Assessment of seminal cell-free DNA as a potential contaminate in studies of human sperm DNA methylation.

BACKGROUND: Human seminal cell-free deoxyribonucleic acid (cfDNA) methylation patterns have not yet been thoroughly explored; however, recent work in mouse has suggested that some cfDNA encountered in the epididymis may contaminate DNA methylation studies assessing the mature spermatozoa. Such contamination could clearly prove to be a significant confounder, for many reasons, in epigenetic studies of male factor infertility. OBJECTIVES: To explore the nature of seminal cfDNA methylation and the likelihood that it would be retained following standard semen sample processing for epigenetic analysis. MATERIALS AND METHODS: We assessed 12 semen samples collected at Utah Fertility Center. For each sample, seminal cfDNA was isolated from the sperm pellet. The spermatozoa was split into three aliquots including one exposed to DNase to remove any additional cfDNA (termed "pure spermatozoa"), one not exposed to DNase, and one exposed to DNase but reintroduced to seminal cfDNA. We additionally assessed blood DNA as our benchmark for somatic cell DNA methylation patterns. DNA methylation was measured via Illumina's 850k array and assessed for differential regional methylation. RESULTS: Forty-six thousand three hundred fifty-two differentially methylated regions (FDR > 40) were identified between pure spermatozoa and seminal cfDNA. We found at these sites that the average DNA methylation in cfDNA always fell somewhere between the average methylation in spermatozoa and blood. We also assessed each sperm treatment groups at all 46,352 regions of interest and found no significant differences at any of these sites. DISCUSSION AND CONCLUSION: Our data suggest that seminal cfDNA is a clear mixture of both somatic and germline DNA and that cfDNA is not a contaminating feature in sperm DNA methylation studies following standard protocols in human sperm DNA extraction.

qDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces.

DNase coatings show great potential to prevent biofilm formation in various applications of the medical implant, food and marine industry. However, straightforward and quantitative methods to characterize the enzymatic activity of these coatings are currently not available. We here introduce the qDNase assay, a quantitative, real-time method to characterize the activity of DNase coatings. The assay combines (1) the use of an oligonucleotide probe, which fluoresces upon cleavage by coated DNases, and (2) the continuous read-out of the fluorescent signal within a microplate fluorometer format. The combination of these two properties results in a real-time fluorescent signal that is used to directly quantify the activity of DNase coatings. As a proof of concept, bovine DNase I coatings were immobilized on titanium by means of chemical grafting and their activity was estimated at 3.87 × 10-4 U. To our knowledge, the qDNase assay provides the first approach to report the activity of a DNase coating in absolute DNase activity units. This assay will not only serve to compare existing DNase coating methods more accurately, but will also enable the rational design of new DNase coating methods in the future.

Nuclease expression in efficient polyhydroxyalkanoates-producing bacteria could yield cost reduction during downstream processing.

Industrial manufacturing of polyhydroxyalkanoates (PHAs) requires purification of PHAs granules from high-cell-density cultures. Cells are broken by homogenization and PHAs granules are cleansed and treated to obtain PHAs latexes. However, cell lysis releases large amounts of DNA which results in an increasing viscosity of the medium, hampering the following downstream steps. Drop in viscosity is generally achieved by costly procedures such as heat treatment or the supplementation of hypochlorite and commercially available nucleases. Searching for a cost-effective solution to this issue, a nuclease gene from Staphylococcus aureus has been integrated into two efficient PHAs-producing bacteria: Cupriavidus necator DSM 545 and Delftia acidovorans DSM 39. Staphylococcal nuclease has been proficiently expressed in both microbial hosts without affecting PHAs production. Moreover, the viscosity of the lysates of recombinant C. necator cells was greatly reduced, indicating that the engineered strain is expected to yield large reduction cost in PHAs downstream processing.

Advances in the application of genetic manipulation methods to apicomplexan parasites.

Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterised parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods, such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation methods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of veterinary interest will ultimately lead to the development of novel and more efficient methods for disease control.

Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models.

Bacteria exploit an arsenal of antimicrobial peptides and proteins to compete with each other. Three main competition systems have been described: type six secretion systems (T6SS); contact dependent inhibition (CDI); and bacteriocins. Unlike T6SS and CDI systems, bacteriocins do not require contact between bacteria but are diffusible toxins released into the environment. Identified almost a century ago, our understanding of bacteriocin distribution and prevalence in bacterial populations remains poor. In the case of protein bacteriocins, this is because of high levels of sequence diversity and difficulties in distinguishing their killing domains from those of other competition systems. Here, we develop a robust bioinformatics pipeline exploiting Hidden Markov Models for the identification of nuclease bacteriocins (NBs) in bacteria of which, to-date, only a handful are known. NBs are large (>60 kDa) toxins that target nucleic acids (DNA, tRNA or rRNA) in the cytoplasm of susceptible bacteria, usually closely related to the producing organism. We identified >3000 NB genes located on plasmids or on the chromosome from 53 bacterial species distributed across different ecological niches, including human, animals, plants, and the environment. A newly identified NB predicted to be specific for Pseudomonas aeruginosa (pyocin Sn) was produced and shown to kill P. aeruginosa thereby validating our pipeline. Intriguingly, while the genes encoding the machinery needed for NB translocation across the cell envelope are widespread in Gram-negative bacteria, NBs are found exclusively in γ-proteobacteria. Similarity network analysis demonstrated that NBs fall into eight groups each with a distinct arrangement of protein domains involved in import. The only structural feature conserved across all groups was a sequence motif critical for cell-killing that is generally not found in bacteriocins targeting the periplasm, implying a specific role in translocating the nuclease to the cytoplasm. Finally, we demonstrate a significant association between nuclease colicins, NBs specific for Escherichia coli, and virulence factors, suggesting NBs play a role in infection processes, most likely by enabling pathogens to outcompete commensal bacteria.

A contaminant-free assessment of Endogenous Retroviral RNA in human plasma.

Endogenous retroviruses (ERVs) comprise 6-8% of the human genome. HERVs are silenced in most normal tissues, up-regulated in stem cells and in placenta but also in cancer and HIV-1 infection. Crucially, there are conflicting reports on detecting HERV RNA in non-cellular clinical samples such as plasma that suggest the study of HERV RNA can be daunting. Indeed, we find that the use of real-time PCR in a quality assured clinical laboratory setting can be sensitive to low-level proviral contamination. We developed a mathematical model for low-level contamination that allowed us to design a laboratory protocol and standard operating procedures for robust measurement of HERV RNA. We focus on one family, HERV-K HML-2 (HK2) that has been most recently active even though they invaded our ancestral genomes almost 30 millions ago. We extensively validated our experimental design on a model cell culture system showing high sensitivity and specificity, totally eliminating the proviral contamination. We then tested 236 plasma samples from patients infected with HIV-1, HCV or HBV and found them to be negative. The study of HERV RNA for human translational studies should be performed with extensively validated protocols and standard operating procedures to control the widespread low-level human DNA contamination.

Assessment of adherence to the guidelines for the management of nausea and vomiting induced by chemotherapy / Avaliação da aderência à diretriz de cuidados para náuseas e vômitos induzidos por quimioterapia

ABSTRACT Objective: To assess adherence of the prescribing physicians in a private cancer care center to the American Society of Clinical Oncology guideline for antiemetic prophylaxis, in the first cycle of antineoplastic chemotherapy. Methods: A total of 139 chemotherapy regimens, of 105 patients, were evaluated retrospectively from 2011 to 2013. Results: We observed 78% of non-adherence to the guideline rate. The main disagreements with the directive were the prescription of higher doses of dexamethasone and excessive use of 5-HT3 antagonist for low risk emetogenic chemotherapy regimens. On univariate analysis, hematological malignancies (p=0.005), the use of two or more chemotherapy (p=0.05) and high emetogenic risk regimes (p=0.012) were factors statistically associated with greater adherence to guidelines. Treatment based on paclitaxel was the only significant risk factor for non-adherence (p=0.02). By multivariate analysis, the chemotherapy of high emetogenic risk most correlated with adherence to guideline (p=0.05). Conclusion: We concluded that the adherence to guidelines is greater if the chemotherapy regime has high emetogenic risk. Educational efforts should focus more intensely on the management of chemotherapy regimens with low and moderate emetogenic potential. Perhaps the development of a computer generated reminder may improve the adherence to guidelines. .

RESUMO Objetivo: Avaliar a adesão dos médicos prescritores, de um centro privado especializado em oncologia, à diretriz de antiêmese profilática da American Society of Clinical Oncology, no primeiro ciclo de quimioterapia antineoplásica. Métodos: Foram avaliados retrospectivamente 139 esquemas de quimioterapia, de 105 pacientes, tratados no período de 2011 a 2013. Resultados: Foram observados 78% de taxa de não adesão à diretriz. As principais discordâncias com a diretriz foram prescrição de doses mais elevadas de dexametasona e uso excessivo de antagonista 5-HT3 para regimes de quimioterapia de risco emetogênico baixo. Pela análise univariada, malignidades hematológicas (p=0,005), uso de dois ou mais quimioterápicos (p=0,05) e regimes de alto risco emetogênico (p=0,012) foram fatores estatisticamente associados a maior adesão à diretriz. O tratamento baseado em paclitaxel foi o único fator estatisticamente significativo para a não adesão (p=0,02). Pela análise multivariada, a quimioterapia de alto risco emetogênico apresentou maior correlação com a adesão à diretriz (p=0,05). Conclusão: Houve maior aderência para a quimioterapia de alto risco emetogênico. Esforços educacionais devem se concentrar mais intensamente na gestão de regimes de quimioterapia com potencial emetogênico baixo e moderado. Talvez o desenvolvimento de lembretes gerados por sistemas informatizados possa melhorar a aderência à diretriz. .

Comparison of extracellular DNase- and protease-producing spoilage bacteria isolated from Delaware pond-sourced and retail channel catfish (Ictalurus punctatus).

BACKGROUND: Spoilage of fishery products begins immediately following filleting due to microbial growth that degrades fish tissue quality prior to consumption. Extensive research has been conducted to identify such bacterial populations. A better understanding of the mechanisms involved in fish spoilage is necessary as a novel remedy for microbial spoilage inhibition has yet to be established for fish tissue. The present study identified, for the first time, bacterial populations that produce extracellular DNase and protease from Delaware and local retail distributed channel catfish (Ictalurus punctatus) fillets. RESULTS: A clear trend was identified between bacteria derived from catfish filleted under aseptic conditions where Pseudomonas was the dominant genus. Bacteria isolated from retail catfish contained high quantities of DNase-producing isolates, in contrast to aseptic-filleted catfish tissue which had none. Both types of catfish sample maintained high populations of protease-producing bacterial colonies throughout the duration of the study. Most bacteria isolated from catfish intestines exhibited DNase production with no protease production. CONCLUSION: Specific spoilage organism populations were significantly higher on retail-derived catfish in comparison to lab-filleted Delaware cultured catfish tissue. It is suggested that DNase production and protease production contribute to the spoilage of fish tissue as a result of mishandling and septic filleting being the major cause of rapid catfish tissue spoilage.

Cross-platform evaluation of commercial real-time reverse transcription PCR master mix kits using a quantitative 5'nuclease assay for Ebola virus.

Selection of optimal reaction master mix reagents is essential to obtain the best performance with diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Every year the number of commercially available master mix kits increases, so it is prudent to periodically evaluate kits on the market. In this study we evaluated five commercial real-time RT-PCR master mix kits, the RealMasterMix RT-PCR ROX kit, the AgPath-ID One-Step RT-PCR kit, the SuperScript III Platinum One-step Quantitative RT-PCR system, the QuantiTect Probe RT-PCR kit, and the LightCycler RNA HybProbe amplification kit, using a 5'nuclease assay for Ebola virus. The kits were evaluated using the manufacturer's recommended conditions, as well as conditions which have been used with the Ebola virus assay during outbreaks. When evaluated for use in Ebola virus RNA detection, the AgPath-ID kit resulted in the greatest sensitivity in comparison to the other four kits. The efficacy of the AgPath-ID kit was instrument-independent in the five real-time PCR platforms tested. This study demonstrated that Ebola virus RNA detection was not equivalent among the master mix reagents studied and, thus, that this variable can affect real-time RT-PCR assay sensitivity. Furthermore, this study rates the master mix reagents for their suitability, providing diagnostic laboratories the option to select from these kits to suit their specific laboratory needs for real-time RT-PCR.

The dynamics of EBV shedding implicate a central role for epithelial cells in amplifying viral output.

To develop more detailed models of EBV persistence we have studied the dynamics of virus shedding in healthy carriers. We demonstrate that EBV shedding into saliva is continuous and rapid such that the virus level is replaced in < or =2 minutes, the average time that a normal individual swallows. Thus, the mouth is not a reservoir of virus but a conduit through which a continuous flow stream of virus passes in saliva. Consequently, virus is being shed at a much higher rate than previously thought, a level too high to be accounted for by replication in B cells in Waldeyer's ring alone. Virus shedding is relatively stable over short periods (hours-days) but varies through 3.5 to 5.5 logs over longer periods, a degree of variation that also cannot be accounted for solely by replication in B cells. This variation means, contrary to what is generally believed, that the definition of high and low shedder is not so much a function of variation between individuals but within individuals over time. The dynamics of shedding describe a process governing virus production that is occurring independently < or =3 times at any moment. This process grows exponentially and is then randomly terminated. We propose that these dynamics are best explained by a model where single B cells sporadically release virus that infects anywhere from 1 to 5 epithelial cells. This infection spreads at a constant exponential rate and is terminated randomly, resulting in infected plaques of epithelial cells ranging in size from 1 to 10(5) cells. At any one time there are a very small number (< or =3) of plaques. We suggest that the final size of these plaques is a function of the rate of infectious spread within the lymphoepithelium which may be governed by the structural complexity of the tissue but is ultimately limited by the immune response.

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.

Assessment of enzyme detection tests useful in identification of campylobacteria.

Twenty-one type or other reference strains, each representing a different Campylobacter, Helicobacter, or Arcobacter taxon, and a reference strain of Staphylococcus aureus were used to assess the reproducibility of nine enzyme detection tests used in the identification of campylobacters. For five of the tests (alkaline phosphatase, DNase, and H2S production, indoxyl acetate hydrolysis, and nitrate reduction), more than one procedure was employed to determine the most suitable method. Alkaline phosphatase test results were better defined and more reproducible if read after 1 h of incubation. Detection of DNase was fully reproducible with each method (except with Helicobacter pylori), but reactions were generally weaker than those of other DNase-producing organisms. Both procedures for determining H2S production were irreproducible for the same strains. The reproducibility of indoxyl acetate hydrolysis was improved by using disks impregnated with 25 microliters of substrate. Reduction of nitrate was best determined by Cook's plate method. Results for the other tests examined (catalase, oxidase, and urease production and hippurate hydrolysis) were both pertinent and fully reproducible for all strains.