The muscle and neural architecture of Taenia crassiceps cysticerci revisited; implications on head-tail polarization of the larvae.

Taenia crassiceps has been used for decades as an experimental model for the study of human and porcine cysticercosis. Even though, its life cycle, tissue organization, ultrastructure and immune response elicited in the host, have been extensively described, there are many other biological questions remaining to be addressed. In the present study we revisited the muscle and neural architecture of cysticerci in two of the most frequently used strains (WFU and ORF), using conventional staining and confocal microscopy imaging, aiming to assemble an updated anatomy. Differences between both strains, including polarization processes during development of the young budding larvae, are emphasized. We also performed a search for genes that have been related to peptidergic neural processes in other related flatworms. These findings can help to understand the anatomical and molecular consequences of the scolex presence or absence in both strains.

Effect of Hydroxyurea on Morphology, Proliferation, and Protein Expression on Taenia crassiceps WFU Strain.

Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.

Towards the development of an epitope-focused vaccine for SARS-CoV-2.

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions. Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

The Genomes of Two Strains of Taenia crassiceps the Animal Model for the Study of Human Cysticercosis.

Human cysticercosis by Taenia solium is the major cause of neurological illness in countries of Africa, Southeast Asia, and the Americas. Publication of four cestode genomes (T. solium, Echinococcus multilocularis, E. granulosus and Hymenolepis microstoma) in the last decade, marked the advent of novel approaches on the study of the host-parasite molecular crosstalk for cestode parasites of importance for human and animal health. Taenia crassiceps is another cestode parasite, closely related to T. solium, which has been used in numerous studies as an animal model for human cysticercosis. Therefore, characterization of the T. crassiceps genome will also contribute to the understanding of the human infection. Here, we report the genome of T. crassiceps WFU strain, reconstructed to a noncontiguous finished resolution and performed a genomic and differential expression comparison analysis against ORF strain. Both strain genomes were sequenced using Oxford Nanopore (MinION) and Illumina technologies, achieving high quality assemblies of about 107 Mb for both strains. Dotplot comparison between WFU and ORF demonstrated that both genomes were extremely similar. Additionally, karyotyping results for both strains failed to demonstrate a difference in chromosome composition. Therefore, our results strongly support the concept that the absence of scolex in the ORF strain of T. crassiceps was not the result of a chromosomal loss as proposed elsewhere. Instead, it appears to be the result of subtle and extensive differences in the regulation of gene expression. Analysis of variants between the two strains identified 2,487 sites with changes distributed in 31 of 65 scaffolds. The differential expression analysis revealed that genes related to development and morphogenesis in the ORF strain might be involved in the lack of scolex formation.

New Insights on NETosis Induced by Entamoeba histolytica: Dependence on ROS from Amoebas and Extracellular MPO Activity.

NETosis is a neutrophil process involving sequential steps from pathogen detection to the release of DNA harboring antimicrobial proteins, including the central generation of NADPH oxidase dependent or independent ROS. Previously, we reported that NETosis triggered by Entamoeba histolytica trophozoites is independent of NADPH oxidase activity in neutrophils, but dependent on the viability of the parasites and no ROS source was identified. Here, we explored the possibility that E. histolytica trophozoites serve as the ROS source for NETosis. NET quantitation was performed using SYTOX® Green assay in the presence of selective inhibitors and scavengers. We observed that respiratory burst in neutrophils was inhibited by trophozoites in a dose dependent manner. Mitochondrial ROS was not also necessary, as the mitochondrial scavenger mitoTEMPO did not affect the process. Surprisingly, ROS-deficient amoebas obtained by pre-treatment with pyrocatechol were less likely to induce NETs. Additionally, we detected the presence of MPO on the cell surface of trophozoites after the interaction with neutrophils and found that luminol and isoluminol, intracellular and extracellular scavengers for MPO derived ROS reduced the amount of NET triggered by amoebas. These data suggest that ROS generated by trophozoites and processed by the extracellular MPO during the contact with neutrophils are required for E. histolytica induced NETosis.

Holocene life and microbiome profiling in ancient tropical Lake Chalco, Mexico.

Metagenomic and traditional paleolimnological approaches are suitable to infer past biological and environmental changes, however, they are often applied independently, especially in tropical regions. We combined both approaches to investigate Holocene Prokaryote and Eukaryote diversity and microbial metabolic pathways in ancient Lake Chalco, Mexico. Here, we report on diversity among a large number of lineages (36,722 OTUs) and functional diversity (27,636,243 non-clustered predicted proteins, and 6,144 annotated protein-family genes). The most abundant domain is Bacteria (81%), followed by Archaea (15%) and Eukarya (3%). We also determined the diversity of protein families and their relationship to metabolic pathways. The early Holocene (> 11,000 cal years BP) lake was characterized by cool, freshwater conditions, which later became warmer and hyposaline (11,000-6,000 cal years BP). We found high abundances of cyanobacteria, and fungi groups associated with mature forests in these sediments. Bacteria and Archaea include mainly anaerobes and extremophiles that are involved in the sulfur, nitrogen, and carbon cycles. We found evidence for early human impacts, including landscape modifications and lake eutrophication, which began ~ 6,000 cal years BP. Subsaline, temperate conditions were inferred for the past 5,000 years. Finally, we found nitrogen-fixing bacteria and protein-family genes that are linked to contaminated environments, as well as several fungal pathogens of crops in near-surface sediments.

Immune modulation by the macrophage migration inhibitory factor (MIF) family: D-dopachrome tautomerase (DDT) is not (always) a backup system.

Human macrophage migration inhibition factor (MIF) is a protein with cytokine and chemokine properties that regulates a diverse range of physiological functions related to innate immunity and inflammation. Most research has focused on the role of MIF in different inflammatory diseases. D-dopachrome tautomerase (DDT), a different molecule with structural similarities to MIF, which shares receptors and biological functions, has recently been reported, but little is known about its roles and mechanisms. In this review, we sought to understand the similarities and differences between these molecules by summarizing what is known about their different structures, receptors and mechanisms regulating their expression and biological activities with an emphasis on immunological aspects.

How to eliminate taeniasis/cysticercosis: porcine vaccination and human chemotherapy (Part 2).

BACKGROUND: The application of effective vaccines against pig cysticercosis and mass chemotherapy against pig cysticercosis and human taeniasis have shown the feasibility of interrupting the parasite's life cycle in endemic areas. METHODS: A mathematical model that divides the population into susceptible, infected, and vaccinated individuals is formulated. The model is based upon the life cycle of the parasite. Computer numerical simulation experiments to evaluate the impact of pig vaccination under different vaccination schedules, and combined intervention strategies including pig vaccination and anthelmintic treatment against human taeniasis are carried out. RESULTS: Vaccination against either pig cysticercosis or against human taeniasis will influence the transmission dynamics not only among vaccinees but also the dynamics of the other hosts as well. When the protective efficacy and/or the coverage rate is less than 100%, different mass interventions like vaccinating the pig population twice in combination with chemotherapeutic treatment against human taeniasis, the elimination of the infection in both pigs and humans can also be achieved. CONCLUSIONS: Our mathematical model has the potential for planning, and designing effective intervention strategies including both mass vaccination and/or chemotherapeutic treatment to eliminate pig cysticercosis, human taeniasis and human neurocysticercosis. The model can be adapted to any given community with mild, moderate endemicity, or even in hyperendemic regions.

Plasminogen-binding proteins as an evasion mechanism of the host's innate immunity in infectious diseases.

Pathogens have developed particular strategies to infect and invade their hosts. Amongst these strategies' figures the modulation of several components of the innate immune system participating in early host defenses, such as the coagulation and complement cascades, as well as the fibrinolytic system. The components of the coagulation cascade and the fibrinolytic system have been proposed to be interfered during host invasion and tissue migration of bacteria, fungi, protozoa, and more recently, helminths. One of the components that has been proposed to facilitate pathogen migration is plasminogen (Plg), a protein found in the host's plasma, which is activated into plasmin (Plm), a serine protease that degrades fibrin networks and promotes degradation of extracellular matrix (ECM), aiding maintenance of homeostasis. However, pathogens possess Plg-binding proteins that can activate it, therefore taking advantage of the fibrin degradation to facilitate establishment in their hosts. Emergence of Plg-binding proteins appears to have occurred in diverse infectious agents along evolutionary history of host-pathogen relationships. The goal of the present review is to list, summarize, and analyze different examples of Plg-binding proteins used by infectious agents to invade and establish in their hosts. Emphasis was placed on mechanisms used by helminth parasites, particularly taeniid cestodes, where enolase has been identified as a major Plg-binding and activating protein. A new picture is starting to arise about how this glycolytic enzyme could acquire an entirely new role as modulator of the innate immune system in the context of the host-parasite relationship.

Entamoeba histolytica Trophozoites Induce a Rapid Non-classical NETosis Mechanism Independent of NOX2-Derived Reactive Oxygen Species and PAD4 Activity.

Neutrophil extracellular traps (NETs) are DNA fibers decorated with histones and antimicrobial proteins from cytoplasmic granules released into the extracellular space in a process denominated NETosis. The molecular pathways involved in NETosis have not been completely understood. Classical NETosis mechanisms involve the neutrophil elastase (NE) translocation to nucleus due to the generation of reactive oxygen species (ROS) by NADPH oxidase (NOX2) or the peptidyl arginine deiminase 4 (PAD4) activation in response to an increase in extracellular calcium influx; both mechanisms result in DNA decondensation. Previously, we reported that trophozoites and lipopeptidophosphoglycan from Entamoeba histolytica trigger NET release in human neutrophils. Here, we demonstrated in a quantitative manner that NETs were rapidly form upon treatment with amoebic trophozoites and involved both nuclear and mitochondrial DNA (mtDNA). NETs formation depended on amoeba viability as heat-inactivated or paraformaldehyde-fixed amoebas were not able to induce NETs. Interestingly, ROS were not detected in neutrophils during their interaction with amoebas, which could explain why NOX2 inhibition using apocynin did not affect this NETosis. Surprisingly, whereas calcium chelation reduced NET release induced by amoebas, PAD4 inhibition by GSK484 failed to block DNA extrusion but, as expected, abolished NETosis induced by the calcium ionophore A23187. Additionally, NE translocation to the nucleus and serine-protease activity were necessary for NET release caused by amoeba. These data support the idea that E. histolytica trophozoites trigger NETosis by a rapid non-classical mechanism and that different mechanisms of NETs release exist depending on the stimuli used.

Identification and characterization of Taenia solium enolase as a plasminogen-binding protein.

The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins.

Effect of Transforming Growth Factor-ß upon Taenia solium and Taenia crassiceps Cysticerci.

Taeniids exhibit a great adaptive plasticity, which facilitates their establishment, growth, and reproduction in a hostile inflammatory microenvironment. Transforming Growth Factor-ß (TGFß), a highly pleiotropic cytokine, plays a critical role in vertebrate morphogenesis, cell differentiation, reproduction, and immune suppression. TGFß is secreted by host cells in sites lodging parasites. The role of TGFß in the outcome of T. solium and T. crassiceps cysticercosis is herein explored. Homologues of the TGFß family receptors (TsRI and TsRII) and several members of the TGFß downstream signal transduction pathway were found in T. solium genome, and the expression of Type-I and -II TGFß receptors was confirmed by RT-PCR. Antibodies against TGFß family receptors recognized cysticercal proteins of the expected molecular weight as determined by Western blot, and different structures in the parasite external tegument. In vitro, TGFß promoted the growth and reproduction of T. crassiceps cysticerci and the survival of T. solium cysticerci. High TGFß levels were found in cerebrospinal fluid from untreated neurocysticercotic patients who eventually failed to respond to the treatment (P = 0.03) pointing to the involvement of TGFß in parasite survival. These results indicate the relevance of TGFß in the infection outcome by promoting cysticercus growth and treatment resistance.

Water-Soluble Ruthenium (II) Chiral Heteroleptic Complexes with Amoebicidal in Vitro and in Vivo Activity.

Three water-soluble Ru(II) chiral heteroleptic coordination compounds [Ru(en)(pdto)]Cl2 (1), [Ru(gly)(pdto)]Cl (2), and [Ru(acac)(pdto)]Cl (3), where pdto = 2,2'-[1,2-ethanediylbis-(sulfanediyl-2,1-ethanediyl)]dipyridine, en = ethylendiamine, gly = glycinate, and acac = acetylacetonate, have been synthezised and fully characterized. The crystal structures of compounds 1-3 are described. The IC50 values for compounds 1-3 are within nanomolar range (14, 12, and 6 nM, respectively). The cytotoxicity for human peripheral blood lymphocytes is extremely low (>100 µM). Selectivity indexes for Ru(II) compounds are in the range 700-1300. Trophozoites exposed to Ru(II) compounds die through an apoptotic pathway triggered by ROS production. The orally administration to infected mice induces a total elimination of the parasite charge in mice faeces 1-2-fold faster than metronidazole. Besides, all compounds inhibit the trophozoite proliferation in amoebic liver abscess induced in hamster. All our results lead us to propose these compounds as promising candidates as antiparasitic agents.

Differential antigenic protein recovery from Taenia solium cyst tissues using several detergents.

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). The protein extracts of T. solium cysts are complex mixtures including cyst's and host proteins. Little is known about the influence of using different detergents in the efficiency of solubilization-extraction of these proteins, including relevant antigens. Here, we describe the use of CHAPS, ASB-14 and Triton X-100, alone or in combination in the extraction buffers, as a strategy to notably increase the recovery of proteins that are usually left aside in insoluble fractions of cysts. Using buffer with CHAPS alone, 315 protein spots were detected through 2D-PAGE. A total of 255 and 258 spots were detected using buffers with Triton X-100 or ASB-14, respectively. More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1% ASB-14 allowed detection of up to 368 spots. Our results indicated that insoluble fractions of T. solium cysts were rich in antigens, including several glycoproteins that were sensitive to metaperiodate treatment. Host proteins, a common component in protein extracts of cysts, were present in larger amounts in soluble than insoluble fractions of cysts proteins. Finally, antigens present in the insoluble fraction were more appropriate as a source of antigens for diagnostic procedures.

Transfection of Platyhelminthes.

Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

Identification and quantification of host proteins in the vesicular fluid of porcine Taenia solium cysticerci.

The host-parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pig's sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3-8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3-8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11-13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.

Evolution, molecular epidemiology and perspectives on the research of taeniid parasites with special emphasis on Taenia solium.

Human cysticercosis is known since old historical times in Greece and China; however, human infections by tapeworms have accompanied human beings for more that hundred thousand years. The disease is tightly bound to poverty and lack of hygiene, and has been eradicated in developed countries, but continues being a public health problem in developing countries of Latin-American, Sub-Saharan Africa and Asia, and is also remerging in a number of non endemic countries. It is considered a neglected disease. Here we revise a number of key scientific contributions on taeniid biology that open new avenues for more effective approaches to the control of cysticercosis. The evolution of flatworms and class Cestoda is analyzed, with special emphasis on the emergence of taeniid parasites and the colonization of the human species by tapeworms. The complex molecular host-parasite interplay in this relationship as result of co-evolution between two distantly related organisms. The relevant host and parasite's factors, in the prospect of identifying species-specific molecular markers useful in epidemiological studies carried out in endemic countries. The new possibilities arising with the characterization of the genomes for several species of tapeworms, including a deeper understanding of these organisms, as well as improved tools for diagnosis, vaccination and drug treatment. The need to revise the current control and management strategies for this tropical neglected disease.

Immunodiagnosis of porcine cysticercosis: identification of candidate antigens through immunoproteomics.

Cysticercosis, caused by the larval stage of Taenia solium, is a zoonotic disease affecting pigs and humans that is endemic to developing countries in Latin America, Africa and South East Asia. The prevalence of infection in pigs, the intermediate host for T. solium, has been used as an indicator for monitoring disease transmission in endemic areas. However, accurate and specific diagnostic tools for porcine cysticercosis remain to be established. Using proteomic approaches and the T. solium genome sequence, seven antigens were identified as specific for porcine cysticercosis, namely, tropomyosin 2, alpha-1 tubulin, beta-tubulin 2, annexin B1, small heat-shock protein, 14-3-3 protein, and cAMP-dependent protein kinase. None of these proteins were cross-reactive when tested with sera from pigs infected with Ascaris spp., Cysticercus tenuicollis and hydatid cysts of Echinococcus spp. or with serum from a Taenia saginata-infected cow. Comparison with orthologues, indicated that the amino acid sequences of annexin B1 and cAMP-dependent protein kinase possessed highly specific regions, which might make them suitable candidates for development of a specific diagnostic assay for porcine cysticercosis.

Health research funding in Mexico: the need for a long-term agenda.

BACKGROUND: The legal framework and funding mechanisms of the national health research system were recently reformed in Mexico. A study of the resource allocation for health research is still missing. We identified the health research areas funded by the National Council on Science and Technology (CONACYT) and examined whether research funding has been aligned to national health problems. METHODS AND FINDINGS: We collected the information to create a database of research grant projects supported through the three main Sectoral Funds managed by CONACYT between 2003 and 2010. The health-related projects were identified and classified according to their methodological approach and research objective. A correlation analysis was carried out to evaluate the association between disease-specific funding and two indicators of disease burden. From 2003 to 2010, research grant funding increased by 32% at a compound annual growth rate of 3.5%. By research objective, the budget fluctuated annually resulting in modest increments or even decrements during the period under analysis. The basic science category received the largest share of funding (29%) while the less funded category was violence and accidents (1.4%). The number of deaths (ρ = 0.51; P<0.001) and disability-adjusted life years (DALYs; ρ = 0.33; P = 0.004) were weakly correlated with the funding for health research. Considering the two indicators, poisonings and infectious and parasitic diseases were among the most overfunded conditions. In contrast, congenital anomalies, road traffic accidents, cerebrovascular disease, and chronic obstructive pulmonary disease were the most underfunded conditions. CONCLUSIONS: Although the health research funding has grown since the creation of CONACYT sectoral funds, the financial effort is still low in comparison to other Latin American countries with similar development. Furthermore, the great diversity of the funded topics compromises the efficacy of the investment. Better mechanisms of research priority-setting are required to adjust the research portfolio to the new health panorama of Mexican population.