Senescent cell population with ZEB1 transcription factor as its main regulator promotes osteoarthritis in cartilage and meniscus.

OBJECTIVES: Single-cell level analysis of articular cartilage and meniscus tissues from human healthy and osteoarthritis (OA) knees. METHODS: Single-cell RNA sequencing (scRNA-seq) analyses were performed on articular cartilage and meniscus tissues from healthy (n=6, n=7) and OA (n=6, n=6) knees. Expression of genes of interest was validated using immunohistochemistry and RNA-seq and function was analysed by gene overexpression and depletion. RESULTS: scRNA-seq analyses of human knee articular cartilage (70 972 cells) and meniscus (78 017 cells) identified a pathogenic subset that is shared between both tissues. This cell population is expanded in OA and has strong OA and senescence gene signatures. Further, this subset has critical roles in extracellular matrix (ECM) and tenascin signalling and is the dominant sender of signals to all other cartilage and meniscus clusters and a receiver of TGFß signalling. Fibroblast activating protein (FAP) is also a dysregulated gene in this cluster and promotes ECM degradation. Regulons that are controlled by transcription factor ZEB1 are shared between the pathogenic subset in articular cartilage and meniscus. In meniscus and cartilage cells, FAP and ZEB1 promote expression of genes that contribute to OA pathogenesis, including senescence. CONCLUSIONS: These single-cell studies identified a senescent pathogenic cell cluster that is present in cartilage and meniscus and has FAP and ZEB1 as main regulators which are novel and promising therapeutic targets for OA-associated pathways in both tissues.

Multiomics analyses reveal the roles of the ASR1 transcription factor in tomato fruits.

The transcription factor ASR1 (ABA, STRESS, RIPENING 1) plays multiple roles in plant responses to abiotic stresses as well as being involved in the regulation of central metabolism in several plant species. However, despite the high expression of ASR1 in tomato fruits, large scale analyses to uncover its function in fruits are still lacking. In order to study its function in the context of fruit ripening, we performed a multiomics analysis of ASR1-antisense transgenic tomato fruits at the transcriptome and metabolome levels. Our results indicate that ASR1 is involved in several pathways implicated in the fruit ripening process, including cell wall, amino acid, and carotenoid metabolism, as well as abiotic stress pathways. Moreover, we found that ASR1-antisense fruits are more susceptible to the infection by the necrotrophic fungus Botrytis cinerea. Given that ASR1 could be regulated by fruit ripening regulators such as FRUITFULL1/FRUITFULL2 (FUL1/FUL2), NON-RIPENING (NOR), and COLORLESS NON-RIPENING (CNR), we positioned it in the regulatory cascade of red ripe tomato fruits. These data extend the known range of functions of ASR1 as an important auxiliary regulator of tomato fruit ripening.

Nuclear receptor HNF4A transrepresses CLOCK:BMAL1 and modulates tissue-specific circadian networks.

Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly transrepresses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK:BMAL1 at a wide array of metabolic genes involved in lipid, glucose, and amino acid homeostasis. Taken together, we establish that HNF4A defines a feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.

FOXO1 transcription factor regulates chondrogenic differentiation through transforming growth factor ß1 signaling.

The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor ß1 (TGFß1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFß1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFß1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9 To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFß1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFß1 signaling.

Changes in ambient temperature are the prevailing cue in determining Brachypodium distachyon diurnal gene regulation.

Plants are continuously exposed to diurnal fluctuations in light and temperature, and spontaneous changes in their physical or biotic environment. The circadian clock coordinates regulation of gene expression with a 24 h period, enabling the anticipation of these events. We used RNA sequencing to characterize the Brachypodium distachyon transcriptome under light and temperature cycles, as well as under constant conditions. Approximately 3% of the transcriptome was regulated by the circadian clock, a smaller proportion than reported in most other species. For most transcripts that were rhythmic under all conditions, including many known clock genes, the period of gene expression lengthened from 24 to 27 h in the absence of external cues. To functionally characterize the cyclic transcriptome in B. distachyon, we used Gene Ontology enrichment analysis, and found several terms significantly associated with peak expression at particular times of the day. Furthermore, we identified sequence motifs enriched in the promoters of similarly phased genes, some potentially associated with transcription factors. When considering the overlap in rhythmic gene expression and specific pathway behavior, thermocycles was the prevailing cue that controlled diurnal gene regulation. Taken together, our characterization of the rhythmic B. distachyon transcriptome represents a foundational resource with implications in other grass species.

Alterations in Placental Gene Expression of Pregnant Women with Chronic Chagas Disease.

Trypanosoma cruzi infection in women of reproductive age is associated with congenital transmission and adverse pregnancy outcomes. The placenta is a key barrier to infection. Gene expression profiles of term placental environment from T. cruzi-seropositive (SP) and -seronegative (SN) mothers were characterized by RNA-Seq. Nine pools of placental RNA paired samples were used: three from SN and six from SP tissues. Each pool consisted of female/male newborns and vaginal/cesarean delivery binomials. No newborn was congenitally infected. T. cruzi satellite DNA quantitative PCR in placental tissues and maternal and neonatal blood, and parasite 18S quantitative RT-PCR from placental RNA were negative, except in three SP women's bloodstream. To identify pathways associated with maternal T. cruzi infection, a gene-set association analysis was implemented: SP placental samples showed overexpression of inflammatory response and lymphocytic activation, whereas numerous biosynthetic processes were down-regulated. About 42 genes showed a significant fold-change between SP and SN groups. KISS1 and CGB5 were down-regulated, whereas KIF12, HLA-G, PRG2, TAC3, FN1, and ATXN3L were up-regulated. Several expressed genes in SP placentas encode proteins associated with preeclampsia and miscarriage. This first transcriptomics study in human term placental environment shows a placental response that may affect the fetus while protecting it from parasite infection; this host response could be responsible for the low rate of congenital transmission in chronic Chagas disease.

Genome-wide data (ChIP-seq) enabled identification of cell wall-related and aquaporin genes as targets of tomato ASR1, a drought stress-responsive transcription factor.

BACKGROUND: Identifying the target genes of transcription factors is important for unraveling regulatory networks in all types of organisms. Our interest was precisely to uncover the spectrum of loci regulated by a widespread plant transcription factor involved in physiological adaptation to drought, a type of stress that plants have encountered since the colonization of land habitats 400 MYA. The regulator under study, named ASR1, is exclusive to the plant kingdom (albeit absent in Arabidopsis) and known to alleviate the stress caused by restricted water availability. As its target genes are still unknown despite the original cloning of Asr1 cDNA 20 years ago, we examined the tomato genome for specific loci interacting in vivo with this conspicuous protein. RESULTS: We performed ChIP followed by high throughput DNA sequencing (ChIP-seq) on leaves from stressed tomato plants, using a high-quality anti-ASR1 antibody. In this way, we unraveled a novel repertoire of target genes, some of which are clearly involved in the response to drought stress. Many of the ASR1-enriched genomic loci we found encode enzymes involved in cell wall synthesis and remodeling as well as channels implicated in water and solute flux, such as aquaporins. In addition, we were able to determine a robust consensus ASR1-binding DNA motif. CONCLUSIONS: The finding of cell wall synthesis and aquaporin genes as targets of ASR1 is consistent with their suggested role in the physiological adaptation of plants to water loss. The results gain insight into the environmental stress-sensing pathways leading to plant tolerance of drought.

Transcriptional regulation of tocopherol biosynthesis in tomato.

Tocopherols, compounds with vitamin E (VTE) activity, are potent lipid-soluble antioxidants synthesized only by photosynthetic organisms. Their biosynthesis requires the condensation of phytyl-diphosphate and homogentisate, derived from the methylerythritol phosphate (MEP) and shikimate pathways (SK), respectively. These metabolic pathways are central in plant chloroplast metabolism and are involved in the biosynthesis of important molecules such as chlorophyll, carotenoids, aromatic amino-acids and prenylquinones. In the last decade, few studies have provided insights into the regulation of VTE biosynthesis and its accumulation. However, the pathway regulatory mechanism/s at mRNA level remains unclear. We have recently identified a collection of tomato genes involved in tocopherol biosynthesis. In this work, by a dedicated qPCR array platform, the transcript levels of 47 genes, including paralogs, were determined in leaves and across fruit development. Expression data were analyzed for correlation with tocopherol profiles by coregulation network and neural clustering approaches. The results showed that tocopherol biosynthesis is controlled both temporally and spatially however total tocopherol content remains constant. These analyses exposed 18 key genes from MEP, SK, phytol recycling and VTE-core pathways highly associated with VTE content in leaves and fruits. Moreover, genomic analyses of promoter regions suggested that the expression of the tocopherol-core pathway genes is trancriptionally coregulated with specific genes of the upstream pathways. Whilst the transcriptional profiles of the precursor pathway genes would suggest an increase in VTE content across fruit development, the data indicate that in the M82 cultivar phytyl diphosphate supply limits tocopherol biosynthesis in later fruit stages. This is in part due to the decreasing transcript levels of geranylgeranyl reductase (GGDR) which restricts the isoprenoid precursor availability. As a proof of concept, by analyzing a collection of Andean landrace tomato genotypes, the role of the pinpointed genes in determining fruit tocopherol content was confirmed. The results uncovered a finely tuned regulation able to shift the precursor pathways controlling substrate influx for VTE biosynthesis and overcoming endogenous competition for intermediates. The whole set of data allowed to propose that 1-deoxy-D-xylulose-5-phosphate synthase and GGDR encoding genes, which determine phytyl-diphosphate availability, together with enzyme encoding genes involved in chlorophyll-derived phytol metabolism appear as the most plausible targets to be engineered aiming to improve tomato fruit nutritional value.

Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation.

BACKGROUND: One hundred years after the discovery of Chagas disease, it remains a major neglected tropical disease. Chronic Chagas heart disease (cChHD) is the most severe manifestation. Heart transplantation is the proper treatment for end-stage heart failure, although reactivation of disease may result after receipt of immunosuppressive therapy. T. cruzi strains cluster into 6 discrete typing units (DTUs; I-VI) associated with different geographical distribution, transmission cycles and varying disease symptoms. In the southern cone of South America, T. cruzi II, V, and VI populations appear to be associated with Chagas disease and T. cruzi I with sylvatic cycles. METHODS: Molecular characterization of DTUs, T. cruzi I genotypes (on the basis of spliced-leader gene polymorphisms), and minicircle signatures was conducted using cardiac explant specimens and blood samples obtained from a cohort of 16 Argentinean patients with cChHD who underwent heart transplantation and from lesion samples obtained from 6 of these patients who presented with clinical reactivation of Chagas disease. RESULTS: Parasite persistence was associated with myocarditis progression, revealing T. cruzi I (genotype Id) in 3 explant samples and T. cruzi II, V, or VI in 5 explant samples. Post-heart transplantation follow-up examination of bloodstream DTUs identified T. cruzi I in 5 patients (genotypes Ia or Id) and T. cruzi II, V, or VI in 7 patients. T. cruzi I, V, and VI were detected in skin chagoma specimens, and T. cruzi V and VI were detected in samples obtained from patients with myocarditis reactivations. Multiple DTUs or genotypes at diverse body sites and polymorphic minicircle signatures at different cardiac regions revealed parasite histotropism. T. cruzi I infections clustered in northern Argentina (latitude, 23 degrees S-27 degrees S), whereas T. cruzi II, V, or VI DTUs were more ubiquitous. CONCLUSIONS: Multiple DTUs coexist in patients with Chagas disease. The frequent finding of T. cruzi I associated with cardiac damage was astounding, revealing its pathogenic role in cChHD at the southern cone.

Genome-Wide Occupancy Profiling Reveals Critical Roles of FoxO1 in Regulating Extracellular Matrix and Circadian Rhythm Genes in Human Chondrocytes.

OBJECTIVE: Osteoarthritis (OA) is the most common age-related joint disease. With aging and in OA, the expression of FoxO transcription factors is reduced, diminishing their chondroprotective actions. In order to elucidate the molecular mechanisms by which FoxO1 protects chondrocytes, we sought to identify the genome-wide occupancy profile of FoxO1. METHODS: We performed FoxO1 chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) on human primary chondrocytes. ChIP-Seq data were integrated with RNA sequencing (RNA-Seq) data sets. Bioinformatics results were confirmed in primary chondrocytes that were treated with a FoxO1 inhibitor. RESULTS: Analysis of FoxO1 ChIP-Seq on human primary chondrocytes showed that pathways implicated in OA pathogenesis are mainly regulated by FoxO1 binding to tissue-specific enhancers with suboptimal binding sites (20% of the peaks), while more ubiquitous FoxO1 pathways are regulated at the promoter level through interaction with its canonical binding motif (7% of the peaks). Integrating FoxO1 occupancy data with RNA-Seq data comparing OA and healthy human cartilage revealed 428 putative FoxO1 target genes that are dysregulated in OA. Pathway analysis showed enrichment for genes belonging to the senescence pathway (logP = -6.73), extracellular matrix (ECM) pathway (logP = -12.97), and circadian clock pathway (logP = -6.30), which suggests that FoxO1 dysregulation plays an important role in their abnormal expression in OA. Using an inhibitor of FoxO1, we confirmed that FoxO1 regulates these pathways in cultured human chondrocytes. CONCLUSION: FoxO1 regulates ubiquitous and cartilage-specific genes in chondrocytes by using different mechanisms. The FoxO1 transcriptional network is a key player in regulating homeostasis, ECM, and circadian clock genes and plays an important role in the abnormal expression of these pathways observed in OA pathogenesis.

Multi-level Modulation of Light Signaling by GIGANTEA Regulates Both the Output and Pace of the Circadian Clock.

Integration of environmental signals with endogenous biological processes is essential for organisms to thrive in their natural environment. Being entrained by periodic environmental changes, the circadian clock incorporates external information to coordinate physiological processes, phasing them to the optimal time of the day and year. Here, we present a pivotal role for the clock component GIGANTEA (GI) as a genome-wide regulator of transcriptional networks mediating growth and adaptive processes in plants. We provide mechanistic details on how GI integrates endogenous timing with light signaling pathways through the global modulation of PHYTOCHROME-INTERACTING FACTORs (PIFs). Gating of the activity of these transcriptional regulators by GI directly affects a wide array of output rhythms, including photoperiodic growth. Furthermore, we uncover a role for PIFs in mediating light input to the circadian oscillator and show how their regulation by GI is required to set the pace of the clock in response to light-dark cycles.

Impairment of infectivity and immunoprotective effect of a LYT1 null mutant of Trypanosoma cruzi.

Trypanosoma cruzi infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of LYT1, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both LYT1 alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant T. cruzi clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 10(3) trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the LYT1 gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated.

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites.

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci--six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.

Molecular identification of Trypanosoma cruzi I tropism for central nervous system in Chagas reactivation due to AIDS.

Trypanosoma cruzi lineages, microsatellite allelic polymorphism, and mithocondrial gene haplotypes were directly typified from peripheral blood and cerebrospinal fluid specimens of a Bolivian patient with Chagas disease with accompanying AIDS and central nervous system severe involvement. Of note, the patient's blood was infected by a mixture of T. cruzi I and T. cruzi IId/e polyclonal populations while the cerebrospinal fluid showed only a monoclonal T. cruzi I population. Our findings do not corroborate the original assumption of innocuity for T. cruzi I in the southern cone of the Americas and highlight lineage I tropism for central nervous system causing lethal Chagas reactivation.

Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease.

Congenital transmission of Trypanosoma cruzi may occur in some or all the gestations from a T. cruzi-infected mother. Variable rates of congenital transmission have been reported in different geographical areas where different parasitic strains predominate, suggesting that parasitic genotypes might play a role in the risk of congenital transmission. Moreover, in cases of transmission it is unknown if the whole maternal T. cruzi population or certain clones are preferentially transmitted by the transplacental route. In this study, bloodstream T. cruzi lineages were identified in blood samples from congenitally infected children, transmitting and non-transmitting mothers and unrelated Chagas disease patients, using improved PCR strategies targeted to nuclear genomic markers. T. cruzi IId was the prevalent genotype among 36/38 PCR-positive congenitally infected infants, 5/5 mothers who transmitted congenital Chagas disease, 12/13 mothers who delivered non-infected children and 28/34 unrelated Chagas disease patients, all coming from endemic localities of Argentina and Bolivia. These figures indicate no association between a particular genotype and vertical transmission. Furthermore, minicircle signatures from the maternal and infants' bloodstream trypanosomes were profiled by restriction fragment length polymorphism of the 330-bp PCR-amplified variable regions in seven cases of mothers and congenitally infected infants. Minicircle signatures were nearly identical between each mother and her infant/s and unique to each mother-infant/s case, a feature that was also observed in twin deliveries. Moreover, allelic size polymorphism analysis of microsatellite loci from populations transmitted to twins showed that all clones from the maternal polyclonal population were equally infective to both siblings.

Molecular diagnosis and typing of Trypanosoma cruzi populations and lineages in cerebral Chagas disease in a patient with AIDS.

Trypanosoma cruzi DNA was amplified from an intracranial biopsy and peripheral blood of an HIV patient with encephalitis; this episode was indicative of AIDS and congenital Chagas disease. The analysis of a micro-satellite locus revealed a multiclonal parasite population at the brain lesion with a more complex minicircle signature than that profiled in blood using restriction fragment length polymorphism (RFLP)-PCR and low stringency single primer (LSSP) PCR. Interestingly, different sublineages of T. cruzi II were detected in blood and brain by means of spliced-leader and 24salpha ribosomal-DNA amplifications. Quantitative-competitive PCR monitored the decrease of parasitic load during treatment and secondary prophylaxis with benznidazole. The synergy between parasiticidal plus anti-retroviral treatments probably allowed the patient a longer survival than usually achieved in similar episodes. This is the first case report demonstrating a differential distribution of natural parasite populations and sublineages in Chagas disease reactivation, showing the proliferation of cerebral variants not detectable in peripheral blood.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

BACKGROUND: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). METHODS/PRINCIPAL FINDINGS: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. CONCLUSIONS/SIGNIFICANCE: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Natural occurring epialleles determine vitamin E accumulation in tomato fruits.

Vitamin E (VTE) content is a low heritability nutritional trait for which the genetic determinants are poorly understood. Here, we focus on a previously detected major tomato VTE quantitative trait loci (QTL; mQTL(9-2-6)) and identify the causal gene as one encoding a 2-methyl-6-phytylquinol methyltransferase (namely VTE3(1)) that catalyses one of the final steps in the biosynthesis of γ- and α-tocopherols, which are the main forms of VTE. By reverse genetic approaches, expression analyses, siRNA profiling and DNA methylation assays, we demonstrate that mQTL(9-2-6) is an expression QTL associated with differential methylation of a SINE retrotransposon located in the promoter region of VTE3(1). Promoter DNA methylation can be spontaneously reverted leading to different epialleles affecting VTE3(1) expression and VTE content in fruits. These findings indicate therefore that naturally occurring epialleles are responsible for regulation of a nutritionally important metabolic QTL and provide direct evidence of a role for epigenetics in the determination of agronomic traits.

Impairment in natural killer cells editing of immature dendritic cells by infection with a virulent Trypanosoma cruzi population.

Early interactions between natural killer (NK) and dendritic cells (DC) shape the immune response at the frontier of innate and adaptive immunity. Activated NK cells participate in maturation or deletion of DCs that remain immature. We previously demonstrated that infection with a high virulence (HV) population of the protozoan parasite Trypanosoma cruzi downmodulates DC maturation and T-cell activation capacity. Here, we evaluated the role of NK cells in regulating the maturation level of DCs. Shortly after infection with HV T. cruzi, DCs in poor maturation status begin to accumulate in mouse spleen. Although infection induces NK cell cytotoxicity and cytokine production, NK cells from mice infected with HV T. cruzi exhibit reduced ability to lyse and fail to induce maturation of bone marrow-derived immature DCs (iDCs). NK-mediated lysis of iDCs is restored by in vitro blockade of the IL-10 receptor during NK-DC interaction or when NK cells are obtained from T. cruzi-infected IL-10 knockout mice. These results suggest that infection with a virulent T. cruzi strain alters NK cell-mediated regulation of the adaptive immune response induced by DCs. This regulatory circuit where IL-10 appears to participate might lead to parasite persistence but can also limit the induction of a vigorous tissue-damaging T-cell response.

Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.

BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.