Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.     

Demographic responses to climate‐driven variation in habitat quality across the annual cycle of a migratory bird species

The demography and dynamics of migratory bird populations depend on patterns of movement and habitat quality across the annual cycle. We leveraged archival GPS‐tagging data, climate data, remote‐sensed vegetation data, and bird‐banding data to better understand the dynamics of black‐headed grosbeak (Pheucticus melanocephalus) populations in two breeding regions, the coast and Central Valley of California (Coastal California) and the Sierra Nevada mountain range (Sierra Nevada), over 28 years (1992–2019). Drought conditions across the annual cycle and rainfall timing on the molting grounds influenced seasonal habitat characteristics, including vegetation greenness and phenology (maturity dates). We developed a novel integrated population model with population state informed by adult capture data, recruitment rates informed by age‐specific capture data and climate covariates, and survival rates informed by adult capture–mark–recapture data and climate covariates. Population size was relatively variable among years for Coastal California, where numbers of recruits and survivors were positively correlated, and years of population increase were largely driven by recruitment. In the Sierra Nevada, population size was more consistent and showed stronger evidence of population regulation (numbers of recruits and survivors negatively correlated). Neither region showed evidence of long‐term population trend. We found only weak support for most climate–demographic rate relationships. However, recruitment rates for the Coastal California region were higher when rainfall was relatively early on the molting grounds and when wintering grounds were relatively cool and wet. We suggest that our approach of integrating movement, climate, and demographic data within a novel modeling framework can provide a useful method for better understanding the dynamics of broadly distributed migratory species.     

Quaternary organization of the Staphylothermus marinus phosphoenolpyruvate synthase: angular reconstitution from cryoelectron micrographs with molecular modeling

Digital electron images of frozen-hydrated preparations of the 2.25-MDa Staphylothermus marinus phosphoenolpyruvate synthase (EC 2.7.9.2) have been analyzed by single-particle classification and averaging and iterative quaternion-based angular reconstitution. Contrast transfer function correction of micrographs obtained at different defocus values was used to improve the informational quality of the projection averages. Three-dimensional reconstructions were obtained to roughly 3-nm spatial resolution, in which the 24 identical subunits were arranged to form an octahedral complex, although the amino-terminal nucleotide-binding domain was not resolved. An atomic model of the subunit was generated by homology modeling using as the reference the known X-ray crystallographic structure of the related enzyme pyruvate orthophosphate dikinase (EC 2.7.9.1) from Clostridium symbiosum (Protein Data Bank entry 1DIK). The S. marinus protein could be arranged into an assembly of 12 homodimers to match the three-dimensional reconstruction in terms of shape and size of the homodimers, as well as overall shape and size of the complex. The quaternary model indicated that active sites of three monomers were localized around cavities (or putative channels) centered at the threefold axes of rotational symmetry and that carboxyl-terminal alpha-helical segments of four monomers were localized at the fourfold axes of rotational symmetry where they could facilitate interdimer interaction. The quaternary arrangement also indicated numerous potential hydrophobic and electrostatic interactions at the interdimer interfaces that could contribute further to structural stability.     

Phenology and productivity in a montane bird assemblage: Trends and responses to elevation and climate variation

Climate variation has been linked to historical and predicted future distributions and dynamics of wildlife populations. However, demographic mechanisms underlying these changes remain poorly understood. Here, we assessed variation and trends in climate (annual snowfall and spring temperature anomalies) and avian demographic variables from mist‐netting data (breeding phenology and productivity) at six sites along an elevation gradient spanning the montane zone of Yosemite National Park between 1993 and 2017. We implemented multi‐species hierarchical models to relate demographic responses to elevation and climate covariates. Annual variation in climate and avian demographic variables was high. Snowfall declined (10 mm/year at the highest site, 2 mm at the lowest site), while spring temperature increased (0.045°C/year) over the study period. Breeding phenology (mean first capture date of juvenile birds) advanced by 0.2 day/year (5 days); and productivity (probability of capturing a juvenile bird) increased by 0.8%/year. Breeding phenology was 12 days earlier at the lowest compared to highest site, 18 days earlier in years with lowest compared to highest snowfall anomalies, and 6 d earlier in relatively warm springs (after controlling for snowfall effects). Productivity was positively related to elevation. However, elevation–productivity responses varied among species; species with higher productivity at higher compared to lower elevations tended to be species with documented range retractions during the past century. Productivity tended to be negatively related to snowfall and was positively related to spring temperature. Overall, our results suggest that birds have tracked the variable climatic conditions in this system and have benefited from a trend toward warmer, drier springs. However, we caution that continued warming and multi‐year drought or extreme weather years may alter these relationships in the future. Multi‐species demographic modeling, such as implemented here, can provide an important tool for guiding conservation of species assemblages under global change.     

Influences of the El Niño/Southern Oscillation and the North Atlantic Oscillation on avian productivity in forests of the Pacific Northwest of North America

To model the effects of global climate phenomena on avian population dynamics, we must identify and quantify the spatial and temporal relationships between climate, weather and bird populations. Previous studies show that in Europe, the North Atlantic Oscillation (NAO) influences winter and spring weather that in turn affects resident and migratory landbird species. Similarly, in North America, the El Niño/Southern Oscillation (ENSO) of the Pacific Ocean reportedly drives weather patterns that affect prey availability and population dynamics of landbird species which winter in the Caribbean. Here we show that ENSO‐ and NAO‐induced seasonal weather conditions differentially affect neotropical‐ and temperate‐wintering landbird species that breed in Pacific North‐west forests of North America. For neotropical species wintering in western Mexico, El Niño conditions correlate with cooler, wetter conditions prior to spring migration, and with high reproductive success the following summer. For temperate wintering species, springtime NAO indices correlate strongly with levels of forest defoliation by the larvae of two moth species and also with annual reproductive success, especially among species known to prey upon those larvae. Generalized linear models incorporating NAO indices and ENSO precipitation indices explain 50–90% of the annual variation in productivity reported for 10 landbird species. These results represent an important step towards spatially explicit modelling of avian population dynamics at regional scales.     

Cold-adapted tubulins in the glacier ice worm, Mesenchytraeus solifugus

Glacier ice worms, Mesenchytraeus solifugus and related species, are the only known annelids that survive obligately in glacier ice and snow. One fundamental component of cold temperature adaptation is the ability to polymerize tubulin, which typically depolymerizes at low physiological temperatures (e.g., <10 degrees C) in most temperate species. In this study, we isolated two alpha-tubulin (Msalpha1, Msalpha2) and two beta-tubulin (Msbeta1, Msbeta2) subunits from an ice worm cDNA library, and compared their predicted amino acid sequences with homologues from other cold-adapted organisms (e.g., Antarctic fish, ciliate) in an effort to identify species-specific amino acid substitutions that contribute to cold temperature-dependent tubulin polymerization. Our comparisons and predicted protein structures suggest that ice worm-specific amino acid substitutions stabilize lateral contact associations, particularly between beta-tubulin protofilaments, but these substitutions occur at different positions in comparison with other cold-adapted tubulins. The ice worm tubulin gene family appears relatively small, comprising one primary alpha- and one primary beta-tubulin monomers, though minor isoforms and psuedogenes were identified. Our analyses suggest that variation occurs in the strategies (i.e., species-specific amino acid substitutions, gene number) by which cold-adapted taxa have evolved the ability to polymerize tubulin at low physiological temperatures.     

The SARS-CoV2 envelope differs from host cells,exposes procoagulant lipids,and is disrupted in vivo by oral rinses

The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes. Unlike cellular membranes, procoagulant amino-PLs were present on the external side of the viral envelope at levels exceeding those on activated platelets. Accordingly, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, povidone-iodine, or chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-s oral rinse with cetylpyridinium chloride mouthwash eliminated live virus in the oral cavity of patients with coronavirus disease 19 for at least 1 h, whereas povidone-iodine and saline mouthwashes were ineffective. We conclude that the SARS-CoV-2 lipid envelope i) is distinct from the host plasma membrane, which may enable design of selective antiviral approaches; ii) contains exposed phosphatidylethanolamine and phosphatidylserine, which may influence thrombosis, pathogenicity, and inflammation; and iii) can be selectively targeted in vivo by specific oral rinses.     

Synthesis of a New Template with a Built-in Adjuvant and Its Use in Constructing Peptide Vaccine Candidates Through Polyoxime Chemistry

Synthetic lipopeptides are showing promise as vaccine candidates, but until now it has been very difficult to prepare them in homogeneous form. We describe the synthesis and characterization of a new water-soluble, four-branched template with a built-in lipophilic adjuvant (Pam₃Cys). Through the use of oxime chemistry, we attached four copies of an unprotected influenza virus peptide and characterized the product (13kDa) by reversed-phase HPLC and electrospray ionization mass spectrometry. Several other such constructions were made using the new template and different peptides. We seem to have a general method for making synthetic lipopeptides in homogeneous form.     

Enhancing Proteomic Throughput in Capillary Electrophoresis–Mass Spectrometry by Sequential Sample Injection

In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis–mass spectrometry for rapid and sensitive proteome characterization of human lymphoblastic T‐cells (line CCRF–CEM). Proteins were extracted, enzymatically digested, and the resulting peptides fractionated by RP–HPLC. Twenty fractions were thereafter analyzed by CE–MS within a single MS analysis. The CE–MS method was designed so that every 10 min a new fraction was injected into the CE system. Without any rinsing or equilibration steps we were able to generate a continuous stream of peptides feeding the mass analyzer. In 250 min, the total analysis time of a single sequential injection experiment, we were able to identify roughly 28 000 peptide sequences counting for 4800 proteins. These numbers could be increased to 62 000 peptides and more than 6100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5 h. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from.