Regulation and function of alternative polyadenylation in development and differentiation.

Alternative processing of nascent mRNAs is widespread in eukaryotic organisms and greatly impacts the output of gene expression. Specifically, alternative cleavage and polyadenylation (APA) is a co-transcriptional molecular process that switches the polyadenylation site (PAS) at which a nascent mRNA is cleaved, resulting in mRNA isoforms with different 3'UTR length and content. APA can potentially affect mRNA translation efficiency, localization, stability, and mRNA seeded protein-protein interactions. APA naturally occurs during development and cellular differentiation, with around 70% of human genes displaying APA in particular tissues and cell types. For example, neurons tend to express mRNAs with long 3'UTRs due to preferential processing at PASs more distal than other PASs used in other cell types. In addition, changes in APA mark a variety of pathological states, including many types of cancer, in which mRNAs are preferentially cleaved at more proximal PASs, causing expression of mRNA isoforms with short 3'UTRs. Although APA has been widely reported, both the function of APA in development and the mechanisms that regulate the choice of 3'end cut sites in normal and pathogenic conditions are still poorly understood. In this review, we summarize current understanding of how APA is regulated during development and cellular differentiation and how the resulting change in 3'UTR content affects multiple aspects of gene expression. With APA being a widespread phenomenon, the advent of cutting-edge scientific techniques and the pressing need for in-vivo studies, there has never been a better time to delve into the intricate mechanisms of alternative cleavage and polyadenylation.

Identification of atlastin genetic modifiers in a model of hereditary spastic paraplegia in Drosophila.

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative disorders characterized by progressive dysfunction of corticospinal motor neurons. Mutations in Atlastin1/Spg3, a small GTPase required for membrane fusion in the endoplasmic reticulum, are responsible for 10% of HSPs. Patients with the same Atlastin1/Spg3 mutation present high variability in age at onset and severity, suggesting a fundamental role of the environment and genetic background. Here, we used a Drosophila model of HSPs to identify genetic modifiers of decreased locomotion associated with atlastin knockdown in motor neurons. First, we screened for genomic regions that modify the climbing performance or viability of flies expressing atl RNAi in motor neurons. We tested 364 deficiencies spanning chromosomes two and three and found 35 enhancer and four suppressor regions of the climbing phenotype. We found that candidate genomic regions can also rescue atlastin effects at synapse morphology, suggesting a role in developing or maintaining the neuromuscular junction. Motor neuron-specific knockdown of 84 genes spanning candidate regions of the second chromosome identified 48 genes required for climbing behavior in motor neurons and 7 for viability, mapping to 11 modifier regions. We found that atl interacts genetically with Su(z)2, a component of the Polycomb repressive complex 1, suggesting that epigenetic regulation plays a role in the variability of HSP-like phenotypes caused by atl alleles. Our results identify new candidate genes and epigenetic regulation as a mechanism modifying neuronal atl pathogenic phenotypes, providing new targets for clinical studies.

Low-nutrient diet in Drosophila larvae stage causes enhancement in dopamine modulation in adult brain due epigenetic imprinting.

Nutrient scarcity is a frequent adverse condition that organisms face during their development. This condition may lead to long-lasting effects on the metabolism and behaviour of adults due to developmental epigenetic modifications. Here, we show that reducing nutrient availability during larval development affects adult spontaneous activity and sleep behaviour, together with changes in gene expression and epigenetic marks in the mushroom bodies (MBs). We found that open chromatin regions map to 100 of 241 transcriptionally upregulated genes in the adult MBs, these new opening zones are preferentially located in regulatory zones such as promoter-TSS and introns. Importantly, opened chromatin at the Dopamine 1-like receptor 2 regulatory zones correlate with increased expression. In consequence, adult administration of a dopamine antagonist reverses increased spontaneous activity and diminished sleep time observed in response to early-life nutrient restriction. In comparison, reducing dop1R2 expression in MBs also ameliorates these effects, albeit to a lesser degree. These results lead to the conclusion that increased dopamine signalling in the MBs of flies reared in a poor nutritional environment underlies the behavioural changes observed due to this condition during development.

Impaired attention mechanisms in confabulating patients: A VLSM and DWI study.

Attention is one of the most studied cognitive functions in brain-damaged populations or neurological syndromes, as its malfunction can be related to deficits in other higher cognitive functions. In the present study, we aimed at delimiting the attention deficits of a sample of brain-injured patients presenting confabulations by assessing their performance on alertness, spatial orienting, and executive control tasks. Confabulating patients, who present false memories or beliefs without intention to deceive, usually show memory deficits and/or executive dysfunction. However, it is also likely that attention processes may be impaired in patients showing confabulations. Here, we compared confabulating patients' attention performance to a lesion control group and a healthy control group. Confabulating patients' mean overall accuracy was lower than the one of healthy and lesion controls along the three experimental tasks. Importantly, confabulators presented a greater Simon congruency effect than both lesion controls and healthy controls in the presence of predictive spatial cues, besides a lower percentage of hits and longer RTs in the Go-NoGo task, demonstrating deficits in executive control. They also showed a higher reliance on alerting and spatially predictive orienting cues in the context of a deficient performance. Grey and white matter analyses showed that patients' percentage of hits in the Go-NoGo task was related to damage to the right inferior frontal gyrus (pars triangularis and pars opercularis), whereas the integrity of the right inferior fronto-occipital fasciculus was negatively correlated with their alertness effect. These results are consistent with previous literature highlighting an executive dysfunction in confabulating patients, and suggest that some additional forms of attention, such as alertness and spatial orienting, could be selectively impaired in this clinical syndrome.

Early-life nutrition interacts with developmental genes to shape the brain and sleep behavior in Drosophila melanogaster.

The mechanisms by which the genotype interacts with nutrition during development to contribute to the variation of complex behaviors and brain morphology of adults are not well understood. Here we use the Drosophila Genetic Reference Panel to identify genes and pathways underlying these interactions in sleep behavior and mushroom body morphology. We show that early-life nutritional restriction effects on sleep behavior and brain morphology depends on the genotype. We mapped genes associated with sleep sensitivity to early-life nutrition, which were enriched for protein-protein interactions responsible for translation, endocytosis regulation, ubiquitination, lipid metabolism, and neural development. By manipulating the expression of candidate genes in the mushroom bodies (MBs) and all neurons, we confirm that genes regulating neural development, translation and insulin signaling contribute to the variable response of sleep and brain morphology to early-life nutrition. We show that the interaction between differential expression of candidate genes with nutritional restriction in early life resides in the MBs or other neurons and that these effects are sex-specific. Natural variations in genes that control the systemic response to nutrition and brain development and function interact with early-life nutrition in different types of neurons to contribute to the variation of brain morphology and adult sleep behavior.

Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage.

Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3' UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3' UTR in proliferating spermatogonia but a short 3' UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3' UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3' UTR versus short 3' UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3' end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

Toward social-ecological coastal zone governance of Chiloé Island (Chile) based on the DPSIR framework.

Coastal zones are complex systems where sustainability needs local participative governance, whose absence may result in conflicts between social actors. In its absence, the first step should be a diagnosis of the current situation based on integrative conceptual frameworks such as the DPSIR. However, in conflicting situations, the generated model needs validation from social actors. Chiloé Island is a critical Chilean coastal fishery and aquaculture area, coexisting with subsistence and cultural uses of marine resources. This article analyzes the current ecological state of the Chiloé coastal zone and its main social-ecological impact using a DPSIR model. We validated its results through a household survey and interviews with local experts and social actors. Results show that increased coastal fisheries and aquaculture generate a critical decrease of coastal species, eutrophication, and pollution that, along with harmful algal blooms, generate conflicts. Social validation showed that experts and local social actors have different perceptions of conflicting parties and conflict solutions. Following a post-normal approach, we propose three ideas to start social-ecological governance of Chiloé coastal marine ecosystems.

Phosphatidic acid increases Notch signalling by affecting Sanpodo trafficking during Drosophila sensory organ development.

Organ cell diversity depends on binary cell-fate decisions mediated by the Notch signalling pathway during development and tissue homeostasis. A clear example is the series of binary cell-fate decisions that take place during asymmetric cell divisions that give rise to the sensory organs of Drosophila melanogaster. The regulated trafficking of Sanpodo, a transmembrane protein that potentiates receptor activity, plays a pivotal role in this process. Membrane lipids can regulate many signalling pathways by affecting receptor and ligand trafficking. It remains unknown, however, whether phosphatidic acid regulates Notch-mediated binary cell-fate decisions during asymmetric cell divisions, and what are the cellular mechanisms involved. Here we show that increased phosphatidic acid derived from Phospholipase D leads to defects in binary cell-fate decisions that are compatible with ectopic Notch activation in precursor cells, where it is normally inactive. Null mutants of numb or the α-subunit of Adaptor Protein complex-2 enhance dominantly this phenotype while removing a copy of Notch or sanpodo suppresses it. In vivo analyses show that Sanpodo localization decreases at acidic compartments, associated with increased internalization of Notch. We propose that Phospholipase D-derived phosphatidic acid promotes ectopic Notch signalling by increasing receptor endocytosis and inhibiting Sanpodo trafficking towards acidic endosomes.

Modeling Parkinson's Disease Heterogeneity to Accelerate Precision Medicine.

A mechanistic understanding of the diverse clinical manifestations of Parkinson's disease (PD) and variable patient response to treatments is lacking. Genetically diverse PD model organisms can be used to map modifier genes and understand clinically relevant phenotypes of varying severity. This strategy can accelerate the pace of discoveries for precision medicine purposes.

DREF Genetically Counteracts Mi-2 and Caf1 to Regulate Adult Stem Cell Maintenance.

Active adult stem cells maintain a bipotential state with progeny able to either self-renew or initiate differentiation depending on extrinsic signals from the surrounding microenvironment. However, the intrinsic gene regulatory networks and chromatin states that allow adult stem cells to make these cell fate choices are not entirely understood. Here we show that the transcription factor DNA Replication-related Element Factor (DREF) regulates adult stem cell maintenance in the Drosophila male germline. A temperature-sensitive allele of DREF described in this study genetically separated a role for DREF in germline stem cell self-renewal from the general roles of DREF in cell proliferation. The DREF temperature-sensitive allele caused defects in germline stem cell self-renewal but allowed viability and division of germline stem cells as well as cell viability, growth and division of somatic cyst stem cells in the testes and cells in the Drosophila eye. Germline stem cells mutant for the temperature sensitive DREF allele exhibited lower activation of a TGF-beta reporter, and their progeny turned on expression of the differentiation factor Bam prematurely. Results of genetic interaction analyses revealed that Mi-2 and Caf1/p55, components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, genetically antagonize the role of DREF in germline stem cell maintenance. Taken together, these data suggest that DREF contributes to intrinsic components of the germline stem cell regulatory network that maintains competence to self-renew.

Conscious perception in patients with prefrontal damage.

We are conscious and verbally report some of the information reaching our senses, although a big amount of information is processed unconsciously. There is no agreement about the neural correlates of consciousness, with low-level theories proposing that neural processing on primary sensory brain regions is the most important neural correlate of consciousness, while high-level theories propose that activity within the fronto-parietal network is the key component of conscious processing (Block, 2009). Contrary to the proposal of high-level theories, patients with prefrontal lobe damage do not present clinical symptoms associated to consciousness deficits. In the present study, we explored the conscious perception of near-threshold stimuli in a group of patients with right prefrontal damage and a group of matched healthy controls. Results demonstrated that perceptual contrast to perceive the near-threshold targets was related to damage to the right dorsolateral prefrontal cortex, and with reduced integrity of the ventral branch of the right superior longitudinal fascicule (SLF III). These results suggest a causal role of the prefrontal lobe in conscious processing.

Jitterbug/Filamin and Myosin-II form a complex in tendon cells required to maintain epithelial shape and polarity during musculoskeletal system development.

During musculoskeletal system development, mechanical tension is generated between muscles and tendon-cells. This tension is required for muscle differentiation and is counterbalanced by tendon-cells avoiding tissue deformation. Both, Jbug/Filamin, an actin-meshwork organizing protein, and non-muscle Myosin-II (Myo-II) are required to maintain the shape and cell orientation of the Drosophila notum epithelium during flight muscle attachment to tendon cells. Here we show that halving the genetic dose of Rho kinase (Drok), the main activator of Myosin-II, enhances the epithelial deformation and bristle orientation defects associated with jbug/Filamin knockdown. Drok and activated Myo-II localize at the apical cell junctions, tendon processes and are associated to the myotendinous junction. Further, we found that Jbug/Filamin co-distribute at tendon cells with activated Myo-II. Finally, we found that Jbug/Filamin and Myo-II are in the same molecular complex and that the actin-binding domain of Jbug/Filamin is necessary for this interaction. These data together suggest that Jbug/Filamin and Myo-II proteins may act together in tendon cells to balance the tension generated during development of muscles-tendon interaction, maintaining the shape and polarity of the Drosophila notum epithelium.

Gaze entropy reflects surgical task load.

BACKGROUND: Task (over-)load imposed on surgeons is a main contributing factor to surgical errors. Recent research has shown that gaze metrics represent a valid and objective index to asses operator task load in non-surgical scenarios. Thus, gaze metrics have the potential to improve workplace safety by providing accurate measurements of task load variations. However, the direct relationship between gaze metrics and surgical task load has not been investigated yet. We studied the effects of surgical task complexity on the gaze metrics of surgical trainees. METHODS: We recorded the eye movements of 18 surgical residents, using a mobile eye tracker system, during the performance of three high-fidelity virtual simulations of laparoscopic exercises of increasing complexity level: Clip Applying exercise, Cutting Big exercise, and Translocation of Objects exercise. We also measured performance accuracy and subjective rating of complexity. RESULTS: Gaze entropy and velocity linearly increased with increased task complexity: Visual exploration pattern became less stereotyped (i.e., more random) and faster during the more complex exercises. Residents performed better the Clip Applying exercise and the Cutting Big exercise than the Translocation of Objects exercise and their perceived task complexity differed accordingly. CONCLUSIONS: Our data show that gaze metrics are a valid and reliable surgical task load index. These findings have potential impacts to improve patient safety by providing accurate measurements of surgeon task (over-)load and might provide future indices to assess residents' learning curves, independently of expensive virtual simulators or time-consuming expert evaluation.

Gated Sensor Fusion: A way to Improve the Precision of Ambulatory Human Body Motion Estimation.

Human body motion is usually variable in terms of intensity and, therefore, any Inertial Measurement Unit attached to a subject will measure both low and high angular rate and accelerations. This can be a problem for the accuracy of orientation estimation algorithms based on adaptive filters such as the Kalman filter, since both the variances of the process noise and the measurement noise are set at the beginning of the algorithm and remain constant during its execution. Setting fixed noise parameters burdens the adaptation capability of the filter if the intensity of the motion changes rapidly. In this work we present a conjoint novel algorithm which uses a motion intensity detector to dynamically vary the noise statistical parameters of different approaches of the Kalman filter. Results show that the precision of the estimated orientation in terms of the RMSE can be improved up to 29% with respect to the standard fixed-parameters approaches.

Automatic determination of validity of input data used in ellipsoid fitting MARG calibration algorithms.

Ellipsoid fitting algorithms are widely used to calibrate Magnetic Angular Rate and Gravity (MARG) sensors. These algorithms are based on the minimization of an error function that optimizes the parameters of a mathematical sensor model that is subsequently applied to calibrate the raw data. The convergence of this kind of algorithms to a correct solution is very sensitive to input data. Input calibration datasets must be properly distributed in space so data can be accurately fitted to the theoretical ellipsoid model. Gathering a well distributed set is not an easy task as it is difficult for the operator carrying out the maneuvers to keep a visual record of all the positions that have already been covered, as well as the remaining ones. It would be then desirable to have a system that gives feedback to the operator when the dataset is ready, or to enable the calibration process in auto-calibrated systems. In this work, we propose two different algorithms that analyze the goodness of the distributions by computing four different indicators. The first approach is based on a thresholding algorithm that uses only one indicator as its input and the second one is based on a Fuzzy Logic System (FLS) that estimates the calibration error for a given calibration set using a weighted combination of two indicators. Very accurate classification between valid and invalid datasets is achieved with average Area Under Curve (AUC) of up to 0:98.

A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage.

In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. Here we show that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism.

Detection of (in)activity periods in human body motion using inertial sensors: a comparative study.

Determination of (in)activity periods when monitoring human body motion is a mandatory preprocessing step in all human inertial navigation and position analysis applications. Distinction of (in)activity needs to be established in order to allow the system to recompute the calibration parameters of the inertial sensors as well as the Zero Velocity Updates (ZUPT) of inertial navigation. The periodical recomputation of these parameters allows the application to maintain a constant degree of precision. This work presents a comparative study among different well known inertial magnitude-based detectors and proposes a new approach by applying spectrum-based detectors and memory-based detectors. A robust statistical comparison is carried out by the use of an accelerometer and angular rate signal synthesizer that mimics the output of accelerometers and gyroscopes when subjects are performing basic activities of daily life. Theoretical results are verified by testing the algorithms over signals gathered using an Inertial Measurement Unit (IMU). Detection accuracy rates of up to 97% are achieved.