Why accelerating partnerships in the Sanitation Economy really matters

Initiated in 2014, the Toilet Board Coalition (TBC) has the ambition to address the global sanitation crisis by accelerating the Sanitation Economy – a market valued up to $12 trillion a year. Business-led, the Toilet Board Coalition believes in market-based solutions, commercially and financially sustainable, to provide universal access to sanitation (SDG No. 6) and reach low-income consumers in emerging markets. To this end, the Coalition brings together large corporations, international development agencies, international organizations, entrepreneurs and NGOs. Through its 12-month accelerator program, the Coalition creates partnerships between sanipreneurs and big corporations to address the numerous barriers preventing innovations to scale. As “Every business’s business”, the Sanitation Economy could unlock promising and profitable opportunities in all sectors, including agriculture and preventive health, even more so in the aftermath of the global COVID-19 pandemic

Profiling G protein-coupled receptors of Fasciola hepatica identifies orphan rhodopsins unique to phylum Platyhelminthes

G protein-coupled receptors (GPCRs) are established drug targets. Despite their considerable appeal as targets for next-generation anthelmintics, poor understanding of their diversity and function in parasitic helminths has thwarted progress towards GPCR-targeted anti-parasite drugs. This study facilitates GPCR research in the liver fluke, Fasciola hepatica, by generating the first profile of GPCRs from the F. hepatica genome. Our dataset describes 147 high confidence GPCRs, representing the largest cohort of GPCRs, and the largest set of in silico ligand-receptor predictions, yet reported in any parasitic helminth. All GPCRs fall within the established GRAFS nomenclature; comprising three glutamate, 135 rhodopsin, two adhesion, five frizzled, one smoothened, and one secretin GPCR. Stringent annotation pipelines identified 18 highly diverged rhodopsins in F. hepatica that maintained core rhodopsin signatures, but lacked significant similarity with non-flatworm sequences, providing a new sub-group of potential flukicide targets. These facilitated identification of a larger cohort of 76 related sequences from available flatworm genomes, representing new members of existing groups (PROF1/Srfb, Rho-L, Rho-R, Srfa, Srfc) of flatworm-specific rhodopsins. These receptors imply flatworm specific GPCR functions, and/or co-evolution with unique flatworm ligands, and could facilitate the development of exquisitely selective anthelmintics. Ligand binding domain sequence conservation relative to deorphanised rhodopsins enabled high confidence ligand-receptor matching of seventeen receptors activated by acetylcholine, neuropeptide F/Y, octopamine or serotonin. RNA-Seq analyses showed expression of 101 GPCRs across various developmental stages, with the majority expressed most highly in the pathogenic intra-mammalian juvenile parasites. These data identify a broad complement of GPCRs in F. hepatica, including rhodopsins likely to have key functions in neuromuscular control and sensory perception, as well as frizzled and adhesion/secretin families implicated, in other species, in growth, development and reproduction. This catalogue of liver fluke GPCRs provides a platform for new avenues into our understanding of flatworm biology and anthelmintic discovery. Keywords: Anthelmintic, Deorphanization, Flukicide, Genome, Invertebrate, Nervous system, Neuropeptide, RNA-Se

Calmodulin disruption impacts growth and motility in juvenile liver fluke

BACKGROUND: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown. METHODS: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays. RESULTS: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity. CONCLUSIONS: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1324-9) contains supplementary material, which is available to authorized users

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke

RNAi dynamics in juvenile Fasciola spp. liver flukes reveals the persistence of gene silencing in vitro

Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200-320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection

Identifying Causes of Electronic Prescription Error: is the Software or Physician at Fault?

Class of 2013 AbstractSpecific Aims: The purpose of this study was to investigate areas of ambiguity or error in the content of prescriptions generated using DrFirst’s electronic prescribing software Rcopia adetermines whether these quality issues are attributed to the software, physician, or both. Methods: Electronic prescriptions generated by DrFirst electronic prescribing software, Rcopia, from July 2012 through September 2012 were analyzed regarding the following metrics: number of free text prescriptions, quantity unit mismatches, and SIG issues. These metrics were expressed as a percentage of the total number of prescriptions generated for each month and used for descriptive analysis. Main Results: The total number of prescriptions generated were 12,043,268, of which 363,142 (3%) were free text (uncoded) and 11,680,126 (97%) were non-free text. SIG as directed was identified in 227,732 prescriptions, of which 11,208 (3.1%) were free text and 216,524(1.9%) were non-free text. Double SIG was identified in 174,625 prescriptions, of which 75,336 (20.1%) were free text and 1,746,250 (14.1%) were non-free text. A total of 830 (0.23%) of free text prescriptions contained a Latin abbreviation. Of 621,816 prescriptions containing a quantity unit error, 7,684 (2.1%) were free text prescriptions and 614,132 (5.3%) were non-free text prescriptions. Conclusion: The authors concluded that the software and physician are responsible for error. There were errors associated with selections made by the prescriber in the drop down menus and coded medications in Rcopia. Furthermore, errors were found in free text prescriptions which must be manually entered by the physician or their staff. This item is part of the Pharmacy Student Research Projects collection, made available by the College of Pharmacy and the University Libraries at the University of Arizona. For more information about items in this collection, please contact Jennifer Martin, Librarian and Clinical Instructor, Pharmacy Practice and Science, [email protected]

Evaluation of Medication Use and Outcomes in Patients Suffering an In-Hospital Cardiac Arrest

Class of 2015 AbstractObjectives: There is limited information regarding medication use during in-hospital cardiac arrest (IHCA). The purpose of this study was to characterize medication use during IHCA, and determine the association between medications used and survival to hospital discharge. Methods: This was a retrospective cohort study conducted in an academic medical center looking at IHCA between October 2009 and December 2013. Data regarding medication use during IHCA and other pertinent predictors of survival were collected. The primary objective was to characterize medications used during IHCA and to assess the relationship between medications used and survival to hospital discharge. Results: There were 171 patients who were included in the study and 44 (26%) survived to hospital discharge. The medications most commonly used were epinephrine, sodium bicarbonate, calcium chloride or gluconate, atropine, amiodarone, vasopressin, magnesium sulfate, and lidocaine. Patients who died were more likely to receive total epinephrine ≥3 mg (53% versus 27%, p=0.005), sodium bicarbonate (73% versus 55%, p=0.025), and calcium (59% versus 27%, p<0.001), compared to survivors, respectively. After adjusting for duration of resuscitation, total epinephrine ≥3 mg (OR 0.38, 95% CI 0.18 to 0.83, p=0.015) and calcium (OR 0.30, 95% CI 0.14 to 0.64, p=0.002) was associated with decreased survival. Conclusions: This study found that 3 mg or more of epinephrine, calcium salts and sodium bicarbonate are linked to decreased survival to hospital discharge. Further research should be done to define the cause of this link.This item is part of the Pharmacy Student Research Projects collection, made available by the College of Pharmacy and the University Libraries at the University of Arizona. For more information about items in this collection, please contact Jennifer Martin, Librarian and Clinical Instructor, Pharmacy Practice and Science, [email protected]