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  • CaneCPI showed strong inhibitory activity against


    CaneCPI-4 showed strong inhibitory activity against recombinant falcipain-2 and falcipain-3, with IC values of 11.9±0.4nM and 42.3±0.8nM, respectively (Table 1, Supplementary material). Falcipains are involved in hemoglobin cleavage, with generation of two fractions: the heme and the globin. The heme group is toxic to the parasite, and the detoxification is mediated through hemozoin formation (malaria pigment) , . As CaneCPI-4 inhibits falcipains, we analyzed the intracellular hemozoin formation in incubated with CaneCPI-4 (2μg/200μL). A significant reduction in hemozoin density was observed in (C) and no changes in morphology of food vacuole were observed in CaneCPI-4 treatment (data not shown). Furthermore, we investigated the effect of CaneCPI-4 in development and/or replication during the erythrocytic cycle (D) and values are summarized in the Table 2 presented in supplementary material. To this end, transgenic parasites expressing the reporter Nano-Luciferase, which is exported to red blood cells , were cultivated in presence of 6μM CaneCPI-4 for up to 96h, which is approximately two reinvasion cycles. As a control, corresponding to 100% growth, parasites were cultivated in the same medium without the cystatin. Growth was reduced by approximately 39% (=0.0003) after 96h but not after 48h or 72h (D). Lower CaneCPI-4 concentrations did not affect growth under the conditions assayed (data not shown). Taken together, our results indicated for the first time that cystatins may be used to inhibit cysteine proteases, as CaneCPI-4 inhibit falcipain activity at nM range and consequently reduce the parasite proteolysis compromising the cell development. The cystatin action identified in the present work involves the inhibition of a key biochemical process, such as the amino Fmoc-Hyp-OH receptor availability from hemoglobin, which directly compromises parasite growth. Furthermore, CaneCPI-4 is also a promising molecule for studies , as it does not present cytotoxicity in HUVECs , which is an important advantage compared to commercial cysteine protease inhibitors. Acknowledgments We thank the Multiuser Multiphoton Confocal Microscopy Laboratory (INFAR-UNIFESP) for the access to confocal microscopy devices. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (13/12913-0; 09/54598-9; 09/53840-0, 11/14403-4, 12/50475-2, 15/06861-3, 15/19316-3, 16/15298-3) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (473.226/2010-3, 311745/2013-0).
    Introduction Naegleria fowleri is a free-living ameba found in warm fresh water lakes, ponds and other warm-bodied waters. It is the cause of primary amebic meningoencephalitis (PAM) in more than 16 countries (Visvesvara and Stehr-Green, 1990). As of 2012, 310 cases had been reported globally with a fatality rate of more than 95% (Gautam et al., 2012). N. fowleri infection is not a notifiable disease in the US and therefore is likely significantly underreported. Based on the free-living ameba registry maintained by the CDC and other data sources, only 143 cases of PAM were reported in the US from 1962 to 2016 (Capewell et al., 2015). Infections occur mostly in warmer, southern-tier states. One reason why the infection is underreported is Minichromosome states differ significantly in their capacity to identify, investigate or report cases (Yoder et al., 2010). Even among the 143 known cases, 139 were fatal. In the USA, especially in the southern warm-weather states, most of the PAM infections occurred after swimming in fresh-water bodies naturally heated by the sun. N. fowleri thrives in warm water and can grow in temperatures of up to 45 °C. A few infections occurred after swimming in geothermal waters or by using tap water originating from warm groundwater (De Jonckheere, 2011). In the US, during 2003–2012, three of 31 persons infected with N. fowleri became infected after performing nasal rinsing with contaminated tap water. Two of the three patients performed nasal rinsing using a neti pot or similar device (Yoder et al., 2012). Recently, ablution has been associated with N. fowleri cases globally. In 2008–2009, 13 cases were reported in Karachi, Pakistan and the infection likely occurred through ablution with tap water (Shakoor et al., 2011). In 2012, a first documented U.S. case of PAM potentially associated with ablution was also reported (Centers for Disease, 2013a).