The initiation and early development of apical-basal polarity in Toxoplasma gondii.

The body plan of the human parasite Toxoplasma gondii has a well-defined polarity. The minus ends of the 22 cortical microtubules are anchored to the apical polar ring, a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end, and is critical for cytokinesis. How this apical-basal polarity is initiated was unknown. Here we examined the development of the apical polar ring and the basal complex using expansion microscopy. We found that substructures in the apical polar ring have different sensitivity to perturbations. In addition, apical-basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the nascent daughter framework grows towards the centrioles, the apical and basal arcs co-develop ahead of the microtubule array. Lastly, two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of individual proteins has modest impact on the lytic cycle. However, the loss of both results in abnormalities in the microtubule array and highly reduced plaquing and invasion efficiency.

The initiation and early development of apical-basal polarity in Toxoplasma gondii.

The human parasite Toxoplasma gondii has a distinctive body plan with a well-defined polarity. In the apical complex, the minus ends of the 22 cortical microtubules are anchored to the apical polar ring, a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end, and is critical for cytokinesis. How this apical-basal polarity axis is initiated was unknown. Here we examined the development of the apical polar ring and the basal complex in nascent daughters using expansion microscopy. We found that different substructures in the apical polar ring have different sensitivity to stress. In addition, apical-basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the construction of the daughter framework progresses towards the centrioles, the apical and the basal arcs co-develop in striking synchrony ahead of the microtubule array, and close into a ring-form before all the microtubules are nucleated. We also found that two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of each protein individually has modest to no impact on the lytic cycle. However, the loss of both results in abnormalities in the microtubule array and highly reduced plaquing and invasion efficiency.

The initiation and early development of the tubulin-containing cytoskeleton in the human parasite Toxoplasma gondii.

The tubulin-containing cytoskeleton of the human parasite Toxoplasma gondii includes several distinct structures: the conoid, formed of 14 ribbon-like tubulin polymers, and the array of 22 cortical microtubules (MTs) rooted in the apical polar ring. Here we analyze the structure of developing daughter parasites using both 3D-SIM and expansion microscopy. Cortical MTs and the conoid start to develop almost simultaneously, but from distinct precursors near the centrioles. Cortical MTs are initiated in a fixed sequence, starting around the periphery of a short arc that extends to become a complete circle. The conoid also develops from an open arc into a full circle, with a fixed spatial relationship to the centrioles. The patterning of the MT array starts from a "blueprint" with ∼five-fold symmetry, switching to 22-fold rotational symmetry in the final product, revealing a major structural rearrangement during daughter growth. The number of MT is essentially invariant in the wild-type array, but is perturbed by the loss of some structural components of the apical polar ring. This study provides insights into the development of tubulin-containing structures that diverge from conventional models, insights that are critical for understanding the evolutionary paths leading to construction and divergence of cytoskeletal frameworks.

The initiation and early development of the tubulin-containing cytoskeleton in the human parasite Toxoplasma gondii.

The tubulin-containing cytoskeleton of the human parasite Toxoplasma gondii includes several distinct structures: the conoid, formed of 14 ribbon-like tubulin polymers, and the array of 22 cortical microtubules (MTs) rooted in the apical polar ring. Here we analyze the structure of developing daughter parasites using both 3D-SIM and expansion microscopy. Cortical MTs and the conoid start to develop almost simultaneously, but from distinct precursors near the centrioles. Cortical MTs are initiated in a fixed sequence, starting around the periphery of a short arc that extends to become a complete circle. The conoid also develops from an open arc into a full circle, with a fixed spatial relationship to the centrioles. The patterning of the MT array starts from a "blueprint" with ∼ 5-fold symmetry, switching to 22-fold rotational symmetry in the final product, revealing a major structural rearrangement during daughter growth. The number of MT is essentially invariant in the wild-type array, but is perturbed by the loss of some structural components of the apical polar ring. This study provides insights into the development of tubulin-containing structures that diverge from conventional models, insights that are critical for understanding the evolutionary paths leading to construction and divergence of cytoskeletal frameworks.

The cortical microtubules of Toxoplasma gondii underlie the helicity of parasite movement.

Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here, we address the role of the cortical microtubules in parasite motility, invasion and egress by utilizing a previously generated mutant (dubbed 'TKO') in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in ∼80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization was further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication was not affected. In a 3D Matrigel matrix, the TKO mutant moved directionally over long distances, but along trajectories that were significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory did not impact either movement speed in the matrix or the speed and behavior of the parasite during entry into and egress from the host cell.

The cortical microtubules of Toxoplasma gondii underlie the helicity of parasite movement.

Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3-D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here we address the role of the cortical microtubules in parasite motility, invasion, and egress by utilizing a previously generated mutant (dubbed "TKO") in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in ~ 80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization is further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication is not affected. In a 3-D Matrigel matrix, the TKO mutant moves directionally over long distances, but along trajectories significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory does not impact either movement speed in the matrix or the speed and behavior of the parasite's entry into and egress from the host cell.

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

ß-Caryophyllene: A Therapeutic Alternative for Intestinal Barrier Dysfunction Caused by Obesity.

Obesity is an excessive accumulation of fat that exacerbates the metabolic and inflammatory processes. Studies associate these processes with conditions and dysregulation in the intestinal tract, increased concentrations of lipopolysaccharides (LPSs) in the blood, differences in the abundance of intestinal microbiota, and the production of secondary metabolites such as short-chain fatty acids. ß-Caryophyllene (BCP) is a natural sesquiterpene with anti-inflammatory properties and with the potential purpose of fighting metabolic diseases. A diet-induced obesity model was performed in 16-week-old C57BL/6 mice administered with BCP [50 mg/kg]. A reduction in the expression of Claudin-1 was observed in the group with a high-fat diet (HFD), which was caused by the administration of BCP; besides BCP, the phylaAkkermansia and Bacteroidetes decreased between the groups with a standard diet (STD) vs. HFD. Nevertheless, the use of BCP in the STD increased the expression of these phyla with respect to fatty acids; a similar effect was observed, in the HFD group that had a decreasing concentration that was restored with the use of BCP. The levels of endotoxemia and serum leptin increased in the HFD group, while in the HFD + BCP group, similar values were found to those of the STD group, attributing the ability to reduce these in conditions of obesity.

An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii.

The phylum Apicomplexa includes thousands of species of unicellular parasites that cause a wide range of human and animal diseases such as malaria and toxoplasmosis. To infect, the parasite must first initiate active movement to disseminate through tissue and invade into a host cell, and then cease moving once inside. The parasite moves by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. One of the most effective invaders in Apicomplexa is Toxoplasma gondii, which can infect any nucleated cell and any warm-blooded animal. During invasion, the parasite first makes contact with the host cell "head-on" with the apical complex, which features an elaborate cytoskeletal apparatus and associated structures. Here we report the identification and characterization of a new component of the apical complex, Preconoidal region protein 2 (Pcr2). Pcr2 knockout parasites replicate normally, but they are severely diminished in their capacity for host tissue destruction due to significantly impaired invasion and egress, two vital steps in the lytic cycle. When stimulated for calcium-induced egress, Pcr2 knockout parasites become active, and secrete effectors to lyse the host cell. Calcium-induced secretion of the major adhesin, MIC2, also appears to be normal. However, the movement of the Pcr2 knockout parasite is spasmodic, which drastically compromises egress. In addition to faulty motility, the ability of the Pcr2 knockout parasite to assemble the moving junction is impaired. Both defects likely contribute to the poor efficiency of invasion. Interestingly, actomyosin activity, as indicated by the motion of mEmerald tagged actin chromobody, appears to be largely unperturbed by the loss of Pcr2, raising the possibility that Pcr2 may act downstream of or in parallel with the actomyosin machinery.

Cystic proliferation of germline stem cells is necessary to reproductive success and normal mating behavior in medaka.

The production of an adequate number of gametes is necessary for normal reproduction, for which the regulation of proliferation from early gonadal development to adulthood is key in both sexes. Cystic proliferation of germline stem cells is an especially important step prior to the beginning of meiosis; however, the molecular regulators of this proliferation remain elusive in vertebrates. Here, we report that ndrg1b is an important regulator of cystic proliferation in medaka. We generated mutants of ndrg1b that led to a disruption of cystic proliferation of germ cells. This loss of cystic proliferation was observed from embryogenic to adult stages, impacting the success of gamete production and reproductive parameters such as spawning and fertilization. Interestingly, the depletion of cystic proliferation also impacted male sexual behavior, with a decrease of mating vigor. These data illustrate why it is also necessary to consider gamete production capacity in order to analyze reproductive behavior.

The central nervous system acts as a transducer of stress-induced masculinization through corticotropin-releasing hormone B.

Exposure to environmental stressors, such as high temperature (HT), during early development of fish induces sex reversal of genotypic females. Nevertheless, the involvement of the brain in this process is not well clarified. In the present work, we investigated the mRNA levels of corticotropin-releasing hormone b (crhb) and its receptors (crhr1 and crhr2), and found that they were upregulated at HT during the crucial period of gonadal sex determination in medaka. In order to clarify their roles in sex reversal, biallelic mutants for crhr1 and crhr2 were produced by CRISPR/Cas9 technology. Remarkably, biallelic mutants of both loci (crhr1 and crhr2) did not undergo female-to-male sex reversal upon exposure to HT. Inhibition of this process in double corticotropin-releasing hormone receptor mutants could be successfully rescued through the administration of the downstream effector of the hypothalamic-pituitary-interrenal axis, cortisol. Taken together, these results reveal for the first time that the CNS acts as a transducer of masculinization induced by thermal stress.

Programa de screening y tratamiento de cáncer de mama / Screening and treatment program of breast cancer

En enero de 1988 comenzamos un programa piloto de screening en cáncer de mama para mujeres asintomáticas. Una mamografía con dos proyecciones se planificó cada 2 años para las mujeres entre 40 y 50 años y anualmente para aquéllas con 50 años o más. Si en esta mamografía se encontraban hallazgos anormales, las mujeres fueron evaluadas con estudios localizados y examen físico realizado por un comité constituído por un radiólogo, un cirujano y un oncólogo. Si luego de esta evaluación la anormalidad del estudio persiste, a juicio del comité, la paciente se planifica para biopsia excisional. Si el resultado de la biopsia es positivo y la enfermedad es localizada, la paciente es tratada conservadoramente con radioterapia. Hasta el 31 de enero de 1990 en 1.742 mujeres se ha realizado este screening y 2.573 mamografías han sido tomadas (en 831 se ha realizado 2 exámenes), 42% de las mujeres tienen 50 años o menos. Después del primer examen 32 mujeres fueron seleccionados para biopsia (24 tenían una lesión no-palpable). Después de la biopsia 4 cánceres fueron diagnosticadas (sólo una palpable) y en otra mujer, una hiperplasia epitelial atípica con lesión no palpable. Después del segundo examen 20 mujeres fueron a biopsia (12 con lesiones no palpables) y 5 cánceres se diagnosticaron (tres no-palpables). De estos 5 cánceres, 2 podrían haber sido vistos en el examen previo, ambos no palpables y tres eran nuevas lesiones. El screening de cáncer de mama es posible de realizar, detecta lesiones pequeñas, permitiendo un tratamiento conservador de las mismas y mejora la sobrevida del cáncer de mama