Expression and purification of recombinant Cathepsin L-like protein.

<p>Protein samples were separated by 12.5% SDS-PAGE gel electrophoresis. (<b>A</b>) Molecular weight standard, (<b>B</b>) lysate of culture before and (<b>C</b>) after induction with IPTG and (<b>D</b>) recombinant Cathepsin L-like protein (MW 47.6 KDa) purified by gel filtration.</p

Immunodepletion assay showing specific IgG antibody recognition of the synthetic peptides with known reactivity to Cathepsin L-like.

<p>Pools of sera (n = 10) from the different groups were depleted with peptide-1 (CT, control group; CD, Chagas disease; CL, cutaneous leishmaniasis; ML, mucosal leishmaniasis; VL, visceral leishmaniasis; CVL, canine visceral leishmaniasis). The mean antibody OD values are shown on the <i>y</i>-axis, and the error bars indicate the standard deviation. Significant differences are indicated on the graphs (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001).</p

Comparison of ROC curves obtained from <i>r</i>CatL, Peptide-1 and SLbA.

<p>The ROC curves were used to determine the ELISA cut-off, sensitivity, specificity and AUC. In case of SLbA for CVL diagnosis (EIE-LVC Kit), ROC curve is not shown (not applicable) in this graph because the cut-off was determined according to the recommendations by the manufacturer (twice the average of the negative control included in kit).</p

Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit.

a<p>Parameters was calculated using all samples presented in this work for TL (CT + CD + CL + ML. n = 135). VL (CT + CD + VL. n = 125) and CVL (CT + CD + CVL. n = 75).</p><p>*<i>Cut-off</i> obtained by ROC curve.</p>#<p><i>Cut off</i> obtained according to the manufacturer.</p><p>Abbreviations: Tse; total sensitivity; TSp: total specificity; CI: confidence interval; PPV: positive predictive value; NPV: negative predictive value; AC: accuracy.</p><p>Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit.</p

Comparison of the ELISA reactivity of <i>r</i>CatL, Peptide-1, SLbA and the EIE-LVC kit against sera from TL and VL patients and from <i>L. infantum</i>-infected dogs.

<p><b>TL and VL</b>: ELISAs were performed on samples from different groups of individuals (CT, control group; CD, Chagas disease patients; CL, cutaneous leishmaniasis; ML, mucosal leishmaniasis; VL, visceral leishmaniasis). <b>CVL</b>: ELISAs were performed on samples from different groups of dogs (CT, control group; CD, <i>T. cruzi</i>-infected dogs; CVL, canine visceral leishmaniasis). ^*Cut-off obtained by a ROC curve. ^#Cut-off suggested by the manufacturer.</p

Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit using ROC curves. Data validation and agreement was confirmed using a kappa index.

a<p>The kappa index was calculated using all samples presented in this work for TL (CT + CD + CL + ML. n = 135), VL (CT + CD + VL. n = 125) and CVL (CT + CD + CVL. n = 75).</p>b<p>Agreement was calculated using parasitological assays as the gold standard.</p><p>*<i>Cut-off</i> obtained by ROC curve.</p>#<p><i>Cut-off</i> suggested by the manufacturer.</p><p>Abbreviations: AUC: area under curve; CI: confidence interval; TP: true positive; TN: true negative; FP: false positive; FN: false negative; κ: kappa index; NA: not applicable.</p><p>Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit using ROC curves. Data validation and agreement was confirmed using a kappa index.</p

Differential expression of small RNA pathway genes associated with the <i>Biomphalaria glabrata/Schistosoma mansoni</i> interaction

<div><p>The World Health Organization (WHO) estimates that approximately 240 million people in 78 countries require treatment for schistosomiasis, an endemic disease caused by trematodes of the genus <i>Schistosoma</i>. In Brazil, <i>Schistosoma mansoni</i> is the only species representative of the genus whose passage through an invertebrate host, snails of the genus <i>Biomphalaria</i>, is obligatory before infecting a mammalian host, including humans. The availability of the genome and transcriptome of <i>B</i>. <i>glabrata</i> makes studying the regulation of gene expression, particularly the regulation of miRNA and piRNA processing pathway genes, possible. This might assist in better understanding the biology of <i>B</i>. <i>glabrata</i> as well as its relationship to the parasite <i>S</i>. <i>mansoni</i>. Some aspects of this interaction are still poorly explored, including the participation of non-coding small RNAs, such as miRNAs and piRNAs, with lengths varying from 18 to 30 nucleotides in mature form, which are potent regulators of gene expression. Using bioinformatics tools and quantitative PCR, we characterized and validated the miRNA and piRNA processing pathway genes in <i>B</i>. <i>glabrata</i>. <i>In silico</i> analyses showed that genes involved in miRNA and piRNA pathways were highly conserved in protein domain distribution, catalytic site residue conservation and phylogenetic analysis. Our study showed differential expression of putative Argonaute, Drosha, Piwi, Exportin-5 and Tudor genes at different snail developmental stages and during infection with <i>S</i>. <i>mansoni</i>, suggesting that the machinery is required for miRNA and piRNA processing in <i>B</i>. <i>glabrata</i> at all stages. These data suggested that the silencing pathway mediated by miRNAs and piRNAs can interfere in snail biology throughout the life cycle of the snail, thereby influencing the <i>B</i>. <i>glabrata/S</i>. <i>mansoni</i> interaction. Further studies are needed to confirm the participation of the small RNA processing pathway proteins in the parasite/host relationship, mainly the effective participation of small RNAs in regulating their target genes.</p></div

Domain structure of small RNA processing pathway proteins in <i>B</i>. <i>glabrata</i>.

<p><b>A—Bgl-Argonaute</b> has the following domains: ArgoN (PF16486—position 52 to 182 and E-value 8.5e^-31), ArgoL1 (PF08699—position 192 to 242 and E-value 9.8e^-24), PAZ (PF02170—position 256 to 364 and E-value 8.7e^-18), ArgoL2 (PF16488—position 373 to 419 and E-value 1.6e^-12), ArgoL2 (PF16488—position 424 to 460 and E-value 4.4e^-12), ArgoMid (PF16487—position 470 to 550 and E-value 2.4e^-34) and PIWI (PF02171—position 557 to 849 and E-value 4.7e^-104); <b>B—Bgl-Piwi</b> has the following domains: PAZ (PF02170 –position 274 to 404 and E-value 1.3e^-32) and PIWI (PF02171—position 547 to 839 and E-value 6.2e^-98); <b>C—Bgl-Drosha</b> has the following domains: Ribonucleas_3_3 (PF14622—position 923 to 1030 and E-value 1.3e^-21) and DSRM (PF00035—position 1081 to 1124 and E-value 2.00e^-06); <b>D—Bgl-Dicer</b> has the following domains: Helicase_C (PF00271—position 423 to 503 and E-value 5.4e^-12), Dicer_dimer (PF03368—position592 to 682 and E-value 1.3e^-24), PAZ (PF02170—position899 to 1050 and E-value 1.2e^-33), Ribonuclease_3 (PF00636 –position 1665 to 1842 and E-value 8.2e^-34) and Ribonuclease_3 (PF00636—position1951 to 2086 and E-value 9.7e^-22).</p

Multiple alignment of the RIBOc domain of Drosha and Dicer proteins.

<p>The highlighted residues, D (aspartic acid) and E (glutamic acid), which are highly conserved in the RIBOc domain of <i>B</i>. <i>glabrata</i>, and their orthologues are responsible for its catalytic activity. The sequences used for the Dicer family were as follows: BGLB002125-PA (<i>Biomphalaria glabrata</i>), XP_005106232.1 (<i>Aplysia californica</i>), NP_524453.1 (<i>Drosophila melanogaster</i>), NP_683750.2 (<i>Mus musculus</i>), XP_006618601.1 (<i>Apis dorsata</i>), XP_004523314.1 (<i>Ceratitis capitata</i>), XP_003745061.1 (<i>Metaseiulus occidentalis</i>), NP_498761.2 (<i>Caenorhabditis elegans</i>), NP_001154925.1 (<i>Danio rerio</i>), XP_868526.3 (<i>Canis lupus familiaris</i>), XP_008774532.1 (<i>Rattus norvegicus</i>), NP_976235.1 (<i>Bos taurus</i>), XP_005179924.1 (<i>Musca domestica</i>) and Smp_169750.2 (<i>Schistosoma mansoni</i>). The sequences used for the Drosha family were as follows: BGLB003167-PA (<i>Biomphalaria glabrata</i>), XP_008199088.1 (<i>Tribolium castaneum</i>), XP_006618766.1 (<i>Apis dorsata</i>), XP_006558454.1 (<i>Apis mellifera</i>), XP_003394274.1 (<i>Bombus terrestris</i>), NP_477436.1 (<i>Drosophila melanogaster</i>), XP_005248351.1 (<i>Homo sapiens</i>), XP_006520084.1 (<i>Mus musculus</i>), EKC20603.1 (<i>Crassostrea gigas</i>), NP_001122460.2 (<i>Caenorhabditis elegans</i>), NP_001103942.1 (<i>Danio rerio</i>), NP_001101125.2 (<i>Rattus norvegicus</i>), XP_854135.2 (<i>Canis lupus familiaris</i>), XP_591998.4 (<i>Bos taurus</i>), XP_005186977.1 (<i>Musca domestica</i>), NP_001006379.1 (<i>Gallus gallus</i>), Smp_142510.2 (<i>Schistosoma mansoni</i>) and Sjp_0048900.1 (<i>Schistosoma japonicum</i>).</p

Phylogenetic tree of Bgl-Argonaute and Bgl-Piwi with their orthologues.

<p>The phylogenetic analysis was performed using Mega 5.2 with bootstrap analysis. Bootstrap percentages are indicated at each branch.</p