A Leishmania secretion system for the expression of major ampullate spidroin mimics

Spider major ampullate silk fibers have been shown to display a unique combination of relatively high fracture strength and toughness compared to other fibers and show potential for tissue engineering scaffolds. While it is not possible to mass produce native spider silks, the potential ability to produce fibers from recombinant spider silk fibers could allow for an increased innovation rate within tissue engineering and regenerative medicine. In this pilot study, we improved upon a prior fabrication route by both changing the expression host and additives to the fiber pulling precursor solution to improve the performance of fibers. The new expression host for producing spidroin protein mimics, protozoan parasite Leishmania tarentolae, has numerous advantages including a relatively low cost of culture, rapid growth rate and a tractable secretion pathway. Tensile testing of hand pulled fibers produced from these spidroin-like proteins demonstrated that additives could significantly modify the fiber’s mechanical and/or antimicrobial properties. Cross-linking the proteins with glutaraldehyde before fiber pulling resulted in a relative increase in tensile strength and decrease in ductility. The addition of ampicillin into the spinning solution resulted in the fibers being able to inhibit bacterial growth

A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity. PLoS Negl Trop. Dis

Abstract Background: The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the c-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

African sleeping sickness is a disease found in sub-Saharan Africa that is caused by the single-celled parasite Trypanosoma brucei. The drugs used widely now to treat infections are 50 years old and notable for their toxicity, emphasizing the need for development of new therapeutics. In the search for potential drug targets, researchers typically focus on enzymes or proteins that are essential to the survival of the infectious agent while being distinct enough from the host to avoid accidental targeting of the host enzyme. This work describes our research on one such trypanosome enzyme, hexokinase, which is a protein that the parasite requires to make energy. Here we describe the results of our search for inhibitors of the parasite enzyme. By screening 220,223 compounds for anti-hexokinase activity, we have identified new inhibitors of the parasite enzyme. Some of these are toxic to trypanosomes while having no effect on mammalian cells, suggesting that they may hold promise for the development of new anti-parasitic compounds

Haematological consequences of acute uncomplicated falciparum malaria: a WorldWide Antimalarial Resistance Network pooled analysis of individual patient data

Background: Plasmodium falciparum malaria is associated with anaemia-related morbidity, attributable to host, parasite and drug factors. We quantified the haematological response following treatment of uncomplicated P. falciparum malaria to identify the factors associated with malarial anaemia. Methods: Individual patient data from eligible antimalarial efficacy studies of uncomplicated P. falciparum malaria, available through the WorldWide Antimalarial Resistance Network data repository prior to August 2015, were pooled using standardised methodology. The haematological response over time was quantified using a multivariable linear mixed effects model with nonlinear terms for time, and the model was then used to estimate the mean haemoglobin at day of nadir and day 7. Multivariable logistic regression quantified risk factors for moderately severe anaemia (haemoglobin < 7 g/dL) at day 0, day 3 and day 7 as well as a fractional fall ≥ 25% at day 3 and day 7. Results: A total of 70,226 patients, recruited into 200 studies between 1991 and 2013, were included in the analysis: 50,859 (72.4%) enrolled in Africa, 18,451 (26.3%) in Asia and 916 (1.3%) in South America. The median haemoglobin concentration at presentation was 9.9 g/dL (range 5.0–19.7 g/dL) in Africa, 11.6 g/dL (range 5.0–20.0 g/dL) in Asia and 12.3 g/dL (range 6.9–17.9 g/dL) in South America. Moderately severe anaemia (Hb < 7g/dl) was present in 8.4% (4284/50,859) of patients from Africa, 3.3% (606/18,451) from Asia and 0.1% (1/916) from South America. The nadir haemoglobin occurred on day 2 post treatment with a mean fall from baseline of 0.57 g/dL in Africa and 1.13 g/dL in Asia. Independent risk factors for moderately severe anaemia on day 7, in both Africa and Asia, included moderately severe anaemia at baseline (adjusted odds ratio (AOR) = 16.10 and AOR = 23.00, respectively), young age (age < 1 compared to ≥ 12 years AOR = 12.81 and AOR = 6.79, respectively), high parasitaemia (AOR = 1.78 and AOR = 1.58, respectively) and delayed parasite clearance (AOR = 2.44 and AOR = 2.59, respectively). In Asia, patients treated with an artemisinin-based regimen were at significantly greater risk of moderately severe anaemia on day 7 compared to those treated with a non-artemisinin-based regimen (AOR = 2.06 [95%CI 1.39–3.05], p < 0.001). Conclusions: In patients with uncomplicated P. falciparum malaria, the nadir haemoglobin occurs 2 days after starting treatment. Although artemisinin-based treatments increase the rate of parasite clearance, in Asia they are associated with a greater risk of anaemia during recovery

Student performance on the Test of Scientific Literacy Skills (TOSLS) does not change with assignment of a low-stakes grade

Abstract Objective Response-validated multiple-choice assessments are used in college courses to assess student learning gains. The ability of a test to accurately reflect student learning gains is highly dependent on the students’ effort. Within our institution, lackluster student effort is common on response-validated multiple-choice concept assessments that are not included as a portion of the semester grade but are used to inform curricular changes. Thus, we set out to determine whether increasing testing stakes by assigning a grade on student performance had an effect on student score and self-reported effort. The Test of Scientific Literacy Skills (TOSLS) is a response-validated multiple-choice assessment used to measure scientific literacy in undergraduates. We administered the TOSLS to students enrolled in a general education Biology course, both during the first 2 weeks (pretest) and the last 2 weeks (posttest) of the course. Results Self-reported effort and TOSLS performance were significantly correlated in the ungraded cohort. This relationship did not exist in the graded sections. Our data indicate that assigning a low-stakes grade has no significant effect on mean student performance or self-reported effort on the TOSLS within our general education course

A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented—one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

Bacterial growth inhibition assays.

<p>Inhibition zone assay with Leishmania derived fibers treated with or without ampicillin. Ampicillin was added to gellan gum solution at 10 mg/mL prior to pulling fibers. Fibers were allowed to air dry for several hours and then placed on LB-agar plates spread with <i>E</i>. <i>coli</i> bacteria. A, an inhibition plate assay using a recombinant MaSp2R8 fiber; B, an inhibition plate assay using a recombinant MaSp2R8 fiber which was treated with ampicillin during the fiber production stage; C, liquid culture showing inhibition of bacterial growth when grown in the presence of a recombinant MaSp2R8 fiber treated with ampicillin during the production process.</p

Primers used in the construction of plasmids containing <i>L</i>. <i>mexicana</i> invertase secretion signal.

<p>The underlined DNA sequences indicate the addition of restriction sites to the primer for use in the molecular cloning process.</p

SEM break point observations of rMaSp2R8 fibers.

<p>Panels A, rMaSp2R8 fiber; Panels B, rMaSp2R8 fiber pretreated with ampicillin before fiber pulling; Panels C, rMaSp2R8 fiber pretreated with glutaraldehyde before fiber pulling. Scale bars show 100 μm or 20 μm lengths.</p