Rapid Method of Processing Sperm for Nucleic Acid Extraction in Clinical Research

Background: Sperm contain highly compact nuclei, inhibiting DNA extraction using traditional techniques. Current methods extracting sperm DNA involve lengthy lysis and no means of stabilizing DNA, hindering clinical research. Objective: We sought to optimize an efficient method of extracting high quality human sperm DNA. Methods: Sperm from three volunteers were isolated using PureCeption. We tested 1) proteinase K with DNA/RNA Shield, 2) DTT and TCEP as reducing agents, 3) QIAshredder homogenization, and 4) stability of sperm DNA fresh (baseline) or after 4 weeks of storage at 4OC in DNA/RNA Shield using modified Quick-gDNA MiniPrep. DNA was PCR amplified using ALU primers and digested with Hinf1 restriction enzymes. DNA methylation was measured by MassARRAY. Results: DNA concentrations were similar with (30.1+0.28ng/μL, 33.4+0.21ng/μL) and without (28.9+0.00ng/μL, 30.9+0.85ng/μL) proteinase K. Sperm cells were lysed after 1 and 20 minutes with 25mM TCEP and 100mM DTT respectively. TCEP (50mM) produced greater DNA concentrations (17.2+0.50ng/μL, 21.3+0.71ng/μL) than 50mM DTT (12.6+0.28ng/μL, 12.3+0.35ng/μL). Adding QIAshredder to 50mM TCEP increased DNA concentrations (25.9+0.35ng/μL, 21.7+0.49ng/μL versus 18.6+0.99ng/μL, 12.3+0.35ng/μL). At baseline and 4 weeks: 1) DNA concentrations were similar (36.2+2.75 ng/μL, 32.2+1.38ng/μL, 44.3+3.93ng/μL versus 40.0+2.98ng/μL, 37.6+1.38ng/μL, 38.7+3.93ng/μL respectively) and 2) DNA was equally amplified by PCR and digested with restriction enzymes. DNA methylation was similar at baseline and 4 weeks for SNURF (1.43+1.02%, 1.55+0.95%), PEG10 (3.69+0.66%, 4.28+1.52%), and H19 (88.93+3.24%, 91.78+2.00%). Conclusions: We stabilized and isolated high quality DNA from human sperm using 5 minute versus \u3e 2 hour lysis in other methods. Our methods may facilitate efficient clinical research

Design of Analog CMOS Circuits for Batteryless Implantable Telemetry Systems

A wireless biomedical telemetry system is a device that collects biomedical signal measurements and transmits data through wireless RF communication. Testing medical treatments often involves experimentation on small laboratory animals, such as genetically modified mice and rats. Using batteries as a power source results in many practical issues, such as increased size of the implant and limited operating lifetime. Wireless power harvesting for implantable biomedical devices removes the need for batteries integrated into the implant. This will reduce device size and remove the need for surgical replacement due to battery depletion. Resonant inductive coupling achieves wireless power transfer in a manner modelled by a step down transformer. With this methodology, power harvesting for an implantable device is realized with the use of a large primary coil external to the subject, and a smaller secondary coil integrated into the implant. The signal received from the secondary coil must be regulated to provide a stable direct current (DC) power supply, which will be used to power the electronics in the implantable device. The focus of this work is on development of an electronic front-end for wireless powering of an implantable biomedical device. The energy harvesting front-end circuit is comprised of a rectifier, LDO regulator, and a temperature insensitive voltage reference. Physical design of the front-end circuit is developed in 0.13um CMOS technology with careful attention to analog layout issues. Post-layout simulation results are presented for each sub-block as well as the full front-end structure. The LDO regulator operates with supply voltages in the range of 1V to 1.5V with quiescent current of 10.5uA The complete power receiver front-end has a power conversion efficiency of up to 29%

The emergence and work processes of executive remuneration consultants

This thesis studies the emergence of executive remuneration consulting as a distinct occupation from the 1990s, and the co-emergence of remuneration consultants and remuneration committees from the early 2000s. These actors, their work processes, norms and interlinkages are studied within the context of key social, economic and political factors, which shape the fields of remuneration consulting work and remuneration governance. In light of recent conflicting governance recommendations, it is important to evaluate the system of governance in relation to the historical reference points which have shaped executive pay practices. In so doing, this thesis analyses the dynamic processes in which numerous actors (remuneration committees, executive directors, Reward/HR directors, remuneration consultants and institutional investors), documents (corporate governance codes, governance guidelines and regulations) and tools (market trends analysis and pay benchmarking) are collectively engaged. Executive remuneration has overwhelmingly been researched from the agency perspective, delineated into two theoretical points of departure: optimal contracting (Jensen and Meckling, 1976) and managerial capture (Bebchuk and Fried, 2003; 2004). Despite managerial capture theorists seeking to address perceived shortcomings in optimal contracting, both result in an undersocialised (Granovetter, 1985; cf. Main, 2006) view of executive pay practices. Drawing on a genealogical approach (Foucault, 1971), Chapter 3 studies the emergence of executive remuneration consulting, while Chapter 4 examines the co-emergence of remuneration consultants and remuneration committees. Drawing on a field-based study at a leading remuneration consultancy, Chapter 5 presents the day-to-day work processes of executive remuneration consultants, and the ways in which consultants have produced their relevance in executive pay design and governance. Chapter 6 problematizes the market for executive talent and presents a conceptualisation of pay benchmarking practice. Chapter 7 argues that a dominant logic of risk has gone undocumented; that it is risk and risk management that ‘percolates and pervades’ (Power, 2004) executive remuneration governance

Disseminated cutaneous Herpes Simplex Virus-1 in a woman with rheumatoid arthritis receiving Infliximab: A case report

<p>Abstract</p> <p>Introduction</p> <p>We present the case of a 49-year-old woman with a seronegative rheumatoid arthritis who developed pustular psoriasis whilst on etanercept and subsequently developed disseminated herpes simplex on infliximab.</p> <p>Case presentation</p> <p>Our patient presented with an inflammatory arthritis which failed to respond to both methotrexate and leflunomide, and sulphasalazine treatment led to side effects. She was started on etanercept but after 8 months of treatment developed scaly pustular lesions on her palms and soles typical of pustular psoriasis. Following the discontinuation of etanercept, our patient required high doses of oral prednisolone to control her inflammatory arthritis. A second biologic agent, infliximab, was introduced in addition to low-dose methotrexate and 15 mg of oral prednisolone. However, after just 3 infusions of infliximab, she was admitted to hospital with a fever, widespread itchy vesicular rash and worsening inflammatory arthritis. Fluid from skin vesicles examined by polymerase chain reaction showed Herpes Simplex Virus type 1. Blood cultures were negative and her chest X-ray was normal. Her infliximab was discontinued and she was started on acyclovir, 800 mg five times daily for 2 weeks. She made a good recovery with improvement in her skin within 48 hours.</p> <p>She continued for 2 months on a prophylactic dose of 400 mg bd. Her rheumatoid arthritis became increasingly active and a decision was made to introduce adalimumab alongside acyclovir. Acyclovir prophylaxis has been continued but the dose tapered so that she is taking only 200 mg of acyclovir on alternate days. There has been no recurrence of Herpes Simplex Virus lesions despite increasing adalimumab to 40 mg weekly 3 months after starting treatment.</p> <p>Conclusion</p> <p>We believe this to be the first reported case of widespread cutaneous Herpes Simplex Virus type 1 infection following treatment with infliximab. We discuss the clinical manifestations of Herpes Simplex Virus infections with particular emphasis on the immunosuppressed patient and the use of prophylactic acyclovir. Pustular psoriasis is now a well recognised but uncommon side effect of antitumour necrosis factor therapy and can lead to cessation of therapy, as in our patient's case.</p

The Integrative Conjugative Element clc (ICEclc) of Pseudomonas aeruginosa JB2

Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds

In situ detection of gliosis and apoptosis in the brains of young rats exposed in utero to a Wi-Fi signal

International audiencePregnant rats were daily whole-body exposed or sham-exposed to a Wi-Fi signal in a free-running reverberation chamber at 0, 0.08, 0.4, and 4 W/kg for 2 h during the last 2 weeks of gestation (5 days/week). Following this in utero exposure, the pups were divided into two groups and 1 group continued exposure for 5 weeks after birth. Several brain areas were examined for gliosis and apoptotic cells. Comparison among sham and exposed groups revealed no significant differences, suggesting that in utero and post-natal exposure to Wi-Fi did not damage the brains of the young rats

Comparative study between radiofrequency-induced and muscimol-induced inhibition of cultured networks of cortical neuron

Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode arrays are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and differences between the inhibitory effect of RF fields (continuous wave, 1800 MHz) to the γ-aminobutyric acid type A (GABAA) receptor agonist muscimol (MU). Inhibition of the network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of ~1°C. Exposure to RF fields preferentially inhibits bursting over spiking activity and exerts fewer constraints on neural network bursting synchrony, differentiating it from a pharmacological inhibition with MU. Network rebound excitation, a phenomenon relying on the intrinsic properties of cortical neurons, was observed following the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure. This implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As changes in AP waveform strongly influence efficacy of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reducing network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of the action of RF fields differ from the ones mediated by the activation of GABAA receptors

Complete genome sequence of the phenanthrene-degrading soil bacterium Delftia acidovorans Cs1-4

Abstract Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and microbial biodegradation is an important means of remediation of PAH-contaminated soil. Delftia acidovorans Cs1-4 (formerly Delftia sp. Cs1-4) was isolated by using phenanthrene as the sole carbon source from PAH contaminated soil in Wisconsin. Its full genome sequence was determined to gain insights into a mechanisms underlying biodegradation of PAH. Three genomic libraries were constructed and sequenced: an Illumina GAii shotgun library (916,416,493 reads), a 454 Titanium standard library (770,171 reads) and one paired-end 454 library (average insert size of 8 kb, 508,092 reads). The initial assembly contained 40 contigs in two scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together and the consensus sequences were computationally shredded into 2 kb overlapping shreds. Illumina sequencing data was assembled, and the consensus sequence was computationally shredded into 1.5 kb overlapping shreds. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 182 additional reactions were needed to close gaps and to raise the quality of the finished sequence. The final assembly is based on 253.3 Mb of 454 draft data (averaging 38.4 X coverage) and 590.2 Mb of Illumina draft data (averaging 89.4 X coverage). The genome of strain Cs1-4 consists of a single circular chromosome of 6,685,842 bp (66.7 %G+C) containing 6,028 predicted genes; 5,931 of these genes were protein-encoding and 4,425 gene products were assigned to a putative function. Genes encoding phenanthrene degradation were localized to a 232 kb genomic island (termed the phn island), which contained near its 3’ end a bacteriophage P4-like integrase, an enzyme often associated with chromosomal integration of mobile genetic elements. Other biodegradation pathways reconstructed from the genome sequence included: benzoate (by the acetyl-CoA pathway), styrene, nicotinic acid (by the maleamate pathway) and the pesticides Dicamba and Fenitrothion. Determination of the complete genome sequence of D. acidovorans Cs1-4 has provided new insights the microbial mechanisms of PAH biodegradation that may shape the process in the environment.http://deepblue.lib.umich.edu/bitstream/2027.42/134560/1/40793_2015_Article_41.pd

Electronic Cigarette Use Among Emerging and Young West Indian Adults

Currently, evidence concerning electronic cigarette (e-cigarette) use in the West Indies is unavailable. This study examines the prevalence and associated factors of e-cigarette use in young Trinidadian adults, 6 years after e-cigarettes were introduced in Trinidad. Young adults between the ages of 18 and 40 years were surveyed from May–June 2016. Based on the survey results, descriptive statistics and logistic regression models were used to identify correlations in e-cigarette use. The prevalence of those who had used e-cigarettes was 24.6%, and 41.9% of these people had used both e-cigarettes and tobacco cigarettes. A high proportion (16.95%) of those who had never used tobacco cigarettes had used e-cigarettes. Males were twice as likely as females to have used e-cigarettes (odds ratio [OR]: 2.60; 95% confidence interval [CI]: 1.85–3.68), and participants aged 18–25 years were more likely than those aged 36–40 years to use e-cigarettes (OR: 0.37; 95% CI: 0.14–0.81). The predictors of e-cigarette use as assessed by univariate analysis were current tobacco cigarette smoking (OR: 9.34; 95% CI: 6.14–14.39; p<0.001) and the belief that e-cigarettes are dangerous to health (OR: 0.61; 95% CI: 0.44–0.85; p=0.004). The predictors as assessed by multivariate logistic regression (adjusted OR) were ethnicity (p=0.043), education (p=0.012), and age group (p=0.007). Those who quit using tobacco cigarettes were 7.98 times more likely to use e-cigarettes (95% CI: 4.21–15.45), and those who knew that e-cigarettes contain nicotine were 2.70 times more likely to use them (95% CI: 1.53–4.86; p<0.001). Two summative scales were constructed that measured knowledge and perception. The perception scale, but not the knowledge scale (Cronbach’s alpha=0.736), was a significant predictor of e-cigarette use. The number of e-cigarette users is high (24.6%) in young adults in Trinidad and in those who have never smoked tobacco (16.95%). Current smokers, as well as those who have quit smoking, are at an increased risk of e-cigarette use. This study established that young adults have a low level of knowledge regarding e-cigarettes and shows that they should be educated on e-cigarette use. Further research to examine the reasons for, and susceptibility to, e-cigarette use is necessary

Manifestation of palmoplantar pustulosis during or after infliximab therapy for plaque-type psoriasis: report on five cases

Infliximab is a monoclonal antibody directed against TNF-α. It has been approved for use in rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriatic arthritis and plaque-type psoriasis. In case reports, positive effects on pustular variants of psoriasis have also been reported. However, paradoxically, manifestation of pustular psoriasis and plaque-type psoriasis has been reported in patients treated with TNF antagonists including infliximab for other indications. Here, we report on 5 patients with chronic plaque-type psoriasis who developed palmoplantar pustulosis during or after discontinuation of infliximab therapy. In two of the five cases, manifestation of palmoplantar pustulosis was not accompanied by worsening of plaque-type psoriasis. Possibly, site-specific factors or a differential contribution of immunological processes modulated by TNF inhibitors to palmoplantar pustulosis and plaque-type psoriasis may have played a role