Alison Roth

Enhancing longevity of Plasmodium vivax and P. falciparum sporozoites after dissection from mosquito salivary glands.

The pre-erythrocytic stages of Plasmodium vivax and Plasmodium falciparum remain challenging for experimental research in part due to limited access to sporozoites. An important factor limiting availability is the laboratory support required for producing infected mosquitoes and the ephemeral nature of isolated extracellular sporozoites. This study was undertaken to investigate methods to improve the availability of this limited resource by extending the longevity of the extracellular sporozoites after mosquito dissection. Our goal in this study was to determine whether buffer conditions more closely mimicking the insect microenvironment could prolong longevity of ex vivo P. vivax and P. falciparum sporozoites. The study compared the current standard dissection buffer RPMI1640 to Hanks Balanced Salt Solution with 1g/L glucose (HBSS-1) or 2g/L glucose (HBSS-2) and Graces Insect Medium for ability to extend longevity of ex vivo P. vivax and P. falciparum sporozoites. The effect of each buffer on sporozoite viability was evaluated by measuring sporozoite gliding motility at 0, 4, 8, and 24h post-dissection from mosquito salivary glands. Comparisons of mean gliding percentages of ex vivo sporozoites in the different buffers and time points found that RPMI and Graces both showed strong gliding at 0h. In contrast, by 4h post-dissection sporozoites in RPMI consistently had the lowest gliding activity, whereas sporozoites in Graces had significantly more gliding compared to all other buffers at almost all time points. Our results indicate that P. vivax and P. falciparum sporozoites maintained in insect media rather than the standard dissection buffer RPMI and HBSS retain viability better over time.

A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.Currently available platforms to study liver stage of Plasmodium species have limitations. Here, the authors show that primary human hepatocyte cultures in 384-well format support hypnozoite and other liver stage development and are suitable for drug and antibody screens.

Multi-wavelength transmission spectroscopy revisited for micron and submicron particle characterization.

Multi-wavelength transmission (MWT) ultraviolet-visible-near-infrared (UV-Vis-NIR) spectroscopy, a technique underappreciated for particle characterization, is systematically explored using a set of NIST traceable standards over the nominal size range of 20 to 20 000 nm. Experimental results demonstrate that the particle size distributions obtained from MWT spectral data are in excellent agreement with the values reported by the manufacturer. In addition, it is shown that quantitative information on the particle concentration can be obtained—which is not currently accessible from commercially available light scattering instrumentation. The results validate that MWT UV-Vis-NIR spectroscopy has a considerable dynamic range for particle size measurements and offers significant advantages over other particle characterization techniques. Among these are the simplicity of the instrumentation and the measurements and the wealth of quantitative information contained in the MWT spectra. Most importantly, with standardized measurement protocols and standardized spectrometer configurations, MWT measurements can be used to provide the user and the manufacturer of particles with traceable data (i.e., the spectra and the quantitative analysis) for quality assurance.

Unraveling the Plasmodium vivax sporozoite transcriptional journey from mosquito vector to human host

Malaria parasites transmitted by mosquito bite are remarkably efficient in establishing human infections. The infection process requires roughly 30 minutes and is highly complex as quiescent sporozoites injected with mosquito saliva must be rapidly activated in the skin, migrate through the body, and infect the liver. This process is poorly understood for Plasmodium vivax due to low infectivity in the in vitro models. To study this skin-to-liver-stage of malaria, we used quantitative bioassays coupled with transcriptomics to evaluate parasite changes linked with mammalian microenvironmental factors. Our in vitro phenotyping and RNA-seq analyses revealed key microenvironmental relationships with distinct biological functions. Most notable, preservation of sporozoite quiescence by exposure to insect-like factors coupled with strategic activation limits untimely activation of invasion-associated genes to dramatically increase hepatocyte invasion rates. We also report the first transcriptomic analysis of the P. vivax sporozoite interaction in salivary glands identifying 118 infection-related differentially-regulated Anopheles dirus genes. These results provide important new insights in malaria parasite biology and identify priority targets for antimalarial therapeutic interventions to block P. vivax infection.

Author Correction: A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum

The original version of this Article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article.

Reagentless Bacterial Identification Using a Combination of Multiwavelength Transmission and Angular Scattering Spectroscopy

Optics based technologies are being advanced by many diagnostic companies around the globe. This resurgence is being driven by several factors including novel materials, enhanced computer power, nonlinear optics, and advances in algorithmic and statistical analysis. This study expands on a previous paper that evaluated the capability of a reagent-free optical profiling platform technology that used multiwavelength transmission spectroscopy to identify bacterial pathogens from pure culture. This study combines multiwavelength angular scattering with transmission based analysis into a single algorithm that will identify bacterial pathogens. Six predominant organisms, S. aureus, E. coli, K. pneumoniae and P. aeruginosa, E. faecalis, and coagulase negative Staphylococcus, were analyzed from a total of 753 clinical isolates received from three large community hospital systems. The bacterial identification method used for comparison in this study was the Vitek-2 (bioMerieux) which utilizes a biochemically based identification system. All of the clinical isolates received were blinded as to their identification until completion of the optical analysis. Sensitivities ranged from 87.7 to 94.6% with specificities ranging from 97.2 to 99.9% indicating that optical profiling is a powerful and exciting new technology that could be developed for the rapid identification of pathogens without the use of chemical reagents.

Subcellular and in-vivo Nano-Endoscopy.

Analysis of individual cells at the subcellular level is important for understanding diseases and accelerating drug discovery. Nanoscale endoscopes allow minimally invasive probing of individual cell interiors. Several such instruments have been presented previously, but they are either too complex to fabricate or require sophisticated external detectors because of low signal collection efficiency. Here we present a nanoendoscope that can locally excite fluorescence in labelled cell organelles and collect the emitted signal for spectral analysis. Finite Difference Time Domain (FDTD) simulations have shown that with an optimized nanoendoscope taper profile, the light emission and collection was localized within ~100 nm. This allows signal detection to be used for nano-photonic sensing of the proximity of fluorophores. Upon insertion into the individual organelles of living cells, the nanoendoscope was fabricated and resultant fluorescent signals collected. This included the signal collection from the nucleus of Acridine orange labelled human fibroblast cells, the nucleus of Hoechst stained live liver cells and the mitochondria of MitoTracker Red labelled MDA-MB-231 cells. The endoscope was also inserted into a live organism, the yellow fluorescent protein producing nematode Caenorhabditis elegans, and a fluorescent signal was collected. To our knowledge this is the first demonstration of in vivo, local fluorescence signal collection on the sub-organelle level.

Multiwavelength Transmission Spectroscopy Revisited for the Characterization of the Protein and Polystyrene Nanoparticle Mixtures

Multiwavelength Transmission (MWT) UV-Vis-NIR spectroscopy, an effective technique often underutilized for the characterization of processes involving particulates, such as protein aggregation, is systematically explored using bovine serum albumin and a set of NIST-traceable particle size (PS) standards having certified particle diameters over the nominal size range of 30 to 100 nm. The PS standards are used as surrogates for protein aggregates and other contaminants such as oils and microbubbles. Therefore, the standards can be used to quantitatively modify the optical properties of protein solutions and thus observe the effect of the presence of aggregates and other particulates on their wavelength-dependent transmission spectra. The experimental results demonstrate that the changes induced in the optical density spectra of proteins due to the presence of PS particles are detectable and consistent with the expectations set by light scattering theory. It is demonstrated that the size and relative concentrations of the particle populations present in the protein samples can be quantified. Because of the considerable dynamic range of MWT UV-Vis-NIR spectroscopy for particle analysis and its real-time measurement capabilities, this type of spectroscopy can be effectively used for the characterization of protein aggregates and for the continuous real-time monitoring of aggregation processes and for the identification and quantification of contaminants in protein-based products.