A meta-analysis of the activity,stability, and mutational characteristics of temperature-adapted enzymes

Understanding the characteristics that define temperature-adapted enzymes has been a major goal of extremophile enzymology in recent decades. In the present study, we explore these characteristics by comparing psychrophilic, mesophilic, and thermophilic enzymes. Through a meta-analysis of existing data, we show that psychrophilic enzymes exhibit a significantly larger gap (T_g) between their optimum and melting temperatures compared with mesophilic and thermophilic enzymes. These results suggest that T_g may be a useful indicator as to whether an enzyme is psychrophilic or not and that models of psychrophilic enzyme catalysis need to account for this gap. Additionally, by using predictive protein stability software, HoTMuSiC and PoPMuSiC, we show that the deleterious nature of amino acid substitutions to protein stability increases from psychrophiles to thermophiles. How this ultimately affects the mutational tolerance and evolutionary rate of temperature adapted organisms is currently unknown.     

Identification of Discrete Sites in Yip1A Necessary for Regulation of Endoplasmic Reticulum Structure

The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.     

Cell type specificity and host genetic polymorphisms influence antibody-dependent enhancement of dengue virus infection

Antibody-dependent enhancement (ADE) is implicated in severe, usually secondary, dengue virus (DV) infections. Preexisting heterotypic antibodies, via their Fc-gamma receptor (FcγR) interactions, may increase disease severity through enhanced target cell infection. Greater numbers of infected target cells may contribute to higher viremia and excess cytokine levels often observed in severe disease. Monocytes, macrophages, and immature and mature dendritic cells (DC) are considered major cellular targets of DV. Apheresis of multiple donors allowed isolation of autologous primary myeloid target cell types for head-to-head comparison of infection rates, viral output, and cytokine production under direct infection (without antibody) or ADE conditions (with antibody). All studied cell types except immature DC supported ADE. All cells undergoing ADE secreted proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) at enhancement titers, but distinct cell-type-specific patterns were observed for other relevant proteins (alpha/beta interferon [IFN-α/β] and IL-10). Macrophages produced type I interferons (IFN-α/β) that were modulated by ADE. Mature DC mainly secreted IFN-β. Interestingly, only monocytes secreted IL-10, and only upon antibody-enhanced infection. While ADE infection rates were remarkably consistent in monocytes (10 to 15%) across donors, IL-10 protein levels varied according to previously described regulatory single nucleotide polymorphisms (SNPs) in the IL-10 promoter region. The homozygous GCC haplotype was associated with high-level IL-10 secretion, while the ACC and ATA haplotypes produced intermediate and low levels of IL-10, respectively. Our data suggest that ADE effects are cell type specific, are influenced by host genetics, and, depending on relative infection rates, may further contribute to the complexity of DV pathogenesis.     

The effect of ecologically relevant variations in light level on the performance of Mongolian gerbils on two visual tasks

Tests of rodent visual capacities are typically performed under standard laboratory illumination. However, light level can have subtle and complex effects on behavior in rodents. We tested Mongolian gerbils (Meriones unguiculatus) – a species that shows individual differences in activity pattern – on visual tasks at three ecologically relevant levels of ambient illuminance: approximating moonlight (1 lx), dawn or dusk (10 lx), and low daylight (100 lx). A jumping task and a grating discrimination quantified depth perception and grating acuity, respectively. Illuminance variations had important effects on behavior. Gerbils jumped faster in lower light on the depth discrimination task, but were also less accurate than in bright light. Daytime activity levels (possibly reflecting variations in activity pattern) mediated these effects, with animals that were awake during a lower proportion of daytime hours exhibiting a trend toward jumping faster with lower light level as compared to more day-active gerbils. Moreover, while illuminance did not affect acuity overall, it interacted with activity levels in a complex way: in both the darkest and lowest light levels, animals that were awake during a greater proportion of daytime hours showed higher acuity levels than animals that were less active during the day. These results indicate that gerbils show behavioral profiles in vision tasks that represent an interaction between visual ability and illuminance-sensitive motivational or emotional actors, perhaps including chronotype. Thus, the most ecologically relevant assessments of the visual performance of rodents will likely be achieved by testing at species-specific preferred light levels.     

Coordinated evolution of brain size,structure, and eye size in Trinidadian killifish

Brain size, brain architecture, and eye size vary extensively in vertebrates. However, the extent to which the evolution of these components is intricately connected remains unclear. Trinidadian killifish, Anablepsoides hartii, are found in sites that differ in the presence and absence of large predatory fish. Decreased rates of predation are associated with evolutionary shifts in brain size; males from sites without predators have evolved a relatively larger brain and eye size than males from sites with predators. Here, we evaluated the extent to which the evolution of brain size, brain structure, and eye size covary in male killifish. We utilized wild‐caught and common garden‐reared specimens to determine whether specific components of the brain have evolved in response to differences in predation and to determine if there is covariation between the evolution of brain size, brain structure, and eye size. We observed consistent shifts in brain architecture in second generation common garden reared, but not wild caught preserved fish. Male killifish from sites that lack predators exhibited a significantly larger telencephalon, optic tectum, cerebellum, and dorsal medulla when compared with fish from sites with predators. We also found positive connections between the evolution of brain structure and eye size but not between overall brain size and eye size. These results provide evidence for evolutionary covariation between the components of the brain and eye size. Such results suggest that selection, directly or indirectly, acts upon specific regions of the brain, rather than overall brain size, to enhance visual capabilities.     

Inhibition of mutant BRAF splice variant signaling by next‐generation,selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAF^V600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF^V600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAF^V600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF^V600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAF^V600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.     

Assessing nutritional diversity of cropping systems in African villages

Background

In Sub-Saharan Africa, 40% of children under five years in age are chronically undernourished. As new investments and attention galvanize action on African agriculture to reduce hunger, there is an urgent need for metrics that monitor agricultural progress beyond calories produced per capita and address nutritional diversity essential for human health. In this study we demonstrate how an ecological tool, functional diversity (FD), has potential to address this need and provide new insights on nutritional diversity of cropping systems in rural Africa.

Methods and Findings

Data on edible plant species diversity, food security and diet diversity were collected for 170 farms in three rural settings in Sub-Saharan Africa. Nutritional FD metrics were calculated based on farm species composition and species nutritional composition. Iron and vitamin A deficiency were determined from blood samples of 90 adult women. Nutritional FD metrics summarized the diversity of nutrients provided by the farm and showed variability between farms and villages. Regression of nutritional FD against species richness and expected FD enabled identification of key species that add nutrient diversity to the system and assessed the degree of redundancy for nutrient traits. Nutritional FD analysis demonstrated that depending on the original composition of species on farm or village, adding or removing individual species can have radically different outcomes for nutritional diversity. While correlations between nutritional FD, food and nutrition indicators were not significant at household level, associations between these variables were observed at village level.

Conclusion

This study provides novel metrics to address nutritional diversity in farming systems and examples of how these metrics can help guide agricultural interventions towards adequate nutrient diversity. New hypotheses on the link between agro-diversity, food security and human nutrition are generated and strategies for future research are suggested calling for integration of agriculture, ecology, nutrition, and socio-economics.     

Constitutive behavior of ocular tissues over a range of strain rates

The constitutive behavior of bovine scleral and corneal tissues is measured in tension and compression, at quasi-static and moderate strain rates. Experiments are conducted at strain rates up to about 50 strain per second by a pneumatic testing system developed to overcome noise and measurement difficulties associated with the time dependent test of low impedance materials. Results for the tissues at room and the natural bovine body temperatures are similar and indicate that ocular tissue exhibits nonlinear stiffening for increasing strain rates, a phenomena termed rate hardening. For example, at a tensile strain rate of 29/s, corneal tissue is found to develop 10 times the stress that it does quasi-statically at the same strain. Thus, conventional constitutive models will grossly underpredict stresses occurring in the corneo-scleral shell due to moderate dynamic events. This has implication to the accuracy of ocular injury models, the study of the stress field in the corneo-scleral shell for glaucoma research and tonometry measurements. The measured data at various strain rates is represented using the general framework of a constitutive model that has been used to represent biological tissue mechanical data. Here it is extended to represent the measured data of the ocular tissues over the range of tested strain rates. Its form allows for straightforward incorporation in various numerical codes. The experimental and analytical methods developed here are felt to be applicable to the test of human ocular tissue.     

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.