Identification and Characterization of Microsatellite Markers Derived from the Whole Genome Analysis of Taenia solium

Background

Infections with Taenia solium are the most common cause of adult acquired seizures worldwide, and are the leading cause of epilepsy in developing countries. A better understanding of the genetic diversity of T. solium will improve parasite diagnostics and transmission pathways in endemic areas thereby facilitating the design of future control measures and interventions. Microsatellite markers are useful genome features, which enable strain typing and identification in complex pathogen genomes. Here we describe microsatellite identification and characterization in T. solium, providing information that will assist in global efforts to control this important pathogen.

Methods

For genome sequencing, T. solium cysts and proglottids were collected from Huancayo and Puno in Peru, respectively. Using next generation sequencing (NGS) and de novo assembly, we assembled two draft genomes and one hybrid genome. Microsatellite sequences were identified and 36 of them were selected for further analysis. Twenty T. solium isolates were collected from Tumbes in the northern region, and twenty from Puno in the southern region of Peru. The size-polymorphism of the selected microsatellites was determined with multi-capillary electrophoresis. We analyzed the association between microsatellite polymorphism and the geographic origin of the samples.

Results

The predicted size of the hybrid (proglottid genome combined with cyst genome) T. solium genome was 111 MB with a GC content of 42.54%. A total of 7,979 contigs (>1,000 nt) were obtained. We identified 9,129 microsatellites in the Puno-proglottid genome and 9,936 in the Huancayo-cyst genome, with 5 or more repeats, ranging from mono- to hexa-nucleotide. Seven microsatellites were polymorphic and 29 were monomorphic within the analyzed isolates. T. solium tapeworms were classified into two genetic groups that correlated with the North/South geographic origin of the parasites.

Conclusions/Significance

The availability of draft genomes for T. solium represents a significant step towards the understanding the biology of the parasite. We report here a set of T. solium polymorphic microsatellite markers that appear promising for genetic epidemiology studies.     

Taenia solium Human Cysticercosis: A Systematic Review of Sero-epidemiological Data from Endemic Zones around the World

Background

Taenia solium cysticercosis is a zoonotic neglected disease responsible for severe health disorders such as seizures and death. Understanding the epidemiology of human cysticercosis (HCC) in endemic regions will help to expose critical information about the transmission of the disease, which could be used to design efficient control programs. This review gathered serological data on apparent prevalence of T. solium circulating antigens and/or seroprevalence of T. solium antibodies, apparent prevalence of human taeniasis and risk factors for HCC from endemic communities in order to understand the differences in exposure to the parasite and active infections with T. solium metacestodes in endemic areas around the world.

Methods

Three databases were used to search sero-epidemiological data from community-based studies conducted between 1989 and 2014 in cysticercosis endemic communities worldwide. The search focused on data obtained from T. solium circulating antigen detection by monoclonal antibody-based sandwich ELISA and/or T. solium antibody seroprevalence determined by Enzyme-linked Immunoelectrotransfer Blot (EITB). A meta-analysis was performed per continent.

Principal Findings

A total of 39,271 participants from 19 countries, described in 37 articles were studied. The estimates for the prevalence of circulating T. solium antigens for Africa, Latin America and Asia were: 7.30% (95% CI [4.23–12.31]), 4.08% (95% CI [2.77–5.95]) and 3.98% (95% CI [2.81–5.61]), respectively. Seroprevalence estimates of T. solium antibodies were 17.37% (95% CI [3.33–56.20]), 13.03% (95% CI [9.95–16.88]) and 15.68% (95% CI [10.25–23.24]) respectively. Taeniasis reported prevalences ranged from 0 (95% CI [0.00–1.62]) to 17.25% (95% CI [14.55–20.23]).

Significance

A significant variation in the sero-epidemiological data was observed within each continent, with African countries reporting the highest apparent prevalences of active infections. Intrinsic factors in the human host such as age and immunity were main determinants for the occurrence of infections, while exposure was mostly related to environmental factors which varied from community to community.     

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines

Objective

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.

Methods

Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.

Results

Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.

Conclusions

We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.     

A Comparative Study of Peripheral Immune Responses to Taenia solium in Individuals with Parenchymal and Subarachnoid Neurocysticercosis

Background

The ability of Taenia solium to modulate the immune system likely contributes to their longevity in the human host. We tested the hypothesis that the nature of the immune response is related to the location of parasite and clinical manifestations of infection.

Methodology

Peripheral blood mononuclear cells (PBMC) were obtained from untreated patients with neurocysticercosis (NCC), categorized as having parenchymal or subarachnoid infection by the presence of cysts exclusively within the parenchyma or in subarachnoid spaces of the brain, and from uninfected (control) individuals matched by age and gender to each patient. Using multiplex detection technology, sera from NCC patients and controls and cytokine production by PBMC after T. solium antigen (TsAg) stimulation were assayed for levels of inflammatory and regulatory cytokines. PBMC were phenotyped by flow cytometry ex vivo and following in vitro stimulation with TsAg.

Principal Findings

Sera from patients with parenchymal NCC demonstrated significantly higher Th1 (IFN-γ/IL-12) and Th2 (IL-4/IL-13) cytokine responses and trends towards higher levels of IL-1β/IL-8/IL-5 than those obtained from patients with subarachnoid NCC. Also higher in vitro antigen-driven TNF-β secretion was detected in PBMC supernatants from parenchymal than in subarachnoid NCC. In contrast, there was a significantly higher IL-10 response to TsAg stimulation in patients with subarachnoid NCC compared to parenchymal NCC. Although no differences in regulatory T cells (Tregs) frequencies were found ex vivo, there was a trend towards greater expansion of Tregs upon TsAg stimulation in subarachnoid than in parenchymal NCC when data were normalized for the corresponding controls.

Conclusions/Significance

T. solium infection of the subarachnoid space is associated with an enhanced regulatory immune response compared to infection in the parenchyma. The resulting anti-inflammatory milieu may represent a parasite strategy to maintain a permissive environment in the host or diminish inflammatory damage from the host immune response in the central nervous system.     

Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda)

Background

Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function.

Methodology/Principal Findings

Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells.

Conclusions/Significance

We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.     

GPS Tracking of Free-Ranging Pigs to Evaluate Ring Strategies for the Control of Cysticercosis/Taeniasis in Peru

Background

Taenia solium, a parasitic cestode that affects humans and pigs, is the leading cause of preventable epilepsy in the developing world. T. solium eggs are released into the environment through the stool of humans infected with an adult intestinal tapeworm (a condition called taeniasis), and cause cysticercosis when ingested by pigs or other humans. A control strategy to intervene within high-risk foci in endemic communities has been proposed as an alternative to mass antihelminthic treatment. In this ring strategy, antihelminthic treatment is targeted to humans and pigs residing within a 100 meter radius of a pig heavily-infected with cysticercosis. Our aim was to describe the roaming ranges of pigs in this region, and to evaluate whether the 100 meter radius rings encompass areas where risk factors for T. solium transmission, such as open human defecation and dense pig activity, are concentrated.

Methodology/Principal Findings

In this study, we used Global Positioning System (GPS) devices to track pig roaming ranges in two rural villages of northern Peru. We selected 41 pigs from two villages to participate in a 48-hour tracking period. Additionally, we surveyed all households to record the locations of open human defecation areas. We found that pigs spent a median of 82.8% (IQR: 73.5, 94.4) of their time roaming within 100 meters of their homes. The size of home ranges varied significantly by pig age, and 93% of the total time spent interacting with open human defecation areas occurred within 100 meters of pig residences.

Conclusions/Significance

These results indicate that 100 meter radius rings around heavily-infected pigs adequately capture the average pig’s roaming area (i.e., home range) and represent an area where the great majority of exposure to human feces occurs.     

Overexpression of Cytoplasmic TcSIR2RP1 and Mitochondrial TcSIR2RP3 Impacts on Trypanosoma cruzi Growth and Cell Invasion

Background

Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi.

Methodology

Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity.

Conclusion

TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.     

Prevalence and Risk Factors Associated with Human Taenia Solium Infections in Mbozi District,Mbeya Region,Tanzania

Background

Taenia solium cysticercosis/taeniosis is emerging as a serious public health and economic problem in many developing countries. This study was conducted to determine prevalence and risk factors of human T. solium infections in Mbeya Region, Tanzania.

Methods and Findings

A cross-sectional survey was conducted in 13 villages of Mbozi district in 2009. Sera of 830 people (mean 37.9±11.3 years (SD); 43% females) were tested for circulating cysticerci antigen (Ag-ELISA) and antibody (Ab-ELISA). A subset of persons found seropositive by Ag-ELISA underwent computed tomography (CT) scan of the brain for evidence of neurocysticercosis. Stool samples from 820 of the same participants were tested for taeniosis by copro-antigens (copro-Ag-ELISA) and formol-ether concentration technique. Cases of T. solium taeniosis were confirmed serologically by EITB assay (rES38). A questionnaire was used for identification of risk factors. Active cysticercosis by positive Ag-ELISA was found in 139 (16.7%) persons while anti-cysticercal antibodies were detected in 376 (45.3%) persons by Ab-ELISA. Among 55 persons positive for Ag-ELISA undergoing CT scan, 30 (54.6%) were found to have structures in the brain suggestive of neurocysticercosis. Using faecal analysis, 43 (5.2%) stool samples tested positive for taeniosis by copro-Ag-ELISA while Taenia eggs were detected in 9 (1.1%) stool samples by routine coprology. Antibodies specifically against adult T. solium were detected in 34 copro-Ag-ELISA positive participants by EITB (rES38) indicating T. solium taeniosis prevalence of 4.1%. Increasing age and hand washing by dipping in contrast to using running water, were found associated with Ag-ELISA seropositivity by logistic regression. Gender (higher risk in females) and water source were risk factors associated with Ab-ELISA seropositivity. Reported symptoms of chronic severe headaches and history of epileptic seizures were found associated with positive Ag-ELISA (p≤0.05).

Conclusion

The present study indicates T. solium infection in humans is highly endemic in the southern highlands of Tanzania.     

New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels

Background

Trypanosoma (T.) evansi is a dyskinetoplastic variant of T. brucei that has gained the ability to be transmitted by all sorts of biting flies. T. evansi can be divided into type A, which is the most abundant and found in Africa, Asia and Latin America and type B, which has so far been isolated only from Kenyan dromedary camels. This study aimed at the isolation and the genetic and phenotypic characterisation of type A and B T. evansi stocks from camels in Northern Ethiopia.

Methodology/principal findings

T. evansi was isolated in mice by inoculation with the cryopreserved buffy coat of parasitologically confirmed animals. Fourteen stocks were thus isolated and subject to genotyping with PCRs targeting type-specific variant surface glycoprotein genes, mitochondrial minicircles and maxicircles, minisatellite markers and the F1-ATP synthase γ subunit gene. Nine stocks corresponded to type A, two stocks were type B and three stocks represented mixed infections between A and B, but not hybrids. One T. evansi type A stock was completely akinetoplastic. Five stocks were adapted to in vitro culture and subjected to a drug sensitivity assay with melarsomine dihydrochloride, diminazene diaceturate, isometamidium chloride and suramin. In vitro adaptation induced some loss of kinetoplasts within 60 days. No correlation between drug sensitivity and absence of the kinetoplast was observed. Sequencing the full coding sequence of the F1-ATP synthase γ subunit revealed new type-specific single nucleotide polymorphisms and deletions.

Conclusions/significance

This study addresses some limitations of current molecular markers for T. evansi genotyping. Polymorphism within the F1-ATP synthase γ subunit gene may provide new markers to identify the T. evansi type that do not rely on variant surface glycoprotein genes or kinetoplast DNA.     

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Background

Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.

Methods

100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (10⁵ CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (T_RLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of T_RLU values was determined. External validation was further done using 50 additional CR-GNB.

Results

Predictive accuracies of T_RLU were high for when all antibiotic combinations and species were collectively analyzed (T_RLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (T_RLU = 0.81, UA = 91%), and lowest for AB (T_RLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.

Conclusion

We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.     

Mitochondrial Dysfunction Induced by N-Butyl-1-(4-Dimethylamino)Phenyl-1,2,3,4-Tetrahydro-β-Carboline-3-Carboxamide Is Required for Cell Death of Trypanosoma cruzi

Background

Chagas’ disease is caused by the protozoan Trypanosoma cruzi and affects thousands of people worldwide. The available treatments are unsatisfactory, and new drugs must be developed. Our group recently reported the trypanocidal activity of the synthetic compound N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxamide (C4), but the mechanism of action of this compound was unclear.

Methodology/Principal Findings

We investigated the mechanism of action of C4 against epimastigote and trypomastigote forms of T. cruzi. The results showed alterations in mitochondrial membrane potential, alterations in cell membrane integrity, an increase in the formation of reactive oxygen species, phosphatidylserine exposure, a reduction of cell volume, DNA fragmentation, and the formation of lipid inclusions.

Conclusion/Significance

These finding suggest that mitochondria are a target of C4, the dysfunction of which can lead to different pathways of cell death.     

Activity Profile of an FDA-Approved Compound Library against Schistosoma mansoni

Background

As plans to expand mass drug treatment campaigns to fight schistosomiasis form, worries about reliance on praziquantel as the sole available treatment motivate the investigation for novel antischistosomal compounds. Drug repurposing might be an inexpensive and effective source of novel antischistosomal leads.

Methodology

1600 FDA approved compounds were first assayed against Schistosoma mansoni schistosomula at a concentration of 10 µM. Active compounds identified from this screen were advanced to the adult worm screen at 33.33 µM, followed by hit characterization. Leads with complementary pharmacokinetic and toxicity profiles were then selected for in vivo studies.

Principal Findings

The in vitro screen identified 121 and 36 compounds active against the schistosomula and adult stage, respectively. Further, in vitro characterization and comparison with already available pharmacokinetic and toxicity data identified 11 in vivo candidates. Doramectin (10 mg/kg) and clofazimine (400 mg/kg) were found to be active in vivo with worm burden reductions of 60.1% and 82.7%, respectively.

Conclusions/Significance

The work presented here expands the knowledge of antischistosomal properties of already approved compounds and underscores variations observed between target-based and phenotypic approaches and among laboratories. The two in vivo-active drugs identified in this study, doramectin and clofazimine are widely available and present as novel drug classes as starting points for further investigation.     

Simultaneous Determination of Glass Transition Temperatures of Several Polymers

Aims

A simple and easy optical method is proposed for the determination of glass transition temperature (T_g) of polymers.

Methods & Results

T_g was determined using the technique of microsphere imaging to monitor the variation of the refractive index of polymer microsphere as a function of temperature. It was demonstrated that the method can eliminate most thermal lag and has sensitivity about six fold higher than the conventional method in T_g determination. So the determined T_g is more accurate and varies less with cooling/heating rate than that obtained by conventional methods. The most attractive character of the method is that it can simultaneously determine the T_g of several polymers in a single experiment, so it can greatly save experimental time and heating energy.

Conclusion

The method is not only applicable for polymer microspheres, but also for the materials with arbitrary shapes. Therefore, it is expected to be broadly applied to different fundamental researches and practical applications of polymers.     

The Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection against Oxidative Stress but Is Not a Determinant of Virulence

Background

The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy.

Methodology/Principal Findings

To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo.

Conclusions/Significance

TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.     

Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion

Background

The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.

Methodology/ Principal Findings

Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca²⁺ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.

Conclusion/Significance

We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.     

Effects of Genotype and Growth Temperature on the Contents of Tannin,Phytate and In Vitro Iron Availability of Sorghum Grains

Background

It has been predicted that the global temperature will rise in the future, which means crops including sorghum will likely be grown under higher temperatures, and consequently may affect the nutritional properties.

Methods

The effects of two growth temperatures (OT, day/night 32/21°C; HT 38/21°C) on tannin, phytate, mineral, and in vitro iron availability of raw and cooked grains (as porridge) of six sorghum genotypes were investigated.

Results

Tannin content significantly decreased across all sorghum genotypes under high growth temperature (P ≤0.05), while the phytate and mineral contents maintained the same level, increased or decreased significantly, depending on the genotype. The in vitro iron availability in most sorghum genotypes was also significantly reduced under high temperature, except for Ai4, which showed a pronounced increase (P ≤0.05). The cooking process significantly reduced tannin content in all sorghum genotypes (P ≤0.05), while the phytate content and in vitro iron availability were not significantly affected.

Conclusions

This research provides some new information on sorghum grain nutritional properties when grown under predicted future higher temperatures, which could be important for humans where sorghum grains are consumed as staple food.     

Trypanosomiasis-Induced Megacolon Illustrates How Myenteric Neurons Modulate the Risk for Colon Cancer in Rats and Humans

Background

Trypanosomiasis induces a remarkable myenteric neuronal degeneration leading to megacolon. Very little is known about the risk for colon cancer in chagasic megacolon patients. To clarify whether chagasic megacolon impacts on colon carcinogenesis, we investigated the risk for colon cancer in Trypanosoma cruzi (T. cruzi) infected patients and rats.

Methods

Colon samples from T. cruzi-infected and uninfected patients and rats were histopathologically investigated with colon cancer biomarkers. An experimental model for chemical myenteric denervation was also performed to verify the myenteric neuronal effects on colon carcinogenesis. All experiments complied the guidelines and approval of ethical institutional review boards.

Results

No colon tumors were found in chagasic megacolon samples. A significant myenteric neuronal denervation was observed. Epithelial cell proliferation and hyperplasia were found increased in chagasic megacolon. Analyzing the argyrophilic nucleolar organiser regions within the cryptal bottom revealed reduced risk for colon cancer in Chagas’ megacolon patients. T. cruzi-infected rats showed a significant myenteric neuronal denervation and decreased numbers of colon preneoplastic lesions. In chemical myenteric denervated rats preneoplastic lesions were reduced from the 2^nd wk onward, which ensued having the colon myenteric denervation significantly induced.

Conclusion/Significance

Our data suggest that the trypanosomiasis-related myenteric neuronal degeneration protects the colon tissue from carcinogenic events. Current findings highlight potential mechanisms in tropical diseases and cancer research.     

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background

Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.

Methodology/Principal Findings

Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP₃R)—an intracellular Ca²⁺ channel and a key molecule involved in Ca²⁺ signaling in the parasite—is important for the transformation process.

Conclusion/Significance

Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.     

The Pathogenesis of Human Cervical Epithelium Cells Induced by Interacting with Trichomonas vaginalis

Background

Trichomonas vaginalis is a protozoan parasite that occurs in the urogenital-vaginal tract and is the primary causative agent of trichomoniasis, a common sexually transmitted disease in humans. The aggregation of this protozoan tends to destroy epithelial cells and induce pathogenesis.

Principal Findings

This study cultured T. vaginalis and human cervical epithelial cells (Z172) under the same conditions in the experiments. Following co-culturing for ten hours, the protozoans became attached to Z172, such that the cells presented a round shape and underwent shrinkage. Time-lapse recording and flow cytometry on interacted Z172 revealed that 70% had been disrupted, 18% presented a necrosis-like morphology and 8% showed signs of apoptosis. Gene expression profiling revealed in the seven inflammatory Z172 genes as well as in T. vaginalis genes that code for adhesion proteins 65 and 65-1.

Significance

These results suggest that cytopathogenic effects progress while Z172 is in contact with T. vaginalis, and the resulting morphological changes can be categorized as disruption.