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Patients with single ventricle heart defects requires a series of staged open-heart procedures, termed Fontan palliation. However, while lifesaving, these operations are associated with significant morbidity and early mortality. The attendant complications are thought to arise in response to the abnormal hemodynamics induced by Fontan palliation, although the pathophysiology underlying these physicochemical changes in cardiovascular and other organs remain unknown. Here, we investigated the microRNA (miRNA) content in serum and serum-derived extracellular vesicles (EVs) by sequencing small RNAs from a physiologically relevant sheep model of the Fontan operation. The differential expression analysis identified the enriched miRNA clusters in (1) serum vs. serum-derived EVs and (2) pre-Fontan EVs vs. post-Fontan EVs. Metascape analysis showed that the overexpressed subset of EV miRNAs by Fontan procedure target liver-specific cells, underscoring a potentially important pathway involved in the liver dysfunction that occurs as a consequence of Fontan palliation. We also found that post-Fontan EV miRNAs were associated with senescence and cell death, whereas pre-Fontan EV miRNAs were associated with stem cell maintenance and epithelial-to-mesenchymal transition. This study shows great potential to identify novel circulating EV biomarkers from Fontan sheep serum that may be used for the diagnosis, prognosis, and therapeutics for patients that have undergone Fontan palliation.

Patch augmentation of the right ventricular outflow tract (RVOT) and pulmonary artery (PA) arterioplasty are relatively common procedures in the surgical treatment of patients with congenital heart disease. To date, several patch materials have been applied with no agreed upon clinical standard. Each patch type has unique performance characteristics, cost, and availability. There are limited data describing the various advantages and disadvantages of different patch materials. We performed a review of studies describing the clinical performance of various RVOT and PA patch materials and found a limited but growing body of literature. Short-term clinical performance has been reported for a multitude of patch types, but comparisons are limited by inconsistent study design and scarce histologic data. Standard clinical criteria for assessment of patch efficacy and criteria for intervention need to be applied across patch types. The field is progressing with improvements in outcomes due to newer patch technologies focused on reducing antigenicity and promoting neotissue formation which may have the ability to grow, remodel, and repair.

Tuberculosis remains one of the leading causes of death from a single infectious agent, surpassing both AIDS and malaria. In recent years, two bicyclic nitroimidazole drugs, delamanid and pretomanid have been approved to treat this airborne infection. This has spurred a renewed interest in developing new and improved nitroimidazole analogs. We have previously identified a new bicyclic heteroaromatic subclass, the nitroimidazopyrazinones, with substituted analogs showing promising activity against Mycobacterium tuberculosis under both aerobic and hypoxic environments. A second generation of nitroimidazopyrazinones with extended biaryl side chain also possessed good antiparasitic activity against Trypanosoma brucei brucei and Trypanosoma cruzi, suggesting the utility of this new scaffold for development into potential candidates against both tuberculosis and the kinetoplastid parasites which cause neglected tropical diseases. In this study, we further evaluated the properties of nitroimidazopyrazinone derivatives by assessing their selectivity against different mycobacterial species, measuring their reduction potential, and determining the kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn), which is the activating enzyme of delamanid and pretomanid in M. tuberculosis. We also conducted an in vivo evaluation of a lead compound, MCC8967 that demonstrated a favorable pharmacokinetic profile, with good oral bioavailability and efficacy in an acute M. tuberculosis infection model. Two other promising compounds MCC9481 and MCC9482, with good in vitro activity (IC50 = 0.016 and 0.10 µM, respectively) against T. cruzi, the causative agent for Chagas diseases, were similarly tested for in vivo activity. These compounds also exhibited good oral bioavailability, and transiently reduced the acute-stage parasite burden by >98‒99% at doses of 50 mg/kg once or twice daily, similar to benznidazole at 100 mg/kg once daily. Overall, we have demonstrated that active nitroimidazopyrazinones have potential to be developed as clinical candidates against both tuberculosis and Chagas disease.Author SummaryTuberculosis and parasitic infections continue to impose a significant threat to public health and economic growth worldwide. Most of the efforts to control these diseases still rely on drug treatments with limited effectiveness and significant side effects. There is now an urgent need to develop new treatments to combat these infections. Here, we report the in vitro and in vivo profile of a new bicyclic nitroimidazole subclass, namely nitroimidazopyrazinones, against mycobacteria and Trypanosoma cruzi. We found that derivatives with monocyclic side chains are selective against Mycobacterium tuberculosis, the causative agent of tuberculosis, but not active against other nontuberculosis mycobacteria. In an acute mouse model, they were able to reduce the bacterial load in lungs via oral administration. From a biochemistry perspective, we demonstrated that deazaflavin-dependent nitroreductase (Ddn) could act effectively on nitroimidazopyrazinones, indicating the potential of Ddn as an activating enzyme for these new compounds in M. tuberculosis. We also showed that derivatives with extended biaryl side chain were effective in suppressing infection in an acute T. cruzi infected murine model, with satisfactory oral bioavailability. These findings improve the understanding of the biological profile of nitroimidazopyrazinones for further development as potential antitubercular and antiparasitic agents.

The shape of a foil undergoing a combined pitching heaving motion is critical to its design in applications that demand high efficiency and thrust. This study focuses on understanding of how the shape of a foil affects its propulsive performance. We perform two-dimensional numerical simulations of fluid flows around a flapping foil for different governing parameters in the range of biological swimmers and bio-inspired underwater vehicles. By varying the foil shape using a class-shape transformation method, we investigate a broad range of foil-like shapes. In the study, we also show consistent results with previous studies that a thicker leading-edge and sharper trailing-edge makes for a more efficient foil shape undergoing a flapping motion. In addition, we explain that the performance of the foil is highly sensitive to its shape, specifically the thickness of the foil between the 18th and 50th percent along the chord of the foil. Moreover, we elucidate the flow mechanisms behind variations in performance metrics, particularly focused on constructive interference between the vortices generated at the leading-edge with the trailing-edge vortex, as well as the pressure field differences that lead to higher power consumption in less efficient foil shapes.

To date, there has been limited investigation of bioabsorbable atrial septal defect (ASD) or patent foramen ovale (PFO) closure devices using clinically relevant large animal models. The purpose of this study is to explore the function and safety of a bioabsorbable ASD occluder (BAO) system for PFO and/or secundum ASD transcatheter closure. Using a sheep model, the intra-atrial septum was evaluated by intracardiac echo (ICE). If a PFO was not present, atrial communication was created via transseptal puncture. Device implantation across the intra-atrial communication was performed with fluoroscopic and ICE guidance. Our 1st generation device consisted of a main structure of thin Poly(L-lactide-co-epsilon-caprolactone) (PLCL) fibers, and an internal Poly glycolic acid (PGA) fabric. Four procedures validated procedure feasibility. Subsequently, device design was modified for improved transcatheter delivery. The 2nd generation device has a two-layered structure and was implanted in six sheep. Results showed procedural success in 9/10 (90%) animals. With deployment, the 1st generation device did not reform into its original disk shape and did not conform nicely along the atrial septum. The 2nd generation device was implanted in six animals, 3 out of 6 survived out to 1 year. At 1 year post implantation, ICE confirmed no residual shunting. By necropsy, biomaterials had partially degraded, and histology of explanted samples revealed significant device endothelialization and biomaterial replacement with a collagen layer. Our results demonstrate that our modified 2nd generation BAO can be deployed via minimally invasive percutaneous transcatheter techniques. The BAO partially degrades over 1 year and is replaced by host native tissues. Future studies are needed prior to clinical trials.

Time-resolved IR spectra of porphyrins bound to biomolecules are reported. DFT calculations aid porphyrin vibration assignment while distinctive IR bands reveal interactions with therapeutic targets such as quadruplex DNA and proteins in HeLa cells.

Probing multiple proprietary pharmaceutical libraries in parallel via virtual screening allowed rapid expansion of the structure-activity relationship (SAR) around hit compounds with moderate efficacy against

In recent years, new vineyards have been established in southwestern Ontario. The open water of Lake Erie provides some winter protection for Vitis hybrids and winter-hardy Vitis vinifera L. cultivars in this area. However, winter damage is possible when vines are grown distant from the open water or when lakes are frozen. To better understand the risks to winter survival, the dormancy and chilling phenology were studied over three winters from 2013–2016. Ten dormant canes of two V. vinifera cultivars, ‘Chardonnay’ and ‘Riesling’, were collected weekly from 1 September until 30 March from the mature vines in a commercial vineyard located at St. Williams (Ontario). The canes defoliated in early October, and the endodormancy was completed by the end of December. The cumulative chilling hours (0–7.2 °C) from defoliation until the completion of endodormancy were averaged 606 hours for ‘Chardonnay’ and 665 hours for ‘Riesling’. ‘Chardonnay’ buds were slightly less hardy than ‘Riesling’ to cold temperatures, with a threshold of about −24 °C for ‘Chardonnay’ and −25 °C for ‘Riesling’. Most primary buds of both cultivars died after 16 February 2015, and more than half died after 12 February 2014, due to severe low temperatures of −33.1 and −26 °C, respectively.

Hazelnut (Corylus avellana L.) is a monoecious, dichogamous plant species that flowers in winter and early spring. In traditional hazelnut growing regions, hazelnut cultivars generally express protandry where the male flowers bloom before the female flowers. Nut set requires that compatible pollen be shed when the stigmas of the main cultivar are receptive. In this study, the floral phenology and date of leaf budbreak of five selections and 19 hazelnut cultivars from Europe and North America were observed over 4 yr in southern Ontario, and results were compared with cumulative growing degree days (GDD). In the continental climate of southern Ontario, most cultivars showed protogyny. Flowering dates varied over the years with pollination occurring in a period of 2–3 wk in early spring. These cultivars were classified into early, mid, and late blooming types. A GDD model was a better tool than average daily temperatures to predict the flowering dates of catkins and female flowers. Averaged over years, most of the cultivars in this study have a female bloom that is likely too early for the pollen shed by most of the other cultivars. This would likely impact yields in a commercial orchard. However, ‘Jefferson’, ‘Gene’, and ‘Epsilon’ have late females that are receptive when other cultivars are shedding pollen.

Digestive Chagas disease (DCD) is an enteric neuropathy caused byTrypanosoma cruziinfection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology, and the impact of treatment with the front-line drug benznidazole. Curative treatment at 6 weeks post-infection resulted in sustained recovery of GI transit function, whereas sub-curative treatment led to infection relapse and gradual return of DCD symptoms. Neuro-immune gene expression profiles shifted from chronic inflammation to a tissue repair signature after cure, accompanied by increased glial cell activity and regenerative neurogenesis in the myenteric neuronal plexus. Delaying treatment until 24 weeks post-infection led to a partial reversal of the DCD phenotype, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronicT. cruziinfection and is not an inevitable consequence of acute stage denervation. The risk that irreversible enteric neuromuscular tissue damage and dysfunction will develop highlights the importance of prompt diagnosis and treatment. Finally, these findings support the concept of treating asymptomaticT. cruziinfected individuals with benznidazole to prevent DCD development.

A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC50 = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC90 = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents., Graphical Abstract

Ultrafast time-resolved infrared (TRIR) is used to report on the binding site of the [Ru(phen)2 (dppz)]2+ "light-switch" complex with both bimolecular (Oxytricha nova telomere) and intramolecular (human telomere) guanine-quadruplex structures in both K+ and Na+ containing solutions. TRIR permits the simultaneous monitoring both of the "dark" and "bright" states of the complex and of the quadruplex nucleobase bases, the latter via a Stark effect induced by the excited state of the complex. These data are used to establish the contribution of guanine base stacking and loop interactions to the binding site of this biologically relevant DNA structure in solution. A particularly striking observation is the strong thymine signal observed for the Na+ form of the human telomere sequence, which is expected to be in the anti-parallel conformation.

The global demand for hazelnuts is increasing steadily, driven by increasing use by chocolate companies, pharmaceuticals, health products, and others. North America only produces 5% of the world crop, of which 99% is produced in Oregon (OR, USA). Most available cultivars are adapted to areas with mild winters and thus do not perform well in Ontario (ON, Canada). Our objective was to identify genotypes capable of supporting the newly formed hazelnut industry in southern Ontario. In the last several decades, selections have been identified in ON, New York (NY, USA), and Michigan (MI, USA) that may be better adapted in ON than cultivars from Europe and OR. To test our hypothesis that these new selections would outperform cultivars from Europe and OR in southern Ontario, two trials were performed to evaluate yield, nut quality, and winter hardiness. As hypothesized, selections from ON and NY such as ‘Gene’, ‘Alex’, and ‘Slate’ were winter hardy and highest yielding, followed by ‘Butler’ and ‘Gamma’ from OR; however, these selections did not perform well in other assessed parameters such as nut quality and catkin survivability. Possible explanations for the poor performance of the European and OR cultivars include (i) longer time needed to acclimatize and enter production phase relative to selections from ON, NY, and MI, and (ii) cold susceptibility during the initial establishment phase. The need to allocate resources towards survival probably delays the ability of the European and OR cultivars to enter the production phase. In conclusion, hazelnut cultivars brought from regions with a different climate need to be tested for cold hardiness.

Ultrafast time resolved infrared (TRIR) is used to report on the binding site of the "light-switch" complex [Ru(phen)2(dppz)]2+1 to i-motif structures in solution. Detailed information is provided due to perturbation of the local base vibrations by a 'Stark-like' effect which is used to establish the contribution of thymine base loop interactions to the binding site of 1 in this increasingly relevant DNA structure.

Trypanosoma cruzi is the etiological agent of Chagas disease. Following T cell mediated suppression of the acute phase infection, this intracellular eukaryotic pathogen persists long-term in a limited sub-set of tissues at extremely low-levels. The reasons for this tissue-specific chronicity are not understood. Using a dual bioluminescent:fluorescent reporter strain and highly sensitive tissue imaging that allows experimental infections to be monitored at single-cell resolution, we have undertaken a systematic analysis of the immunological micro-environments of rare parasitized cells in the mouse colon, a key site of persistence. We demonstrate that incomplete recruitment of T cells to a subset of colonic infection foci permits the occurrence of repeated cycles of intracellular parasite replication and differentiation to motile trypomastigotes at a frequency sufficient to perpetuate chronic infections. The life-long persistence of parasites in this tissue site continues despite the presence, at a systemic level, of a highly effective T cell response. Overcoming this low-level dynamic host:parasite equilibrium represents a major challenge for vaccine development.

A family of six Ru(II) polypyridyl complexes (

A 15-year-old girl with a history of complex congenital heart disease and prior pulmonary valve replacement presented with suspected endocarditis. PET/CT imaging with 18F-FDG was performed to evaluate the potential presence of intracardiac vegetations after previously inconclusive findings from CT angiography, transthoracic echocardiography, and transesophageal echocardiography. PET/CT detected heterogeneous, asymmetric, increased 18F-FDG uptake in the region of the pulmonary valve prosthesis, typical for infection, and confirmed diagnosis of bacterial infective endocarditis. This report highlights the utility of 18F-FDG PET/CT imaging to complement the Duke criteria for determining the diagnosis and therapeutic management of pediatric patients with infective endocarditis.

Infections with Flaviviridae viruses, such as hepatitis C virus (HCV) and dengue virus (DENV) pose global health threats. Infected individuals are at risk of developing chronic liver failure or haemorrhagic fever respectively, often with a fatal outcome if left untreated. Diseases caused by tropical parasites of the Trypanosoma species, T. brucei and T. cruzi, constitute significant socioeconomic burden in sub-Saharan Africa and continental Latin America, yet drug development is under-funded. Anti-HCV chemotherapy is associated with severe side effects and high cost, while dengue has no clinically approved therapy and antiparasitic drugs are outdated and difficult to administer. Moreover, drug resistance is an emerging concern. Consequently, the need for new revolutionary chemotherapies is urgent. By utilizing a molecular framework combination approach, we combined two distinct chemical entities with proven antiviral and trypanocidal activity into a novel hybrid scaffold attached by an acetohydroxamic acid group (CH2CONHOH), aiming at derivatives with dual activity. The novel spiro-carbocyclic substituted hydantoin analogues were rationally designed, synthesized and evaluated for their potency against three HCV genotypes (1b, 3a, 4a), DENV and two Trypanosoma species (T. brucei, T. cruzi). They exhibited significant EC50 values and remarkable selectivity indices. Several modifications were undertaken to further explore the structure activity relationships (SARs) and confirm the pivotal role of the acetohydroxamic acid metal binding group.

Trypanosoma cruzi is the etiological agent of Chagas disease. Following T cell mediated suppression of the acute phase infection, this intracellular eukaryotic pathogen persists in a limited sub-set of tissues at extremely low-levels. The reasons for this tissue-specific chronicity are not understood. Using a dual bioluminescent:fluorescent reporter strain, which allows experimental infections to be imaged at single-cell resolution, we have characterised the ‘hyper-local’ immunological microenvironment of rare parasitized cells in the mouse colon, a key site of persistence. We demonstrate that incomplete recruitment of T cells to infection foci permits the occurrence of repeated cycles of intracellular parasite replication and differentiation to motile trypomastigotes at a frequency sufficient to perpetuate chronic infections. The life-long persistence of parasites in this tissue site continues despite the presence, at a systemic level, of a highly effective T cell response. Overcoming this low-level dynamic host:parasite equilibrium represents a major challenge for vaccine development.

Made available in DSpace on 2022-04-29T08:45:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Hydroxymethylnitrofurazone (NFOH) is a therapeutic candidate for Chagas disease (CD). It has negligible hepatotoxicity in a murine model compared to the front-line drug benznidazole (BZN). Here, using Trypanosoma cruzi strains that express bioluminescent and/or fluorescent reporter proteins, we further investigated the in vitro and in vivo activity of NFOH to define whether the compound is trypanocidal or trypanostatic. The in vitro activity was assessed by exploiting the fluorescent reporter strain using wash-out assays and real-time microscopy. For animal experi-mentation, BALB/c mice were inoculated with the bioluminescent reporter strain and assessed by highly sensitive in vivo and ex vivo imaging. Cyclophosphamide treatment was used to promote parasite relapse in the chronic stage of infection. Our data show that NFOH acts by a trypanostatic mechanism, and that it is more active than BZN in vitro against the infectious trypomastigote form of Trypanosoma cruzi. We also found that it is more effective at curing experimental infections in the chronic stage, compared with the acute stage, a feature that it shares with BZN. Therefore, given its reduced toxicity, enhanced anti-trypomastigote activity, and curative properties, NFOH can be considered as a potential therapeutic option for Chagas disease, perhaps in combination with other trypanocidal agents. School of Pharmaceutical Sciences Sao Paulo State University (UNESP) Department of Infection Biology Faculty of Infectious and Tropical Diseases London School of Hygiene and Tropical Medicine, Keppel Street LAPEN—Laboratory of Design and Synthesis of Chemotherapeutic Agents Potentially Active on Neglected Diseases Department of Pharmacy School of Pharmaceutical Sciences University of Sao Paulo (USP) Advanced Research Center in Medicine School of Medicine Union of the Colleges of the Great Lakes (UNILAGO) School of Pharmaceutical Sciences Sao Paulo State University (UNESP) FAPESP: 2016/10847-9

In this work, we present the synthesis and the anti-trypanosomal activity of the 2-(4-(adamant-1-yl)phenyl)-N-hydroxyarylamides, 1a,b and the 2-(4-(adamant-1-yl)phenoxy)-N-hydroxyacetamide, 1c. The 4-(adamant-1-yl)phenyl- and 4-(adamant-1-yl)phenoxy- moieties, which are endowed with promising drug-like properties, are functionalized at the side chain termini as hydroxamic acids. The phenoxy acetohydroxamic derivative, 1c, shows the most interesting profile in terms of activity and toxicity against trypanosomes and merits further investigation.

The development and translation of regenerative medicine approaches for the treatment of hypoplastic left heart syndrome (HLHS) provides a promising alternative to the current standard of care. We review the strategies that have been pursued to date and those that hold the greatest promise in moving forward. Significant challenges remain. Continued scientific advances and technological breakthroughs will be required if we are to translate this technology to the clinic and move from palliative to curative treatment.

Two new dinuclear Ru(ii) polypyridyl complexes containing an alkyl disulphide functionalised bipyridine-based ligand and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesised and characterised. Their attachment onto the surface of gold nanoparticles (AuNPs, average diameter of ca. 2.5 nm) resulted in the formation of two new water-soluble Ru(ii)-AuNP conjugates that combine the advantageous properties of both moieties. Both free complexes show the attractive photophysical properties of Ru(ii) polypyridyl complexes and a rapid cellular uptake in HeLa cervical cancer cells. However, their corresponding gold conjugates displayed lower quantum yields than those determined for the free complexes presumed to be due to an energy transfer quenching of the Ru(ii) luminescence by interaction with the gold surface. Despite their diminished luminescence, confocal fluorescence microscopy studies revealed that the Ru(ii)-AuNP conjugates are successfully internalised into HeLa cells and better tolerated than their free complex counterparts after 24 h incubation, which makes them potential luminescent nanomaterials for bioimaging applications.

Chronic Trypanosoma cruzi infections are typically life-long, with small numbers of parasites surviving in restricted tissue sites, which include the gastro-intestinal tract. There is considerable debate about the replicative status of these persistent parasites. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2’deoxyuridine incorporation into DNA to provide “snapshots” of parasite replication. Highly sensitive imaging of infection foci at single cell resolution revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections are associated with a reduced rate of parasite replication. Despite this, very few host cells survive infection for >14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. Therefore, long-term persistence in the colon is more likely to be associated with reduced proliferation than with dormancy.

Ultrafast time resolved infrared (TRIR) is used to report on the binding site of the "light-switch" complex [Ru(phen)

Two water-soluble amphiphilic Ru(ii) polypyridyl complexes containing N-1,10-phenanthrolin-5-yldocosanamide and 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ligands were synthesised and their photophysical and photobiological properties evaluated; both complexes showed a rapid cellular uptake and displayed phototoxicity against HeLa cervical cancer cells.

Infections with Trypanosoma cruzi are usually life-long despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understand how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which has allowed us to visualise infected host cells in murine tissue, with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of 200 parasites, that we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites found in large mega-nests within the muscle fibres. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission, and have implications for drug development.IMPORTANCETrypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence in experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon, and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a life-long infection, despite a vigorous immune response. It also informs drug-development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

Chagas disease results from infection with the trypanosomatid parasite Trypanosoma cruzi. Progress in developing new drugs has been hampered by the long term and complex nature of the condition and by our limited understanding of parasite biology. Technical difficulties in assessing the parasite burden during the chronic stage of infection have also proven to be a particular challenge. In this context, the development of noninvasive, highly sensitive bioluminescence imaging procedures based on parasites that express a red-shifted luciferase has greatly enhanced our ability to monitor infections in experimental models. Applications of this methodology have led to new insights into tissue tropism and infection dynamics and have been a major driver in drug development. The system has been further modified by the generation of parasite reporter lines that express bioluminescent:fluorescent fusion proteins, an advancement that has allowed chronic infections in mice to be examined at a cellular level. By exploiting bioluminescence, to identify the rare sites of tissue infection, and fluorescence to detect T. cruzi at the level of individual host cells in histological sections, it has been possible to investigate the replication and differentiation status of parasites in vivo and to examine the cellular environment of infection foci. In combination, these data provide a framework for the detailed dissection of disease pathogenesis and drug activity.

Ultrafast time-resolved infrared (TRIR) is used to report on the binding site of the [Ru(phen)

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge., Author summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is becoming an emerging threat in non-endemic countries and establishing new foci in endemic countries. The treatment available has not changed significantly in over 40 years. Therefore, there is an urgent need for a greater understanding of parasite biology and disease pathogenesis to identify new therapeutic targets and to maximise the efficient use of existing drugs. We have used genetically modified strains of T. cruzi carrying a bioluminescence/fluorescence dual reporter fusion gene to monitor parasite replication in vivo during both acute and chronic infections in a mouse model. Utilising TUNEL assays for mitochondrial DNA replication and EdU incorporation for total DNA replication, we have found that parasite division within infected cells is asynchronous in all phases of infection. Differentiation also appears to be uncoordinated, with replicating amastigotes co-existing with non-dividing trypomastigotes in the same host cell.

Recent advances in the use of transient absorption (TA) and time-resolved vibrational spectroscopies (TRIR and TR3) to study both excited states and reaction intermediates in metal complexes and porphyrins which intercalate into DNA are reviewed. A particularly well-studied class of compounds, which nicely illustrates the comparative advantages of these techniques, is that of ruthenium dppz complexes where the complex might show light-switching or photo-oxidising behaviour depending on the nature of the ancillary ligand. Comparative data on Re- and Cr-dppz complexes are also considered. In the second part of this review transient studies of porphyrins, which are known to intercalate into DNA, are considered with particular emphasis on tetramethyl-pyridiniumporphyrins, where the photophysical behaviour of the metal-free and various metal derivatives are compared.

We have previously described a number of lipophilic conformationally constrained spiro carbocyclic 2,6-diketopiperazine (2,6-DKP)-1-acetohydroxamic acids as potent anti-trypanosomal agents. In this report, we extend the SAR analysis in this class of compounds with respect to in vitro growth inhibition of Trypanosoma and Leishmania parasites. Introduction of bulky hydrophobic substituents at the vicinal position of the basic nitrogen atom in the spiro carbocyclic 2,6-DKP ring system can provide analogues which are potently active against bloodstream-form T. brucei and exhibit significant activities towards T. cruzi epimastogotes and L. infantum promastogotes and intracellular amastigotes. In particular, compounds possessing a benzyl or 4-chlorobenzyl substituent were found to be the most active growth inhibitors, with activities in the low nanomolar and low micromolar ranges for T. brucei and L. infantum, respectively. The benzyl substituted (S)-enantiomer was the most potent derivative against T. brucei (IC50 =6.8 nM), T. cruzi (IC50 =0.21 μΜ) and L. infantum promastigotes (IC50 =2.67 μM) and intracellular amastigotes (IC50 =2.60 μM). Moreover, the (R)-chiral benzyl substituted derivative and its racemic counterpart displayed significant activities against L. donovani. Importantly, the active compounds show high selectivity in comparison with two mammalian cell lines. This article is protected by copyright. All rights reserved.

Weinheim Key to the development of DNA-targeting phototherapeutic drugs is determining the interplay between the photoactivity of the drug and its binding preference for a target sequence. For the photo-oxidising lambda-[Ru(TAP)2(dppz)]2+ (Λ-1) (dppz=dipyridophenazine) complex bound to either d{T1C2G3G4C5G6C7C8G9A10}2 (G9) or d{TCGGCGCCIA}2 (I9), the X-ray crystal structures show the dppz intercalated at the terminal T1C2;G9A10 step or T1C2;I9A10 step. Thus substitution of the G9 nucleobase by inosine does not affect intercalation in the solid state although with I9 the dppz is more deeply inserted. In solution it is found that the extent of guanine photo-oxidation, and the rate of back electron-transfer, as determined by pico- and nanosecond time-resolved infrared and transient visible absorption spectroscopy, is enhanced in I9, despite it containing the less oxidisable inosine. This is attributed to the nature of the binding in the minor groove due to the absence of an NH2 group. Similar behaviour and the same binding site in the crystal are found for d{TTGGCGCCAA}2 (A9). In solution, we propose that intercalation occurs at the C2G3;C8I9 or T2G3;C8A9 steps, respectively, with G3 the likely target for photo-oxidation. This demonstrates how changes in the minor groove (in this case removal of an NH2 group) can facilitate binding of RuIIdppz complexes and hence influence any sensitised reactions occurring at these sites. No similar enhancement of photooxidation on binding to I9 is found for the delta enantiomer. Irish Research Council Science Foundation Ireland University College Dublin Biotechnology and Biological Sciences Research Council Royal Irish Academy/Royal Society

The performance of photocatalytic treatment processes were assessed using different photocatalysts against E. coli and bacteriophages MS2, ΦX174 and PR772, in a recirculating continuous flow compound parabolic collector system under real sunlight conditions. Suspended TiO2 and ZnO nanoparticle powders and Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate in solution were tested separately, as well as in combination, using E. coli. For a 3-log reduction of E. coli in distilled water, inactivation rates in terms of cumulative dose were in the order Ru(bpy)3Cl2>(TiO2 & Ru(bpy)3Cl2)>(ZnO & Ru(bpy)3Cl2)>ZnO>TiO2>photolysis. Reactivation of E. coli was observed following all trials despite the detection limit being reached, although the reactivated colonies were observed to be under stress and much slower growing when compared to original colonies. Treatment with Ru(bpy)3Cl2 was also compared against standard photolysis of bacteriophages MS2, ΦX174 and PR772 with the order of photolytic inactivation for a 3-log reduction in terms of cumulative UV-A dose being ΦX174>PR772>MS2. However, MS2 was found to be the most susceptible bacteriophage to treatment with Ru(bpy)3Cl2, with complete removal of the phage observed within the first 15min of exposure. Ru(bpy)3Cl2 also significantly improved inactivation rates for PR772 and ΦX174.

Ruthenium(II) [Ru(II)] polypyridyl complexes have been the focus of intense investigations since work began exploring their supramolecular interactions with DNA. In recent years, there have been considerable efforts to translate this solution-based research into a biological environment with the intention of developing new classes of probes, luminescent imaging agents, therapeutics and theranostics. In only 10 years the field has expanded with diverse applications for these complexes as imaging agents and promising candidates for therapeutics. In light of these efforts this review exclusively focuses on the developments of these complexes in biological systems, both in cells and in vivo, and hopes to communicate to readers the diversity of applications within which these complexes have found use, as well as new insights gained along the way and challenges that researchers in this field still face.

The Monhantic Fort site is a Mashantucket Pequot fortified village occupied during King Philip’s War (1675–1677). Archaeological investigations resulted in the recovery of over 900 pieces of European flint (English and continental European) derived from ballast cobbles, believed to be primarily associated with the wartime production of gunflints. The majority of the gunflints are bifacial (86%), indicating a process of manufacture distinct from contemporaneous English manufacture. While many lithic tools had been replaced with European metal tools by this time, the flint assemblage also contains a number of objects indicating use of lithic tools in domestic contexts. Spatial analysis of the flint assemblage in the context of domestic and nondomestic spaces indicates individual production of gunflints and tools, rather than specialized production confined to a single workshop area by a limited number of individuals. Comparison of the flint assemblage at Monhantic Fort with a contemporaneous domestic site at Mashantucket and the Aptucxet Trading Post in Bourne, Massachusetts, supports the contention that the fort assemblage was the result of the wartime production of gunflints.

In this report, we extend the SAR analysis of a number of lipophilic guanylhydrazone analogues with respect to in vitro growth inhibition of Trypanosoma brucei and Trypanosoma cruzi. Sleeping sickness and Chagas disease, caused by the tropical parasites T. brucei and T. cruzi, constitute a significant socioeconomic burden in low-income countries of sub-Saharan Africa and Latin America, respectively. Drug development is underfunded. Moreover, current treatments are outdated and difficult to administer, while drug resistance is an emerging concern. The synthesis of adamantane-based compounds that have potential as antitrypanosomal agents is extensively reviewed. The critical role of the adamantane ring was further investigated by synthesizing and testing a number of novel lipophilic guanylhydrazones. The introduction of hydrophobic bulky substituents onto the adamantane ring generated the most active analogues, illustrating the synergistic effect of the lipophilic character of the C1 side chain and guanylhydrazone moiety on trypanocidal activity. The n-decyl C1-substituted compound G8 proved to be the most potent adamantane derivative against T. brucei with activity in the nanomolar range (EC50=90 nM). Molecular simulations were also performed to better understand the structure-activity relationships between the studied guanylhydrazone analogues and their potential enzyme target.

INTRODUCTION: Chagas disease, the most important parasitic infection in Latin America, is caused by the intracellular protozoan Trypanosoma cruzi. To treat this disease, only two nitroheterocyclic compounds with toxic side effects exist and frequent treatment failures are reported. Hence there is an urgent need to develop new drugs. Recently, metabolomics has become an efficient and cost-effective strategy for dissecting drug mode of action, which has been applied to bacteria as well as parasites, such as different Trypanosome species and forms. OBJECTIVES: We assessed if the metabolomics approach can be applied to study drug action of the intracellular amastigote form of T. cruzi in a parasite-host cell system. METHODS: We applied a metabolic fingerprinting approach (DI-MS and NMR) to evaluate metabolic changes induced by six different (candidate) drugs in a parasite-host cell system. In a second part of our study, we analyzed the impact of two drugs on polar metabolites, lipid and proteins to evaluate if affected pathways can be identified. RESULTS: Metabolic signatures, obtained by the fingerprinting approach, resulted in three different clusters. Two can be explained by already known of mode actions, whereas the three experimental drugs formed a separate cluster. Significant changes induced by drug action were observed in all the three metabolic fractions (polar metabolites, lipids and proteins). We identified a general impact on the TCA cycle, but no specific pathways could be attributed to drug action, which might be caused by a high percentage of common metabolome between a eukaryotic host cell and a eukaryotic parasite. Additionally, ion suppression effects due to differences in abundance between host cells and parasites may have occurred. CONCLUSION: We validated the metabolic fingerprinting approach to a complex host-cell parasite system. This technique can potentially be applied in the early stage of drug discovery and could help to prioritize early leads or reconfirmed hits for further development.

Infections with Flaviviridae viruses, such as hepatitis C virus (HCV) and dengue virus (DENV) pose global health threats. Infected individuals are at risk of developing chronic liver failure or haemorrhagic fever respectively, often with a fatal outcome if left untreated. Diseases caused by tropical parasites of the Trypanosoma species, T. brucei and T. cruzi, constitute significant socioeconomic burden in sub-Saharan Africa and continental Latin America, yet drug development is under-funded. Anti-HCV chemotherapy is associated with severe side effects and high cost, while dengue has no clinically approved therapy and antiparasitic drugs are outdated and difficult to administer. Moreover, drug resistance is an emerging concern. Consequently, the need for new revolutionary chemotherapies is urgent. By utilizing a molecular framework combination approach, we combined two distinct chemical entities with proven antiviral and trypanocidal activity into a novel hybrid scaffold attached by an acetohydroxamic acid group (CH(2)CONHOH), aiming at derivatives with dual activity. The novel spiro-carbocyclic substituted hydantoin analogues were rationally designed, synthesized and evaluated for their potency against three HCV genotypes (1b, 3a, 4a), DENV and two Trypanosoma species (T. brucei, T. cruzi). They exhibited significant EC(50) values and remarkable selectivity indices. Several modifications were undertaken to further explore the structure activity relationships (SARs) and confirm the pivotal role of the acetohydroxamic acid metal binding group.

Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. However, a major drawback of bioluminescence imaging is that it does not allow visualization of host-parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualizing T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterize their immunological context.

The synthesis and pharmacological evaluation of C1-substituted adamantane hydrazones, their C2-substituted isomers, and C1-substituted adamantane furanoic carboxamides is described. These new adamantane derivatives exhibited an interesting pharmacological profile in terms of trypanocidal activity and selectivity. The most active adduct with the best selectivity in this study was found to be the phenylacetoxy hydrazone 1 b (2-[4-(tricyclo[3.3.1.13,7 ]dec-1-yl)phenyl]-N'-[(5-nitrofuran-2-yl)methylene]acetohydrazide; EC50 =11±0.9 nm, SITb =770).

In this work, the synthesis and the pharmacological evaluation of diphenoxyadamantane alkylamines Ia-f and IIa-f is described. The new diphenoxy-substituted adamantanes share structural features present in trypanocidal and antitubercular agents. 1-Methylpiperazine derivative Ia is the most potent against T. brucei compound, whilst its hexylamine congener IIf exhibits a significant antimycobacterial activity.

Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. However, a major drawback of bioluminescence imaging is that it does not allow visualization of host-parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualizing T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterize their immunological context.

[Ru(TAP)

Novel imidazoline benzimidazole derivatives containing diversely substituted phenoxy moieties were synthesized with the aim of evaluating their antitrypanosomal activity, DNA/RNA binding affinity and in vitro ADME properties. The presence of the diethylaminoethyl subunit in 18a–18c led to enhanced antitrypanosomal potency, particularly for 18a and 18c, which contain unsubstituted and methoxy-substituted phenoxy moieties. They were found to be > 2-fold more potent against African trypanosomes than nifurtimox. Fluorescence and CD spectroscopy, thermal denaturation assays and computational analysis indicated a preference of 18a–18c toward AT-rich DNA and their minor groove binding mode. Replacement of the amidine group with less basic and ionisable nitrogen-containing moieties failed to improve membrane permeability of the investigated compounds. Due to structural diversification, the compounds displayed a range of physico-chemical features resulting in variable in vitro ADME properties, leaving space for further optimization of the biological profiles.