Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.     

Systematic review of effective strategies for reducing screen time among young children

Screen-media use among young children is highly prevalent, disproportionately high among children from lower-income families and racial/ethnic minorities, and may have adverse effects on obesity risk. Few systematic reviews have examined early intervention strategies to limit TV or total screen time; none have examined strategies to discourage parents from putting TVs in their children's bedrooms or remove TVs if they are already there. In order to identify strategies to reduce TV viewing or total screen time among children <12 years of age, we conducted a systematic review of seven electronic databases to June 2011, using the terms "intervention" and "television," "media," or "screen time." Peer-reviewed intervention studies that reported frequencies of TV viewing or screen-media use in children under age 12 were eligible for inclusion. We identified 144 studies; 47 met our inclusion criteria. Twenty-nine achieved significant reductions in TV viewing or screen-media use. Studies utilizing electronic TV monitoring devices, contingent feedback systems, and clinic-based counseling were most effective. While studies have reduced screen-media use in children, there are several research gaps, including a relative paucity of studies targeting young children (n = 13) or minorities (n = 14), limited long-term (>6 month) follow-up data (n = 5), and few (n = 4) targeting removing TVs from children's bedrooms. Attention to these issues may help increase the effectiveness of existing strategies for screen time reduction and extend them to different populations.     

Improving predictions of tropical forest response to climate change through integration of field studies and ecosystem modeling

Tropical forests play a critical role in carbon and water cycles at a global scale. Rapid climate change is anticipated in tropical regions over the coming decades and, under a warmer and drier climate, tropical forests are likely to be net sources of carbon rather than sinks. However, our understanding of tropical forest response and feedback to climate change is very limited. Efforts to model climate change impacts on carbon fluxes in tropical forests have not reached a consensus. Here, we use the Ecosystem Demography model (ED2) to predict carbon fluxes of a Puerto Rican tropical forest under realistic climate change scenarios. We parameterized ED2 with species‐specific tree physiological data using the Predictive Ecosystem Analyzer workflow and projected the fate of this ecosystem under five future climate scenarios. The model successfully captured interannual variability in the dynamics of this tropical forest. Model predictions closely followed observed values across a wide range of metrics including aboveground biomass, tree diameter growth, tree size class distributions, and leaf area index. Under a future warming and drying climate scenario, the model predicted reductions in carbon storage and tree growth, together with large shifts in forest community composition and structure. Such rapid changes in climate led the forest to transition from a sink to a source of carbon. Growth respiration and root allocation parameters were responsible for the highest fraction of predictive uncertainty in modeled biomass, highlighting the need to target these processes in future data collection. Our study is the first effort to rely on Bayesian model calibration and synthesis to elucidate the key physiological parameters that drive uncertainty in tropical forests responses to climatic change. We propose a new path forward for model‐data synthesis that can substantially reduce uncertainty in our ability to model tropical forest responses to future climate.     

Molecular insights into the evolution of an enzyme; esterase6 in Drosophila

It is still a suspicion among some evolutionary biologists that the incursion of molecular biology into their field will do little more than determine the DNA sequence differences underlying evolutionary changes already evident at the organismal level. Work on an esterase enzyme involved in the reproductive biology of Drosophila belies this view. Although it is already one of the most intensively studied gene - enzyme systems at an organismal level, recent molecular invetigations reveal several unexpected, and, in some cases, still inexplicable phenomena in its evolutionary history.     

Molecular and genetic studies on the euchromatin-heterochromatin transition region of the X chromosome of Drosophila melanogaster

A recombinant Charon 4 bacteriophage has been isolated on the basis of RNAs which are enriched in the head of the adult Drosophila melanogaster and hence are likely to be of neural origin. The cloned insert maps to the near vicinity of the uncoordinated locus in polytene chromosome band 19E8. This band is within the transition zone between the euchromatic and heterochromatic regions of the X chromosome, a region which has been well characterized cytogenetically. The insert contains both repetitious and low copy number sequences, some of which vary extensively in both frequency and restriction fragment size between different laboratory strains. One particular family of moderately repeated sequences occurs predominantly in divisions 19 and 20 of the X chromosome and perhaps the distally located X heterochromatin. The molecular landscape surrounding the initial entry point contains many repeated sequences and is thus unlike those observed in most published chromosomal walks. The possible significance of the presence of repeated sequence families in the distinct properties of this region are discussed.     

AN IMPROVED CHEMICALLY DEFINED BASAL MEDIUM (CMRL-1415) FOR NEWLY EXPLANTED MOUSE EMBRYO CELLS

An improved chemically defined medium, CMRL-1415, has been devised by testing the response of trypsinized, newly explanted mouse embryo cells in stationary cultures to various modifications of an earlier medium, CMRL-1066. The improvements are attributed to changes in amino acid levels, in the vitamin-coenzyme composition, and to an enhanced buffering capacity resulting from the use of free base amino acids, galactose and pyruvate, together with greatly reduced levels of glucose and sodium bicarbonate and the inclusion of both monobasic and dibasic sodium phosphate in the ratio 1:4. When the medium is equilibrated with 5% CO₂ in air, an initial pH of 7.2–7.4 is achieved, with excellent buffering capacity. CMRL-1415 contains 50 ingredients (9 fewer than CMRL-1066) and is prepared from six stable stock concentrates. By omitting sodium bicarbonate (to give CMRL-1415-ATM), the medium may be used in unsealed cultures in free gas exchange with air. CMRL-1415 and CMRL-1415-ATM are intended for use with and without serum protein and other supplements; and by preparing them double strength they may be combined with agar or other gelling agents to provide a semi-solid substrate.     

Restoring depleted coral‐reef fish populations through recruitment enhancement: a proof of concept

To determine whether enhancing the survival of new recruits is a sensible target for the restorative management of depleted coral‐reef fish populations, settlement‐stage ambon damsel fish Pomacentrus amboinensis were captured, tagged and then either released immediately onto small artificial reefs or held in aquaria for 1 week prior to release. Holding conditions were varied to determine whether they affected survival of fish: half the fish were held in bare tanks (non‐enriched) and the other half in tanks containing coral and sand (enriched). Holding fish for this short period had a significantly positive effect on survivorship relative to the settlement‐stage treatment group that were released immediately. The enrichment of holding conditions made no appreciable difference on the survival of fish once released onto the reef. It did, however, have a positive effect on the survival of fish while in captivity, thus supporting the case for the provision of simple environmental enrichment in fish husbandry. Collecting and holding settlement‐stage fish for at least a week before release appear to increase the short‐term survival of released fish; whether it is an effective method for longer‐term enhancement of locally depleted coral‐reef fish populations will require further study.     

Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance because of frequent natural exposure and their use in medical applications. Depending on the preparation studied and the end point measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. We prepared highly purified chitosan and chitin and examined the capacity of these glycans to stimulate murine macrophages to release the inflammasome-associated cytokine IL-1β. We found that although chitosan was a potent NLRP3 inflammasome activator, acetylation of the chitosan to chitin resulted in a near total loss of activity. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL-1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. In contrast, chitin is relatively inert.     

Molecular Analysis of the P-M Gonadal Dysgenesis Cline in Eastern Australian Drosophila Melanogaster

The latitudinal cline in P-M gonadal dysgenesis potential in eastern Australia has been shown to comprise three regions which are, from north to south respectively, P, Q, and M, with the P-to-Q and Q-to-M transitions occurring over relatively short distances. The P element complements of 30 lines from different regions of the cline were determined by molecular techniques. The total amount of P element-hybridizing DNA was high in all lines, and it did not correlate in any obvious way with the P-M phenotypes of individual lines. The number of potentially full-sized P elements per genome was high in lines from the P regions, but variable or low among lines from the Q and M regions, and thus declined overall from north to south. A particular P element deletion-derivative, the KP element, occurred in all the tested lines. The number of KP elements was low in lines from the P region, much higher in lines from the Q region, and highest among lines from the M region, thus forming a cline reciprocal to that of the full-sized P elements. Another transposable element, hobo, which has been described as causing dysgenic traits similar to those of P-M hybrid dysgenesis, was shown to be present in all lines and to vary among them in number, but not in any latitudinal pattern. The P-M cline in gonadal dysgenesis potential can be inferred to be based on underlying clinal patterns of genomic P element complements. P activity of a line was positively correlated with the number of full-sized P elements in the line, and negatively correlated with the number of KP elements. Among Q and M lines, regulatory ability was not correlated with numbers of KP elements.     

Human Dendritic Cell DC-SIGN and TLR-2 Mediate Complementary Immune Regulatory Activities in Response to Lactobacillus rhamnosus JB-1

The microbiota is required for optimal host development and ongoing immune homeostasis. Lactobacilli are common inhabitants of the mammalian large intestine and immunoregulatory effects have been described for certain, but not all, strains. The mechanisms underpinning these protective effects are beginning to be elucidated. One such protective organism is Lactobacillus rhamnosus JB-1 (Lb. rhamnosus JB-1). Lb. murinus has no such anti-inflammatory protective effects and was used as a comparator organism. Human monocyte-derived dendritic cells (MDDCs) were co-incubated with bacteria and analysed over time for bacterial adhesion and intracellular processing, costimulatory molecule expression, cytokine secretion and induction of lymphocyte polarization. Neutralising antibodies were utilized to identify the responsible MDDC receptors. Lb. rhamnosus JB-1 adhered to MDDCs, but internalization and intracellular processing was significantly delayed, compared to Lb. murinus which was rapidly internalized and processed. Lb. murinus induced CD80 and CD86 expression, accompanied by high levels of cytokine secretion, while Lb. rhamnosus JB-1 was a poor inducer of costimulatory molecule expression and cytokine secretion. Lb. rhamnosus JB-1 primed MDDCs induced Foxp3 expression in autologous lymphocytes, while Lb. murinus primed MDDCs induced Foxp3, T-bet and Ror-γt expression. DC-SIGN was required for Lb. rhamnosus JB-1 adhesion and influenced IL-12 secretion, while TLR-2 influenced IL-10 and IL-12 secretion. Here we demonstrate that the delayed kinetics of bacterial processing by MDDCs correlates with MDDC activation and stimulation of lymphocytes. Thus, inhibition or delay of intracellular processing may be a novel strategy by which certain commensals may avoid the induction of proinflammatory responses.