A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

<p>Abstract</p> <p>Background</p> <p>Human African Trypanosomiasis (HAT) is caused by two trypanosome species, <it>Trypanosoma brucei rhodesiense </it>and <it>Trypanosoma brucei gambiense</it>. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness across species and disease stages, thus there is a considerable need to find alternative drugs. A well recognised approach to identify new drug candidates is high throughput screening (HTS) of large compound library collections.</p> <p>Results</p> <p>We describe here the development of a luciferase based viability assay in 384-well plate format suitable for HTS of <it>T.b.brucei</it>. The parameters that were explored to determine the final HTS assay conditions are described in detail and include DMSO tolerability, Z', diluents and cell inoculum density. Reference compound activities were determined for diminazene, staurosporine and pentamidine and compared to previously published IC₅₀data obtained. The assay has a comparable sensitivity to reference drugs and is more cost effective than the 96-well format currently reported for <it>T.b.brucei</it>.</p> <p>Conclusion</p> <p>Due to the reproducibility and sensitivity of this assay it is recommended for potential HTS application. As it is commercially available this assay can also be utilised in many laboratories for both large and small scale screening.</p

The Small and the Dead: A Review of Ancient DNA Studies Analysing Micromammal Species

The file attached is the Published/publisher’s pdf version of the article

Tripanocidno i citotoksično djelovanje etanolskoga iscrpka lišća Psidium guajava određivano bojanjem alamarskim plavilom

Ethanolic extracts prepared from the leaves of Psidium guajava were evaluated for anti-trypanosoma and cytotoxicity activity in the bloodstream species of Trypanosoma brucei brucei (BS427) and HEK293 in 384-well Alamar Blue assays respectively. Cytotoxicity activity in HEK293 cells was subsequently used to estimate the selectivity index of the extracts. The activities of the plant extracts were determined to evaluate if further chemical and biological profi ling may be warranted for potential development in early drug discovery for African Sleeping Sickness. Two trypanocides, pentamidine and diminazene, were employed as reference drugs, while puromycin was also included as control for general cell growth inhibition. The results show that the extracts inhibited growth of T. b. brucei with an IC50 of 6.3 μg/mL and 48.9 μg/mL for 80% and 20% ethanolic preparations respectively, with corresponding activity of less than 50% against HEK293 at the highest screening dose of 238.10 μg/mL. The estimated selectivity index of the extracts compares favourably with pentamidine and diminazene. Meanwhile the reference compounds were found to have activities in agreement with published sensitivities at the doses screened. The lack of cytotoxicity at the doses screened and direct activity against T. b. brucei whole cells, make these extracts suitable candidates for further chemical elucidation and biological profiling.Istraženo je tripanocidno djelovanje etanolskog iscrpka lišća Psidium guajava na vrstu Trypanosoma brucei brucei (BS427) i njegova citotoksičnost na stanice HEK293 bojanjem alamarskim plavilom u 384 jažice. Citotoksični učinak na stanice HEK293 rabljen je za procjenu indeksa selektivnosti. Učinkovitost biljnih iscrpaka određivana je da bi se procijenila svrhovitost budućih kemijskih i bioloških istraživanja potencijalnoga lijeka za afričku bolest spavanja. U istraživanju su rabljena dva tripanocida, pentamidin i diminazen, te puromicin kao sredstvo koje usporava rast stanica. Rezultati su pokazali da 80% etanolskih pripravaka s IC50 od 6,3 μg/mL koči rast i razvoj tripanosoma, a samo 20% onih s IC50 od 48,9 μg/mL, s odgovarajućom aktivnosti manjom od 50% na stanice HEK293 u najvećoj dozi od 238,10 μg/mL. Indeks selektivnosti iscrpaka bio je sukladan s aktivnošću pentamidina i diminazena. Aktivnost istraživanih sastojaka bila je sukladna s razinom prije objavljene osjetljivosti. Izostanak citotoksičnosti na razini rabljenih koncentracija i izravna djelotvornost na stanice T. b. brucei daju osnovu za daljnja kemijska i biološka istraživanja predmetnih pripravaka

Burden of respiratory viral infection in persons with human immunodeficiency virus

This study was conducted to determine the prevalence of respiratory viral infections (RVI) in persons living with HIV (PLH) admitted with a respiratory complaint using real-time reverse transcription polymerase chain reaction and primer-independent next-generation sequencing (NGS). Of 82 subjects, respiratory viruses were the most common pathogen identified in 27 (33%), followed by fungus and bacteria in 8 (10%) and 4 (5%) subjects, respectively. Among subjects with RVI, 11 (41%) required ICU admission and 16 (59%) required mechanical ventilation. The proportion of respiratory viruses identified, and the associated complicated hospital course highlights the significant role that RVIs play in the lung health of PLH

Randomized trial examining the effect of exercise and wellness interventions on preventing postpartum depression and perceived stress

Background Approximately 13–19% of postpartum women experience postpartum depression and a majority report at least some stress during the postpartum phase. Traditional interventions such as psychotherapy and antidepressant medications are often not feasible or desirable. The purpose of this study was to examine two low cost, brief, accessible interventions designed to prevent postpartum depression and perceived stress among women at high risk. Methods Participants (n = 450) who were on average four weeks postpartum, had a history of depression before pregnancy, and exercised less than 60 min per week were randomly assigned to one of the following three conditions: (1) 6-month telephone-based exercise intervention; (2) 6-month telephone-based wellness/support intervention (e.g., healthy eating, sleep, and perceived stress); or (3) usual care. Results Overall, 2.4% of participants met criteria for depression at 6 months and 3.6% at 9 months with no differences between groups. At 6 months following randomization, median symptoms of depression were significantly lower among wellness participants compared to usual care participants (b = − 1.00, SE = 0.46, p = .03). Perceived stress at 6 months post-randomization was significantly lower among exercise vs. usual care participants (b = − 2.00, SE = .98, p = .04) and exercise vs. wellness participants (b = − 2.20, SE = 1.11, p = .04). Conclusions The wellness intervention was efficacious for preventing symptoms of depression; however, postpartum depression that met the diagnostic criteria was surprisingly low in all conditions among this at risk sample of postpartum women. Exercise interventions may have a protective effect on perceived stress among women at risk for postpartum depression. Practitioners should consider integrating exercise and wellness interventions into postpartum care. Trial registration Clinical Trials Number: NCT01883479 (06/21/2013)

Re-evaluating pretomanid analogues for Chagas disease:Hit-to-lead studies reveal both in vitro and in vivo trypanocidal efficacy

Phenotypic screening of a 900 compound library of antitubercular nitroimidazole derivatives related to pretomanid against the protozoan parasite Trypanosoma cruzi (the causative agent for Chagas disease) identified several structurally diverse hits with an unknown mode of action. Following initial profiling, a first proof-of-concept in vivo study was undertaken, in which once daily oral dosing of a 7-substituted 2-nitroimidazooxazine analogue suppressed blood parasitemia to low or undetectable levels, although sterile cure was not achieved. Limited hit expansion studies alongside counter-screening of new compounds targeted at visceral leishmaniasis laid the foundation for a more in-depth assessment of the best leads, focusing on both drug-like attributes (solubility, metabolic stability and safety) and maximal killing of the parasite in a shorter timeframe. Comparative appraisal of one preferred lead (58) in a chronic infection mouse model, monitored by highly sensitive bioluminescence imaging, provided the first definitive evidence of (partial) curative efficacy with this promising nitroimidazooxazine class