Developing novel ways of studying motility in Schistosoma mansoni and its potential contribution towards inhibiting Schistosomiasis.

Schistosomiasis, caused by Schistosoma mansoni, is responsible for infecting approximately 200 million people worldwide, mostly from low-income and middle-income populations; it is a key neglected tropical parasitic disease, second only to malaria as the most devastating parasitic disease in the world. An infection is initiated when the cercarial form of the parasite is released from its intermediary invertebrate host, a Biomphalaria snail, into the surrounding fresh water. Cercariae are non-feeding, free swimming, extremely infectious, highly motile schistosomal stage with bifurcated tails and they penetrate the mammalian skin tail-first, thus infecting the human host. Post attachment, the cercariae sheds its tail and the resulting schistosomule continues to develop within the host circulatory system. The parasites travel to the hepatic system, where they transform into adult worms, mate and lay eggs, most of which are excreted through the host’s excretory system and the rest accumulating within the internal organs of the body. The spread of schistosomiasis relies heavily on the motility of the cercariae before human infection, as well as the movement of the schistosomules through the human body, post infection. For my doctoral dissertation, I have focused on the aspect of motility of the S.mansoni worms pre and post infection. The first part of my research deals with the design and development of a sensitive, simple, cheap biological assay i.e. a microfluidic platform to study the movement of the schistosomules as they travel through the host circulatory system. The complete navigation and the kinetics of the movement of the juvenile worms through the convoluted pulmonary, blood and hepatic vessels within the host remains largely unexplored. We believe that this novel approach will provide a highly efficient method for screening potential anti-schistosomal compounds and improving motility assays. The second part of my research concentrates on the qualitative and quantitative proteomic analysis of the cercarial tails and cercarial bodies. Using mechanical separation of cercarial tails and bodies, and mass spectrometric analyses, we have identified a total of 945 proteins in the combined cercarial proteome from 4 independent samples: 791 proteins in the cercarial tails and 645 proteins from the somule bodies. Gene oncology analysis was conducted on the obtained proteomic data, and the peptide hits were classified based on molecular function, biological function and subcellular location. In conclusion, I believe that by preventing the motility of the parasitic worms at different stages of the life cycle is a novel, previously unexplored route for investigating potential drug targets which could interrupt the spread of the disease

A Miniaturized Screen of a Schistosoma mansoni Serotonergic G Protein-Coupled Receptor Identifies Novel Classes of Parasite-Selective Inhibitors

Schistosomiasis is a tropical parasitic disease afflicting ~200 million people worldwide and current therapy depends on a single drug (praziquantel) which exhibits several non-optimal features. These shortcomings underpin the need for next generation anthelmintics, but the process of validating physiologically relevant targets (‘target selection’) and pharmacologically profiling them is challenging. Remarkably, even though over a quarter of current human therapeutics target rhodopsin-like G protein coupled receptors (GPCRs), no library screen of a flatworm GPCR has yet been reported. Here, we have pharmacologically profiled a schistosome serotonergic GPCR (Sm.5HTR) implicated as a downstream modulator of PZQ efficacy, in a miniaturized screening assay compatible with high content screening. This approach employs a split luciferase based biosensor sensitive to cellular cAMP levels that resolves the proximal kinetics of GPCR modulation in intact cells. Data evidence a divergent pharmacological signature between the parasitic serotonergic receptor and the closest human GPCR homolog (Hs.5HTR7), supporting the feasibility of optimizing parasitic selective pharmacophores. New ligands, and chemical series, with potency and selectivity for Sm.5HTR over Hs.5HTR7 are identified in vitro and validated for in vivo efficacy against schistosomules and adult worms. Sm.5HTR also displayed a property resembling irreversible inactivation, a phenomenon discovered at Hs.5HTR7, which enhances the appeal of this abundantly expressed parasite GPCR as a target for anthelmintic ligand design. Overall, these data underscore the feasibility of profiling flatworm GPCRs in a high throughput screening format competent to resolve different classes of GPCR modulators. Further, these data underscore the promise of Sm.5HTR as a chemotherapeutically vulnerable node for development of next generation anthelmintics

Theoretical Engineering of the Gut Micro biome for the Purpose of Creating Superior Soldiers

The purpose of this review is to highlight research raising the possibility of exploiting the host-microbiome gut axis for military purposes. Through optimizing the gut-microbiome environment it is possible to enhance nutritional access to indigestible material, provide local and systemic analgesia, enhance psychological robustness to battlefield stress, produce endogenous steroids, reduce muscle fatigue, and promote peripheral wound healing. However, this approach is still in its early stages and thus has not been explored to its full potential. The challenges that are currently preventing the practical use of gut bacteria include the following: inconsistency of clinical outcomes, transient effects requiring continuous supplementation, the type of regimen selected, the initiation and cessation of regimen, and the broader clinical studies needed to validate this research. This review is intended to shed light on the numerous and varied positive impacts such an approach could have for the military if further developed

Veterinary Considerations for the Theoretical Resurrection of Extinct Species

The de-extinction of the dinosaur is a dubious possibility but its consideration brings forth some issues that are at least worthy of scientific discussion. In this review, we discuss two distinct issues that have implications for a de-extinct species such as a dinosaur: the ability, or lack thereof, to safely sedate a rare and potentially fractious animal capable of harming the veterinary staff tasked with its care; and, disease risks associated with a species that has been extinct for millions of years. To identify potential sedatives, comparative pharmacology will be needed to uncover the links between receptor pharmacology and the desired clinical outcomes of activating established alpha-2 adrenergic, opioid, and benzodiazepine receptors. Specific to disease control, it will be necessary to understand the unique susceptibility of the new species to current diseases as well as predicting their reservoir capacity for potential human and veterinary pandemic diseases. While the topics presented herein are not exhaustive, this review highlights some of the foremost research that should be conducted in order to serve the unique veterinary needs of a de-extinct species using the dinosaur as a paradigm. Addressing these issues should be considered if an intact dinosaur genome becomes available, regardless of the feasibility of dinosaur resurrection

Developing novel ways of studying motility in Schistosoma mansoni and its potential contribution towards inhibiting Schistosomiasis.

Schistosomiasis, caused by Schistosoma mansoni, is responsible for infecting approximately 200 million people worldwide, mostly from low-income and middle-income populations; it is a key neglected tropical parasitic disease, second only to malaria as the most devastating parasitic disease in the world. An infection is initiated when the cercarial form of the parasite is released from its intermediary invertebrate host, a Biomphalaria snail, into the surrounding fresh water. Cercariae are non-feeding, free swimming, extremely infectious, highly motile schistosomal stage with bifurcated tails and they penetrate the mammalian skin tail-first, thus infecting the human host. Post attachment, the cercariae sheds its tail and the resulting schistosomule continues to develop within the host circulatory system. The parasites travel to the hepatic system, where they transform into adult worms, mate and lay eggs, most of which are excreted through the host’s excretory system and the rest accumulating within the internal organs of the body. The spread of schistosomiasis relies heavily on the motility of the cercariae before human infection, as well as the movement of the schistosomules through the human body, post infection. For my doctoral dissertation, I have focused on the aspect of motility of the S.mansoni worms pre and post infection. The first part of my research deals with the design and development of a sensitive, simple, cheap biological assay i.e. a microfluidic platform to study the movement of the schistosomules as they travel through the host circulatory system. The complete navigation and the kinetics of the movement of the juvenile worms through the convoluted pulmonary, blood and hepatic vessels within the host remains largely unexplored. We believe that this novel approach will provide a highly efficient method for screening potential anti-schistosomal compounds and improving motility assays. The second part of my research concentrates on the qualitative and quantitative proteomic analysis of the cercarial tails and cercarial bodies. Using mechanical separation of cercarial tails and bodies, and mass spectrometric analyses, we have identified a total of 945 proteins in the combined cercarial proteome from 4 independent samples: 791 proteins in the cercarial tails and 645 proteins from the somule bodies. Gene oncology analysis was conducted on the obtained proteomic data, and the peptide hits were classified based on molecular function, biological function and subcellular location. In conclusion, I believe that by preventing the motility of the parasitic worms at different stages of the life cycle is a novel, previously unexplored route for investigating potential drug targets which could interrupt the spread of the disease.</p

Pharmacological profiling an abundantly expressed schistosome serotonergic GPCR identifies nuciferine as a potent antagonist

5-hydroxytryptamine (5-HT) is a key regulator of muscle contraction in parasitic flatworms. In Schistosoma mansoni, the myoexcitatory action of 5-HT is effected through activation of a serotonergic GPCR (Sm.5HTRL), prioritizing pharmacological characterization of this target for anthelmintic drug discovery. Here, we have examined the effects of several aporphine alkaloids on the signaling activity of a heterologously expressed Sm.5HTRL construct using a cAMP biosensor assay. Four structurally related natural products – nuciferine, D-glaucine, boldine and bulbocapnine – were demonstrated to block Sm.5HTRL evoked cAMP generation with the potency of GPCR blockade correlating well with the ability of each drug to inhibit contractility of schistosomule larvae. Nuciferine was also effective at inhibiting both basal and 5-HT evoked motility of adult schistosomes. These data advance our understanding of structure-affinity relationships at Sm.5HTRL, and demonstrate the effectiveness of Sm.5HTRL antagonists as hypomotility-evoking drugs across different parasite life cycle stages

Pharmacological profiling of Sm.5HTR.

<p>(<b>A</b>) Schematic of assay workflow for screening a library of known GPCR ligands against HEK293 cells expressing either Sm.5HTR or Hs.5HT7. Cells transfected with either 5HTR and F22 cAMP biosensor were plated in 96-well format and exposed to test compounds (10μM). 5-HT was added after 30mins (at an EC₈₀ concentration) after which luminescence values were recorded (time = 60min). (<b>B</b>) Scatter plots summarizing effects of test compounds on the Sm.5HTR response to 5-HT (dotted line highlights threshold for defining compound ‘hits’). Hits were defined at a threshold of ≥50% inhibition relative to control wells (DMSO only, open symbols). (<b>C</b>) Compounds were also screened against HEK293 cells expressing the F22 sensor alone (no Sm.5HTR) to screen for cAMP generation at endogenous receptors. For reference, a forskolin data point is shown in red. (<b>D</b>) Compounds were also screened against forskolin (20μM) evoked changes in luminescence relative to control samples (DMSO, open circles). (<b>E</b>) Heat map of all test compounds screened against <i>S</i>. <i>mansoni</i> Sm.5HTR (left) and human Hs.5HT7 (middle). Each colored box represents the fold change in luminescence in response to an individual test compound (253 in total) keyed by the pseudocolor scale. Compounds showing activity against endogenous receptors in cells transfected with the 22F biosensor alone (21 compounds total) were masked (black). Right, Venn diagram summarizing selectivity of antagonist ‘hits’ against either Sm.5HTR, Hs.5HT7R, or both 5-HT receptors. In total, 23 ligands were classified as potential ‘hits’ at Sm.5HTR and 31 ligands as ‘hits’ at Hs.5HTR7, with 7 in common.</p

Long lasting inhibition of schistosome movement caused by bromocriptine.

<p>Time course of schistosome recovery from exposure to Sm.5HTR antagonists (black = DMSO control, blue = rotundine, red = bromocriptine). Mobility of worms was recorded following exposure to antagonist (10μM, ‘drug’, 2 hour exposure) and addition of 5-HT (100μM, ‘drug + 5-HT’). Media was then exchanged, and recordings were subsequently made on the same worms stimulated with 5-HT (100μM) at the indicated timepoints (3, 6, 24hrs after drug washout). Data for males (A) and females (B) is presented normalized to basal movement of control (dmso) worms in the absence of 5-HT.</p

Structure activity relationships for various drug classes against Hs.5HT7R and Sm.5HTR.

<p>Comparison of IC₅₀s for compounds inhibiting cAMP generation via Hs.5HT7R (abscissa) or Sm.5HTR (ordinate). Compounds with no preferential selectivity for either receptor, showing similar IC₅₀s, would cluster along the solid line. Hits in the lower right quadrant (red square) show sub-μM potency at Sm.5HTR but supra-μM potency at Hs.5HT7R. Four compounds meet this criterion (bromocriptine = ‘39’, rotundine = ‘37’, tetrandrine = ‘31’ and tetrabenazine = ‘32’). Compound classes are indicated as follows: ergot alkaloids (green), isoquinolines (blue), tricyclic and tetracyclic antidepressants (magenta), sulfonyl compounds (orange), miscellaneous structures (open). Individual compounds are: 1, SB269970; 2, amisulpride; 3, SB742457; 4, olanzapine; 5, mianserin; 6, quetiapine; 7, clozapine; 8, cyproheptadine; 9, ketotifen; 10, loratadine; 11, maprotiline; 12, clomipramine; 13, desloratadine; 14, rupatadine; 15, vortioxetine; 16, amitriptyline; 17, risperidone; 18, domperidone; 19, chlorprothixene; 20, clemastine; 21, aripiprazole; 22, ketanserin; 23, ifenprodil; 24, tripelennamine; 25, fluoxetine; 26, atomoxetine; 27, orphenadrine; 28, lisuride; 29, benztropine; 30, cyclizine; 31, tetrandrine; 32, tetrabenazine; 33, berberine; 34, 6, 7-diethoxy-1, 2, 3, 4-tetrahydroisoquinoline; 35, corynoline; 36, alfuzosin; 37, rotundine; 38, fanchinoline; 39, bromocriptine; 40, metergoline; 41, LY215840; 42, nicergoline; 43, mesulergine; 44, dihydroergocristine.</p

Long lasting inhibition of Sm.5HT7R evoked by a subset of ligands.

<p>Sm.5HTR displays an inactivating antagonist property reported for human 5HT7R. (<b>A</b>) Both the ‘inactivating antagonist’ bromocriptine (10μM, blue) and the competitive antagonist cyproheptadine (10μM, grey) acutely inhibit the effect of 5-HT (10μM) at Sm.5HTR (5-HT alone, black). (<b>B</b>) Sm.5HTR remains insensitive to 5-HT following washout of bromocriptine but not cyproheptadine. Cells were pre-incubated with antagonists as in (A) for 30 mins, followed by solution exchange, and the assay for 5-HT responsiveness 1hr later. (<b>C</b>) Inhibition of 5-HT response at Sm.5HTR by both ‘inactivating antagonists’ established at Hs.5HT7R (methiothepin, bromocriptine, lisuride, risperidone, metergoline) and competitive antagonists (clozapine, cyproheptadine). All drugs were tested at 10μM for 30mins. **, p <0.01. (<b>D</b>) Persistent effects of antagonists (10μM) shown in (C) after washout and subsequent assay for 5-HT response (1hr later). **, p <0.01, *, p <0.05. (<b>E</b>) Titration of these ‘inactivating antagonists’ revealed the dose-response relationship for Sm.5HTR inhibition after washout. Colors correspond to drug identity in C&D. Data represent mean±s.e.m., n = 3 (C-E).</p