When small non-coding RNAs meet the ribosome: Tuning the translational machinery

The functions of ribosomes in translation are complex and involve different types of activities critical for decoding the genetic code, linkage of amino acids via amide bonds to form polypeptide chains, as well as the release and proper targeting of the synthesized protein. Non-protein-coding RNAs (ncRNAs) have been recognized to be crucial in establishing regulatory networks.1 However all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. The main goal of this project is to identify potential novel ncRNAs that directly bind and possibly regulate the ribosome during protein biosynthesis. To address this question we applied various stress conditions to the archaeal model organism Haloferax volcanii and deep-sequenced the ribosome-associated small ncRNA interactome. In total we identified 6.250 ncRNA candidates. Significantly, we observed the emersed presence of tRNA-derived fragments (tRFs). These tRFs have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNAs. Here we present evidence that tRFs from H. volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome a 26 residue long fragment originating from the 5’ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production.2 Currently we are investigating the binding site of this tRF on the 30S subunit in more detail.

An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

Psychosocial influences on fruit, vegetable and milk consumption among a cohort of Houston-area 5th graders

Epidemiological evidence suggests that fruit and vegetable intake is negatively associated with the development of several chronic diseases, including heart disease, some cancers and diabetes mellitus. Inadequate consumption of milk during developmental years is associated with osteoporosis. Consumption of fruit, vegetable and milk (FVM) declines from childhood to adolescence. Adolescent eating habits persist into adulthood; thus, understanding psychosocial factors such as self-efficacy, norms and preferences, is important for developing effective interventions. Preferences, one of the most consistent correlates of fruit and vegetable consumption in children and adolescents, may mediate the relationships between self-efficacy and norms and fruit and vegetable consumption. ^ Fifth grade students from one middle school in South Texas were followed for two years. Students completed lunch food records and questionnaires assessing fruit, vegetable and milk self-efficacy and norms and fruit and vegetable preferences. Principal component analyses identified four scales: Fruit Self-Efficacy, Vegetable Self-Efficacy, Fruit and Vegetable Norms, and Milk Influences. Reliability and validity of the four scales and emerging subscales were assessed using Cronbach's alpha and consumption data, respectively. Associations between longitudinal FVM consumption and self-efficacy and norms were tested. Additionally, the influence of preferences on the relationship of self-efficacy, norms and fruit and vegetable consumption was examined. ^ Confirmatory factor analyses confirmed four scales and subscales. Internal consistency and test-retest reliabilities were acceptable. Self-efficacy and norms were related to FVM consumption and changes in fruit and high fat vegetable consumption over the two-year period. While intake over the two-year period differed statistically, eating patterns were stable. Preferences mediated the relation between fruit self-efficacy and FV norms and fruit consumption. ^ In conclusion, self-efficacy and norms about consuming FVM at school appear to influence FVM consumption. Because eating patterns were similar over the two-year period, establishing healthy eating habits in elementary school is important. While FVM self-efficacy and norms influenced FVM consumption in children, only fruit preferences mediated the relationship of fruit consumption, self-efficacy and norms. Since the influences of FVM consumption appear to differ, interventions designed to increase consumption should target these differences and consider the specificity of self-efficacy and norms. ^

Body mass index trajectories and predictors among 3rd to 12th graders using growth curve mixture modeling the Child and Adolescent Trial for Cardiovascular Health (CATCH) study

This dissertation examined body mass index (BMI) growth trajectories and the effects of gender, ethnicity, dietary intake, and physical activity (PA) on BMI growth trajectories among 3rd to 12th graders (9-18 years of age). Growth curve model analysis was performed using data from The Child and Adolescent Trial for Cardiovascular Health (CATCH) study. The study population included 2909 students who were followed up from grades 3-12. The main outcome was BMI at grades 3, 4, 5, 8, and 12. ^ The results revealed that BMI growth differed across two distinct developmental periods of childhood and adolescence. Rate of BMI growth was faster in middle childhood (9-11 years old or 3rd - 5th grades) than in adolescence (11-18 years old or 5th - 12th grades). Students with higher BMI at 3rd grade (baseline) had faster rates of BMI growth. Three groups of students with distinct BMI growth trajectories were identified: high, average, and low. ^ Black and Hispanic children were more likely to be in the groups with higher baseline BMI and faster rates of BMI growth over time. The effects of gender or ethnicity on BMI growth differed across the three groups. The effects of ethnicity on BMI growth were weakened as the children aged. The effects of gender on BMI growth were attenuated in the groups with a large proportion of black and Hispanic children, i.e., “high” or “average” BMI trajectory group. After controlling for gender, ethnicity, and age at baseline, in the “high BMI trajectory”, rate of yearly BMI growth in middle childhood increased 0.102 for every 500 Kcals increase (p=0.049). No significant effects of percentage of energy from total fat and saturated fat on BMI growth were found. Baseline BMI increased 0.041 for every 30 minutes increased in moderate-to-vigorous PA (MVPA) in the “low BMI trajectory”, while Baseline BMI decreased 0.345 for every 30 minutes increased in vigorous PA (VPA) in the “high BMI trajectory”. ^ Childhood overweight and obesity interventions should start at the earliest possible ages, prior to 3rd grade and continue through grade school. Interventions should focus on all children, but specifically black and Hispanic children, who are more likely to be highest at-risk. Promoting VPA earlier in childhood is important for preventing overweight and obesity among children and adolescents. Interventions should target total energy intake, rather than only percentage of energy from total fat or saturated fat. ^

The effects of ethnicity and ethnic composition of schools on physical activity and nutrition behaviors of 8th and 11th graders in Texas

With rates of obesity and overweight continuing to increase in the US, the attention of public health researchers has focused on nutrition and physical activity behaviors. However, attempts to explain the disparate rates of obesity and overweight between whites and Hispanics have often proven inadequate. Indeed, the nebulous term ‘ethnicity’ provides little important detail in addressing potential biological, behavioral, and environmental factors that may affect rates of obesity and overweight. In response to this, the present research seeks to test the explanatory powers of ethnicity by situating the nutrition and physical activity behaviors of whites and Hispanic into their broader social contexts. It is hypothesized that a student's gender and grade level, as well as the socioeconomic status and ethnic composition of their school, will have more predictive power for these behaviors than will self-reported ethnicity. ^ Analyses revealed that while ethnicity did not seem to impact nutrition behaviors among the wealthier schools and those with fewer Hispanics, ethnicity was relevant in explaining these behaviors in the poorest tertile of schools and those with the highest number of Hispanics. With respect to physical activity behaviors, the results were mixed. The variables representing regular physical activity, participation in extracurricular physical activities, and performance of strengthening and toning exercises were more likely to be determined by SES and ethnic composition than ethnicity, especially among 8th grade males. However, school sports team and physical education participation continued to vary by ethnicity, even after controlling for SES and ethnic composition of schools. In conclusion then, it is important to understand the intersecting demographic and social variables that define and surround the individual in order to understand nutrition and physical activity behaviors and thus overweight and obesity.^

Possible selves of inner city fourth graders: What do they believe about their future education and occupation?

Possible selves, a component of self-concept, are the thoughts people have about their future. The overall purpose of this study was to examine the educational and occupational possible selves of inner city children. Specific aims of the study were to: 1) Assess the similarities and differences between the level of schooling inner city children hope to complete and expect to complete, and 2) Assess the similarities and differences between the jobs inner city children hope to have in the future and expect to have. As part of a larger study designed to pilot test a health promotion intervention, fourth grade students (n = 19) completed two measures: the Level of Schooling Questionnaire and the Jobs Questionnaire. No significant differences were found between students' hoped-for and expected educational possible selves or their hoped-for and expected occupational possible selves. Future studies can explore methods and strategies to support, encourage, and cultivate inner city children's possible selves.^

Neural bruise prediction models for fruit handling and machinery evaluation.

Some neural bruise prediction models have been implemented in the laboratory, for the most traded fruit species and varieties, allowing the prediction of the acceptability or rejectability for damages, with respect to the EC Standards. Different models have been built for both quasi-static (compression) and dynamic (impact) loads covering the whole commercial ripening period of fruits. A simulation process has been developed gathering the information on laboratory bruise models and load sensor calibrations for different electronic devices (IS-100 and DEA-1, for impact and compression loads respectively). Some evaluation methodology has been designed gathering the information on the mechanical properties of fruits and the loading records of electronic devices. The evaluation system allows to determine the current stage of fruit handling process and machinery.

Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.