Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.     

Insulin binding to the analytical antibody sandwich pair OXI‐005 and HUI‐018: Epitope mapping and binding properties

The insulin epitopes for two monoclonal antibodies (mAbs), OXI‐005 and HUI‐018, commonly used in combination for insulin concentration determination in sandwich assays, were determined using X‐ray crystallography. The crystal structure of the HUI‐018 Fab in complex with human insulin (HI) was determined and OXI‐005 Fab crystal structures were determined in complex with HI and porcine insulin (PI) as well as on its own. The OXI‐005 epitope comprises insulin residues 1,3,4,19–21 (A‐chain) and 25–30 (B‐chain) and for HUI‐018 residues 7,8,10–14,17 (A‐chain) and 5–7, 10, 14 (B‐chain). The areas of insulin involved in interactions with the mAb are 20% (OXI‐005) and 24% (HUI‐018) of the total insulin surface. Based on the Fab complex crystal structures with the insulins a molecular model for simultaneous binding of the Fabs to PI was built and this model was validated by small angle X‐ray scattering measurements for the ternary complex. The epitopes for the mAbs on insulin were found well separated from each other as expected from luminiscent oxygen channeling immunoassay results for different insulins (HI, PI, bovine insulin, DesB30 HI, insulin glargine, insulin lispro). The affinities of the OXI‐005 and HUI‐018 Fabs for HI, PI, and DesB30 HI were determined using surface plasmon resonance. The K _Ds were found to be in the range of 1–4 nM for the HUI‐018 Fab, while more different for the OXI‐005 Fab (50 nM for HI, 20 nM for PI and 400 nM for DesB30 HI) supporting the importance of residue B30 for binding to OXI‐005.     

Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron‐ and sulfur‐oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af‐Tth) was determined by X‐ray crystallography to a resolution of 1.95 Å. Af‐Tth is a homodimer, and its monomer structure exhibits an eight‐bladed β‐propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β‐propeller region. We observed unexplained electron densities in this cavity of the substrate‐soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site‐specific Af‐Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af‐Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.     

Aptamer‐mediated synthesis of multifunctional nano‐hydroxyapatite for active tumour bioimaging and treatment

ObjectivesThe nano‐hydroxyapatite (nHAp) is widely used to develop imaging probes and drug carriers due to its excellent bioactivity and biocompatibility. However, traditional methods usually need cumbersome and stringent conditions such as high temperature and post‐modification to prepare the functionalized nHAp, which do not benefit the particles to enter cells due to the increased particle size. Herein, a biomimetic synthesis strategy was explored to achieve the AS1411‐targeted tumour dual‐model bioimaging using DNA aptamer AS1411 as a template. Then, the imaging properties and the biocompatibility of the synthesized AS‐nFAp:Gd/Tb were further investigated.Materials and methodsThe AS‐nFAp:Gd/Tb was prepared under mild conditions through a one‐pot procedure with AS1411 as a template. Besides, the anticancer drug DOX was loaded to AS‐nFAp:Gd/Tb so as to achieve the establishment of a multifunctional nano‐probe that integrated the tumour diagnosis and treatment. The AS‐nFAp:Gd/Tb was characterized by transmission electron microscopy (TEM), energy disperse X‐ray Spectroscopy (EDS) mapping, X‐ray photoelectron spectroscopy (XPS) spectrum, X‐ray diffraction (XRD), fourier‐transformed infrared (FTIR) spectroscopy, capillary electrophoresis analyses, zeta potential and particle sizes. The in vitro magnetic resonance imaging (MRI) and fluorescence imaging were performed on an MRI system and a confocal laser scanning microscope, respectively. The potential of the prepared multifunctional nHAp for a targeted tumour therapy was investigated by a CCK‐8 kit. And the animal experiments were conducted on the basis of the guidelines approved by the Animal Care and Use Committee of Sichuan University, China.ResultsIn the presence of AS1411, the as‐prepared AS‐nFAp:Gd/Tb presented a needle‐like morphology with good monodispersity and improved imaging performance. Furthermore, due to the specific binding between AS1411 and nucleolin up‐expressed in cancer cells, the AS‐nFAp:Gd/Tb possessed excellent AS1411‐targeted fluorescence and MRI imaging properties. Moreover, after loading chemotherapy drug DOX, in vitro and in vivo studies showed that DOX@AS‐nFAp:Gd/Tb could effectively deliver DOX to tumour tissues and exert a highly effective tumour inhibition without systemic toxicity compared with pure DOX.ConclusionsThe results indicated that the prepared multifunctional nHAp synthesized by a novel biomimetic strategy had outstanding capabilities of recognition and treatment for the tumour and had good biocompatibility; hence, it might have a potential clinical application in the future.     

Extension of a de novo TIM barrel with a rationally designed secondary structure element

The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recent de novo design of a four‐fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein, we developed a design pipeline based on computational ab initio folding that solves a less complex sub‐problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high‐resolution X‐ray structure for one variant and compare it to our design model. The successful extension of this robust TIM‐barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.     

EFAMIX,a tool to decompose inline chromatography SAXS data from partially overlapping components

Small‐angle X‐ray scattering (SAXS) is an established technique for structural analysis of biological macromolecules in solution. During the last decade, inline chromatography setups coupling SAXS with size exclusion (SEC‐SAXS) or ion exchange (IEC‐SAXS) have become popular in the community. These setups allow one to separate individual components in the sample and to record SAXS data from isolated fractions, which is extremely important for subsequent data interpretation, analysis, and structural modeling. However, in case of partially overlapping elution peaks, inline chromatography SAXS may still yield scattering profiles from mixtures of components. The deconvolution of these scattering data into the individual fractions is nontrivial and potentially ambiguous. We describe a cross‐platform computer program, EFAMIX, for restoring the scattering and concentration profiles of the components based on the evolving factor analysis (EFA). The efficiency of the program is demonstrated in a number of simulated and experimental SEC‐SAXS data sets. Sensitivity and limitations of the method are explored, and its applicability to IEC‐SAXS data is discussed. EFAMIX requires minimal user intervention and is available to academic users through the program package ATSAS as from release 3.1.     

X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non‐homologous end joining (NHEJ) as the primary conserved DNA double‐strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB‐inducing agents, generation of antibody and T‐cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X‐ray scattering (SAXS) results combined with X‐ray crystallography (MX) and cryo‐electron microscopy (cryo‐EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra‐molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo‐EM or MX structures. In the long‐range synaptic complex, X‐ray repair cross‐complementing 4 (XRCC4) plus XRCC4‐like‐factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA‐PKcs (DNA‐dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto‐phosphorylation opens DNA‐PKcs dimer licensing NHEJ via concerted conformational transformations of XLF‐XRCC4, XLF–Ku80, and LigIV^BRCT–Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short‐range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation‐of‐function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.     

Protein Data Bank Japan: Celebrating our 20th anniversary during a global pandemic as the Asian hub of three dimensional macromolecular structural data

Protein Data Bank Japan (PDBj), a founding member of the worldwide Protein Data Bank (wwPDB) has accepted, processed and distributed experimentally determined biological macromolecular structures for 20 years. During that time, we have continuously made major improvements to our query search interface of PDBj Mine 2, the BMRBj web interface, and EM Navigator for PDB/BMRB/EMDB entries. PDBj also serves PDB‐related secondary database data, original web‐based modeling services such as Homology modeling of complex structure (HOMCOS), visualization services and utility tools, which we have continuously enhanced and expanded throughout the years. In addition, we have recently developed several unique archives, BSM‐Arc for computational structure models, and XRDa for raw X‐ray diffraction images, both of which promote open science in the structural biology community. During the COVID‐19 pandemic, PDBj has also started to provide feature pages for COVID‐19 related entries across all available archives at PDBj from raw experimental data and PDB structural data to computationally predicted models, while also providing COVID‐19 outreach content for high school students and teachers.     

Variation in heteroploid reproduction and gene flow across a polyploid complex: One size does not fit all

Whole‐genome duplication is considered an important speciation mechanism in plants. However, its effect on reproductive isolation between higher cytotypes is not well understood. We used backcrosses between different ploidy levels and surveys of mixed‐ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. We backcrossed F1 hybrids derived from 2X‐4X and 4X‐6X crosses in the Campanula rotundifolia autopolyploid complex, measured backcross fitness, and estimated backcross DNA cytotype. We then sampled four natural mixed‐ploidy contact zones (two 2X‐4X and two 4X‐6X), estimated ploidy, and genotyped individuals across each contact zone. Reproductive success and capacity for gene flow was markedly lower for 2X‐4X than 4X‐6X hybrids. In fact, 3X hybrids could not backcross; all 2X‐4X backcross progeny resulted from neotetraploid F1 hybrids. Further, no 3X individuals were found in 2X‐4X contact zones, and 2X and 4X individuals were genetically distinct. By contrast, backcrosses of 5X hybrids were relatively successful, particularly when crossed to 6X individuals. In 4X‐6X contact zones, 5X individuals and aneuploids were common and all cytotypes were largely genetically similar and spatially intermixed. Taken together, these results provide strong evidence that reproduction is low between 2X and 4X cytotypes, primarily occurring via unreduced gamete production, but that reproduction and gene flow are ongoing between 4X and 6X cytotypes. Further, it suggests whole‐genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes.

To assess the role of ploidy in determining reproductive isolation and speciation in polyploid contact zones, we used backcrosses between different ploidy levels and surveys of mixed‐ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. Reproductive success and capacity for gene flow was markedly higher for 4X‐6X hybrids than 2X‐4X hybrids, which was also seen in natural mixed‐ploidy contact zones. Our results suggest whole‐genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes.     

A new l‐arginine oxidase engineered from l‐glutamate oxidase

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l‐Glutamate oxidase (LGOX) from Streptomyces sp. X‐119‐6 catalyzes the oxidative deamination of l‐glutamate to produce 2‐ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l‐glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l‐glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l‐arginine. The oxidative deamination activity of LGOX to l‐arginine is higher than that of l‐arginine oxidase form from Pseudomonas sp. TPU 7192. X‐ray crystal structure analysis revealed that the guanidino group of l‐arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild‐type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l‐arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l‐arginine by LGOX R305E, is proportional to the concentration of l‐arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l‐arginine. Thus, LGOX R305E is suitable for the determination of l‐arginine.     

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4‐amino‐4,6‐dideoxy‐d‐glucose, also known as d‐viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide‐linked sugar, which in the Mimivirus is thought to be UDP‐d‐glucose. The enzyme required for the installment of the amino group at the C‐4′ position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5′‐phosphate‐dependent enzyme, referred to as L136. For this analysis, three high‐resolution X‐ray structures were determined: the wildtype enzyme/pyridoxamine 5′‐phosphate/dTDP complex and the site‐directed mutant variant K185A in the presence of either UDP‐4‐amino‐4,6‐dideoxy‐d‐glucose or dTDP‐4‐amino‐4,6‐dideoxy‐d‐glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP‐d‐glucose or dTDP‐d‐glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three‐dimensional architecture was previously reported by this laboratory. As determined in this investigation,DesI shows a profound preference in its catalytic efficiency for the dTDP‐linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three‐dimensional model for a virally encoded PLP‐dependent enzyme and thus provides new information on sugar aminotransferases in general.     

Microcomputed tomography used to link head morphology and observed feeding behavior in cichlids of Lake Malaŵi

Cichlids inhabiting the Great Lakes of Africa have radiated extremely rapidly, with Lake Malaŵi harboring some 850 species. This rapid radiation may be linked to the diversity in behaviors, sexual selection, and phenotypic plasticity. To determine the relationships between morphology and behaviors, microcomputed tomography (microCT) was used to observe internal morphological structures. Observed morphological adaptations were linked with observed behavior of cichlids in Lake Malaŵi with respect to the various available food resources. Many of these adaptations have parallels, sometimes into the finest details, in other drainage systems and can thus be considered as variations of how cichlids in general respond to environmental opportunities and challenges. Variations in the structure and teeth of the pharyngeal jaws and the oral jaws allowed for fine tuning of specializations, so that various species can utilize the same source without direct competition. We suggested that high‐resolution X‐ray computed tomography will permit scientists to infer life history and behavior characters of rare or extinct taxa from a detailed examination of morphology and linkages between morphology and behavior found in extant species.     

Neutralization of SARS‐CoV‐2 by highly potent,hyperthermostable, and mutation‐tolerant nanobodies

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.     

Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes

The reactivation of X‐linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X‐linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist‐inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X‐linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X‐linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.     

Site‐directed mutagenesis of Serine‐72 reveals the location of the fructose 6‐phosphate regulatory site of the Agrobacterium tumefaciens ADP‐glucose pyrophosphorylase

The allosteric regulation of ADP–glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6‐phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP‐glucose pyrophosphorylase. By X‐ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate‐binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP‐glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.     

Space‐for‐time is not necessarily a substitution when monitoring the distribution of pelagic fishes in the San Francisco Bay‐Delta

Occupancy models are often used to analyze long‐term monitoring data to better understand how and why species redistribute across dynamic landscapes while accounting for incomplete capture. However, this approach requires replicate detection/non‐detection data at a sample unit and many long‐term monitoring programs lack temporal replicate surveys. In such cases, it has been suggested that surveying subunits within a larger sample unit may be an efficient substitution (i.e., space‐for‐time substitution). Still, the efficacy of fitting occupancy models using a space‐for‐time substitution has not been fully explored and is likely context dependent. Herein, we fit occupancy models to Delta Smelt (Hypomesus transpacificus) and Longfin Smelt (Spirinchus thaleichthys) catch data collected by two different monitoring programs that use the same sampling gear in the San Francisco Bay‐Delta, USA. We demonstrate how our inferences concerning the distribution of these species changes when using a space‐for‐time substitution. Specifically, we found the probability that a sample unit was occupied was much greater when using a space‐for‐time substitution, presumably due to the change in the spatial scale of our inferences. Furthermore, we observed that as the spatial scale of our inferences increased, our ability to detect environmental effects on system dynamics was obscured, which we suspect is related to the tradeoffs associated with spatial grain and extent. Overall, our findings highlight the importance of considering how the unique characteristics of monitoring programs influences inferences, which has broad implications for how to appropriately leverage existing long‐term monitoring data to understand the distribution of species.     

Structural and functional understanding of the toll‐like receptors

Recognition of invading pathogens by the innate immune system is essential to initiate antimicrobial responses and trigger adaptive immunity. This is largely mediated by an array of pattern‐recognition receptor families that are essential for recognizing conserved molecular motifs characteristic of pathogenic microbes. One such family is the Toll‐like receptors (TLRs). Activation of TLRs induces production of pro‐inflammatory cytokines and type I interferons: the former triggers the synthesis of inflammatory mediators which cause fever, pain and other inflammation, and the latter mediates antiviral responses. Over the past decade, significant progress has been made in structural elucidation of TLRs in higher eukaryotes. The TLR structures with and without agonist and antagonist have been revealed by X‐ray crystallography and cryo‐electron microscopy studies, demonstrating the activated dimer formation induced by the agonistic ligand and the inhibition mechanism of the antagonistic ligand. Intracellular assembled structures and the TLR‐chaperone complex are also reported. As the structural understanding of TLRs becomes better integrated with biochemical and immunological studies, a more comprehensive picture of their architectural and functional properties will emerge. This review summarizes recent advances in structural biological and mechanistic studies on TLRs.     

Classifying behavior from short‐interval biologging data: An example with GPS tracking of birds

1. Recent advances in digital data collection have spurred accumulation of immense quantities of data that have potential to lead to remarkable ecological insight, but that also present analytic challenges. In the case of biologging data from birds, common analytical approaches to classifying movement behaviors are largely inappropriate for these massive data sets.
2. We apply a framework for using K‐means clustering to classify bird behavior using points from short time interval GPS tracks. K‐means clustering is a well‐known and computationally efficient statistical tool that has been used in animal movement studies primarily for clustering segments of consecutive points. To illustrate the utility of our approach, we apply K‐means clustering to six focal variables derived from GPS data collected at 1–11 s intervals from free‐flying bald eagles (Haliaeetus leucocephalus) throughout the state of Iowa, USA. We illustrate how these data can be used to identify behaviors and life‐stage‐ and age‐related variation in behavior.
3. After filtering for data quality, the K‐means algorithm identified four clusters in >2 million GPS telemetry data points. These four clusters corresponded to three movement states: ascending, flapping, and gliding flight; and one non‐moving state: perching. Mapping these states illustrated how they corresponded tightly to expectations derived from natural history observations; for example, long periods of ascending flight were often followed by long gliding descents, birds alternated between flapping and gliding flight.
4. The K‐means clustering approach we applied is both an efficient and effective mechanism to classify and interpret short‐interval biologging data to understand movement behaviors. Furthermore, because it can apply to an abundance of very short, irregular, and high‐dimensional movement data, it provides insight into small‐scale variation in behavior that would not be possible with many other analytical approaches.