Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9- RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability, and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryote

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death

Simultaneous transcriptional profiling of Leishmania major and its murine macrophage host cell reveals insights into host-pathogen interactions

Parasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The life cycle of Leishmania parasites is split between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must evade or deactivate the host's innate and adaptive immune responses in order to survive and replicate. We performed transcriptome profiling using RNA-seq to simultaneously identify global changes in murine macrophage and L. major gene expression as the parasite entered and persisted within murine macrophages during the first 72 h of an infection. Differential gene expression, pathway, and gene ontology analyses enabled us to identify modulations in host and parasite responses during an infection. The most substantial and dynamic gene expression responses by both macrophage and parasite were observed during early infection. Murine genes related to both pro- and anti-inflammatory immune responses and glycolysis were substantially upregulated and genes related to lipid metabolism, biogenesis, and Fc gamma receptor-mediated phagocytosis were downregulated. Upregulated parasite genes included those aimed at mitigating the effects of an oxidative response by the host immune system while downregulated genes were related to translation, cell signaling, fatty acid biosynthesis, and flagellum structure. The gene expression patterns identified in this work yield signatures that characterize multiple developmental stages of L. major parasites and the coordinated response of Leishmania-infected macrophages in the real-time setting of a dual biological system. This comprehensive dataset offers a clearer and more sensitive picture of the interplay between host and parasite during intracellular infection, providing additional insights into how pathogens are able to evade host defenses and modulate the biological functions of the cell in order to survive in the mammalian environment.https://doi.org/10.1186/s12864-015-2237-

Capacidad discriminante de los hallazgos en tomografía para la identificación de lesiones pulmonares por infección con nocardia

Objective: Evaluate the diagnostic value of some findings in thoracic computed tomography, in patients with confirmed pulmonary nocardiosis, so that, taken together, they may help distinguish it from other conditions that may show superimposable radiological findings. Material and methods: Subjects with microbiological diagnosis of nocardiosis in our hospital over a 5-year period were selected. The findings observed in tomography were collected and compared with those observed in three comparison groups made up by pathologies which produce superimposable image manifestations (tuberculosis, pulmonary neoplasms, and pneumonias). We sought to establish a model that would help distinguish, based on image findings, pulmonary nocardiosis from other groups. Results: We established a model which combines three variables (age, adenopathies, and heterogeneous size of nodules) and obtained a dis- criminatory capacity of 79 % compared with other alternative diagnoses. Conclusions: although the findings in image techniques are not specific for pulmonary nocardiosis, the grouping of findings in our model, in a proper clinical context, may be useful in establishing a provisional diagnosis of pulmonary nocardiosis, to the detriment of other possibili- ties, and help manage therapeutic approaches pending microbiological confirmation.Objetivo: evaluar el valor diagnóstico de algunos hallazgos en tomo - grafía computada de tórax, en pacientes con nocardiosis pulmonar confirmada, con el fin de que en conjunto permitan diferenciarla de otras afecciones que pueden mostrar hallazgos radiológicos superponibles. Material y métodos: se seleccionaron los sujetos con diagnóstico micro - biológico de nocardiosis en nuestro hospital en un periodo de 5 años. Se recogieron los hallazgos observados en tomografía y se compararon con los observados en tres grupos de comparación constituidos por patologías que producen manifestaciones de imagen superponibles (tuberculosis, neoplasias pulmonares y neumonías). Se intentó estable- cer un modelo que permitiera diferenciar, por imagen, la nocardiosis pulmonar de los otros grupos. Resultados: establecimos un modelo que combina tres variables (edad, adenopatías y tamaño heterogéneo de los nódulos) y obtuvimos una ca - pacidad discriminativa de 79% frente a otros diagnósticos alternativos. Conclusiones: a pesar de que los hallazgos en técnicas de imagen no son específicos para nocardiosis pulmonar, la agrupación de los ha- llazgos de nuestro modelo, en un contexto clínico adecuado, puede ser de utilidad a la hora de establecer un diagnóstico provisional de nocardiosis pulmonar, en detrimento de otras posibilidades, y permite gestionar conductas terapéuticas mientras se espera la confirmación microbiológica

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5

Background: Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. Results: We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (ΔLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of ΔLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving ΔLicpa::CPA) was sufficient to complement the reduced infectivity of both ΔLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone ΔLicpaC1::CPA compared with the CPA-deficient mutant ΔLicpaC1. Conclusion: The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the ΔLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters

Studies on the CPA cysteine peptidase in the <i>Leishmania infantum</i> genome strain JPCM5

Background Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. Results We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (ΔLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of ΔLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving ΔLicpa::CPA) was sufficient to complement the reduced infectivity of both ΔLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone ΔLicpaC1::CPA compared with the CPA-deficient mutant ΔLicpaC1. Conclusion The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the ΔLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters

Validity and Reliability of a New Optoelectronic System for Measuring Active Range of Motion of Upper Limb Joints in Asymptomatic and Symptomatic Subjects

The aim of this study was to evaluate the validity of the Veloflex infrared dynamic angle-meter (Veloflex-IDA) and the intra- and inter-rater reliability when measuring the ranges of motion (ROMs) of the upper limb joints. Thirty-five healthy and 20 symptomatic participants were evaluated. Twelve upper limb movements were measured in two sessions with the Veloflex-IDA, which is a device composed of a camera that tracks the trajectory of retro-reflective markers. In addition, a goniometer was used in the first session to evaluate concurrent validity. Validity and agreement were evaluated by Pearson correlation coefficient (r) and Bland&ndash;Altmann plots. Intra- and inter-rater reliability were evaluated using intra-class correlation (ICC), standard error of measurement (SEM), and minimal detectable change (MDC). Both instruments showed excellent correlation for all movements (r range from 0.992 to 0.999). The intra- and inter-rater reliability were excellent (ICC range from 0.95 to 0.99 and 0.90 to 0.98, respectively). Intra-rater reliability showed SEMs &lt;1.38% and &lt;5.19% and inter-rater reliability SEMs &lt;2.26% and &lt;5.22% for asymptomatic and symptomatic, respectively. Veloflex-IDA is a valid and reliable alternative to measure the upper limb joints&rsquo; ROM and it can be used in clinical practice and research after basic training

An Optoelectronic System for Measuring the Range of Motion in Healthy Volunteers: A Cross-Sectional Study

Background and Objectives: Within the clinical evaluation of multiple pathologies of the lower limb, the measurement of range of motion (ROM) of its joints is fundamental. To this end, there are various tools, from the goniometer as a reference to more recent devices such as inclinometry-based applications, photo capture applications, or motion capture systems. This study aimed to assess the validity, intra-rater, and inter-rater reliability of the VeloFlex system (VS), which is a new camera-based tool designed for tracking joint trajectories and measuring joint ROM. Materials and Methods: Thirty-five healthy volunteers (16 females; aged 18&ndash;61 years) participated in this study. All participants were assessed on two separate occasions, one week apart. During the first assessment session, measurements were obtained using a goniometer and the VS, whereas, in the second session, only the VS was used. In each assessment session, nine active movements were examined. For each movement, three trials were tested, and the mean of these three measures was used for analysis. To evaluate the concurrent validity and agreement, the Pearson correlation coefficient (r) and Bland-Altmann plots were used. Intra-rater and inter-rater reliability were evaluated using intra-class correlation (ICC), standard error of measurement (SEM), and minimal detectable change (MDC). Results: Both devices showed excellent correlations for all movements (r ranged from 0.992 to 0.999). The intra-rater reliability of the VS was excellent (ICC ranged from 0.93 to 0.99), SEMs ranged from 0.53% to 2.61% and the MDC ranged from 0.68&deg; to 3.26&deg;. The inter-rater reliability of the VS was also excellent (ICC ranged from 0.88 to 0.98), SEMs ranged from 0.81% to 4.76% and the MDC ranged from 2.27&deg; to 4.42&deg;. Conclusions: The VS is a valid and reliable tool for the measurement of ROM of lower limb joints in healthy subjects