Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

Elevated summer temperatures delay spawning and reduce redd construction for resident brook trout (Salvelinus fontinalis)

Redd (nest) surveys for resident brook trout (Salvelinus fontinalis) were conducted annually in a mountain lake in northern New York for 11 years with multiple surveys conducted during the spawning season in eight of those years. Repeated surveys throughout the spawning season allowed us to fit an individually based parametric model and estimate the day of year on which spawning was initiated, reached its midpoint, and ended during each year. Spawning phenology was then assessed relative to (1) mean of maximum daily air temperature and (2) mean of maximum daily water temperature at the lake bottom during summer in each year using a linear model. Elevated temperatures in summer were correlated with a delay in spawning and a reduction in the total number of redds constructed. Increasing the summer mean of maximum daily air temperatures by 1 °C delayed spawning by approximately 1 week and decreased the total number of redds constructed by nearly 65. Lake spawning brook trout select redd sites based on the presence of discharging groundwater that is relatively constant in temperature within and across years, leading to relatively consistent egg incubation times. Therefore, delayed spawning is likely to delay fry emergence, which could influence emergence synchrony with prey items. This work highlights non‐lethal and sub‐lethal effects of elevated summer temperatures on native resident salmonids in aquatic environments with limited thermal refugia.     

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein's activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests to determine whether specific proteins colocalize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein colocalization with specific lipid species.     

The Aromatase Gene (CYP19A1) Variants and Circulating Hepatocyte Growth Factor in Postmenopausal Women

Background

Estrogen and androgen have been linked to the regulation of circulating hepatocyte growth factor (HGF), an adipose tissue-derived cytokine. It is possible that the CYP19A1 gene which alters sex hormones production may influence HGF levels. We examined the association between the CYP19A1 gene variants and plasma HGF concentrations.

Design

We evaluated 45 common and putative functional variants of CYP19A1 and circulating levels of HGF among 260 postmenopausal women who later developed colorectal cancer from the Women''s Health Initiative Observational Cohort. As the distribution of HGF levels was highly skewed, we transformed HGF concentrations for all women into a log-, ranked-, or normal score-scale value. Multiple linear regression with adjustment for age was used to evaluate the associations.

Results

We observed an association between the rs7172156, rs1008805, rs6493494, rs749292, and rs11636639 variants and HGF levels in ranked and normal score scales (corrected p values ≤0.02), although the association of these 5 SNPs with log-scale HGF was not significant (corrected p values ≥0.16). The associations remained unchanged after additional adjustment for hormone therapy use and estradiol levels. These 5 SNPs, which were in linkage disequilibrium (pairwise D′≥97%, r²≥56%), constituted a block with 2 common haplotypes accounting for 82% frequency. The most common haplotype, TCCCA, was associated with lower ranked- or normal score-transformed HGF levels (corrected p values ≤0.001), whereas the second most common haplotype, CTTCA, was associated with higher ranked- or normal score-transformed HGF levels (corrected p values ≤0.02).

Conclusion

Our findings of a potential association between the CYP19A1 variants and circulating HGF levels warrant confirmation in studies with larger sample size.     

Genome-wide association study of circulating estradiol, testosterone, and sex hormone-binding globulin in postmenopausal women

Genome-wide association studies (GWAS) have successfully identified common genetic variants that contribute to breast cancer risk. Discovering additional variants has become difficult, as power to detect variants of weaker effect with present sample sizes is limited. An alternative approach is to look for variants associated with quantitative traits that in turn affect disease risk. As exposure to high circulating estradiol and testosterone, and low sex hormone-binding globulin (SHBG) levels is implicated in breast cancer etiology, we conducted GWAS analyses of plasma estradiol, testosterone, and SHBG to identify new susceptibility alleles. Cancer Genetic Markers of Susceptibility (CGEMS) data from the Nurses' Health Study (NHS), and Sisters in Breast Cancer Screening data were used to carry out primary meta-analyses among ~1600 postmenopausal women who were not taking postmenopausal hormones at blood draw. We observed a genome-wide significant association between SHBG levels and rs727428 (joint β = -0.126; joint P = 2.09 × 10(-16)), downstream of the SHBG gene. No genome-wide significant associations were observed with estradiol or testosterone levels. Among variants that were suggestively associated with estradiol (P<10(-5)), several were located at the CYP19A1 gene locus. Overall results were similar in secondary meta-analyses that included ~900 NHS current postmenopausal hormone users. No variant associated with estradiol, testosterone, or SHBG at P<10(-5) was associated with postmenopausal breast cancer risk among CGEMS participants. Our results suggest that the small magnitude of difference in hormone levels associated with common genetic variants is likely insufficient to detectably contribute to breast cancer risk.     

Persurf,a New Method to Improve Surfactant Delivery: A Study in Surfactant Depleted Rats

Purpose

Exogenous surfactant is not very effective in adults with ARDS, since surfactant does not reach atelectatic alveoli. Perfluorocarbons (PFC) can recruit atelectatic areas but do not replace impaired endogenous surfactant. A surfactant-PFC-mixture could combine benefits of both therapies. The aim of the proof-of-principal-study was to produce a PFC-in-surfactant emulsion (Persurf) and to test in surfactant depleted Wistar rats whether Persurf achieves I.) a more homogenous pulmonary distribution and II.) a more homogenous recruitment of alveoli when compared with surfactant or PFC alone.

Methods

Three different PFC were mixed with surfactant and phospholipid concentration in the emulsion was measured. After surfactant depletion, animals either received 30 ml/kg of PF5080, 100 mg/kg of stained (green dye) Curosurf™ or 30 ml/kg of Persurf. Lungs were fixated after 1 hour of ventilation and alveolar aeration and surfactant distribution was estimated by a stereological approach.

Results

Persurf contained 3 mg/ml phospholipids and was stable for more than 48 hours. Persurf-administration improved oxygenation. Histological evaluation revealed a more homogenous surfactant distribution and alveolar inflation when compared with surfactant treated animals.

Conclusions

In surfactant depleted rats administration of PFC-in-surfactant emulsion leads to a more homogenous distribution and aeration of the lung than surfactant alone.     

Combining the Finite Element Method with Structural Connectome-based Analysis for Modeling Neurotrauma: Connectome Neurotrauma Mechanics

This article presents the integration of brain injury biomechanics and graph theoretical analysis of neuronal connections, or connectomics, to form a neurocomputational model that captures spatiotemporal characteristics of trauma. We relate localized mechanical brain damage predicted from biofidelic finite element simulations of the human head subjected to impact with degradation in the structural connectome for a single individual. The finite element model incorporates various length scales into the full head simulations by including anisotropic constitutive laws informed by diffusion tensor imaging. Coupling between the finite element analysis and network-based tools is established through experimentally-based cellular injury thresholds for white matter regions. Once edges are degraded, graph theoretical measures are computed on the "damaged" network. For a frontal impact, the simulations predict that the temporal and occipital regions undergo the most axonal strain and strain rate at short times (less than 24 hrs), which leads to cellular death initiation, which results in damage that shows dependence on angle of impact and underlying microstructure of brain tissue. The monotonic cellular death relationships predict a spatiotemporal change of structural damage. Interestingly, at 96 hrs post-impact, computations predict no network nodes were completely disconnected from the network, despite significant damage to network edges. At early times ([Formula: see text]) network measures of global and local efficiency were degraded little; however, as time increased to 96 hrs the network properties were significantly reduced. In the future, this computational framework could help inform functional networks from physics-based structural brain biomechanics to obtain not only a biomechanics-based understanding of injury, but also neurophysiological insight.