Ancient DNA reveals differences in behaviour and sociality between brown bears and extinct cave bears

Ancient DNA studies have revolutionized the study of extinct species and populations, providing insights on phylogeny, phylogeography, admixture and demographic history. However, inferences on behaviour and sociality have been far less frequent. Here, we investigate the complete mitochondrial genomes of extinct Late Pleistocene cave bears and middle Holocene brown bears that each inhabited multiple geographically proximate caves in northern Spain. In cave bears, we find that, although most caves were occupied simultaneously, each cave almost exclusively contains a unique lineage of closely related haplotypes. This remarkable pattern suggests extreme fidelity to their birth site in cave bears, best described as homing behaviour, and that cave bears formed stable maternal social groups at least for hibernation. In contrast, brown bears do not show any strong association of mitochondrial lineage and cave, suggesting that these two closely related species differed in aspects of their behaviour and sociality. This difference is likely to have contributed to cave bear extinction, which occurred at a time in which competition for caves between bears and humans was likely intense and the ability to rapidly colonize new hibernation sites would have been crucial for the survival of a species so dependent on caves for hibernation as cave bears. Our study demonstrates the potential of ancient DNA to uncover patterns of behaviour and sociality in ancient species and populations, even those that went extinct many tens of thousands of years ago.     

Early Evaluation and Monitoring of Critical Patients with Acute Respiratory Distress Syndrome (ARDS) Using Specific Genetic Polymorphisms

A high percentage of critical patients are found to develop acute respiratory distress syndrome (ARDS). Several studies have reported high mortality rates in these cases which are most frequently associated with multiple organ dysfunctions syndrome. Lately, many efforts have been made to evaluate and monitor ARDS in critical patients. In this regard, the assessment of genetic polymorphisms responsible for developing ARDS present as a challenge and are considered future biomarkers. Early detection of the specific polymorphic gene responsible for ARDS in critically ill patients can prove to be a useful tool in the future, able to help decrease the mortality rates in these cases. Moreover, identifying the genetic polymorphism in these patients can help in the implementation of a personalized intensive therapy scheme for every type of patient, based on its genotype.     

Stabilization of Metals in Subsurface by Biopolymers: Laboratory Drainage Flow Studies

Environmental contamination with heavy metals and radionuclides remains a major problem worldwide. The current clean-up methodologies are based on energy-intensive engineering processes, which are disruptive and costly. A new universal technology targeted for the permanent enclosure and fixation of nuclear and other extreme hazardous metallic wastes in subsurface sites is needed. Such technology will be useful in treating contamination at many sites in the U.S., with specific applications to Department of Energy (DOE) sites. Biopolymers are potential tools for such an innovative technology. Biopolymers have repeated sequences, and therefore provide ample opportunity for chemical reactions with metals, soil particles, and other biopolymers. They also have the additional ability of creating cross-linking interpenetrating networks that can encapsulate the contaminants. Based on this concept, in the present work five biopolymers (xanthan, chitosan, polyhydroxy butyrate, guar gum, polyglutamic acid) were investigated for potential use in the stabilization of metals in the subsurface. The effects of these biopolymers (used alone and in combinations) on soil characteristics (permeability, shear strength) and their metal uptake ability have been studied using laboratory drainage flow systems. Biopolymer solutions were run through the experimental sandpack columns, followed by copper solution and leaching agents (distilled water and hydrochloric acid). The permeability and shear strength of sand were evaluated. Copper uptake capacity of each biopolymer and combination of biopolymers was also studied along with subsequent leaching. All biopolymers tested improved sand characteristics (by decreasing permeability and increasing shear strength) and had good metal uptake ability (60–90%) with relatively low leachability (10–22%). While biopolymers used alone were more efficient in metal uptake, the combination of two biopolymers (xanthan and chitosan) had an increasing plugging effect. These results show the potential of using biopolymers in subsurface metal stabilization.     

A Unitary Anesthetic Binding Site at High Resolution

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA_A receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA_A receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA_A receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.Most general anesthetics alter the activity of ligand-gated ion channels, and electrophysiology, photolabeling, and transgenic animal experiments imply that this effect contributes to the mechanism of anesthesia (1–9). Although the molecular mechanism for this effect is not yet clear, photolabeling studies indicate that anesthetics bind within the transmembrane regions of Cys-loop ligand-gated ion channels such as the nicotinic acetylcholine and the γ-aminobutyric acid (GABA)² type A receptors (2, 9–11). Practical difficulties associated with overexpression, purification, and crystallization of ion channels have thus far stymied investigation of the structural and energetic bases underlying anesthetic recognition. However, general anesthetics also bind specifically to sites in soluble proteins, including firefly luciferase, human serum albumin (HSA), and horse spleen apoferritin (HSAF) (12–14), and x-ray crystal structures have been determined for complexes of these proteins with several general anesthetics (14–16). In particular, HSAF is an attractive model for studying anesthetic-protein interactions because it has the highest affinity for anesthetics of any protein studied to date, has a unique anesthetic binding site, and is a multimer of 4-helix bundles, much like the putative anesthetic binding regions in ligand-gated channels. In addition, apoferritin is commercially available and crystallizes readily. Most importantly, however, the affinity of HSAF for a broad range of general anesthetics is highly correlated with anesthetic potency, confirming the utility and relevance of this model system (17).Ferritin is a 24-mer iron-binding protein. It sequesters free iron ions, thereby helping to maintain non-toxic levels of iron in the cell and functioning as a cellular iron reservoir (18, 19). Each subunit has a molecular mass of ∼20 kDa and adopts a 4-helix bundle fold. The 24-mer forms a hollow, roughly spherical particle with 432 symmetry. Two ferritin isoforms are found in mammals, heavy (H) and light (L), and 24-mers can contain all H chains, all L chains, or mixtures of varying stoichiometry; the biological significance of the H/L ratio is not yet clear (20).In addition to the large central cavity, the apoferritin 24-mer contains additional, smaller cavities at the dimer interfaces; these smaller cavities are of an appropriate size to accommodate anesthetics. X-ray crystallography has confirmed that this interfacial cavity is the binding site for the inhalational anesthetics halothane and isoflurane, and isothermal titration calorimetry (ITC) measurements have shown that this interfacial site has a relatively high affinity for these anesthetics (K_a values ∼10⁵ m⁻¹) (14).General anesthetics fall into at least two broad classes, inhalational and injectable. Whereas both classes of drugs can induce the amnesia, immobility, and hypnosis associated with anesthesia, molecules in the two classes differ substantially in their chemical and physical properties. Prior to this work, only one crystal structure has been available for an injectable general anesthetic complexed with a protein-propofol, bound to HSA (16). This structure revealed that the propofol binding sites on this protein do not, by and large, overlap with the binding sites for inhalational anesthetics. This raises the question of whether the two types of drug invariably bind to separate sets of targets, or whether they could possibly transduce their effects by binding to a single protein site. To address this question we assessed whether propofol binds to the apoferritin site that had been previously identified as the binding site for inhalational anesthetics. Using x-ray crystallography, calorimetry, and molecular modeling, we show that the two types of anesthetics do indeed share a common binding site. We also investigated structure-binding relationships for a homologous series of propofol-like compounds and found that, remarkably, the energetics of binding to apoferritin precisely match the compound''s abilities to potentiate GABA effects at GABA_A receptors, suggesting that similar structural and physicochemical factors mediate anesthetic recognition by both apoferritin and ligand-gated ion channels. This argues for the possibility that anesthetic binding might trigger structural and dynamic alterations in GABA_A receptors similar to those observed in apoferritin, and that these changes underlie anesthetic effects.     

Predictors of major infections in systemic lupus erythematosus

Introduction  

Infections commonly complicate the course of systemic lupus erythematosus (SLE). Our aim is to investigate the clinical predictors of major infections in patients with SLE.     

Regulation of the Na+/K+ ATPase by Klotho

Klotho-hypomorphic (Klotho(hm)) mice suffer from renal salt wasting and hypovolemia despite hyperaldosteronism. The present study explored the effect of Klotho on renal Na(+)/K(+) ATPase activity. According to immunohistochemistry and confocal microscopy Na(+)/K(+) ATPase protein abundance in isolated collecting ducts was lower in Klotho(hm) mice than in their wild type littermates (Klotho(+/+)). Analysis with dual electrode voltage clamp recording showed that expression of Klotho in Xenopus oocytes increased the Na(+)/K(+) ATPase pump current. Treatment of Xenopus oocytes with Klotho protein similarly increased the pump current. In conclusion, Klotho increases the membrane abundance and activity of the Na(+)/K(+) ATPase. Decreased Na(+)/K(+) ATPase activity could thus contribute to the volume-depletion of klotho(hm) mice.     

Enantioselective Surface Plasmon Resonance Sensor Based on C60 Fullerene‐Glutathione Self‐Assembled Monolayer (SAM)

A novel enantioselective surface plasmon resonance (SPR) sensor based on a self‐assembled monolayer of C₆₀ fullerene as the chiral selector is proposed. A binding assay, apparent affinity constant, and apparent dissociation binding constant were used to analyze and study the enantioselectivity of C₆₀ fullerene‐glutathione film for L‐histidine, which was chosen as the model analyte. The apparent affinity constant for the complex formed by L‐histidine with C₆₀ fullerene‐glutathione film was 5.2 x 10⁹ M^‐1. The proposed SPR sensor can be used for the assay of L‐histidine in the 10^‐10 – 10^‐7 mol/L concentration range. Chirality 26:129–131, 2014. © 2014 Wiley Periodicals, Inc.     

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.