Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles.

Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.

Adaptive evolution of the enigmatic Takakia now facing climate change in Tibet.

The most extreme environments are the most vulnerable to transformation under a rapidly changing climate. These ecosystems harbor some of the most specialized species, which will likely suffer the highest extinction rates. We document the steepest temperature increase (2010-2021) on record at altitudes of above 4,000 m, triggering a decline of the relictual and highly adapted moss Takakia lepidozioides. Its de-novo-sequenced genome with 27,467 protein-coding genes includes distinct adaptations to abiotic stresses and comprises the largest number of fast-evolving genes under positive selection. The uplift of the study site in the last 65 million years has resulted in life-threatening UV-B radiation and drastically reduced temperatures, and we detected several of the molecular adaptations of Takakia to these environmental changes. Surprisingly, specific morphological features likely occurred earlier than 165 mya in much warmer environments. Following nearly 400 million years of evolution and resilience, this species is now facing extinction.

Structure and mechanism of human cystine exporter cystinosin.

Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of these transporters, we focus on cystinosin, a prototypical lysosomal amino acid transporter that exports cystine to the cytosol, where its reduction to cysteine supplies this limiting amino acid for diverse fundamental processes and controlling nutrient adaptation. Cystinosin mutations cause cystinosis, a devastating lysosomal storage disease. Here, we present structures of human cystinosin in lumen-open, cytosol-open, and cystine-bound states, which uncover the cystine recognition mechanism and capture the key conformational states of the transport cycle. Our structures, along with functional studies and double electron-electron resonance spectroscopic investigations, reveal the molecular basis for the transporter's conformational transitions and protonation switch, show conformation-dependent Ragulator-Rag complex engagement, and demonstrate an unexpected activation mechanism. These findings provide molecular insights into lysosomal amino acid efflux and a potential therapeutic strategy.

Open-state structure and pore gating mechanism of the cardiac sodium channel.

The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.

Structure of the Cardiac Sodium Channel.

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.

Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells.

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.

Structure of the Nav1.4-ß1 Complex from Electric Eel.

Voltage-gated sodium (Nav) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNav1.4, the Nav channel from electric eel, in complex with the ß1 subunit at 4.0 Å resolution. The immunoglobulin domain of ß1 docks onto the extracellular L5I and L6IV loops of EeNav1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSDIII). The VSDs exhibit "up" conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule. Structural comparison with closed NavPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.

Chronic Stress Alters Striosome-Circuit Dynamics, Leading to Aberrant Decision-Making.

Effective evaluation of costs and benefits is a core survival capacity that in humans is considered as optimal, "rational" decision-making. This capacity is vulnerable in neuropsychiatric disorders and in the aftermath of chronic stress, in which aberrant choices and high-risk behaviors occur. We report that chronic stress exposure in rodents produces abnormal evaluation of costs and benefits resembling non-optimal decision-making in which choices of high-cost/high-reward options are sharply increased. Concomitantly, alterations in the task-related spike activity of medial prefrontal neurons correspond with increased activity of their striosome-predominant striatal projection neuron targets and with decreased and delayed striatal fast-firing interneuron activity. These effects of chronic stress on prefronto-striatal circuit dynamics could be blocked or be mimicked by selective optogenetic manipulation of these circuits. We suggest that altered excitation-inhibition dynamics of striosome-based circuit function could be an underlying mechanism by which chronic stress contributes to disorders characterized by aberrant decision-making under conflict. VIDEO ABSTRACT.

Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish.

Drastic increases in myofiber number and size are essential to support vertebrate post-embryonic growth. However, the collective cellular behaviors that enable these increases have remained elusive. Here, we created the palmuscle myofiber tagging and tracking system for in toto monitoring of the growth and fates of ~5000 fast myofibers in developing zebrafish larvae. Through live tracking of individual myofibers within the same individuals over extended periods, we found that many larval myofibers readily dissolved during development, enabling the on-site addition of new and more myofibers. Remarkably, whole-body surveillance of multicolor-barcoded myofibers further unveiled a gradual yet extensive elimination of larval myofiber populations, resulting in near-total replacement by late juvenile stages. The subsequently emerging adult myofibers are not only long-lasting, but also morphologically and functionally distinct from the larval populations. Furthermore, we determined that the elimination-replacement process is dependent on and driven by the autophagy pathway. Altogether, we propose that the whole-body replacement of larval myofibers is an inherent yet previously unnoticed process driving organismic muscle growth during vertebrate post-embryonic development.

Refractive lensing of scintillating FRBs by subparsec cloudlets in the multiphase CGM.

We consider the refractive lensing effects of ionized cool ([Formula: see text]) gas cloudlets in the circumgalactic medium (CGM) of galaxies. In particular, we discuss the combined effects of lensing from these cloudlets and scintillation from plasma screens in the Milky Way interstellar medium (ISM). We show that, if the CGM comprises a mist of subparsec cloudlets with column densities of order [Formula: see text] (as predicted by [M. McCourt, S. P. Oh, R. O'Leary, A. M. Madigan, MNRAS 473, 5407-5431 (2018)]), then fast radio bursts (FRBs) whose sightlines pass within a virial radius of a CGM halo will may be lensed into tens of refractive images with a ∼10 ms scattering timescale. When these images are formed, they will be resolved by scintillating screens in the Milky Way ISM and will suppress the observed scintillation. We illustrate this effect in refractive lensing and argue that positive detections of FRB scintillation may constrain the properties of these cool-gas cloudlets, with current scintillation observation weakly disfavoring the cloudlet model. We propose that sheet-like geometries for the cool gas in the CGM can reconcile quasar absorption measurements (from which we infer the presence of the cool gas with structure on subparsec scales) and the unexpected lack of lensing signals from this gas thus far observed.

Influence of lipid bilayer on the structure of the muscle-type nicotinic acetylcholine receptor.

The muscle-type nicotinic acetylcholine receptor is a transmitter-gated ion channel residing in the plasma membrane of electrocytes and striated muscle cells. It is present predominantly at synaptic junctions, where it effects rapid depolarization of the postsynaptic membrane in response to acetylcholine released into the synaptic cleft. Previously, cryo-EM of intact membrane from Torpedo revealed that the lipid bilayer surrounding the junctional receptor has a uniquely asymmetric and ordered structure, due to a high concentration of cholesterol. It is now shown that this special lipid environment influences the transmembrane (TM) folding of the protein. All five submembrane MX helices of the membrane-intact junctional receptor align parallel to the surface of the cholesterol-ordered lipids in the inner leaflet of the bilayer; also, the TM helices in the outer leaflet are splayed apart. However in the structure obtained from the same protein after extraction and incorporation in nanodiscs, the MX helices do not align to a planar surface, and the TM helices arrange compactly in the outer leaflet. Realignment of the MX helices of the nanodisc-solved structure to a planar surface converts their adjoining TM helices into an obligatory splayed configuration, characteristic of the junctional receptor. Thus, the form of the receptor sustained by the special lipid environment of the synaptic junction is the one that mediates fast synaptic transmission; whereas, the nanodisc-embedded protein may be like the extrajunctional form, existing in a disordered lipid environment.

The development and applications of multidimensional biomolecular spectroscopy illustrated by photosynthetic light harvesting.

The parallel and synergistic developments of atomic resolution structural information, new spectroscopic methods, their underpinning formalism, and the application of sophisticated theoretical methods have led to a step function change in our understanding of photosynthetic light harvesting, the process by which photosynthetic organisms collect solar energy and supply it to their reaction centers to initiate the chemistry of photosynthesis. The new spectroscopic methods, in particular multidimensional spectroscopies, have enabled a transition from recording rates of processes to focusing on mechanism. We discuss two ultrafast spectroscopies - two-dimensional electronic spectroscopy and two-dimensional electronic-vibrational spectroscopy - and illustrate their development through the lens of photosynthetic light harvesting. Both spectroscopies provide enhanced spectral resolution and, in different ways, reveal pathways of energy flow and coherent oscillations which relate to the quantum mechanical mixing of, for example, electronic excitations (excitons) and nuclear motions. The new types of information present in these spectra provoked the application of sophisticated quantum dynamical theories to describe the temporal evolution of the spectra and provide new questions for experimental investigation. While multidimensional spectroscopies have applications in many other areas of science, we feel that the investigation of photosynthetic light harvesting has had the largest influence on the development of spectroscopic and theoretical methods for the study of quantum dynamics in biology, hence the focus of this review. We conclude with key questions for the next decade of this review.

Mapping cellular responses to DNA double-strand breaks using CRISPR technologies.

DNA double-strand breaks (DSBs) are one of the most genotoxic DNA lesions, driving a range of pathological defects from cancers to immunodeficiencies. To combat genomic instability caused by DSBs, evolution has outfitted cells with an intricate protein network dedicated to the rapid and accurate repair of these lesions. Pioneering studies have identified and characterized many crucial repair factors in this network, while the advent of genome manipulation tools like clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has reinvigorated interest in DSB repair mechanisms. This review surveys the latest methodological advances and biological insights gained by utilizing Cas9 as a precise 'damage inducer' for the study of DSB repair. We highlight rapidly inducible Cas9 systems that enable synchronized and efficient break induction. When combined with sequencing and genome-specific imaging approaches, inducible Cas9 systems greatly expand our capability to spatiotemporally characterize cellular responses to DSB at specific genomic coordinates, providing mechanistic insights that were previously unobtainable.

Sarcopenia phenotype and impaired muscle function in male mice with fast-twitch muscle-specific knockout of the androgen receptor.

Sarcopenia is distinct from normal muscle atrophy in that it is closely related to a shift in the muscle fiber type. Deficiency of the anabolic action of androgen on skeletal muscles is associated with sarcopenia; however, the function of the androgen receptor (AR) pathway in sarcopenia remains poorly understood. We generated a mouse model (fast-twitch muscle-specific AR knockout [fmARKO] mice) in which the AR was selectively deleted in the fast-twitch muscle fibers. In young male mice, the deletion caused no change in muscle mass, but it reduced muscle strength and fatigue resistance and induced a shift in the soleus muscles from fast-twitch fibers to slow-twitch fibers (14% increase, P = 0.02). After middle age, with the control mice, the male fmARKO mice showed much less muscle function, accompanied by lower hindlimb muscle mass; this phenotype was similar to the progression of sarcopenia. The bone mineral density of the femur was significantly reduced in the fmARKO mice, indicating possible osteosarcopenia. Microarray and gene ontology analyses revealed that in male fmARKO mice, there was downregulation of polyamine biosynthesis-related geneswhich was confirmed by liquid chromatography-tandem mass spectrometry assay and the primary cultured myofibers. None of the AR deletion-related phenotypes were observed in female fmARKO mice. Our findings showed that the AR pathway had essential muscle type- and sex-specific roles in the differentiation toward fast-twitch fibers and in the maintenance of muscle composition and function. The AR in fast-twitch muscles was the dominant regulator of muscle fiber-type composition and muscle function, including the muscle-bone relationship.

An endoscopically compatible fast-gelation powder forms Janus-adhesive hydrogel barrier to prevent postoperative adhesions.

Postoperative adhesions occur widely in various tissues, bringing the risk of secondary surgery and increased medical burden. Hydrogel barriers with Janus-adhesive ability can achieve physical isolation of adjacent tissues and are therefore considered an ideal solution. However, integrating endoscopic delivery convenience and viscoelastic Janus hydrogel formation remains a great challenge. Here, we present a report of the in situ formation of Janus-adhesive hydrogel barrier using a sprayable fast-Janus-gelation (FJG) powder. We first methacrylate the polysaccharide macromolecules to break the intermolecular hydrogen bonds and impart the ability of rapid hydration. FJG powder can rapidly absorb interfacial water and crosslink through borate ester bonds, forming a toughly adhesive viscoelastic hydrogel. The Janus barrier can be simply formed by further hydrating the upper powder with cationic solution. We construct rat models to demonstrate the antiadhesions efficiency of viscoelastic FJG hydrogels in organs with different motion modalities (e.g., intestine, heart, liver). We also developed a low-cost delivery device with a standardized surgical procedure and further validated the feasibility and effectiveness of FJG powder in minimally invasive surgery using a preclinical translational porcine model. Considering the advantages in terms of therapeutic efficacy, clinical convenience, and commercialization, our results reveal the great potential of Janus-gelation powder materials as a next-generation antiadhesions barrier.

High time-resolved studies of stick-slip show similar dilatancy to fast and slow earthquakes.

Fast and slow earthquakes are two modes of energy release by the slip in tectonic fault rupture. Although fast and slow slips were observed in the laboratory stick-slip experiments, due to the sampling rate limitation, the details of the fault thickness variation were poorly understood. Especially, why a single fault would show different modes of slip remains elusive. Herein, we report on ring shear experiments with an ultrahigh sampling rate (10 MHz) that illuminate the different physical processes between fast and slow slip events. We show that the duration of slips ranged from dozens to hundreds of milliseconds. Fast slip events are characterized by continuous large-amplitude AE (acoustic emission) and somewhat intricate variation of the sample thickness: A short compaction pulse during the rapid release of stress is followed by dilation and vibrations of the sample thickness. As the slip ends, the thickness of the sample first recovers by slow compaction and then dilates again before nucleation of the following slip event. In contrast, during slow slip events, the shear stress reduction is accompanied by intermittent bursts of low-amplitude AE and sample dilation. We observed the detailed thickness variation during slips and found that dilation occurs during both fast and slow slips, which is consistent with natural observations of coseismic dilatation. This study may be used to reveal the mechanism of fault slips during fast and slow earthquakes, which explain the potential effect of fast and slow slips on stress redistribution and structural rearrangement in faults.

Airborne disease transmission during indoor gatherings over multiple time scales: Modeling framework and policy implications.

Indoor superspreading events are significant drivers of transmission of respiratory diseases. In this work, we study the dynamics of airborne transmission in consecutive meetings of individuals in enclosed spaces. In contrast to the usual pairwise-interaction models of infection where effective contacts transmit the disease, we focus on group interactions where individuals with distinct health states meet simultaneously. Specifically, the disease is transmitted by infected individuals exhaling droplets (contributing to the viral load in the closed space) and susceptible ones inhaling the contaminated air. We propose a modeling framework that couples the fast dynamics of the viral load attained over meetings in enclosed spaces and the slow dynamics of disease progression at the population level. Our modeling framework incorporates the multiple time scales involved in different setups in which indoor events may happen, from single-time events to events hosting multiple meetings per day, over many days. We present theoretical and numerical results of trade-offs between the room characteristics (ventilation system efficiency and air mass) and the group's behavioral and composition characteristics (group size, mask compliance, testing, meeting time, and break times), that inform indoor policies to achieve disease control in closed environments through different pathways. Our results emphasize the impact of break times, mask-wearing, and testing on facilitating the conditions to achieve disease control. We study scenarios of different break times, mask compliance, and testing. We also derive policy guidelines to contain the infection rate under a certain threshold.

Computing geodesic paths encoding a curvature prior for curvilinear structure tracking.

In this paper, we introduce an efficient method for computing curves minimizing a variant of the Euler-Mumford elastica energy, with fixed endpoints and tangents at these endpoints, where the bending energy is enhanced with a user-defined and data-driven scalar-valued term referred to as the curvature prior. In order to guarantee that the globally optimal curve is extracted, the proposed method involves the numerical computation of the viscosity solution to a specific static Hamilton-Jacobi-Bellman (HJB) partial differential equation (PDE). For that purpose, we derive the explicit Hamiltonian associated with this variant model equipped with a curvature prior, discretize the resulting HJB PDE using an adaptive finite difference scheme, and solve it in a single pass using a generalized fast-marching method. In addition, we also present a practical method for estimating the curvature prior values from image data, designed for the task of accurately tracking curvilinear structure centerlines. Numerical experiments on synthetic and real-image data illustrate the advantages of the considered variant of the elastica model with a prior curvature enhancement in complex scenarios where challenging geometric structures appear.

Sex Differences in Dopamine Release in Nucleus Accumbens and Dorsal Striatum Determined by Chronic Fast-Scan Cyclic Voltammetry: Effects of Social Housing and Repeated Stimulation.

We investigated sex differences in dopamine (DA) release in the nucleus accumbens (NAc) and dorsolateral striatum (DLS) using a chronic 16-channel carbon fiber electrode and fast-scan cyclic voltammetry (FSCV). Electrical stimulation-induced (ES; 60 Hz) DA release was recorded in the NAc of single- or pair-housed male and female rats. When core (NAcC) and shell (NAcS) were recorded simultaneously, there was greater ES DA release in NAcC of pair-housed females compared with single females and males. Housing did not affect ES NAc DA release in males. In contrast, there was significantly more ES DA release from the DLS of female rats than male rats. This was true prior to and after treatment with methamphetamine. Furthermore, in castrated (CAST) males and ovariectomized (OVX) females, there were no sex differences in ES DA release from the DLS, demonstrating the hormone dependence of this sex difference. However, in the DLS of both intact and gonadectomized rats, DA reuptake was slower in females than that in males. Finally, DA release following ES of the medial forebrain bundle at 60 Hz was studied over 4 weeks. ES DA release increased over time for both CAST males and OVX females, demonstrating sensitization. Using this novel 16-channel chronic FSCV electrode, we found sex differences in the effects of social housing in the NAcS, sex differences in DA release from intact rats in DLS, and sex differences in DA reuptake in DLS of intake and gonadectomized rats, and we report sensitization of ES-induced DA release in DLS in vivo.

Time-efficient filtering of imaging polarimetric data by checking physical realizability of experimental mueller matrices.

MOTIVATION: Imaging Mueller polarimetry has already proved its potential for biomedicine, remote sensing and metrology. The real-time applications of this modality require both video rate image acquisition and fast data post-processing algorithms. First, one must check the physical realizability of the experimental Mueller matrices in order to filter out non-physical data, ie to test the positive semi-definiteness of the 4 × 4 Hermitian coherency matrix calculated from the elements of corresponding Mueller matrix pixel-wise. For this purpose, we compared the execution time for the calculations of i) eigenvalues, ii) Cholesky decomposition, iii) Sylvester's criterion, and iv) coefficients of the characteristic polynomial (two different approaches) of the Hermitian coherency matrix, all calculated for the experimental Mueller matrix images (600 pixels × 700 pixels) of mouse uterine cervix. The calculations were performed using C ++ and Julia programming languages. RESULTS: Our results showed the superiority of the algorithm iv) based on the simplification via Pauli matrices over other algorithms for our dataset. The sequential implementation of latter algorithm on a single core already satisfies the requirements of real-time polarimetric imaging. This can be further amplified by the proposed parallelization (e.g., we achieve a 5-fold speed up on 6 cores). AVAILABILITY AND IMPLEMENTATION: The source codes of the algorithms and experimental data are available at https://github.com/pogudingleb/mueller_matrices.