Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture

Quantification of Epstein-Barr virus DNA load, interleukin-6, interleukin-10, transforming growth factor-β1 and stem cell factor in plasma of patients with nasopharyngeal carcinoma

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a common epithelial neoplasm among the Chinese populations in Southern China and South East Asia. Epstein-Barr virus (EBV) is known to be an important etiologic agent of NPC and the viral gene products are frequently detected in NPC tissues along with elevated antibody titres to the viral proteins (VCA and EA) in a majority of patients. Elevated plasma EBV DNA load is regarded as an important marker for the presence of the disease and for the monitoring of disease progression. However, other serum/plasma parameters such as the levels of certain interleukins and growth factors have also been implicated in NPC. The objectives of the present study are, 1) to investigate the correlations between plasma EBV DNA load and the levels of interleukin (IL)-6, IL-10, TGF-β1 and SCF (steel factor) and 2) to relate these parameters to the stages of NPC and the effect of treatment. METHODS: A total of 78 untreated NPC patients were enrolled in this study. Of these, 51 were followed-up after treatment. The remaining patients had irregular or were lost to follow-up. Plasma EBV DNA was quantified using real-time quantitative PCR. The levels of plasma interleukins and growth factors were quantified using ELISA. RESULTS: A significant decrease in EBV DNA load was detected in plasma of untreated NPC patients (1669 ± 637 copies/mL; n = 51) following treatment (57 ± 37 copies/mL, p < 0.05); n = 51). Plasma EBV DNA load was shown to be a good prognosticator for disease progression and clinical outcome in five of the follow-up patients. A significant difference in IL-6 levels was noted between the untreated patients (164 ± 37 pg/mL; n = 51) and following treatment (58 ± 16 pg/mL, p < 0.05; n = 51). Positive correlations between EBV DNA load and IL-10 (r(49) = 0.535, p < 0.01), between IL6 and IL-10 (r(49) = 0.474, p < 0.01) and between TGF and SCF (r(49) = 0.464, p < 0.01) were observed in patients following treatment. None of the parameters tested including IgA-VCA were associated with tumour stages. CONCLUSION: We conclude that among the parameters investigated, EBV DNA load and IL-6 levels were promising markers for the presence of NPC and for the assessment of treatment outcome

The future of EPAC-targeted therapies: agonism versus antagonism

yesPharmaceutical manipulation of cAMP levels exerts beneficial effects through the regulation of the exchange protein activated by cAMP (EPAC) and protein kinase A (PKA) signalling routes. Recent attention has turned to the specific regulation of EPAC isoforms (EPAC1 and EPAC2) as a more targeted approach to cAMP-based therapies. For example, EPAC2-selective agonists could promote insulin secretion from pancreatic β cells, whereas EPAC1-selective agonists may be useful in the treatment of vascular inflammation. By contrast, EPAC1 and EPAC2 antagonists could both be useful in the treatment of heart failure. Here we discuss whether the best way forward is to design EPAC-selective agonists or antagonists and the current strategies being used to develop isoform-selective, small-molecule regulators of EPAC1 and EPAC2 activity

Leadership As We Know It

Leadership as We Know it is a collection of insights into modern leadership compiled by graduate students in Winona State University’s Leadership Education program during the Spring 2019 semester in a course aptly titled, Change Leadership. Each chapter was penned by one of 20 unique class members who offer their vision of leadership based upon their eclectic personal backgrounds and professional experiences, whose fields include athletics, business, education, and more. These diverse narratives offer something for everyone; whether it be a veteran or blossoming leader eager to continue their growth and evolution. Leadership as We Know it provides accounts from seasoned professionals who oversee their own organizational departments as well as emerging leaders just beginning their careers. Throughout these unique stories, clear patterns will emerge for the reader in what it takes to inspire change and provide authentic leadership for followers.https://openriver.winona.edu/leadershipeducationbooks/1003/thumbnail.jp

Reliability of clinical tests to evaluate nerve function and mechanosensitivity of the upper limb peripheral nervous system

BACKGROUND: Clinical tests to assess peripheral nerve disorders can be classified into two categories: tests for afferent/efferent nerve function such as nerve conduction (bedside neurological examination) and tests for increased mechanosensitivity (e.g. upper limb neurodynamic tests (ULNTs) and nerve palpation). Reliability reports of nerve palpation and the interpretation of neurodynamic tests are scarce. This study therefore investigated the intertester reliability of nerve palpation and ULNTs. ULNTs were interpreted based on symptom reproduction and structural differentiation. To put the reliability of these tests in perspective, a comparison with the reliability of clinical tests for nerve function was made. METHODS: Two experienced clinicians examined 31 patients with unilateral arm and/or neck pain. The examination included clinical tests for nerve function (sensory testing, reflexes and manual muscle testing (MMT)) and mechanosensitivity (ULNTs and palpation of the median, radial and ulnar nerve). Kappa statistics were calculated to evaluate intertester reliability. A meta-analysis determined an overall kappa for the domains with multiple kappa values (MMT, ULNT, palpation). We then compared the difference in reliability between the tests of mechanosensitivity and nerve function using a one-sample t-test. RESULTS: We observed moderate to substantial reliability for the tests for afferent/efferent nerve function (sensory testing: kappa = 0.53; MMT: kappa = 0.68; no kappa was calculated for reflexes due to a lack of variation). Tests to investigate mechanosensitivity demonstrated moderate reliability (ULNT: kappa = 0.45; palpation: kappa = 0.59). When compared statistically, there was no difference in reliability for tests for nerve function and mechanosensitivity (p = 0.06). CONCLUSION: This study demonstrates that clinical tests which evaluate increased nerve mechanosensitivity and afferent/efferent nerve function have comparable moderate to substantial reliability. To further investigate the clinometric properties of these tests, more studies are needed to evaluate their validity

Review of the cultivation program within the National Alliance for Advanced Biofuels and Bioproducts

The cultivation efforts within the National Alliance for Advanced Biofuels and Bioproducts (NAABB)were developed to provide four major goals for the consortium, which included biomass production for downstream experimentation, development of new assessment tools for cultivation, development of new cultivation reactor technologies, and development of methods for robust cultivation. The NAABB consortium test beds produced over 1500 kg of biomass for downstream processing. The biomass production included a number of model production strains, but also took into production some of the more promising strains found through the prospecting efforts of the consortium. Cultivation efforts at large scale are intensive and costly, therefore the consortium developed tools and models to assess the productivity of strains under various environmental conditions, at lab scale, and validated these against scaled outdoor production systems. Two new pond-based bioreactor designs were tested for their ability to minimize energy consumption while maintaining, and even exceeding, the productivity of algae cultivation compared to traditional systems. Also, molecular markers were developed for quality control and to facilitate detection of bacterial communities associated with cultivated algal species, including the Chlorella spp. pathogen, Vampirovibrio chlorellavorus,which was identified in at least two test site locations in Arizona and New Mexico. Finally, the consortium worked on understanding methods to utilize compromised municipal waste water streams for cultivation. This review provides an overview of the cultivation methods and tools developed by the NAABB consortium to produce algae biomass, in robust low energy systems, for biofuel production

The implementation of a service-learning component in an organic chemistry laboratory course

Education institutions globally are increasingly expected to explore avenues for the implementation of service-learning into their curricula. A second-year undergraduate organic chemistry laboratory experiment, in which the undergraduate students make azo dyes, can provide a vehicle for a service-learning module in which university undergraduate students then teach students from resource-limited secondary schools how to make azo dyes. Evidence is provided to show how the theory is reinforced for both sets of students through a shared practical experience. The practical application of chemistry is conveyed through the use of the synthetic azo dyes to dye tshirts. The results of this study show that the service-learning experience clearly assists undergraduate students to appreciate the role of chemists in the broader society while at the same time increasing awareness of the inequalities in school education systems

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

10.1371/journal.pone.0074040PLoS ONE89-POLN

A multicentre evaluation and expert recommendations of use of the newly developed BioFire Joint Infection polymerase chain reaction panel

Septic arthritis is a serious condition with significant morbidity and mortality, routinely diagnosed using culture. The FDA has recently approved the rapid molecular BioFire® Joint Infection Panel (BJIP) for synovial fluid. We aimed to evaluate the BJIP compared to culture and its potential use in patient management. A multicentre retrospective evaluation of BJIP was conducted in the UK and Ireland. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated between the BJIP and routine culture. A multidisciplinary team (MDT) discussion addressing the optimal or potential case use of the assay practice was facilitated. Three hundred ninety-nine surplus synovial fluid samples (~ 70% from native joints) from eight centres were processed using BJIP in addition to routine culture. An increased yield of positive results was detected using BJIP compared to routine culture (98 vs 83), giving an overall PPA of 91.6% and overall NPA of 93% for the BJIP compared to culture results. The BJIP detected resistant markers and additional organisms that could influence antibiotic choices including Neisseria gonorrhoeae and Kingella kingae. The MDT agreed that the assay could be used, in addition to standard methods, in adult and children patients with specialist advice use based on local needs. Rapid results from BJIP were assessed as having potential clinical impact on patient management. Organisms not included in the panel may be clinically significant and may limit the value of this test for PJI