Gene discovery: Polymorphism association with obsessive-compulsive disorder and proteome analysis of Artemisia annua.

PART I: Polymorphism association with Obsessive-Compulsive Disorder In light of the putative role of the serotonergic, dopaminergic and possibly (nor)adrenergic system in OCD, following polymorphisms were analysed in a sample of >100 OCD patients and a control sample of >100 ethnically matched Caucasian subjects by means of a case-control study: Taq IA polymorphism in the non-coding region flanking the 3’ end of the dopamine D2 receptor (DRD2) gene; Catechol-O-Methyl Transferase (COMT) NlaIII high/low activity polymorphism; 5-HT2A 1438 A/G polymorphism; 5-HT1Dβ G861C polymorphism and the 5-HTTLPR 44-bp deletioninsertion. There was a higher frequency of the DRD2 A2 allele (p = 0.020) and a higher frequency of the low-activity COMT allele (p = 0.035) in male OCD patients compared to male controls. In addition, we observed an association of the DRD2 A2A2 genotype in patients with an early onset of OCD (p= 0.033). We studied whether the serotonin polymorphisms affect the efficacy of venlafaxine and paroxetine treatment in OCD. The response in paroxetine treated patients is associated with the GG genotype of the 5-HT2A polymorphism (χ2 = 8.66, df=2, p=0.013). In venlafaxine treated patients, response is associated with the SL genotype of the 5-HTTLPR polymorphism (χ2 = 9.71, df=2, p=0.008). PART II: proteomics on Artemisia annua Three strategies were followed to discover genes of the plant Artemisia annua L. that are involved in the production of the antimalarial artemisinin: a proteome analysis, a quantitative cDNA amplified fragment length polymorphism analysis (cDNA AFLP) and the construction of full length Expressed Sequence Tag (EST) cDNA libraries. We identified proteins that were differentially expressed between trichomes and whole leaf tissue. Proteins that are upregulated in the trichomes might be involved in artemisinin production. To narrow down the results to the most valid gene candidates, the proteome data was compared with a cDNA AFLP analysis that investigated samples of A. annua leafs, taken at different time points during a 72h time period after exposure to jasmonic acid. We were able to compile a list of EST candidates, which could be useful for further investigation

Effect of multiple allelic drop-outs in forensic RMNE calculations

Technological advances such as massively parallel sequencing enable increasing amounts of genetic information to be obtained from increasingly challenging samples. Certainly on low template, degraded and multi-contributor samples, drop-outs will increase in number for many profiles simply by analyzing more loci, making it difficult to probabilistically assess how many drop-outs have occurred and at which loci they might have occurred. Previously we developed a Random Man Not Excluded (RMNE) method that can take into account allelic drop-out while avoiding detailed estimations of the probability that drop-outs have occurred, nor making assumptions about at which loci these drop-outs might have occurred. The number of alleles that have dropped out, does not need to be exactly known. Here we report a generic Python algorithm to calculate the RMNE probabilities for any given number of loci. The number of allowed drop-outs can be set between 0 and twice the number of analyzed loci. The source code has been made available on https://github.com/fvnieuwe/rmne. An online web-based RMNE calculation tool has been made available on http://forensic.ugent.be/rmne. The tool can calculate these RMNE probabilities from a custom list of probabilities of the observed and non-observed alleles from any given number of loci. Using this tool, we explored the effect of allowing allelic drop-outs on the evidential value of random forensic profiles with a varying number of loci. Our results give insight into how the number of allowed drop-outs affects the evidential value of a profile and how drop-out can be managed in the RMNE approach

Automatic detection of spermatozoa for laser capture microdissection

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER (TM). Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery

Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SpIF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, spIF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved

My-Forensic-Loci-queries (MyFLq) framework for analysis of forensic STR data generated by massive parallel sequencing

Forensic scientists are currently investigating how to transition from capillary electrophoresis (CE) to massive parallel sequencing (MPS) for analysis of forensic DNA profiles. MPS offers several advantages over CE such as virtually unlimited multiplexy of loci, combining both short tandem repeat (STR) and single nucleotide polymorphism (SNP) loci, small amplicons without constraints of size separation, more discrimination power, deep mixture resolution and sample multiplexing. We present our bioinformatic framework My-Forensic-Loci-queries (MyFLq) for analysis of MPS forensic data. For allele calling, the framework uses a MySQL reference allele database with automatically determined regions of interest (ROIs) by a generic maximal flanking algorithm which makes it possible to use any STR or SNP forensic locus. Python scripts were designed to automatically make allele calls starting from raw MPS data. We also present a method to assess the usefulness and overall performance of a forensic locus with respect to MPS, as well as methods to estimate whether an unknown allele, which sequence is not present in the MySQL database, is in fact a new allele or a sequencing error. The MyFLq framework was applied to an Illumina MiSeq dataset of a forensic Illumina amplicon library, generated from multilocus STR polymerase chain reaction (PCR) on both single contributor samples and multiple person DNA mixtures. Although the multilocus PCR was not yet optimized for MPS in terms of amplicon length or locus selection, the results show excellent results for most loci. The results show a high signal-to-noise ratio, correct allele calls, and a low limit of detection for minor DNA contributors in mixed DNA samples. Technically, forensic MPS affords great promise for routine implementation in forensic genomics. The method is also applicable to adjacent disciplines such as mitochondrial DNA research

Draft genome sequence of methicillin-resistant Staphylococcus epidermidis strain ET-024, isolated from an endotracheal tube biofilm of a mechanically ventilated patient

Staphylococcus epidermidis strain ET-024 was isolated from a biofilm on an endotracheal tube of a mechanically ventilated patient. This strain is resistant to methicillin and the draft genome sequence shares some characteristics with other nosocomial S. epidermidis strains (such as S. epidermidis RP62A)

Forensic SNP genotyping using nanopore MinION sequencing

One of the latest developments in next generation sequencing is the Oxford Nanopore Technologies' (ONT) MinION nanopore sequencer. We studied the applicability of this system to perform forensic genotyping of the forensic female DNA standard 9947 A using the 52 SNP-plex assay developed by the SNPforID consortium. All but one of the loci were correctly genotyped. Several SNP loci were identified as problematic for correct and robust genotyping using nanopore sequencing. All these loci contained homopolymers in the sequence flanking the forensic SNP and most of them were already reported as problematic in studies using other sequencing technologies. When these problematic loci are avoided, correct forensic genotyping using nanopore sequencing is technically feasible

Rejections in an non-purpose bred assistance dog population : reasons, consequences and methods for screening

Assistance dogs aid people with various impairments on a daily basis. To become an assistance dog, a strict selection procedure and intensive training period must be successfully completed. Consequently, not every dog acquired for this purpose, becomes an assistance dog. The purpose of this study was to investigate reasons for failure and the financial consequences thereof for assistance dog associations that do not have a dedicated breeding program for their dogs. Data were collected for a total of 537 dogs enlisted between 2001 and 2015 and purchased out of the general dog population by five Belgian assistance dog associations. Only 60 percent of the dogs actually became an assistance dog and the main reasons for failure were related to undesirable behavioural characteristics and orthopaedic disorders. The estimated average financial loss per rejected dog was found to be 10524 euro. A detailed comparison of the two most popular breeds (Golden Retriever and Labrador Retriever) within the guide dogs and mobility assistance dogs revealed no significant difference in probability of successfully completing the training. However, a comparison of orthopaedic screening methods revealed a higher rejection with computed tomography for elbow dysplasia and laxity-based radiographical techniques for hip dysplasia compared to radiography and the standard ventrodorsal hip extend radiograph alone, respectively. Based on these results, we provide several suggestions to increase the probability of success