whole genome DASL. Globin reduction further increases the detection sensitivity and reliability of both whole genome DASL and IVT-based direct hybridization with little effect on raw intensity correlations. Raw intensity correlations between total RNA and globin reduced RNA were 0.955 for IVT-based direct hybridization and 0.979 for whole genome DASL.</p> <p>Conclusions</p> <p>Overall, the detection sensitivity of the whole genome DASL assay is higher than the IVT-based direct hybridization assay, with or without globin reduction, and should be considered in conjunction with globin reduction methods for future blood-based gene expression studies.</p

Genetic Annotation of Bacteriophages MScarn, Knocker, and Neos5

We annotated the genomes of three recently discovered bacteriophages to learn more about their genetic composition. MScarn is a lytic bacteriophage that infects Gordonia terrae 3612. It was discovered and purified from soil collected in Iroquois, SD. MScarn is a cluster CT phage, one of only 37 discovered to date. Its genome is 45,677 base pairs long and has 10-nucleotide 3’ sticky overhanging ends. Its GC content is 60.3% which is typical of CT cluster members. Knocker is a cluster B9 phage that was isolated on the host Mycobacterium smegmatis mc²155 from soil collected in Watertown, SD. Its circularly permuted genome contains 71,459 base pairs, and it has a high GC content of 69.7%. Similar to the other three members of the B9 cluster, it exhibits a lytic life cycle. Neos5, a lytic bacteriophage, was also isolated on Mycobacterium smegmatis mc²155 from soil collected in Baltimore, MD. It is a cluster B3 phage with a circularly permuted genome of 68,886 base-pairs and a 67.5% GC content, synonymous to the other 37 members of the cluster. All three phages were discovered, purified, and annotated by Northwestern College students

Outcome Measures in Rheumatology - Interventions for medication Adherence (OMERACT-Adherence) Core Domain Set for Trials of Interventions for Medication Adherence in Rheumatology: 5 Phase Study Protocol

Background: Over the last 20 years, there have been marked improvements in the availability of effective medications for rheumatic conditions such as gout, osteoporosis and rheumatoid arthritis (RA), which have led to a reduction in disease flares and the risk of re-fracture in osteoporosis, and the slowing of disease progression in RA. However, medication adherence remains suboptimal, as treatment regimens can be complex and difficult to continue long term. Many trials have been conducted to improve adherence to medication. Core domains, which are the outcomes of most relevance to patients and clinicians, are a pivotal component of any trial. These core domains should be measured consistently, so that all relevant trials can be combined in systematic reviews and meta-analyses to reach conclusions that are more valid. Failure to do this severely limits the potential for trial-based evidence to inform decisions on how to support medication adherence. The Outcome Measures in Rheumatology (OMERACT) - Interventions for Medication Adherence study by the OMERACT-Adherence Group aims to develop a core domain set for interventions that aim to support medication adherence in rheumatology. Methods/design: This OMERACT-Adherence study has five phases: (1) a systematic review to identify outcome domains that have been reported in interventions focused on supporting medication adherence in rheumatology; (2) semi-structured stakeholder interviews with patients and caregivers to determine their views on the core domains; (3) focus groups using the nominal group technique with patients and caregivers to identify and rank domains that are relevant to them, including the reasons for their choices; (4) an international three-round modified Delphi survey involving patients with diverse rheumatic conditions, caregivers, health professionals, researchers and other stakeholders to develop a preliminary core domain set; and (5) a stakeholder workshop with OMERACT members to review, vote on and reach a consensus on the core domain set for interventions to support medication adherence in rheumatology. Discussion: Establishing a core domain set to be reported in all intervention studies undertaken to support patients with medication adherence will enhance the relevance and the impact of these results and improve the lives of people with rheumatic conditions.The OMERACT-Adherence Group receives funding from OMERACT, which will be used to support a patient research partner in the OMERACT-Adherence Group to attend the OMERACT conference. OMERACT (http://www.omeract.org, contact: secretariat [email protected]) is the primary sponsor responsible for approving the initiation and overviewing the ongoing progress and management of the study. OMERACT mentors overview the design and conduct of the studies, including the interpretation of data and preparation, and review and approval of manuscripts. The following funding organisations had no role in the design and conduct of the studies; collection, management, analysis and interpretation of the data; or preparation, review or approval of manuscripts. AK is supported by the Arthritis Australia Scholarship funded by the Allan and Beryl Stephens Grant from the Estate of the Late Beryl Stephens. AT is supported by a National Health and Medical Research Council Fellowship (1037162). RC’s employer, the Parker Institute, Bispebjerg, and Frederiksberg Hospital, is supported by a core grant (OCAY-13-309) from the Oak Foundation. Phases 1–3 of the OMERACT-Adherence study were funded by a 2018 Arthritis Australia project grant (major funder), and a private research grant provided by Professor Stephen Hall