bluuebunny/crossref_metadata_2025 huggingface dataset:
PLCγ1 promotes phase separation of the T cell signaling clusters
Authors: Zeng Longhui , Palaia Ivan , Šarić Anđela , Su Xiaolei
Published: July 2020

The T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here we report that phospholipase PLCγ1 promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly crosslinks LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK and SLP76 activation. Intriguingly, a non-monotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction.
High-resolution line-scanning reveals distinct visual response properties across human cortical layers
Authors: Morgan Andrew T. , Nothnagel Nils , Petro Lucy. S. , Goense Jozien , Muckli Lars
Published: July 2020

Our understanding of the human brain relies on advancing noninvasive brain imaging approaches. Characterization of the function of brain circuitry depends on the spatiotemporal correspondence at which recorded signals can be mapped onto underlying neuronal structures and processes. Here we aimed to address key first-stage questions of feasibility, reliability, and utility of line-scanning fMRI as a next generation non-invasive imaging method for human neuroscience research at the mesoscopic scale. Line-scanning can achieve high spatial resolution by employing anisotropic voxels aligned to cortical layers. The method can simultaneously achieve high temporal resolution by limiting acquisition to a very small patch of cortex which is repeatedly acquired as a single frequency-encoded k-space line. We developed multi-echo line-scanning procedures to record cortical layers in humans at high spatial (200 μm) and temporal resolution (100 ms) using ultra high-field 7T fMRI. Quantitative mapping allowed us to identify cortical layers in primary visual cortex (V1) and record functional signals from them while participants viewed movie clips. Analysis of these recordings revealed layer-specific V1 spatial and orientation tuning properties analogous to those previously observed in electrophysiological recordings of non-human primates. We have consequently demonstrated that line-scanning is a powerful non-invasive imaging technique for investigating mesoscopic functional circuits in human cortex.

Graphical Abstract
Quantitative profiling of protease specificity
Authors: Ratnikov B. I. , Cieplak P. , Remacle A. G. , Nguyen E. , Smith J. W.
Published: July 2020

Proteases comprise an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methodologies have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of “selectome”, which defines the set of substrates that uniquely represents specificity of a protease. We applied it to two closely related members of the Matrixin family – MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the numbers and relative catalytic efficiencies of substrates. Using these advances greatly facilitates uncovering selectivity between closely related members of protease families and provides insight into to the degree of contribution of catalytic cleft specificity to protein substrate recognition, thus providing basis to overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes and inhibitors for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.
Type I Interferon regulates cytokine-delayed neutrophil apoptosis, reactive oxygen species production and chemokine expression via activation of p38 MAPK
Authors: Glennon-Alty Laurence , Moots Robert J , Edwards Steven W , Wright Helen L
Published: July 2020

Interferons (IFNs) are key regulators of a number of inflammatory conditions in which neutrophils play an important role in pathology, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), where Type I IFNs are implicated in disease pathology. However, IFNs are usually generatedin vivotogether with other cytokines that also have immunoregulatory functions but such interactions are poorly-defined experimentally. We measured the effects of Type-I IFN (IFNα), elevated in both RA and SLE, on the functions of healthy neutrophils incubatedin vitroin the absence and presence of pro-inflammatory cytokines typically elevated in inflammatory diseases (TNFα, GM-CSF). IFNαalone had no effect on neutrophil apoptosis, however it did abrogate the anti-apoptotic effect of GM-CSF (18h, p< 0.01). The enhanced stabilty of the anti-apoptotic protein Mcl-1 and delayed activation of caspase activation normally regulated by GM-CSF were blocked by IFNα: this effect was mediated, in part, by activation of p38 MAPK, increased turnover of the anti-apoptotic protein Mcl-1 and cleavage of caspases. IFNαalone also primed ROS production alone and maintained the transient priming effect of TNF for up to 4h: it also down-regulated GM-CSF and TNFα-activated expression of CXCL1, CXCL2, CXCL3, CXCL8, CCL3 and CCL4, but in contrast increased the expression of CXCL10. These novel data identify complex regulatory signalling networks in which Type I IFNs profoundly alter the response of neutrophils to inflammatory cytokines. This is likely to have important consequencesin vivoand may explain the complexity and heterogeneity of inflammatory diseases such as RA, in which multiple cytokine cascades have been activated.
Comparative whole-genome approach to identify bacterial traits for microbial interactions
Authors: Zoccarato Luca , Sher Daniel , Miki Takeshi , Segrè Daniel , Grossart Hans-Peter
Published: July 2020

Microbial interactions shape the structure and function of microbial communities with profound consequences for biogeochemical cycles and ecosystem health. Yet, most interaction mechanisms are studied only in model systems and their prevalence is unknown. To systematically explore the functional and interaction potential of sequenced marine bacteria, we developed a new trait-based approach, and applied it to 473 complete genomes (248 genera), representing a significant fraction of marine microbial communities. We identified genome functional clusters (GFCs) which condense bacterial diversity into groups with potentially common ecology and life history. Most GFCs revealed unique combinations of interaction traits, some widely distributed (e.g., antimicrobial activity), others less common (e.g., production of siderophores and phytohormones, biosynthesis and export of specific B vitamins). Specific GFCs, comprising Alpha- and Gammaproteobacteria, are predicted to preferentially interact synergistically and/or antagonistically with bacteria and phytoplankton. Moreover, linked trait clusters (LTCs) identify traits that may have evolved together and point to specific modes of interactions. Our approach translates multidimensional genomic information into an atlas of marine bacteria and their ecosystem functions, relevant for understanding the fundamental rules that govern community assembly and dynamics.
Epithelial layer unjamming shifts energy metabolism toward glycolysis
Authors: DeCamp Stephen J. , Tsuda Victor M.K. , Ferruzzi Jacopo , Koehler Stephan A. , Giblin John T. , Roblyer Darren , Zaman Muhammad H. , Weiss Scott T. , DeMarzio Margherita , Park Chan Young , Ogassavara Nicolas Chiu , Mitchel Jennifer , Butler James P. , Fredberg Jeffrey J.
Published: July 2020

In development of an embryo, healing of a wound, or progression of a carcinoma, a requisite event is collective epithelial cellular migration. For example, cells at the advancing front of a wound edge tend to migrate collectively, elongate substantially, and exert tractions more forcefully compared with cells many ranks behind. With regards to energy metabolism, striking spatial gradients have recently been reported in the wounded epithelium, as well as in the tumor, but within the wounded cell layer little is known about the link between mechanical events and underlying energy metabolism. Using the advancing confluent monolayer of MDCKII cells as a model system, here we report at single cell resolution the evolving spatiotemporal fields of cell migration speeds, cell shapes, and traction forces measured simultaneously with fields of multiple indices of cellular energy metabolism. Compared with the epithelial layer that is unwounded, which is non-migratory, solid-like and jammed, the leading edge of the advancing cell layer is shown to become progressively more migratory, fluid-like, and unjammed. In doing so the cytoplasmic redox ratio becomes progressively smaller, the NADH lifetime becomes progressively shorter, and the mitochondrial membrane potential and glucose uptake become progressively larger. These observations indicate that a metabolic shift toward glycolysis accompanies collective cellular migration but show, further, that this shift occurs throughout the cell layer, even in regions where associated changes in cell shapes, traction forces, and migration velocities have yet to penetrate. In characterizing the wound healing process these morphological, mechanical, and metabolic observations, taken on a cell-by-cell basis, comprise the most comprehensive set of biophysical data yet reported. Together, these data suggest the novel hypothesis that the unjammed phase evolved to accommodate fluid-like migratory dynamics during episodes of tissue wound healing, development, and plasticity, but is more energetically expensive compared with the jammed phase, which evolved to maintain a solid-like non-migratory state that is more energetically economical.

Two sentence summary

At the leading front of an advancing confluent epithelial layer, each cell tends to migrate, elongate, and pull on its substrate far more than do cells many ranks behind, but little is known about underlying metabolic events. Using the advancing monolayer of MDCKII cells as a model of wound healing, here we show at single cell resolution that physical changes associated with epithelial layer unjamming are accompanied by an overall shift toward glycolytic metabolism.
Single neurons throughout human memory regions phase-lock to hippocampal theta
Authors: Schonhaut Daniel R. , Ramayya Ashwin G. , Solomon Ethan A. , Herweg Nora A. , Fried Itzhak , Kahana Michael J.
Published: July 2020

Functional interactions between the hippocampus and cortex are critical for episodic memory. Neural oscillations are believed to coordinate these interactions, and in rodents, prefrontal neurons phase-lock to hippocampal theta oscillations during memory-guided behavior. We assessed inter-regional phase-locking to hippocampal oscillations in humans by recording 1,233 cortical and amygdala neurons and simultaneous hippocampal local field potentials in 18 neurosurgical patients. We identified 362 neurons (29.4%) from multiple regions that phase-locked to rhythmic hippocampal activity, predominantly at theta (2-8Hz) frequencies. Compared to baseline spiking, strong theta phase-locking coincided with regionally-specific increases in hippocampal theta power, local and hippocampal high frequency activity, and cross-frequency power correlations between the hippocampus and a phase-locked neuron’s local region. These results reveal that spike-time synchrony with hippocampal theta is a defining feature of cortico-hippocampal functional connections in humans. We propose that theta phase-locking could mediate flexible inter-regional communication to shape the content and quality of episodic memories.
Organic Dust Exposure Induces Stress Response and Mitochondrial Dysfunction in Monocytic Cells
Authors: Mahadev-Bhat Sanjana , Shrestha Denusha , Massey Nyzil , Karriker Locke A. , Kanthasamy Anumantha G. , Charavaryamath Chandrashekhar
Published: July 2020

Exposure to airborne organic dust (OD), rich in microbial pathogen-associated molecular patterns, has been shown to induce inflammatory responses in the lung resulting in changes in airway structure and function. A common manifestation in lung inflammation is the occurrence of altered mitochondrial structure and bioenergetics, consequently regulating mitochondrial ROS (mROS) and creating a vicious cycle of mitochondrial dysfunction.

The role of mitochondrial dysfunction in airway diseases such as COPD and asthma is well known. However, whether OD exposure induces mitochondrial dysfunction largely remains unknown. Therefore, in this study, we tested a hypothesis that OD exposure induces mitochondrial stress using a human monocytic cell line (THP-1). We examined the mechanisms of organic dust extract (ODE) exposure-induced mitochondrial structural and functional changes in THP-1 cells.

In addition, the effect of co-exposure to ethyl pyruvate (EP), a known anti-inflammatory agent, or mitoapocynin (MA), a mitochondria targeting NOX2 inhibitor was examined. Transmission electron microscopy images showed significant changes in cellular and organelle morphology upon ODE exposure. ODE exposure with and without EP co-treatment increased the mtDNA leakage into the cytosol. Next, ODE exposure increased the PINK1 and Parkin expression, cytoplasmic cytochrome c levels and reduced mitochondrial mass and cell viability, indicating mitophagy. MA treatment was partially protective by decreasing Parkin expression, mtDNA and cytochrome c release and increasing cell viability.
Revival of light-evoked neuronal signals in the post-mortem mouse and human retina
Authors: Abbas Fatima , Becker Silke , Jones Bryan W. , Mure Ludovic S. , Panda Satchidananda , Hanneken Anne , Vinberg Frans
Published: July 2020

The retina, a highly metabolic tissue in the central nervous system, consumes the most oxygen and energy stores in the body by tissue mass/volume. Consequently, it is not surprising that retinal ischemia leads to a rapid loss of retinal light responses and electrical transmission. In this study, we show that despite a swift decline of retinal light responses after circulatory death (decay time constant τ = ∼1-2 min), we were able to restore mouse rod and cone photoreceptor light signals from enucleated eyes up to 3 h postmortem with significantly better postmortem recovery of cone versus rod light responses. We also demonstrate that both rod and cone phototransduction are more resistant to postmortem enucleation delay than synaptic transmission to second order neurons (bipolar cells). Encouraged by these analyses and the lack of previously successful postmortem retinal light recordings from human foveal/macular photoreceptors, we attempted to restore light responses in the human macula using donor eyes harvested 0.5 – 5 hours postmortem. Here we show successful recordings of human macular cone photoresponses in samples obtained from eyes enucleated up to 5 hours postmortem. Comparing ‘freshly’ enucleated non-human primate eyes with human eyes enucleated and reoxygenated at different times after death, we show the exponential decay of the light response amplitudes has a time constant of 74 min. We find that both cause of death and donor age are useful parameters for predicting the recovery of retinal light responses in postmortem human macular tissue. Moreover, we present evidence that hypoxia and secondary acidosis, two modifiable factors, are primary contributors to the rapid loss of retinal light signaling after death. Finally, we show postmortem hypoxia rather than acidosis is the major cause of irreversible decay of the light response amplitudes. The criteria and methodology that we have established here for reviving electrical photoresponses in the postmortem human eye will serve as a new starting point for studying neurophysiology and disease in the human retina.
Striatal circuits support broadly opponent aspects of action suppression and production
Authors: Cruz Bruno F. , Soares Sofia , Paton Joseph J.
Published: July 2020

Imbalance between action suppression and production characterizes several basal ganglia (BG) disorders. Relatedly, the direct and indirect pathways of the BG are hypothesized to promote and suppress actions, respectively. Yet striatal direct (dMSNs) and indirect (iMSNs) medium spiny neurons are coactive around movement, apparently contradicting direct-indirect functional opponency. In the dorsolateral striatum of mice, we observed coactivation around movements, but elevated and diminished activity of iMSNs and dMSNs, respectively, during action suppression. Furthermore, relative activity of the two hemispheres evolved in opposite directions in the two pathways as the need to suppress movements to either side of the body developed over time. Lastly, optogenetic inhibition experiments revealed the necessity of iMSNs but not dMSNs for the proactive suppression of specific actions, and dMSNs but not iMSNs for generalized action vigor. These data demonstrate distinct yet still broadly opponent roles for the direct and indirect pathways in behavioral control.
Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels
Authors: Yarishkin Oleg , Phuong Tam T. T. , Baumann Jackson M. , De Ieso Michael L. , Vazquez-Chona Felix , Rudzitis Christopher N. , Sundberg Chad , Lakk Monika , Stamer W. Daniel , Križaj David
Published: July 2020

Mechanosensitivity of the trabecular meshwork (TM) is a key determinant of intraocular pressure (IOP) yet our understanding of the molecular mechanisms that subserve it remains in its infancy. Here, we show that mechanosensitive Piezo1 channels modulate the TM pressure response via calcium signaling and dynamics of the conventional outflow pathway. Pressure steps evoked fast, inactivating cation currents and calcium signals that were inhibited by Ruthenium Red, GsMTx4 and Piezo1 shRNA. Piezo1 expression was confirmed by transcript and protein analysis, and by visualizing Yoda1-mediated currents and [Ca2+]ielevations in primary human TM cells. Piezo1 activation was obligatory for transduction of physiological shear stress and was coupled to reorganization of F-actin cytoskeleton and focal adhesions. The importance of Piezo1 channels as pressure sensors was shown by the GsMTx4 -dependence of the pressure-evoked current and conventional outflow function. We also demonstrate that Piezo1 collaborates with the stretch-activated TRPV4 channel, which mediated slow, delayed currents to pressure steps. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests a novel potential therapeutic target for treating glaucoma.

Significance Statement

Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humor drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humor outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts, and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. These findings provide a new mechanistic framework for trabecular mechanotransduction and its role in the regulation of fast fluctuations in ocular pressure, as well as chronic remodeling of TM architecture that epitomizes glaucoma.
Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1
Authors: Liu Yanxin , Sun Ming , Myasnikov Alexander G. , Elnatan Daniel , Delaeter Nicolas , Nguyenquang Michael , Agard David A.
Published: July 2020

Hsp90 is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of cellular “client” proteins. The ATP-driven client maturation process is regulated by a large number of co-chaperones. Among them, Aha1 is the most potent activator of Hsp90 ATPase activity and thus dramatically affects Hsp90’s client proteins. To understand the Aha1 activation mechanism, we determined full-length Hsp90:Aha1 structures in six different states by cryo-electron microscopy, including nucleotide-free semi-closed, nucleotide-bound pre-hydrolysis, and semi-hydrolyzed states. Our structures demonstrate that the two Aha1 domains can each interact with Hsp90 in two different modes, uncovering a complex multistep activation mechanism. The results show that Aha1 accelerates the chemical step of ATP hydrolysis like a conventional enzyme, but most unusually, catalyzes the rate-limiting large-scale conformational changes of Hsp90 fundamentally required for ATP hydrolysis. Our work provides a structural framework to guide small molecule development targeting this critical modulator of client protein maturation.
On the role of phase lag in multi-appendage metachronal swimming of euphausiids
Authors: Ford Mitchell P. , Santhanakrishnan Arvind
Published: July 2020

Metachronal paddling is a common method of drag-based aquatic propulsion, in which a series of swimming appendages are oscillated, with the motion of each appendage phase-shifted relative to the neighboring appendages. Ecologically and economically important euphausiid species such as Antarctic krill (E. superba) swim constantly by stroking their paddling appendages (pleopods), with locomotion accounting for the bulk of their metabolic expenditure. They tailor their swimming gaits for behavioral and energetic needs by changing pleopod kinematics. The functional importance of inter-pleopod phase lag (ϕ) to metachronal swimming performance and wake structure is unknown. To examine this relation, we developed a geometrically and dynamically scaled robot (‘krillbot’) capable of self-propulsion. Krillbot pleopods were prescribed to mimic published kinematics of fast-forward swimming (FFW) and hovering (HOV) gaits ofE. superba, and the Reynolds number and Strouhal number of the krillbot matched well with those calculated for freely-swimmingE. superba. In addition to examining published kinematics with unevenϕbetween pleopod pairs, we modifiedE. superbakinematics to uniformly varyϕfrom 0% to 50% of the cycle. Swimming speed and thrust were largest for FFW withϕbetween 15%-25%, coincident withϕrange observed in FFW gait of E. superba. In contrast to synchronous rowing (ϕ=0%) where distances between hinged joints of adjacent pleopods were nearly constant throughout the cycle, metachronal rowing (ϕ>0%) brought adjacent pleopods closer together and moved them farther apart. This factor minimized body position fluctuation and augmented metachronal swimming speed. Though swimming speed was lowest for HOV, a ventrally angled downward jet was generated that can assist with weight support during feeding. In summary, our findings show that inter-appendage phase lag can drastically alter both metachronal swimming speed and the large-scale wake structure.
Mechanism and effects of the skeletal muscle Nav1.4 inhibition by cannabidiol
Authors: Ghovanloo Mohammad-Reza , Choudhury Koushik , Bandaru Tagore S. , Fouda Mohamed A. , Rayani Kaveh , Rusinova Radda , Phaterpekar Tejas , Nelkenbrecher Karen , Watkins Abeline R. , Poburko Damon , Thewalt Jenifer , Andersen Olaf S. , Delemotte Lucie , Goodchild Samuel J. , Ruben Peter C.
Published: July 2020

Cannabis sativacontains active constituents called phytocannabinoids. Some phytocannabinoids are psychotropic and others are not. The primary non-psychotropic phytocannabinoid is cannabidiol (CBD), which is proposed to be therapeutic against many conditions, including muscle spasms. Mechanisms have been proposed for the action of CBD on different systems, involving multiple targets, including the voltage-gated sodium channel (Nav) family, which are heavily implicated in many of the conditions CBD has been reported to relieve. In this study, we investigated the modulatory mechanism of CBD on Nav1.4. Based on previous results, we tested the hypothesis that CBD mechanism of action involves: 1) modulation of membrane elasticity, which indirectly contributes to Nav inhibition; and 2) physical block of the Nav pore. We first performed molecular dynamic (MD) simulations to visualize CBD effects and localization inside the membrane, and then performed NMR to verify the MD results, showing CBD localizes below membrane headgroups. Then, we performed a gramicidin-based fluorescence (GFA) assay that showed CBD alters membrane elasticity. Next, we used site-directed mutagenesis in (F1586A) and around (WWWW) the Nav1.4 pore. Removing the local anesthetic binding site with F1586A reduced CBD block of INa. Occluding the fenestrations with WWWW blocked CBD access from the membrane into the Nav1.4 pore. However, stabilization of inactivation, via CBD-induced changes in membrane elasticity persisted, in WWWW. To investigate the potential therapeutic value of CBD against some Nav1.4 channelopathies, we used a pathogenic variant of Nav1.4, P1158S, known to cause myotonia and periodic paralysis. We found CBD reduces excitability in both wild-type and the mixed myotonia/periodic paralysis variant. Ourin-vitro/in-silicoresults suggest that CBD may have therapeutic value against myotonia. Because Nav1.4 is crucial to skeletal muscle contraction, we used rat diaphragm myography and found the presence of saturating levels of CBD reduces skeletal muscle contraction.

We used multidisciplinary approaches to show the mechanism and pathway by which CBD inhibits the skeletal muscle, Nav1.4. Our results suggest CBD modulates membrane elasticity and directly interacts with Nav1.4 within its pore.
Soft ticks perform evaporative cooling during blood-feeding
Authors: Lazzari Claudio R. , Fauquet Aurélie , Lahondère Chloé , Araújo Ricardo N. , Pereira Marcos H.
Published: July 2020

Feeding on the blood of warm-blooded vertebrates is associated to thermal stress in haematophagous arthropods. It has been demonstrated that blood-sucking insects protect their physiological integrity either by synthesising heat-shock proteins or by means of thermoregulatory mechanisms. In this work, we describe the first thermoregulatory mechanism in a tick species,Ornithodoros rostratus. By performing real-time infrared thermography during feeding on mice we found that this acarian eliminates big amounts of fluid (urine) through their coxal glands; this fluid quickly spreads over the cuticular surface and its evaporation cools-down the body of the tick. The spread of the fluid is possible thanks to capillary diffusion through the sculptured exoskeleton ofOrnithodoros. We discuss our findings in the frame of the adaptive strategies to cope with the thermal stress experienced by blood-sucking arthropods at each feeding event warm-blooded hosts.
Assessment of Proton-Coupled Conformational Dynamics of SARS and MERS Coronavirus Papain-like Proteases: Implication for Designing Broad-Spectrum Antiviral Inhibitors
Authors: Henderson Jack A. , Verma Neha , Shen Jana
Published: July 2020

Broad-spectrum antiviral drugs are urgently needed to stop the COVID-19 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines and their protonation/deprotonation modulates catalysis and conformational plasticity. Here we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using a newly developed GPU-accelerated implicit-solvent continuous constant pH molecular dynamics method with an asynchronous replica-exchange scheme. The calculated pKa’s support the catalytic roles of the Cys-His-Asp triad. We also found that several residues can switch protonation states at physiological pH, among which is C270/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2/CoV PLpro. Simulations revealed that the BL2 conformational dynamics is coupled to the titration of C271/270, in agreement with the crystal structures of SARS-CoV-2 PLpro. Simulations also revealed that BL2 in MERS-CoV PLpro is very flexible, sampling both open and closed states despite the lack of an analogous cysteine. Our work provides a starting point for more detailed mechanistic studies to assist structure-based design of broad-spectrum inhibitors against CoV PLpros.
Phase 1/2 Study to Describe the Safety and Immunogenicity of a COVID-19 RNA Vaccine Candidate (BNT162b1) in Adults 18 to 55 Years of Age: Interim Report
Authors: Mulligan Mark J. , Lyke Kirsten E. , Kitchin Nicholas , Absalon Judith , Gurtman Alejandra , Lockhart Stephen , Neuzil Kathleen , Raabe Vanessa , Bailey Ruth , Swanson Kena A. , Li Ping , Koury Kenneth , Kalina Warren , Cooper David , Fontes-Garfias Camila , Shi Pei-Yong , Türeci Ӧzlem , Tompkins Kristin R. , Walsh Edward E. , Frenck Robert , Falsey Ann R. , Dormitzer Philip R. , Gruber William C. , Şahin Uğur , Jansen Kathrin U.
Published: July 2020

In March 2020, the WHO declared a pandemic of coronavirus disease 2019 (COVID-19), due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 With >8.8 million cases and >450,000 deaths reported globally, a vaccine is urgently needed. We report the available safety, tolerability, and immunogenicity data from an ongoing placebo-controlled, observer-blinded dose escalation study among healthy adults, 18-55 years of age, randomized to receive 2 doses, separated by 21 days, of 10 μg, 30 μg, or 100 μg of BNT162b1, a lipid nanoparticle-formulated, nucleoside-modified, mRNA vaccine that encodes trimerized SARS-CoV-2 spike glycoprotein RBD. Local reactions and systemic events were dose-dependent, generally mild to moderate, and transient. RBD-binding IgG concentrations and SARS-CoV-2 neutralizing titers in sera increased with dose level and after a second dose. Geometric mean neutralizing titers reached 1.8-to 2.8-fold that of a panel of COVID-19 convalescent human sera. These results support further evaluation of this mRNA vaccine candidate.
A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 within nasopharyngeal and oropharyngeal swabs at Hampshire Hospitals NHS Foundation Trust
Authors: Fowler Veronica L. , Armson Bryony , Gonzales Jose L. , Wise Emma L. , Howson Emma L. A. , Vincent-Mistiaen Zoe , Fouch Sarah , Maltby Connor J. , Grippon Seden , Munro Simon , Jones Lisa , Holmes Tom , Tillyer Claire , Elwell Joanne , Sowood Amy , Santos Helio , de Peyer Oliver , Dixon Sophie , Hatcher Thomas , Sivanesan Suvetha , Patrick Helen , Laxman Shailen , Walsh Charlotte , Andreou Michael , Morant Nick , Clark Duncan , Moore Nathan , Houghton Rebecca , Cortes Nicholas , Kidd Stephen P.
Published: July 2020

The COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed on extracted RNA (RNA RT-LAMP) or directly from swab (Direct RT-LAMP). Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1⨯101and 1⨯102copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a CTcut-off of 33 was employed, above which increasingly evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT-LAMP was 67% and 97%, respectively. When setting CTcut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (CT< 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify super shedders. Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by screening out those strong positives from diagnostic pipelines.
Handyfuge-LAMP: low-cost and electricity-free centrifugation for isothermal SARS-CoV-2 detection in saliva
Authors: Li Ethan , Larson Adam , Kothari Anesta , Prakash Manu
Published: July 2020

Point of care diagnostics for COVID-19 detection are vital to assess infection quickly and at the source so appropriate measures can be taken. The loop-mediated isothermal amplification (LAMP) assay has proven to be a reliable and simple protocol that can detect small amounts of viral RNA in patient samples (<10 genomes per μL) (Nagamine, Hase, and Notomi 2002). Recently, Rabe and Cepko at Harvard published a sensitive and simple protocol for COVID-19 RNA detection in saliva using an optimized LAMP assay (Rabe and Cepko, 2020).

This LAMP protocol has the benefits of being simple, requiring no specialized equipment; rapid, requiring less than an hour from sample collection to readout; and cheap, costing around $1 per reaction using commercial reagents. The pH based colorimetric readout also leaves little ambiguity and is intuitive. However, a shortfall in many nucleic acid-based methods for detection in saliva samples has been the variability in output due to the presence of inhibitory substances in saliva. Centrifugation to separate the reaction inhibitors from inactivated sample was shown to be an effective way to ensure reliable LAMP amplification. However, a centrifuge capable of safely achieving the necessary speeds of 2000 RPM for several minutes often costs hundreds of dollars and requires a power supply.

We present here an open hardware solution- Handyfuge - that can be assembled with readily available components for the cost of <5 dollars a unit and could be used together with the LAMP assay for point of care detection of COVID-19 RNA from saliva. The device is then validated using the LAMP protocol from Rabe and Cepko. With the use of insulated coolers for reagent supply chain and delivery, the assay presented can be completed without the need for electricity or any laboratory scale infrastructure.
The structure of enteric human adenovirus 41 - a leading cause of diarrhea in children
Authors: Rafie K. , Lenman A. , Fuchs J. , Rajan A. , Arnberg N. , Carlson L.-A.
Published: July 2020

Human adenovirus (HAdV) types F40 and F41 are a prominent cause of diarrhea and diarrhea-associated mortality in young children worldwide. These enteric HAdVs differ strikingly in tissue tropism and pathogenicity from respiratory and ocular adenoviruses, but the structural basis for this divergence has been unknown. Here we present the first structure of an enteric HAdV - HAdV-F41 - determined by cryo-EM to a resolution of 3.8Å. The structure reveals extensive alterations to the virion exterior as compared to non-enteric HAdVs, including a unique arrangement of capsid protein IX. The structure also provides new insights into conserved aspects of HAdV architecture such as a proposed location of protein V, which links the viral DNA to the capsid, and assembly-induced conformational changes in the penton base protein. Our findings provide the structural basis for adaptation to a fundamentally different tissue tropism of enteric HAdVs.
Inhibitory ultrapotent chemogenetics activate dopamine D1 receptor-expressing medium spiny neurons
Authors: Gantz Stephanie C. , Ortiz Maria M. , Belilos Andrew J. , Moussawi Khaled
Published: July 2020

Ultrapotent chemogenetics, including the chloride-permeable inhibitory PSAM4-GlyR receptor, were recently proposed as a powerful strategy to selectively control neuronal activity in awake, behaving animals. We aimed to validate the inhibitory function of PSAM4-GlyR in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) in the ventral striatum. Activation of PSAM4-GlyR with the uPSEM792ligand enhanced rather than suppressed the activity of D1-MSNsin vivoas indicated by increased c-fos expression in D1-MSNs. Whole-cell recordings in mouse brain slices showed that activation of PSAM4-GlyR did not inhibit firing of action potentials in D1-MSNs. Activation of PSAM4-GlyR depolarized D1-MSNs, attenuated GABAergic inhibition, and shifted the reversal potential of PSAM4-GlyR current to more depolarized potentials, perpetuating the depolarizing effect of receptor activation. The data show that ‘inhibitory’ PSAM4-GlyR chemogenetics may actually activate certain cell types, and highlight the pitfalls of utilizing chloride conductances to inhibit neurons.
The association of Plk1 with the Astrin-Kinastrin complex promotes formation and maintenance of a metaphase plate
Authors: Geraghty Zoë , Barnard Christina , Uluocak Pelin , Gruneberg Ulrike
Published: July 2020

Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The Astrin-Kinastrin/SKAP complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which Astrin-Kinastrin fulfils these diverse roles are not fully understood. Here we characterise a direct interaction between Astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on Astrin as well as four Plk1 phosphorylation sites on Astrin. Regulation of Astrin-Kinastrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that Astrin phosphorylation by Plk1 contributes to this process.

We demonstrate that Plk1 binds to and phosphorylates the N-terminus of Astrin. This interaction promotes recruitment of the Astrin-complex to kinetochores and stabilises microtubule-kinetochore-attachments in situations when mitosis is delayed.
A genome-wide and cotranscriptional suppressor of R loops
Authors: Matson Jacob P. , Zou Lee
Published: July 2020

R loops arise from hybridization of RNA transcripts with template DNA during transcription. Unrepaired R loops lead to transcription–replication collisions, causing DNA damage and genomic instability. In this issue of Genes & Development, Pérez-Calero and colleagues (pp. 898–912) identify UAP56 as a cotranscriptional RNA–DNA helicase that unwinds R loops. They found that UAP56 helicase activity is required to remove R loops formed from different sources and prevent R-loop accumulation genome-wide at actively transcribed genes.
Purification of Labeled Antibodies Using Size-Exclusion Chromatography
Authors: Berg Eric A. , Fishman Jordan B.
Published: July 2020

Many antibody labeling procedures call for a desalting or purification step requiring size-exclusion chromatography (SEC). The method outlined here contains information needed to desalt an antibody conjugate. Similar procedures would be used for ion-exchange chromatography using a gradient of increasing ionic strength. Resins can be purchased in bulk (as in this protocol), or commercially available columns are available.
Labeling of DNA Probes by Nick Translation
Authors: Green Michael R. , Sambrook Joseph
Published: July 2020

In this method, E. coli DNA Pol I binds to a nick or short gap in duplex DNA. The 5′ → 3′ exonuclease activity of Pol I then removes nucleotides from one strand of the DNA, creating a template for the synthesis of DNA by the 5′ → 3′ polymerase activity of Pol I. The simultaneous elimination of nucleotides from the 5′ side and the addition of nucleotides to the 3′ side result in movement of the nick (nick translation) along the DNA, which becomes labeled to high specific activity.
Labeling Antibodies
Authors: Berg Eric A. , Fishman Jordan B.
Published: July 2020

This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific site of modification, cross-linker options, types of labels, and postlabeling cleanup methodology, along with the advantages and disadvantages of each method. In general, polyclonal antibodies are more versatile and resistant to activity loss than are monoclonal antibodies. Greater care must be taken when labeling monoclonal antibodies to ensure a quality conjugate. The methods outlined here can be adapted for a variety of labels including multiple labels on the same immunoglobulin. The most important consideration when undertaking an antibody labeling experiment is to maintain the activity of the antibody. This is an empirical process and will often require additional experiments to optimize the label of a particular antibody. When successful, these reagents are very useful and adaptable biomolecules. This introduction provides the reader with methods and options for producing a variety of labeled immunological tools.
Structural basis of self-assembly in the lipid-binding domain of mycobacterial polar growth factor Wag31
Authors: Choukate Komal , Chaudhuri Barnali
Published: July 2020

Wag31, or DivIVA, is an essential protein and a drug target in the human pathogen Mycobacterium tuberculosis that self-assembles at the negatively curved membrane surface to form a higher-order structural scaffold, maintains rod-shaped cellular morphology and localizes key cell-wall synthesizing enzymes at the pole for exclusive polar growth. The crystal structure of the N-terminal lipid-binding domain of mycobacterial Wag31 was determined at 2.3 Å resolution. The structure revealed a highly polar surface lined with several conserved charged residues that suggest probable sites for interactions with membrane lipids. Crystal-packing analysis revealed a previously unseen `dimer-of-dimers' assembly state of N-terminal Wag31, which is formed by antiparallel stacking of two coiled-coil dimers. Size-exclusion column-chromatography-coupled small-angle solution X-ray scattering data revealed a tetrameric form as a major assembly state of N-terminal Wag31 in solution, further supporting the crystal structure. The results suggest that, in addition to lipid binding, the N-terminal Wag31 can participate in self-assembly to form filamentous structures. Plausible models of linear self-assembly and branching of Wag31 filaments consistent with available data are suggested.
Tartryl-CoA inhibits succinyl-CoA synthetase
Authors: Huang Ji , Fraser Marie E.
Published: July 2020

Succinyl-CoA synthetase (SCS) catalyzes the only substrate-level phosphorylation step in the tricarboxylic acid cycle. Human GTP-specific SCS (GTPSCS), an αβ-heterodimer, was produced inEscherichia coli. The purified protein crystallized from a solution containing tartrate, CoA and magnesium chloride, and a crystal diffracted to 1.52 Å resolution. Tartryl-CoA was discovered to be bound to GTPSCS. The CoA portion lies in the amino-terminal domain of the α-subunit and the tartryl end extends towards the catalytic histidine residue. The terminal carboxylate binds to the phosphate-binding site of GTPSCS.
Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization
Authors: Schiefner André , Walser Rebecca , Gebauer Michaela , Skerra Arne
Published: July 2020

Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and theFragaria × ananassaO-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.
Structural characterization of borneol dehydrogenase from Pseudomonas sp. TCU-HL1
Authors: Khine Aye Aye , Chen Hao-Ping , Huang Kai-Fa , Ko Tzu-Ping
Published: July 2020

During the microbial degradation of borneol, a bicyclic plant monoterpene, it is first converted into camphor by borneol dehydrogenase (BDH) and then enters a known camphor-degradation pathway. Previously, a recombinant Pseudomonas BDH was found in inclusion bodies when expressed in Escherichia coli. After refolding, it was still unstable and was difficult to concentrate. Here, the protein-expression conditions were improved by changing the medium from lysogeny broth to Terrific Broth, yielding a soluble form of the enzyme with higher activity. The protein was crystallized and its 3D structure was determined by X-ray diffraction. Like other known homologues such as quinuclidinone reductase, the protein forms a tetramer with subunits containing Rossmann folds. Structural comparison revealed major differences in the C-terminal helices and the associated loops. It is likely that these regions contain the determinants for substrate recognition.