Leukocyte proliferation mediates disease pathogenesis in the Ndufs4(KO) mouse model of Leigh syndrome

Symmetric, progressive, necrotizing lesions in the brainstem are a defining feature of Leigh syndrome (LS). A mechanistic understanding of the pathogenesis of these lesions has been elusive. Here, we report that leukocyte proliferation is causally involved in the pathogenesis of Leigh syndrome. Directly depleting leukocytes with a colony-stimulating factor 1 receptor (CSF1R) inhibitor dramatically attenuates disease, including complete prevention of CNS lesion formation and substantial extension of survival. Leukocyte depletion rescues a range of symptoms including hyperlactemia, seizures, respiratory function, and neurologic symptoms. These data provide a mechanistic explanation for the beneficial effects of mTOR inhibition. More importantly, these findings dramatically alter our understanding of the pathogenesis of LS, demonstrating that immune involvement directly drives disease. These findings have significant implication for the mechanisms of disease resulting from mitochondrial dysfunction, and may lead to novel therapeutic strategies.

Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low-parasitaemia samples

Background Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined. Methods DNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA. Results MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI+sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration + sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. Conclusion This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies. Keywords Plasmodium falciparum , malaria, whole genome sequencing, selective whole genome amplification, vacuum filtration

Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low-parasitemia samples

Background: Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitemia samples. In this study, we evaluated whether enzymatic digestion with MspJI prior to sWGA improved genome coverage compared to sWGA alone. We also examined the potential of sWGA to cause amplification bias in polyclonal infections. Methods: DNA extracted from lab-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA. Results: MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI+sWGA and sWGA alone, particularly for low parasitemia samples. The optimized sWGA (filtration + sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. Conclusion: This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.

Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low-parasitemia samples

Background: Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA, reducing the need for pre-processing of samples. However, sWGA has had limited success in generating reliable sequencing data from low parasitemia samples. In this study, we evaluated whether enzymatic digestion with MspJI prior to sWGA and whole genome sequencing improves genome coverage compared to sWGA alone when applied to samples representing a range of parasitemias. We also examined the potential of sWGA to cause amplification bias in polyclonal infections. Results: MspJI digestion did not enrich for parasite DNA. Samples that underwent filtration prior to sWGA had the highest parasite DNA yield and displayed higher genome coverage compared to MspJI+sWGA and sWGA only, particularly for low parasitemia samples. The optimized sWGA approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasite isolates from the same geographic region generated using the optimized sWGA did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. Conclusion: The optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.

Integrated Microfluidic Device for Enrichment and Identification of Circulating Tumor Cells from the Blood of Patients with Colorectal Cancer

Integrated device with high purity for circulating tumor cell (CTC) identification has been regarded as a key goal to make CTC analysis a “bench-to-bedside” technology. Here, we have developed a novel integrated microfluidic device that can enrich and identify the CTCs from the blood of patients with colorectal cancer. To enrich CTCs from whole blood, microfabricated trapping chambers were included in the miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and normal blood cells. Microvalves were also introduced sequentially in the device, enabling automatic CTC enrichment as well as immunostaining reagent delivery. Under optimized conditions, the whole blood spiked with caco-2 cells passing through the microfluidic device after leukocyte depletion and approximately 73% of caco-2 cells were identified by epithelial cell adhesion molecule (EpCAM) staining. In clinical samples, CTCs were detectable from all patients with advanced colorectal cancer within 3 h. In contrast, the number of CTCs captured on the device from the blood of healthy donors was significantly lower than that from the patients, suggesting the utilization of the integrated device for further molecular analyses of CTCs.

Self-Seeding Microwells to Isolate and Assess the Viability of Single Circulating Tumor Cells

The availability of viable tumor cells could significantly improve the disease management of cancer patients. Here we developed and evaluated a method using self-seeding microwells to obtain single circulating tumor cells (CTC) and assess their potential to expand. Conditions were optimized using cells from the breast cancer cell line MCF-7 and blood from healthy volunteers collected in EDTA blood collection tubes. 43% of the MCF-7 cells (nucleus+, Ethidium homodimer-1-, Calcein AM+, α-EpCAM+, α-CD45-) spiked into 7.5 mL of blood could be recovered with 67% viability and these could be further expanded. The same procedure tested in metastatic breast and prostate cancer patients resulted in a CTC recovery of only 0–5% as compared with CTC counts obtained with the CellSearch® system. Viability of the detected CTC ranged from 0–36%. Cell losses could be mainly contributed to the smaller size and greater flexibility of CTC as compared to cultured cells from cell lines and loss during leukocyte depletion prior to cell seeding. Although CTC losses can be reduced by fixation, to obtain viable CTC no fixatives can be used and pore size in the bottom of microwells will need to be reduced, filtration conditions adapted and pre-enrichment improved to reduce CTC losses.