The Light Responsive Transcriptome of the Zebrafish: Function and Regulation

Most organisms possess circadian clocks that are able to anticipate the day/night cycle and are reset or “entrained” by the ambient light. In the zebrafish, many organs and even cultured cell lines are directly light responsive, allowing for direct entrainment of the clock by light. Here, we have characterized light induced gene transcription in the zebrafish at several organizational levels. Larvae, heart organ cultures and cell cultures were exposed to 1- or 3-hour light pulses, and changes in gene expression were compared with controls kept in the dark. We identified 117 light regulated genes, with the majority being induced and some repressed by light. Cluster analysis groups the genes into five major classes that show regulation at all levels of organization or in different subset combinations. The regulated genes cover a variety of functions, and the analysis of gene ontology categories reveals an enrichment of genes involved in circadian rhythms, stress response and DNA repair, consistent with the exposure to visible wavelengths of light priming cells for UV-induced damage repair. Promoter analysis of the induced genes shows an enrichment of various short sequence motifs, including E- and D-box enhancers that have previously been implicated in light regulation of the zebrafish period2 gene. Heterologous reporter constructs with sequences matching these motifs reveal light regulation of D-box elements in both cells and larvae. Morpholino-mediated knock-down studies of two homologues of the D-box binding factor Tef indicate that these are differentially involved in the cell autonomous light induction in a gene-specific manner. These findings suggest that the mechanisms involved in period2 regulation might represent a more general pathway leading to light induced gene expression

Complement C1 Esterase Inhibitor Levels Linked to Infections and Contaminated Heparin-Associated Adverse Events

Activation of kinin-kallikrein and complement pathways by oversulfated-chondroitin-sulfate (OSCS) has been linked with recent heparin-associated adverse clinical events. Given the fact that the majority of patients who received contaminated heparin did not experience an adverse event, it is of particular importance to determine the circumstances that increase the risk of a clinical reaction. In this study, we demonstrated by both the addition and affinity depletion of C1inh from normal human plasma, that the level of C1inh in the plasma has a great impact on the OSCS-induced kallikrein activity and its kinetics. OSCS-induced kallikrein activity was dramatically increased after C1inh was depleted, while the addition of C1inh completely attenuated kallikrein activity. In addition, actual clinical infection can lead to increased C1inh levels. Plasma from patients with sepsis had higher average levels of functional C1inh and decreased OSCS-induced kallikrein activity. Lastly, descriptive data on adverse event reports suggest cases likely to be associated with contaminated heparin are inversely correlated with infection. Our data suggest that low C1inh levels can be a risk factor and high levels can be protective. The identification of risk factors for contact system-mediated adverse events may allow for patient screening and clinical development of prophylaxis and treatments

Impact of Daily Thermocycles on Hatching Rhythms, Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled "gating" mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation

Rate-invariant analysis of covariance trajectories

Statistical analysis of dynamic systems, such as videos and dynamic functional connectivity, is often translated into a problem of analyzing trajectories of relevant features, particularly covariance matrices. As an example, in video-based action recognition, a natural mathematical representation of activity videos is as parameterized trajectories on the set of symmetric, positive-definite matrices (SPDMs). The variable execution-rates of actions, implying arbitrary parameterizations of trajectories, complicates their analysis and classification. To handle this challenge, we represent covariance trajectories using transported square-root vector fields (TSRVFs), constructed by parallel translating scaled-velocity vectors of trajectories to their starting points. The space of such representations forms a vector bundle on the SPDM manifold. Using a natural Riemannian metric on this vector bundle, we approximate geodesic paths and geodesic distances between trajectories in the quotient space of this vector bundle. This metric is invariant to the action of the reparameterization group, and leads to a rate-invariant analysis of trajectories. In the process, we remove the parameterization variability and temporally register trajectories during analysis. We demonstrate this framework in multiple contexts, using both generative statistical models and discriminative data analysis. The latter is illustrated using several applications involving video-based action recognition and dynamic functional connectivity analysis

Barrier Tissue Macrophages: Functional Adaptation to Environmental Challenges

Macrophages are found throughout the body, where they have crucial roles in tissue development, homeostasis and remodeling, as well as being sentinels of the innate immune system that can contribute to protective immunity and inflammation. Barrier tissues, such as the intestine, lung, skin and liver, are exposed constantly to the outside world, which places special demands on resident cell populations such as macrophages. Here we review the mounting evidence that although macrophages in different barrier tissues may be derived from distinct progenitors, their highly specific properties are shaped by the local environment, which allows them to adapt precisely to the needs of their anatomical niche. We discuss the properties of macrophages in steady-state barrier tissues, outline the factors that shape their differentiation and behavior and describe how macrophages change during protective immunity and inflammation

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Sensitive valence structures of [(pap)(2)ru(q)](n) (n =+2,+1, 0,-1,-2) with two different redox noninnocent ligands, q=3,5-di-tert-butyl-n-aryl-1,2-benzoquinonemonoimine and pap=2-phenylazopyridine

The complexes [(pap)2Ru(Q)]ClO4, [1]ClO4-[4]ClO4, with two different redox noninnocent ligands, Q = 3,5-di-tert-butyl-N-aryl-1,2-benzoquinonemonoimine (-aryl = m-(Cl)2C6H3 (1+), C6H5 (2+), m-(OCH3)2C6H3 (3+), and m-(tBu)2C6H3 (4+)) and pap = 2-phenylazopyridine, have been synthesized and characterized using various analytical techniques. The single-crystal X-ray structure of the representative [2]ClO4.C7H8 exhibits multiple intermolecular C-H...O hydrogen bondings and C-H...p interactions. The C1-O1 = 1.287(4) (density functional theory, DFT, 1.311) and C6-N1 = 1.320(4) (DFT, 1.353) Å and intraring bond distances associated with the sensitive quinine (Q) moiety along with the azo(pap) bond distances, N3-N4 = 1.278(4) (DFT, 1.297) and N6-N7 = 1.271(4) (DFT, 1.289)Å , in 2+ justify the [(pap)2RuII(Q·-)]+ valence configuration at the native state of 1+-4+. Consequently, Mulliken spin densities on Q, pap, and Ru in 2+ are calculated to be 0.8636, 0.1040, and 0.0187, respectively, and 1+-4+ exhibit free radical sharp EPR spectra and one weak and broad transition around 1000 nm in CH3CN due to interligand transition involving a singly occupied molecular orbital (SOMO) of Q·- and the vacant π orbital of pap. Compounds 1+-4+ undergo a quasi-reversible oxidation and three successive reductions. The valence structure of the electron paramagnetic resonance (EPR)-inactive oxidized state in 12+-42+ has been established as [(pap)2RuII(Q°)]2+ instead of the alternate formalism of antiferromagnetically coupled [(pap)2RuIII(Q·-)]2+ on the basis of the DFT calculations on the optimized 2+, which predict that the singly occupied molecular orbital is primarily composed of Q with 77% contribution. Accordingly, the optimized structure of 22+ predicts shorter C1-O1 (1.264) and C6-N1 (1.317 Å) distances and longer Ru1-O1 (2.080) and Ru1-N1 (2.088 Å) distances. Compounds 12+-42+ exhibit the lowest energy transitions around 600 nm, corresponding to Ru(dπ )/Q(π )→pap(π). The presence of two sets of strongly p-acceptor ligands, pap and Q, in 12+-42+ stabilizes the Ru(II) state to a large extent such that the further oxidation of {RuII-Q°}→{RuIII-Q°} has not been detected within +2.0 V versus a saturated calomel electrode. The EPR-inactive reduced states 1-4 have been formulated as [(pap)2RuII(Q2-)] over the antiferromagnetically coupled alternate configuration, [(pap)(pap·-)RuII(Q·-)]. The optimized structure of 2 predicts sensitive C1-O1 and C6-N1 bond distances of 1.337 and 1.390 Å, respectively, close to the doubly reduced Q2- state, whereas the N-N distances of pap, N3-N4 = 1.299 and N6-N7 = 1.306 Å, remain close to the neutral state. In corroboration with the doubly reduced Q2- state, 1-4 exhibit a moderately strong interligand p(Q2-) → π (pap) transition in the near-IR region near 1300 nm. The subsequent two reductions are naturally centered around the azo functions of the pap ligands