From Genes to Ecosystems: Resource Availability and DNA Methylation Drive the Diversity and Abundance of Restriction Modification Systems in Prokaryotes

Together, prokaryotic hosts and their viruses numerically dominate the planet and are engaged in an eternal struggle of hosts evading viral predation and viruses overcoming defensive mechanisms employed by their hosts. Prokaryotic hosts have been found to carry several viral defense systems in recent years with Restriction Modification systems (RMs) were the first discovered in the 1950s. While we have biochemically elucidated many of these systems in the last 70 years, we still struggle to understand what drives their gain and loss in prokaryotic genomes. In this work, we take a computational approach to understand the underlying evolutionary drivers of RMs by assessing ‘big data’ signals of RMs in prokaryotic genomes and incorporating molecular data in trait-based mathematical models. Focusing on the Cyanobacteria, we found a large discrepancy in the frequency of RMs per genome in different environmental contexts, where Cyanobacteria that live in oligotrophic nutrient conditions have few to no RMs and those in nutrient-rich conditions consistently have many RMs. While our models agree with the observation that increased nutrient inputs make the selective pressure of RMs more intense, they were unable to reconcile the high numbers of RMs per genome with their potent defensive properties- a situation of apparent overkill. By incorporating viral methylation, an unavoidable effect of RMs, we were able to explain how organisms could carry over 15 RMs. With this discovery, we then tried and reassess the distribution of methyltransferases, an essential component of RMs that can also have alternate physiological rolls in the cell. We expand on conventional wisdom, that methyltransferases that are widely phylogenetically conserved are associated with global cellular regulation. However, we also find that organisms with high numbers of RMs also have a surprising amount of conservation in the methyltransferases that they carry. This data suggests caution should be used in associating phylogenic signals with functional rolls in methyltransferases as different functional rolls seem to overlap in their phylogenetic signal. Indeed, we suggest trait-based modeling may be the best tool in elucidating why organisms with a high selective pressure to maintain RMs appear to have conserved methyltransferase

Anger Induction and Ambient Interventions: Effects on Cardiovascular Activity and Frontal EEG Asymmetry

Background and aims: The experience of anger could affect cardiovascular (CV) and electro-encephalographic (EEG) parameters but such parameters could vary within the motivational context. Although models of motivational contexts were proposed by CV literature as challenge/threat (Blascovich & Tomaka, 1996) and by frontal EEG asymmetry literature as approach/avoidance (Harmon-Jones, 2004a) little is known whether a negative emotion such as anger could be indexed by CV and EEG responses within motivational contexts. Anger in a threat context may be particularly detrimental for health due to low control compared to anger in a challenge context, where control is high. Hence, the aim of the research was twofold: 1. to investigate how a motivational context (challenge vs. threat) influences the cardiovascular system and frontal EEG asymmetry during anger induction protocols and 2. to analyse the efficacy of ambient interventions (music, light) to reduce the impact of anger on cardiovascular responses. Affective computing through the use of ambient intelligence technology could be used to promote positive emotion or to ameliorate negative moods. Method: There were two anger induction protocols within the thesis. Firstly, anger was manipulated using an experimenter effect (i.e., rude vs. polite experimenter). Participants were exposed to a computer-based problem-solving task under conditions of control and no control which represented the motivational contexts of challenge/threat. Secondly, anger was induced by exposing participants to a time constrained driving schedule on a simulated route with financial penalties for any delays to arrive to the destination. Motivation was manipulated by exposing participants to traffic delays at an early (challenge) and later point (threat) on a simulated driving route. STAXI-2 (Spielberg, 1999) was used to measure anger states and motivation was measured by Confidence and Perceived Control Scale from Dundee Stress State Questionnaire (Matthews & Desmond, 1998). Psychophysiological variables included: blood pressure (BP), cardiovascular impedance (ICG), frontal EEG asymmetry, and facial electromyography (fEMG). Results: The cardiovascular and EEG results of the present thesis pointed to a circumplex model of anger with quadruplet facets along cardiovascular responses to challenge/threat contexts in conjunction with approach/avoidance tendencies where a threat motivation with avoidance was indexed by increased blood pressure and cardiac output and by greater right frontal activation. The difference in the approach-threat responses was the activation of the left hemisphere. The challenge-avoidance state was defined by increased total peripheral resistance (TPR), systolic blood pressure (SBP), heart rate (HR), mean arterial pressure (MAP) and greater right frontal hemisphere activation. No frontal asymmetric activity was identified in the challenge-approach, but increased TPR, SBP, HR and MAP were observed. The ambient intervention results suggested that cardiovascular responses (e.g., SBP) could be reduced by low activation music or blue ambient light. Discussion and conclusions: Anger in the context of challenge can be distinguished from anger in the context of threat via a specific pattern of CV (systolic BP) and EEG measures (frontal peripheral brain site). Ambient interventions (low activation music or blue light) could be factors in modulating physiological reactions while driving; discrepancies between self-report measures and physiological responses, low sensitivity of impendence data to manipulations and low impact of various colour ambient lights on cardiovascular responses were addressed within a theoretical and methodological

The Insertion/Deletion Polymorphism of the Angiotensin Converting Enzyme (ACE) in Parkinson’s Disease

Parkinson’s disease (PDI is a neurodegenerative disorder of unknown etiology. Both genetic and environmental factors are thought to be implicated to some extent. The ACE gene insertion/deletion (I/D) polymorphism has been associated with common neurodegenerative disorders that share similar clinical and neuropathological features with PD (Alzheimer’s disease). In this study we set out to examine the role of the ACE gene insertion/deletion (I/D) polymorphism in Parkinson’s disease (PD)

The Insertion/Deletion Polymorphism of the Angiotensin Converting Enzyme (ACE) in Parkinson’s Disease

Parkinson’s disease (PDI is a neurodegenerative disorder of unknown etiology. Both genetic and environmental factors are thought to be implicated to some extent. The ACE gene insertion/deletion (I/D) polymorphism has been associated with common neurodegenerative disorders that share similar clinical and neuropathological features with PD (Alzheimer’s disease). In this study we set out to examine the role of the ACE gene insertion/deletion (I/D) polymorphism in Parkinson’s disease (PD)

Interface Analysis of the Complex between ERK2 and PTP-SL

The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2–PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex

A Caenorhabditis elegans Wild Type Defies the Temperature–Size Rule Owing to a Single Nucleotide Polymorphism in tra-3

Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature–size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature–size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature–size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 × CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature–size rule, which has puzzled biologists for decades

SMRT Sequencing of Paramecium Bursaria Chlorella Virus-1 Reveals Diverse Methylation Stability in Adenines Targeted by Restriction Modification Systems

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives

Bite-sized and peer-assisted video-based learning in statistics education: benefits on attainment, attitudes and preferences of university students

The use and acceptance of online learning have increased following the COVID-19 pandemic. This mixed-methods study examined learners' preferences and performance in online learning interactions in relation to two factors: 'bite-sized' learning and 'presenter status' in instructional videos. University students (N = 18) without a mathematical background utilised bite-sized online learning episodes focusing on statistics. Each episode included a 10-minute instructional video followed by an assessment. The videos implemented three alternative 'presenter-status' conditions: lecturer, student-imitating-lecturer or student-peer-tutor. Individual students completed three episodes, one from each presenter-status condition (counterbalanced). Participants presented high performance in the post-episode assessments, irrespective of presenter status. Students also reported remarkably positive views towards bite-sized learning in user-satisfaction questionnaires. Finally, qualitative analysis of open-ended responses and interviews uncovered three main themes: positive learning experiences, divergent attitudes towards video-based learning, and differential preferences for presenter status. These findings have clear implications for teaching and learning in higher education