Seasonal Dynamics of Atlantic Herring (Clupea harengus L.) Populations Spawning in the Vicinity of Marginal Habitats

Gillnet sampling and analyses of otolith shape, vertebral count and growth indicated the presence of three putative Atlantic herring (Clupea harengus L.) populations mixing together over the spawning season February–June inside and outside an inland brackish water lake (Landvikvannet) in southern Norway. Peak spawning of oceanic Norwegian spring spawners and coastal Skagerrak spring spawners occurred in March–April with small proportions of spawners entering the lake. In comparison, spawning of Landvik herring peaked in May–June with high proportions found inside the lake, which could be explained by local adaptations to the environmental conditions and seasonal changes of this marginal habitat. The 1.85 km² lake was characterized by oxygen depletion occurring between 2.5 and 5 m depth between March and June. This was followed by changes in salinity from 1–7‰ in the 0–1 m surface layer to levels of 20–25‰ deeper than 10 m. In comparison, outside the 3 km long narrow channel connecting the lake with the neighboring fjord, no anoxic conditions were found. Here salinity in the surface layer increased over the season from 10 to 25‰, whereas deeper than 5 m it was stable at around 35‰. Temperature at 0–5 m depth increased significantly over the season in both habitats, from 7 to 14°C outside and 5 to 17°C inside the lake. Despite differences in peak spawning and utilization of the lake habitat between the three putative populations, there was an apparent temporal and spatial overlap in spawning stages suggesting potential interbreeding in accordance with the metapopulation concept.     

FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvβ3 integrin

Osteopontin (OPN) is a ubiquitously expressed, multifunctional, and highly phosphorylated protein. OPN contains two neighboring integrin-binding motifs, RGD and SVVYGLR, which mediate interaction with cells. Phosphorylation and proteolytic processing affect the integrin-binding activities of OPN. Here we report that the kinase, FAM20C, phosphorylates Ser¹⁴⁶ in the ¹⁴³RGDSVVYGLR¹⁵² motif of OPN and that Ser¹⁴⁶ is phosphorylated in vivo in human and bovine milk. Ser¹⁴⁶ is located right next to the RGD motif and close by the regulatory thrombin and plasmin cleavage sites in the OPN sequence. Phosphorylation of Ser¹⁴⁶ could potentially affect the proteolytic processing and the integrin-binding activities of OPN. We show that phosphorylation of Ser¹⁴⁶ does not affect the susceptibility of OPN for thrombin or plasmin cleavage. However, phosphorylation of Ser¹⁴⁶ significantly reduces the RGD-mediated interaction with the α_vβ₃ integrin in MDA-MB-435 and Moαv cells. This suggests a new mechanism by which specific phosphorylation of OPN can regulate interaction with the α_vβ₃ integrin and thereby affect OPN-cell interaction.     

High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

Background

Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.

Methods

Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).

Results

To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.

Conclusions

We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.

General significance

The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.     

Effects of exercise on VLDL-triglyceride oxidation and turnover

Lipids are important substrates for oxidation at rest and during exercise. Aerobic exercise mediates a delayed onset decrease in total and VLDL-triglyceride (TG) plasma concentration. However, the acute effects of exercise on VLDL-TG oxidation and turnover remain unclear. Here, we studied the acute effects of 90 min of moderate-intensity exercise in healthy women and men. VLDL-TG kinetics were assessed using a primed constant infusion of ex vivo labeled [1-(14)C]triolein VLDL-TG. Fractional VLDL-TG-derived fatty acid oxidation was measured from (14)CO(2) specific activity in expired air. VLDL-TG concentration was unaltered during exercise and early recovery, whereas non-VLDL-TG concentration decreased significantly.VLDL-TG secretion rate decreased significantly during exercise and remained suppressed during recovery. Total VLDL-TG oxidation rate was unaffected by exercise. However, the contribution of VLDL-TG oxidation to total energy expenditure fell from 14 ± 9% at rest to 3 ± 4% during exercise. We conclude that VLDL-TG fatty acids are quantitatively important oxidative substrates under basal postabsorptive conditions but remain unaffected during 90-min moderate-intensity exercise and, thus, become relatively less important during exercise. Lower VLDL secretion rate during exercise may contribute to the decrease in TG concentrations during and after exercise.     

Fitness landscape of Atlantic cod shaped by harvest selection and natural selection

Harvesting may lead to evolutionary changes in life histories on a contemporary time scale, changes that could be maladaptive in natural contexts. However, our understanding of the strength and direction of harvest-induced selection versus natural selection is still limited, partly due to the difficulty of tracking the fate of individuals in the wild. Here, we present direct estimates of harvest mortality, natural mortality and site fidelity of coastal Atlantic cod (Gadus morhua) from the Norwegian Skagerrak coast. Furthermore, we present standardised selection differentials for fish body size. Estimates are obtained from acoustic telemetry, where we continuously monitored fish (n = 60) within a semi-sheltered area using a network of 25 listening stations. To obtain additional information about harvested cod, all fish (body size: 30–66 cm) were also tagged with traditional T-bar tags with a printed reward of 500 NOK (60 E). We estimate that 75% of the fish died within the study area during 1 year. Fishing mortality was markedly higher than natural mortality. Together, recreational fishers and commercial fishers caught at least 50% of the tagged fish during 1 year. Standardised selection differentials showed that fisheries targeted larger fish (i.e. favoured the survival of smaller fish), while natural selection favoured the survival of larger fish. Albeit on a small scale, we provide empirical evidence that harvesting can have a dominant influence on the fitness landscape experienced by a marine fish such as the Atlantic cod. We suggest that no-take marine reserves may help to counter evolutionary impacts of harvesting in the ocean.     

Analysis of mortality metrics associated with a comprehensive range of disorders in Denmark, 2000 to 2018: A population-based cohort study

BackgroundThe provision of different types of mortality metrics (e.g., mortality rate ratios [MRRs] and life expectancy) allows the research community to access a more informative set of health metrics. The aim of this study was to provide a panel of mortality metrics associated with a comprehensive range of disorders and to design a web page to visualize all results.Methods and findingsIn a population-based cohort of all 7,378,598 persons living in Denmark at some point between 2000 and 2018, we identified individuals diagnosed at hospitals with 1,803 specific categories of disorders through the International Classification of Diseases-10th Revision (ICD-10) in the National Patient Register. Information on date and cause of death was obtained from the Registry of Causes of Death. For each of the disorders, a panel of epidemiological and mortality metrics was estimated, including incidence rates, age-of-onset distributions, MRRs, and differences in life expectancy (estimated as life years lost [LYLs]). Additionally, we examined models that adjusted for measures of air pollution to explore potential associations with MRRs. We focus on 39 general medical conditions to simplify the presentation of results, which cover 10 broad categories: circulatory, endocrine, pulmonary, gastrointestinal, urogenital, musculoskeletal, hematologic, mental, and neurologic conditions and cancer. A total of 3,676,694 males and 3,701,904 females were followed up for 101.7 million person-years. During the 19-year follow-up period, 1,034,273 persons (14.0%) died. For 37 of the 39 selected medical conditions, mortality rates were larger and life expectancy shorter compared to the Danish general population. For these 37 disorders, MRRs ranged from 1.09 (95% confidence interval [CI]: 1.09 to 1.10) for vision problems to 7.85 (7.77 to 7.93) for chronic liver disease, while LYLs ranged from 0.31 (0.14 to 0.47) years (approximately 16 weeks) for allergy to 17.05 (16.95 to 17.15) years for chronic liver disease. Adjustment for air pollution had very little impact on the estimates; however, a limitation of the study is the possibility that the association between the different disorders and mortality could be explained by other underlying factors associated with both the disorder and mortality.ConclusionsIn this study, we show estimates of incidence, age of onset, age of death, and mortality metrics (both MRRs and LYLs) for a comprehensive range of disorders. The interactive data visualization site (https://nbepi.com/atlas) allows more fine-grained analysis of the link between a range of disorders and key mortality estimates.

In a population-based study, Oleguer Plana-Ripoll and colleagues report on and develop an online resource to study mortality metrics and life expectancy associated with different health conditions among individuals living in Denmark.     

Increased hypothalamic-pituitary-adrenal axis activity and hepatic insulin resistance in low-birth-weight rats

Individuals born with a low birth weight (LBW) have an increased prevalence of type 2 diabetes, but the mechanisms responsible for this association are unknown. Given the important role of insulin resistance in the pathogenesis of type 2 diabetes, we examined insulin sensitivity in a rat model of LBW due to intrauterine fetal stress. During the last 7 days of gestation, rat dams were treated with dexamethasone and insulin sensitivity was assessed in the LBW offspring by a hyperinsulinemic euglycemic clamp. The LBW group had liver-specific insulin resistance associated with increased levels of PEPCK expression. These changes were associated with pituitary hyperplasia of the ACTH-secreting cells, increased morning plasma ACTH concentrations, elevated corticosterone secretion during restraint stress, and an approximately 70% increase in 24-h urine corticosterone excretion. These data support the hypothesis that prenatal stress can result in chronic hyperactivity of the hypothalamic-pituitary-adrenal axis, resulting in increased plasma corticosterone concentrations, upregulation of hepatic gluconeogenesis, and hepatic insulin resistance.     

Evidence for Restricted Reactivity of ADAMDEC1 with Protein Substrates and Endogenous Inhibitors

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1′ position. Cys³⁹², present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys³⁹² for a Ser increased the reactivity with α₂-macroglobulin but not with casein or Cm-Tf. Substitution of Asp³⁶² for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.     

Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52_330–370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52_281–329 in a complex that interacts directly with IFT70/IFT52_330–381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.     

Revisiting the structure of the Vps10 domain of human sortilin and its interaction with neurotensin

Sortilin is a multifunctional receptor involved in sorting and apoptosis. We have previously reported a 2.0‐Å structure of the Vps10 ectodomain in complex with one of its ligands, the tridecapeptide neurotensin. Here we set out to further characterize the structural properties of sortilin and its interaction with neurotensin. To this end, we have determined a new 2.7 Å structure using a crystal grown with a 10‐fold increased concentration of neurotensin. Here a second peptide fragment was observed within the Vps10 β‐propeller, which may in principle either represent a second molecule of neurotensin or the N‐terminal part of the molecule bound at the previously identified binding site. However, in vitro binding experiments strongly favor the latter hypothesis. Neurotensin thus appears to bind with a 1:1 stoichiometry, and whereas the N‐terminus does not bind on its own, it enhances the affinity in context of full‐length neurotensin. We conclude that the N‐terminus of neurotensin probably functions as an affinity enhancer for binding to sortilin by engaging the second binding site. Crystal packing differs partly from the previous structure, which may be due to variations in the degree and pattern of glycosylations. Consequently, a notable hydrophobic loop, not modeled previously, could now be traced. A computational analysis suggests that this and a neighboring loop may insert into the membrane and thus restrain movement of the Vps10 domain. We have, furthermore, mapped all N‐linked glycosylations of CHO‐expressed human sortilin by mass spectrometry and find that their locations are compatible with membrane insertion of the hydrophobic loops.