Differences in transcription between free-living and CO_2-activated third-stage larvae of Haemonchus contortus

Background: The disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development. Results: Totals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma-secreted proteins (ASPs). Using an in silico-subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development. Conclusion: The present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention

Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease

Biomarkers are becoming increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered 'synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: they target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that these agents can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and substantially improve early detection of cancer compared with current clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and point-of-care diagnostics.National Institutes of Health (U.S.) (Bioengineering Research Partnership R01 CA124427)Kathy and Curt Marble Cancer Research FundNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32CA159496-01

Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease

Biomarkers are increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered `synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that they can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and can substantially improve early detection of cancer compared with clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and to point-of-care diagnostics

Molecular Mechanisms Regulating Chronological Aging and Cell Death in the Toxic Dinoflagellate, Karenia brevis

The toxic dinoflagellate, Karenia brevis, forms nearly annual blooms in the Gulf of Mexico that persist for many months in coastal waters, causing extensive marine animal mortalities and human health impacts. The molecular mechanisms that contribute to cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. The studies presented in this dissertation investigate the existence and involvement of a programmed cell death-like (PCD-like) pathway in the demise of K. brevis cultures following oxidative stress and chronological aging. Firstly, to gain an understanding of the molecular processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out and identified extensive transcriptomic remodeling during the transition into stationary phase indicative of a shift in the metabolic and signaling requirements for survival in a quiescent non-dividing phase. To better understand the connection between the transcriptomic context identified in the microarray study and the presence of a PCO-like pathway in K. brevis, hallmark morphological and biochemical changes (DNA fragmentation, caspase-like activity, and caspase 3-like protein expression) were used to define PCD-like morphological changes following chronological aging and oxidative stress. Targeted in silico bioinformatic mining was used to identify enzymes potentially responsible for the activities observed, as well as the substrates. Finally, K. brevis S-adenosylmethionine synthetase (KbAdoMetS), a putative caspase substrate predicted from the bioinformatics screen, was examined using MALDI-TOF MS to confirm the validity of the bioinformatics approach. Taken together, this work identified that K. brevis contains morphological changes indicative of a caspase-dependent PCD-like pathway and that KbAdoMetS is a caspase 3-like substrate. Finally, we sought to characterize the presence of metacaspases in Karenia brevis, and specifically evaluated the role of metacaspase 1 (KbMC1) during chronological aging and death in culture. Immunocytochemistry, subcellular fractionation, and western blotting results using a custom KbMC1 peptide antibody indicate that KbMC1 may be involved in PCD-like execution through its chloroplastic localization with proposed interactions with the photosynthetic machinery. This study provides the first comprehensive investigation of the molecular processes regulating chronological aging and execution of PCD-like death in a toxic dinoflagellate