CONSTANS, a HUB for all seasons: How photoperiod pervades plant physiology regulatory circuits.

How does a plant detect the changing seasons and make important developmental decisions accordingly? How do they incorporate daylength information into their routine physiological processes? Photoperiodism, or the capacity to measure the daylength, is a crucial aspect of plant development that helps plants determine the best time of the year to make vital decisions, such as flowering. The protein CONSTANS (CO) constitutes the central regulator of this sensing mechanism, not only activating florigen production in the leaves but also participating in many physiological aspects in which seasonality is important. Recent discoveries place CO in the center of a gene network that can determine the length of the day and confer seasonal input to aspects of plant development and physiology as important as senescence, seed size, or circadian rhythms. In this review, we discuss the importance of CO protein structure, function, and evolutionary mechanisms that embryophytes have developed to incorporate annual information into their physiology.

New indices in predicting cardiometabolic risk and its relation to endothelial dysfunction in adolescents: The HELENA study.

BACKGROUND AND AIMS: Blood pressure (BP) changes and insulin resistance (IR) are important cardiometabolic risk (CMR) factors; their early identification can contribute to the reduction of cardiovascular events in adulthood. This necessitates the search for more accessible and easily applied indicators for their prediction. Therefore, this study aimed to evaluate the predictive power of the indices, TyG, TG/HDL-c, height-corrected lipid accumulation product (HLAP), and visceral adiposity index (VAI), in identifying the CMR obtained by high BP and IR and to verify their relationship with biomarkers of endothelial dysfunction (ED) in European adolescents. METHODS AND RESULTS: The anthropometric data and blood biomarkers of 744 adolescents (343 boys and 401 girls) from the Healthy Lifestyle in Europe by Nutrition in Adolescence Cross-Sectional Study (HELENA-CSS), with a mean age of 14.67 (SD 1.15) years, were assessed. The adolescents were then classified according to the presence or absence of high BP and IR. The cut-off points of the indices evaluated for the identification of CMR were determined. The relationship between CMR diagnosed using these indices and ED biomarkers was tested. The HLAP and TG/HDL-c were fair predictors of CMR obtained by IR in male adolescents. These indices showed association with hsCRP in sVCAM-1 in boys, but it lost significance after adjusting for age and body mass index. CONCLUSION: TG/HDL-c and HLAP indices showed a fair performance in predicting CMR, obtained by IR, in male adolescents. ED showed no association with the CMR identified by the indices.

Longitudinal associations between cardiovascular biomarkers and metabolic syndrome during puberty: the PUBMEP study.

Puberty has been described as a life stage of considerable metabolic risk specially for those with obesity. The low-grade systemic inflammatory status associated with obesity could be one of the connections with metabolic syndrome (MetS). Thus, we aimed to assess the relationship between inflammatory and cardiovascular biomarkers and the development of MetS during puberty. Seventy-five children from the PUBMEP study (33 females), aged 4-18 years, were included. Cardiovascular and inflammatory biomarkers were measured in the prepubertal and pubertal stage, including high-sensitivity C-reactive protein (CRP), leptin, tumor necrosis factor-alpha (TNFα), interleukin 8 (IL8), monocyte chemoattractant protein 1 (MCP-1), total plasminogen activator inhibitor-1 (tPAI), resistin, adiponectin, myeloperoxidase (MPO), and soluble intercellular adhesion molecule-1 (sICAM-1). MetS was diagnosed at each measurement point. Mixed-effects and logistic regressions were performed. Those children with MetS in puberty presented higher prepubertal values of several cardiometabolic biomarkers in comparison to those without MetS (z-score body mass index (zBMI), waist circumference, insulin, HOMA-IR, leptin, and tPAI (p < 0.05)). For prepubertal children with obesity, the odds of developing MetS in puberty were significantly higher in those having high zBMI (OR = 4.27; CI: 1.39-22.59) or high concentrations of tPAI (OR = 1.19; CI: 1.06-1.43). CONCLUSION: Those with obesity with higher prepubertal tPAI plasma levels had 19% higher odds of having MetS at puberty highlighting the existence of association between MetS, obesity, and inflammation already in puberty. Thus, assessing cardiometabolic and inflammatory status in children with obesity already at prepuberty is key to avoiding future comorbidities. WHAT IS KNOWN: • Inflammation, metabolic syndrome, and obesity may have their onset in childhood. • Puberty is a life stage characterized for an increased cardiovascular risk. WHAT IS NEW: • Prepuberty state could be an early indicator of future cardiometabolic risk. • Children with obesity and high total plasminogen have higher odds of future metabolic syndrome.

Differences in the Immune Response of the Nonmetastatic Axillary Lymph Nodes between Triple-Negative and Luminal A Breast Cancer Surrogate Subtypes.

Breast cancer (BC) comprises four immunohistochemical surrogate subtypes of which triple-negative breast cancer (TNBC) has the highest risk of mortality. Axillary lymph nodes (ALNs) are the regions where BC cells first establish before distant metastasis, and the presence of tumor cells in the ALN causes an immune tolerance profile that contrasts with that of the nonmetastatic ALN (ALN-). However, few studies have compared the immune components of the ALNs- in BC subtypes. The present study aimed to determine whether differences between immune populations in the primary tumor and ALNs- were associated with the luminal A or TNBC subtype. We evaluated a retrospective cohort of 144 patients using paraffin-embedded biopsies. The TNBC samples tended to have a higher histologic grade and proliferation index and had higher levels of immune markers compared with luminal A in primary tumors and ALNs-. Two methods for validating the multivariate analysis found that histologic grade, intratumoral S100 dendritic cells, and CD8 T lymphocytes and CD57 natural killer cells in the ALNs- were factors associated with TNBC, whereas CD83 dendritic cells in the ALNs- were associated with the luminal A subtype. In conclusion, we found that intratumoral regions and ALNs- of TNBC contained higher concentrations of markers related to immune tolerance than luminal A. This finding partially explains the worse prognosis of patients with TNBC.

CONSTANS-FKBP12 interaction contributes to modulation of photoperiodic flowering in Arabidopsis.

Flowering time is a key process in plant development. Photoperiodic signals play a crucial role in the floral transition in Arabidopsis thaliana, and the protein CONSTANS (CO) has a central regulatory function that is tightly regulated at the transcriptional and post-translational levels. The stability of CO protein depends on a light-driven proteasome process that optimizes its accumulation in the evening to promote the production of the florigen FLOWERING LOCUS T (FT) and induce seasonal flowering. To further investigate the post-translational regulation of CO protein we have dissected its interactome network employing in vivo and in vitro assays and molecular genetics approaches. The immunophilin FKBP12 has been identified in Arabidopsis as a CO interactor that regulates its accumulation and activity. FKBP12 and CO interact through the CCT domain, affecting the stability and function of CO. fkbp12 insertion mutants show a delay in flowering time, while FKBP12 overexpression accelerates flowering, and these phenotypes can be directly related to a change in accumulation of FT protein. The interaction is conserved between the Chlamydomonas algal orthologs CrCO-CrFKBP12, revealing an ancient regulatory step in photoperiod regulation of plant development.

The Immune Response in Nonmetastatic Axillary Lymph Nodes Is Associated with the Presence of Axillary Metastasis and Breast Cancer Patient Outcome.

Tumor cells can modify the immune response in primary tumors and in the axillary lymph nodes with metastasis (ALN+) in breast cancer (BC), influencing patient outcome. We investigated whether patterns of immune cells in the primary tumor and in the axillary lymph nodes without metastasis (ALN-) differed between patients diagnosed without ALN+ (diagnosed-ALN-) and with ALN+ (diagnosed-ALN+) and the implications for clinical outcome. Eleven immune markers were studied using immunohistochemistry, tissue microarray, and digital image analysis in 141 BC patient samples (75 diagnosed-ALN+ and 66 diagnosed-ALN-). Two logistic regression models were derived to identify the clinical, pathologic, and immunologic variables associated with the presence of ALN+ at diagnosis. There are immune patterns in the ALN- associated with the presence of ALN+ at diagnosis. The regression models revealed a small subgroup of diagnosed-ALN+ with ALN- immune patterns that were more similar to those of the ALN- of the diagnosed-ALN-. This small subgroup also showed similar clinical behavior to that of the diagnosed-ALN-. Another small subgroup of diagnosed-ALN- with ALN- immune patterns was found whose members were more similar to those of the ALN- of the diagnosed-ALN+. This small subgroup had similar clinical behavior to the diagnosed-ALN+. These data suggest that the immune response present in ALN- at diagnosis could influence the clinical outcome of BC patients.

Approaching Adversarial Example Classification with Chaos Theory.

Adversarial examples are one of the most intriguing topics in modern deep learning. Imperceptible perturbations to the input can fool robust models. In relation to this problem, attack and defense methods are being developed almost on a daily basis. In parallel, efforts are being made to simply pointing out when an input image is an adversarial example. This can help prevent potential issues, as the failure cases are easily recognizable by humans. The proposal in this work is to study how chaos theory methods can help distinguish adversarial examples from regular images. Our work is based on the assumption that deep networks behave as chaotic systems, and adversarial examples are the main manifestation of it (in the sense that a slight input variation produces a totally different output). In our experiments, we show that the Lyapunov exponents (an established measure of chaoticity), which have been recently proposed for classification of adversarial examples, are not robust to image processing transformations that alter image entropy. Furthermore, we show that entropy can complement Lyapunov exponents in such a way that the discriminating power is significantly enhanced. The proposed method achieves 65% to 100% accuracy detecting adversarials with a wide range of attacks (for example: CW, PGD, Spatial, HopSkip) for the MNIST dataset, with similar results when entropy-changing image processing methods (such as Equalization, Speckle and Gaussian noise) are applied. This is also corroborated with two other datasets, Fashion-MNIST and CIFAR 19. These results indicate that classifiers can enhance their robustness against the adversarial phenomenon, being applied in a wide variety of conditions that potentially matches real world cases and also other threatening scenarios.

Immune response profile of primary tumour, sentinel and non-sentinel axillary lymph nodes related to metastasis in breast cancer: an immunohistochemical point of view.

Approximately 1.67 million new cases of breast cancer (BC) are diagnosed annually, and patient survival significantly decreases when the disease metastasizes. The axillary lymph nodes (ALNs) are the main doorway for BC tumoral cell escape, through which cells can disseminate to distant organs. The immune response, which principally develops in the lymph nodes, is linked to cancer progression, and its efficacy at controlling tumoral growth is compromised during the disease. Immunohistochemistry (IHC) is one of the most widely used research techniques for studying the immune response. It allows the measurement of the expression of particular markers related to the immune populations. This review focuses on the role of the immune populations in the primary tumour in the locoregional metastasis of the ALN, and the relationship of the immune response in these regions to distant metastasis. We considered only studies of immune cells using IHC techniques. In particular, lymphocytes, macrophages and dendritic cells all play important roles in BC and have been extensively studied. Although further research is needed, there is much evidence of their role in the invasion of the ALN and distant organs. Their association with tumoral growth or protection has not yet been demonstrated decisively and is very likely to be determined by a combination of factors. Moreover, even though IHC is a widely used technique in cancer diagnosis and research, there is still room for improvement, since its quantification needs to be properly standardized.

Adherence to the Mediterranean diet in metabolically healthy and unhealthy overweight and obese European adolescents: the HELENA study.

PURPOSE: To examine the adherence to the Mediterranean dietary pattern (MDP) in metabolically healthy overweight or obese (MHO) and metabolically unhealthy obese (MUO) European adolescents. METHODS: In this cross-sectional study, 137 overweight/obese adolescents aged 12-17 years old from the HELENA study were included. Height, weight, waist circumference and skinfold thickness were measured and body mass index and body fat percent were calculated. Systolic and diastolic blood pressure, glucose, HDL cholesterol, triglycerides and cardiorespiratory fitness (20 m shuttle run test) were measured. MHO and MUO phenotypes were categorized following the Jolliffe and Janssen criteria. Two non-consecutive 24 h recalls were used for dietary intake assessment and the adherence to the MDP was calculated using the Mediterranean dietary pattern score (MDP score) (range 0-9). RESULTS: A total of 45 (22 girls) adolescents (32.8%) were categorized as MHO. The adherence to the MDP was significantly higher in MHO than in MUO adolescents regardless of age, sex, body fat percentage, energy intake and center (MDP score: 4.6 ± 1.6 vs. 3.9 ± 1.5, p = 0.036), but this difference became non-significant after further adjustment for cardiorespiratory fitness. Participants who had a low adherence to the MDP (MDP score ≤ 4) had a higher likelihood of having MUO phenotype regardless of sex, age, energy intake, center and body fat percentage (OR 2.2; 95% CI 1.01-4.81, p = 0.048). CONCLUSIONS: Adherence to the MDP might be beneficial to maintain metabolic health in overweight/obese adolescents, yet cardiorespiratory fitness seems to play a key role on the metabolic phenotype.

HER2 challenge contest: a detailed assessment of automated HER2 scoring algorithms in whole slide images of breast cancer tissues.

AIMS: Evaluating expression of the human epidermal growth factor receptor 2 (HER2) by visual examination of immunohistochemistry (IHC) on invasive breast cancer (BCa) is a key part of the diagnostic assessment of BCa due to its recognized importance as a predictive and prognostic marker in clinical practice. However, visual scoring of HER2 is subjective, and consequently prone to interobserver variability. Given the prognostic and therapeutic implications of HER2 scoring, a more objective method is required. In this paper, we report on a recent automated HER2 scoring contest, held in conjunction with the annual PathSoc meeting held in Nottingham in June 2016, aimed at systematically comparing and advancing the state-of-the-art artificial intelligence (AI)-based automated methods for HER2 scoring. METHODS AND RESULTS: The contest data set comprised digitized whole slide images (WSI) of sections from 86 cases of invasive breast carcinoma stained with both haematoxylin and eosin (H&E) and IHC for HER2. The contesting algorithms predicted scores of the IHC slides automatically for an unseen subset of the data set and the predicted scores were compared with the 'ground truth' (a consensus score from at least two experts). We also report on a simple 'Man versus Machine' contest for the scoring of HER2 and show that the automated methods could beat the pathology experts on this contest data set. CONCLUSIONS: This paper presents a benchmark for comparing the performance of automated algorithms for scoring of HER2. It also demonstrates the enormous potential of automated algorithms in assisting the pathologist with objective IHC scoring.

Eyes of Things.

Embedded systems control and monitor a great deal of our reality. While some "classic" features are intrinsically necessary, such as low power consumption, rugged operating ranges, fast response and low cost, these systems have evolved in the last few years to emphasize connectivity functions, thus contributing to the Internet of Things paradigm. A myriad of sensing/computing devices are being attached to everyday objects, each able to send and receive data and to act as a unique node in the Internet. Apart from the obvious necessity to process at least some data at the edge (to increase security and reduce power consumption and latency), a major breakthrough will arguably come when such devices are endowed with some level of autonomous "intelligence". Intelligent computing aims to solve problems for which no efficient exact algorithm can exist or for which we cannot conceive an exact algorithm. Central to such intelligence is Computer Vision (CV), i.e., extracting meaning from images and video. While not everything needs CV, visual information is the richest source of information about the real world: people, places and things. The possibilities of embedded CV are endless if we consider new applications and technologies, such as deep learning, drones, home robotics, intelligent surveillance, intelligent toys, wearable cameras, etc. This paper describes the Eyes of Things (EoT) platform, a versatile computer vision platform tackling those challenges and opportunities.

8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae.

8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2Δ cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H(+)-pumping pyrophosphatase as an alternative H(+)-pump relieves Vma(-)-like phenotypes in erg2Δ-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H(+)-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2Δ mutants accumulate autophagic bodies under nitrogen starvation similar to Vma(-) mutants. In contrast to classical Atg(-) mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8Δ60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2Δ cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg(-) defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants.

New Trends of Emerging Technologies in Digital Pathology.

The future paradigm of pathology will be digital. Instead of conventional microscopy, a pathologist will perform a diagnosis through interacting with images on computer screens and performing quantitative analysis. The fourth generation of virtual slide telepathology systems, so-called virtual microscopy and whole-slide imaging (WSI), has allowed for the storage and fast dissemination of image data in pathology and other biomedical areas. These novel digital imaging modalities encompass high-resolution scanning of tissue slides and derived technologies, including automatic digitization and computational processing of whole microscopic slides. Moreover, automated image analysis with WSI can extract specific diagnostic features of diseases and quantify individual components of these features to support diagnoses and provide informative clinical measures of disease. Therefore, the challenge is to apply information technology and image analysis methods to exploit the new and emerging digital pathology technologies effectively in order to process and model all the data and information contained in WSI. The final objective is to support the complex workflow from specimen receipt to anatomic pathology report transmission, that is, to improve diagnosis both in terms of pathologists' efficiency and with new information. This article reviews the main concerns about and novel methods of digital pathology discussed at the latest workshop in the field carried out within the European project AIDPATH (Academia and Industry Collaboration for Digital Pathology).

Evaluation of cytokeratin-19 in breast cancer tissue samples: a comparison of automatic and manual evaluations of scanned tissue microarray cylinders.

BACKGROUND: Digital image (DI) analysis avoids visual subjectivity in interpreting immunohistochemical stains and provides more reproducible results. An automated procedure consisting of two variant methods for quantifying the cytokeratin-19 (CK19) marker in breast cancer tissues is presented. METHODS: The first method (A) excludes the holes inside selected CK19 stained areas, and the second (B) includes them. 93 DIs scanned from complete cylinders of tissue microarrays were evaluated visually by two pathologists and by the automated procedures. RESULTS AND CONCLUSIONS: There was good concordance between the two automated methods, both of which tended to identify a smaller CK19-positive area than did the pathologists. The results obtained with method B were more similar to those of the pathologists; probably because it takes into account the entire positive tumoural area, including the holes. However, the pathologists overestimated the positive area of CK19. Further studies are needed to confirm the utility of this automated procedure in prognostic studies.

A Continuous Metabolic Syndrome Score Is Associated with Specific Biomarkers of Inflammation and CVD Risk in Prepubertal Children.

BACKGROUND/AIMS: We aimed to evaluate the use of a continuous metabolic syndrome (MetS) score and to assess the associations of this score with risk biomarkers of inflammation, endothelial damage and cardiovascular disease (CVD) in prepubertal children. METHODS: A total of 677 prepubertal children (295 obese, 146 overweight, and 236 normal-weight) were recruited. MetS traits, markers of inflammation, endothelial damage and CVD risk were measured, and a continuous MetS score was calculated, consisting of the sum/5 of the standardised scores of the MetS components. RESULTS: The continuous MetS score was significantly associated with active plasminogen activator inhibitor-1 (r = 0.406, p < 0.001), adiponectin (r = -0.212, p < 0.001), resistin (r = 0.263, p < 0.001), C-reactive protein (r = 0.254, p < 0.001), tumour necrosis factor alpha (r = 0.120, p = 0.003), myeloperoxidase (r = 0.188, p < 0.001) and sE-selectin (r = 0.278, p < 0.001). Children in the normal-weight, overweight and obese groups with MetS totalled 0 (0%), 1 (0.7%) and 24 (8.7%), respectively, whereas the at-risk children identified using the continuous MetS score in each group totalled 2 (0.85%), 17 (11.6%) and 167 (56.6%), respectively. CONCLUSIONS: The association of the continuous MetS score with specific risk biomarkers of inflammation, endothelial damage and CVD supports its use in the early identification of children at increased risk of metabolic dysfunction.

Inorganic pyrophosphatase defects lead to cell cycle arrest and autophagic cell death through NAD+ depletion in fermenting yeast.

Inorganic pyrophosphatases are required for anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although their exact nature has not been studied at the cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S phase upon Ipp1p deficiency, but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolization and eventual cell death by autophagy. Impaired NAD(+) metabolism is a major determinant of cell death in this scenario because demise can be avoided under conditions favoring accumulation of the oxidized pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the energy metabolism of the cell.

Waist-to-height ratio, inflammation and CVD risk in obese children.

OBJECTIVE: To evaluate the association between waist-to-height ratio (WHtR) and specific biomarkers of inflammation, CVD risk and endothelial dysfunction in prepubertal obese children. DESIGN: Prospective, multicentre case-control study matched by age and sex. SETTING: Children were recruited between May 2007 and May 2010 from primary-care centres and schools in three cities in Spain (Cordoba, Santiago de Compostela and Zaragoza). SUBJECTS: Four hundred and forty-six (223 normal weight and 223 obese) Caucasian prepubertal children aged 6-12 years. RESULTS: WHtR was higher in the obese than in the normal-weight children. Blood pressure, waist circumference, weight, height, insulin, plasma lipids, leptin, resistin, abnormal neutrophil and monocyte counts, C-reactive protein, IL-6, IL-8, TNF-α, myeloperoxidase, soluble intercellular adhesion molecule-1, selectin and plasminogen activator inhibitor-1 levels were higher in the obese than in the normal-weight group. Adiponectin and HDL-cholesterol were lower and glucose and metalloproteinase-9 showed no differences. Resistin, TNF-α and active plasminogen activator inhibitor-1 were associated with WHtR, a sensitive indicator of central obesity. CONCLUSIONS: Our results lead to the hypothesis that changes in biomarker levels of insulin resistance, inflammation and CVD risk before puberty might induce metabolic consequences of obesity in obese children before reaching adulthood.

Firearm-related action recognition and object detection dataset for video surveillance systems.

The proposed dataset is comprised of 398 videos, each featuring an individual engaged in specific video surveillance actions. The ground truth for this dataset was expertly curated and is presented in JSON format (standard COCO), offering vital information about the dataset, video frames, and annotations, including precise bounding boxes outlining detected objects. The dataset encompasses three distinct categories for object detection: "Handgun", "Machine_Gun", and "No_Gun", dependent on the video's content. This dataset serves as a resource for research in firearm-related action recognition, firearm detection, security, and surveillance applications, enabling researchers and practitioners to develop and evaluate machine learning models for the detection of handguns and rifles across various scenarios. The meticulous ground truth annotations facilitate precise model evaluation and performance analysis, making this dataset an asset in the field of computer vision and public safety.

CONSTANS alters the circadian clock in Arabidopsis thaliana.

Plants are sessile organisms that have acquired highly plastic developmental strategies to adapt to the environment. Among these processes, the floral transition is essential to ensure reproductive success and is finely regulated by several internal and external genetic networks. The photoperiodic pathway, which controls the plant response to day length, is one of the most important pathways controlling flowering. In Arabidopsis photoperiodic flowering, CONSTANS (CO) is the central gene activating the expression of the florigen FLOWERING LOCUS T (FT) in the leaves at the end of a long day. The circadian clock strongly regulates CO expression. However, to date, no evidence has been reported regarding a feedback loop from the photoperiod pathway back to the circadian clock. Using transcriptional networks, we have identified relevant network motifs regulating the interplay between the circadian clock and the photoperiod pathway. Gene expression, chromatin immunoprecipitation experiments, and phenotypic analysis allowed us to elucidate the role of CO over the circadian clock. Plants with altered CO expression showed a different internal clock period, measured by daily leaf rhythmic movements. We show that CO can activate key genes related to the circadian clock, such as CCA1, LHY, PRR5, and GI, at the end of a long day by binding to specific sites on their promoters. Moreover, a high number of PRR5 repressed target genes are upregulated by CO, and this could explain the phase transition promoted by CO. The CO-PRR5 complex interacts with the bZIP transcription factor HY5 and helps to localize the complex in the promoters of clock genes. Our results indicate that there may be a feedback loop in which CO communicates back to the circadian clock, providing seasonal information to the circadian system.

Analysis of aberrant splicing and nonsense-mediated decay of the stop codon mutations c.109G>T and c.504_505delCT in 7 patients with HMG-CoA lyase deficiency.

Eukaryotic cells can be protected against mutations that generate stop codons by nonsense-mediated mRNA decay (NMD) and/or nonsense-associated altered splicing (NAS). However, the processes are only partially understood and do not always occur. In this work, we study these phenomena in the stop codon mutations c.109G>T (p.Glu37*) and c.504_505delCT; the second and third most frequent mutations in HMG-CoA lyase deficiency (MIM #246450). The deficiency affects the synthesis of ketone bodies and produces severe disorders during early childhood. We used a minigene approach, real-time quantitative PCR and the inhibition of NMD by puromycin treatment, to study the effect of stop codons on splicing (NAS) and NMD in seven patients. Surprisingly, none of the stop codons studied appears to be the direct cause of aberrant splicing. In the mutation c.109G>T, the splicing is due to the base change G>T at position 109, which is critical and cannot be explained by disruption of exonic splicing enhancer (ESE) elements, by the appearance of exonic splicing silencer (ESS) elements which were predicted by bioinformatic tools or by the stop codons. Moreover, the mutation c.504_505delCT produces two mRNA transcripts both with stop codons that generate simultaneous NMD phenomena. The effects of the mutations studied on splicing seemed to be similar in all the patients. Furthermore, we report a Spanish patient with 3-hydroxy-3-methylglutaric aciduria and a novel missense mutation: c.825C>G (p.Asn275Lys).