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Molecular and Biochemical Parasitology

Lucas L Maldonado, Georgina Stegmayer, Diego H Milone, Guilherme Oliveira, Mara Rosenzvit, Laura Kamenetzky
The species of the genus Echinococcus are parasitic platyhelminths that cause echinococcosis and exert a global burden on public and animal health. Here we performed codon usage bias and comparative genomic analyses using whole genome and expression data of three Echinococcus species. The study of 4,710,883 codons, two orders of magnitude more than in previous research works, showed that the codon usage in Echinococcus genes is biased towards the pyrimidines T and C ending codons, with an average effective number of codons equal to 57 revealing a low codon usage bias...
August 3, 2018: Molecular and Biochemical Parasitology
María Victoria Solana, María Fernanda Domínguez, Silvana Scarcella, Santiago Radio, Pablo Smircich, Silvina Fernández, Hugo Solana, José F Tort
The use of Triclabendazole for controlling fasciolosis is compromised by increased drug resistance affecting livestock and humans. Although the mode of action of TCBZ is still unknown, putative candidates and markers of resistance have been advanced. A single nucleotide polymorphism (T687 G) in F. hepatica PGP was proposed as marker of resistance in a small scale study of European susceptible and resistant flukes, but the association was not found in Australian samples. The T687 G SNP was absent in more than 40 samples from 2 TCBZ-resistant and 3 susceptible isolates across Latin America here analyzed...
July 25, 2018: Molecular and Biochemical Parasitology
Huafang Shi, Kiantra Butler, Christian Tschudi
We previously established an in vitro differentiation system based on the inducible expression of the RNA binding protein 6 (RBP6), which initiated differentiation of Trypanosoma brucei non-infectious procyclics to infectious metacyclics (MFs). However, further differentiation to bloodstream forms (BFs) required infection of mice. Here we report the serendipitous isolation of a single point mutation in RBP6 (Q109K), whose expression not only generated MFs, but purified MFs continued the developmental cycle in vitro to BFs expressing variant surface glycoprotein-2 (VSG-2), formerly known as VSG 221...
July 25, 2018: Molecular and Biochemical Parasitology
Ahmad Syibli Othman, Jing-Wen Lin, Blandine M Franke-Fayard, Hans Kroeze, Fiona J A van Pul, Séverine Chevalley-Maurel, Jai Ramesar, Catherin Marin-Mogollon, Matthijs M Jore, Merribeth J Morin, Carole A Long, Robert Sauerwein, Ashley Birkett, Kazutoyo Miura, Chris J Janse, Shahid M Khan
The transmission-blocking vaccine candidate Pfs48/45 from the human malaria parasite Plasmodium falciparum is known to be difficult to express in heterologous systems, either as full-length protein or as correctly folded protein fragments that retain conformational epitopes. In this study we express full-length Pfs48/45 in the rodent parasite P. berghei. Pfs48/45 is expressed as a transgene under control of the strong P. berghei schizont-specific msp1 gene promoter (Pfs48/45@PbMSP1). Pfs48/45@PbMSP1 schizont-infected red blood cells produced full-length Pfs48/45 and the structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes...
July 24, 2018: Molecular and Biochemical Parasitology
Luciana Baroni, Luiz M Pereira, Sutherland K Maciver, Ana P Yatsuda
Neospora caninum is an apicomplexan parasite that causes infectious abortion in cows. As an obligate intracellular parasite, N. caninum requires a host cell environment to survive and replicate. The locomotion and invasion mechanisms of apicomplexan parasites are centred on the actin-myosin system to propel the parasite forwards and into the host cell. The functions of actin, an intrinsically dynamic protein, are modulated by actin-binding proteins (ABPs). Actin-depolymerising factor (ADF) is a ubiquitous ABP responsible for accelerating actin turnover in eukaryotic cells and is one of the few known conserved ABPs from apicomplexan parasites...
July 21, 2018: Molecular and Biochemical Parasitology
Cong Liu, Zhenkui Li, Yuanyuan Jiang, Huiting Cui, Jing Yuan
Male and female gametocytes are the infectious forms critical for malaria transmission and targets of intervention. Gametocytes are generally produced in relatively small numbers, and it has been difficult to obtain pure male and female gametocytes for various studies. Male and female gametocytes expressing unique fluorescence reporters have been generated for both Plasmodium falciparum and Plasmodium berghei parasites, which allows isolation of large numbers of pure male and female gametocytes and has greatly contributed to our understanding of gametocyte biology...
July 21, 2018: Molecular and Biochemical Parasitology
Analía G Níttolo, Carolina P Bañuelos, Juan I Saborit, Valeria Tekiel, Daniel O Sánchez, Gabriela V Levy
TbRRM1, an SR-related protein, is involved in transcriptional and post-transcriptional gene expression regulation in procyclic T. brucei. In previous work, we found that TbRRM1 is essential and its depletion leads to cell cycle impairment, aberrant phenotypes and cell loss by apoptotic-like death. Here, we report the findings obtained after TbRRM1 knockdown in bloodstream parasites. Depletion of TbRRM1 in this cell stage led also to growth arrest and cell loss by apoptosis-like death. However, microscopic analysis showed aberrant cell morphology with parasites displaying flagellum detachment and cytokinesis impairment after RNAi induction, suggesting that TbRRM1 could play different roles depending on parasite stage...
July 19, 2018: Molecular and Biochemical Parasitology
Miria G Pereira, Gonzalo Visbal, Tatiana F R Costa, Susana Frases, Wanderley de Souza, Geórgia Atella, Narcisa Cunha-E-Silva
The Chagas disease agent Trypanosoma cruzi proliferates in the insect vector as highly endocytic epimastigotes that store nutrients, including lipids in reservosomes (lysosome related compartments). Although nutrient storage is important for epimastigote transformation into infective metacyclics, the epimastigote lipid droplets (LDs) remain uncharacterized. Here, we characterized the epimastigote LDs and examined their relationship with the endocytic pathway. Fluorescence microscopy using BODIPY showed that LDs have high neutral lipid content and harbor Rab18, differently from other lipid-rich organelles (such as reservosomes)...
July 19, 2018: Molecular and Biochemical Parasitology
Flávia S Damasceno, María Julia Barisón, Marcell Crispim, Rodolpho O O Souza, Letícia Marchese, Ariel M Silber
Trypanosoma cruzi, the aetiological agent of Chagas disease, can obtain L-glutamine (Gln) through the enzyme glutamine synthetase (GS) using glutamate (Glu) and ammonia as substrates. In this work, we show additional non-canonical roles for this amino acid: its involvement in ATP maintenance and parasite survival under severe metabolic stress conditions and its participation in the differentiation process occurring in the insect vector (metacyclogenesis). These roles are dependent on the supply of Gln from an extracellular source...
July 17, 2018: Molecular and Biochemical Parasitology
Julia A Loos, María Celeste Nicolao, Andrea C Cumino
Cystic echinococcosis is a neglected parasitic disease caused by the larval stage of Echinococcus granulosus for which an effective treatment is not yet available. Since autophagy constitutes a homeostatic mechanism during stress, either inhibition or activation of its activity might be detrimental for survival of the parasite. Amongst the critical molecules that regulate autophagy, TOR, AMPK and sirtuins are the best characterized ones. Previously, we have identified the autophagic machinery, the occurrence of TORC1-controlled events, and the correlation between autophagy and the activation of the unfolded protein response in E...
July 11, 2018: Molecular and Biochemical Parasitology
Francisco Olmo, Fernanda C Costa, Gurdip Singh Mann, Martin C Taylor, John M Kelly
The limited flexibility and time-consuming nature of the genetic manipulation procedures applicable to Trypanosoma cruzi continue to restrict the functional dissection of this parasite. We hypothesised that transformation efficiency could be enhanced if electroporation was timed to coincide with DNA replication. To test this, we generated epimastigote cultures enriched at the G1/S boundary using hydroxyurea-induced cell-cycle synchronisation, and then electroporated parasites at various time points after release from the cell-cycle block...
July 7, 2018: Molecular and Biochemical Parasitology
Lauren E-A Eyssen, Perina Vather, Laurelle Jackson, Phindile Ximba, Nicolas Biteau, Théo Baltz, Alain Boulangé, Philippe Büscher, Theresa H T Coetzer
African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATLcat , was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S2 pocket...
July 2018: Molecular and Biochemical Parasitology
Pranay Jakkula, Rahila Qureshi, Atif Iqbal, S R Sagurthi, Insaf A Qureshi
Most of the signaling pathways are regulated by reversible phosphorylation-dephosphorylation which involves enzymes- kinases and phosphatases. Current knowledge about the protein phosphatases in parasites like Trypanosoma and Leishmania is very minimal despite their enormousity. In present study, full length ORF of Leishmania donovani PP2C was cloned into expression vector followed by purification and molecular weight determination using Ni-NTA affinity and gel giltration chromatography respectively. Purified LdPP2C was found to be enzymatically active, while inhibition study suggested that sanguinarine acts as a non-competitive inhibitor...
July 2018: Molecular and Biochemical Parasitology
Weerachai Saijuntha, Chairat Tantrawatpan, Takeshi Agatsuma, Chennan Wang, Pewpan M Intapan, Wanchai Maleewong, Trevor N Petney
Fasciola hepatica and F. gigantica are ruminant liver flukes that are found worldwide. They can occur sympatrically and hybridize, consequently producing an intermediate form (Fasciola sp.), especially in Africa and Asia. The bridge intron (TkBridgeInt) and intron 4 of domain 2 (TkD2Int4) of the taurocyamine kinase gene were cloned and sequenced from 14 F. hepatica, 18 F. gigantica, and 12 Fasciola sp. These intron regions could be used to differentiate F. hepatica from F. gigantica and revealed genetic hybridization and DNA recombination between F...
July 2018: Molecular and Biochemical Parasitology
Juliette Morlon-Guyot, Laurence Berry, Isabelle Sauquet, Gurman Singh Pall, Hiba El Hajj, Markus Meissner, Wassim Daher
Toxoplasma gondii which is a member of the coccidian parasites owns a spatially polarized secretory system, which synthesizes de novo micronemes and rhoptries. These apical secretory organelles discharge their contents into host cells promoting invasion and survival. Herein, we identified a novel Coccidian Specific CORVET/HOPS Associated Protein (CSCHAP) belonging to the interaction network of both tethering complexes. CSCHAP is associated with the endomembrane system, rhoptries, micronemes and probably to the inner core of the conoid...
July 2018: Molecular and Biochemical Parasitology
A Taubert, L M R Silva, Z D Velásquez, C Larrazabal, D Lütjohann, C Hermosilla
Obligate intracellular apicomplexan parasites are considered as deficient in cholesterol biosynthesis and scavenge cholesterol from their host cell in a parasite-specific manner. Compared to fast proliferating apicomplexan species producing low numbers of merozoites per host cell, (e. g. Toxoplasma gondii), the macromeront-forming protozoa Eimeria bovis is in extraordinary need for cholesterol for offspring production (≥ 170,000 merozoites I/macromeront). Interestingly, optimized in vitro E. bovis merozoite I production occurs under low foetal calf serum (FCS, 1...
July 2018: Molecular and Biochemical Parasitology
Kirti Gupta, Ankit Gupta, Saman Habib
The ribosomal RNA adenine dimethyltransferases (rAD) of KsgA/Dim1 family are universally conserved with eukaryotic rADs separated into distinct cytosolic Dim1 and organellar KsgA/TFB homologs. Among the two putative KsgA proteins encoded by the Plasmodium falciparum genome, we found that PfKsgA1 was dually localised in the cytoplasm and the mitochondrion. The protein interacted specifically with small ribosomal subunit as detected by ribosome pull-down using anti-PfKsgA1 antibodies. Recombinant PfKsgA1 exhibited methyltransferase activity which was further confirmed by complementation in an Escherichia coli KsgA knockout strain...
July 2018: Molecular and Biochemical Parasitology
Wakisa Kipandula, Simon A Young, Stuart A MacNeill, Terry K Smith
Diseases caused by the pathogenic kinetoplastids continue to incapacitate and kill hundreds of thousands of people annually throughout the tropics and sub-tropics. Unfortunately, in the countries where these neglected diseases occur, financial obstacles to drug discovery and technical limitations associated with biochemical studies impede the development of new, safe, easy to administer and effective drugs. Here we report the development and optimisation of a Crithidia fasciculata resazurin viability assay, which is subsequently used for screening and identification of anti-crithidial compounds in the MMV and GSK open access chemical boxes...
June 2018: Molecular and Biochemical Parasitology
Divya N Nair, Rajesh Prasad, Neha Singhal, Manish Bhattacharjee, Renu Sudhakar, Pushpa Singh, Subramonian Thanumalayan, Uday Kiran, Yogendra Sharma, Puran Singh Sijwali
Plasmodium falciparum DJ1 (PfDJ1) belongs to the DJ-1/ThiJ/PfpI superfamily whose members are present in all the kingdoms of life and exhibit diverse cellular functions and biochemical activities. The common feature of the superfamily is the class I glutamine amidotransferase domain with a conserved redox-active cysteine residue, which mediates various activities of the superfamily members, including anti-oxidative activity in PfDJ1 and human DJ1 (hDJ1). As the superfamily members represent diverse functional classes, to investigate if there is any sequence feature unique to hDJ1-like proteins, sequences of the representative proteins of different functional classes were compared and analysed...
June 2018: Molecular and Biochemical Parasitology
José Rubén Ramírez-Iglesias, María Carolina Pérez-Gordones, Jesús Rafael Del Castillo, Alfredo Mijares, Gustavo Benaim, Marta Mendoza
The plasma membrane Ca2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain...
June 2018: Molecular and Biochemical Parasitology
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