Mega-scale experimental analysis of protein folding stability in biology and design.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

Structure and receptor recognition by the Lassa virus spike complex.

Lassa virus (LASV) is a human pathogen, causing substantial morbidity and mortality1,2. Similar to other Arenaviridae, it presents a class-I spike complex on its surface that facilitates cell entry. The virus's cellular receptor is matriglycan, a linear carbohydrate that is present on α-dystroglycan3,4, but the molecular mechanism that LASV uses to recognize this glycan is unknown. In addition, LASV and other arenaviruses have a unique signal peptide that forms an integral and functionally important part of the mature spike5-8; yet the structure, function and topology of the signal peptide in the membrane remain uncertain9-11. Here we solve the structure of a complete native LASV spike complex, finding that the signal peptide crosses the membrane once and that its amino terminus is located in the extracellular region. Together with a double-sided domain-switching mechanism, the signal peptide helps to stabilize the spike complex in its native conformation. This structure reveals that the LASV spike complex is preloaded with matriglycan, suggesting the mechanism of binding and rationalizing receptor recognition by α-dystroglycan-tropic arenaviruses. This discovery further informs us about the mechanism of viral egress and may facilitate the rational design of novel therapeutics that exploit this binding site.

Computational design of BclxL inhibitors that target transmembrane domain interactions.

Several methods have been developed to explore interactions among water-soluble proteins or regions of proteins. However, techniques to target transmembrane domains (TMDs) have not been examined thoroughly despite their importance. Here, we developed a computational approach to design sequences that specifically modulate protein-protein interactions in the membrane. To illustrate this method, we demonstrated that BclxL can interact with other members of the B cell lymphoma 2 (Bcl2) family through the TMD and that these interactions are required for BclxL control of cell death. Next, we designed sequences that specifically recognize and sequester the TMD of BclxL. Hence, we were able to prevent BclxL intramembrane interactions and cancel its antiapoptotic effect. These results advance our understanding of protein-protein interactions in membranes and provide a means to modulate them. Moreover, the success of our approach may trigger the development of a generation of inhibitors targeting interactions between TMDs.

Glial 'omics in ischemia: Acute stroke and chronic cerebral small vessel disease.

Vascular injury and pathologies underlie common diseases including ischemic stroke and cerebral small vessel disease (CSVD). Prior work has identified a key role for glial cells, including microglia, in the multifaceted and temporally evolving neuroimmune response to both stroke and CSVD. Transcriptional profiling has led to important advances including identification of distinct gene expression signatures in ischemia-exposed, flow cytometrically sorted microglia and more recently single cell RNA sequencing-identified microglial subpopulations or clusters. There is a reassuring degree of overlap in the results from these two distinct methodologies with both identifying a proliferative and a separate type I interferon responsive microglial element. Similar patterns were later seen using multimodal and spatial transcriptomal profiling in ischemia-exposed microglia and astrocytes. Methodological advances including enrichment of specific neuroanatomic/functional regions (such as the neurovascular unit) prior to single cell RNA sequencing has led to identification of novel cellular subtypes and generation of new credible hypotheses as to cellular function based on the enhanced cell sub-type specific gene expression patterns. A ribosomal tagging strategy focusing on the cellular translatome analyses carried out in the acute phases post stroke has revealed distinct inflammation-regulating roles for microglia and astrocytes in this setting. Early spatial transcriptomics experiments using cerebral ischemia models have identified regionally distinct microglial cell clusters in ischemic core versus penumbra. There is great potential for combination of these methods for multi-omics approaches to further elucidate glial responses in the context of both acute ischemic stroke and chronic CSVD.

Characterizing Trends in Chronic Superficial Venous Disease Treatment among Medicare Beneficiaries from 2010 to 2018.

This study describes trends in surgical versus endovascular interventions for treatment of chronic superficial venous disease (SVD) in the Medicare population. Medicare Part B data from 2010 to 2018 were obtained. Claims for SVD treatment were identified using Healthcare Common Procedure Coding System codes. Total percentage change in utilization rates and market share was determined for each provider group. Utilization of SVD treatments increased by 58%, mostly owing to growing utilization of endovascular treatments. There was a 66% decrease in surgical treatments. The utilization of ablation and sclerotherapy plateaued in 2016 and decreased in 2017-2018 with the advent of mechanochemical ablation, endovenous microfoam, and cyanoacrylate adhesive, respectively. Analysis showed that endovascular utilization increased across most specialties, with the largest growth seen in cardiology by 427%. Radiologists showed utilization growth of 125%, encompassing 11% of the market share. Endovascular treatment for SVD remains predominant, with increased utilization and concomitant decrease in surgical methods.

Pancreatic Walled-Off Necrosis: Cross-Sectional Imaging Depiction of Debris Predicts the Success of Endoscopic Drainage Using Lumen-Apposing Metal Stents.

INTRODUCTION: The use of endoscopic ultrasound (EUS)-guided transmural stent placement for pancreatic walled-off necrosis (WON) drainage is widespread. This study retrospectively analyzed imaging parameters predicting the outcomes of WON endoscopic drainage using lumen-apposing metal stents (LAMS). METHODS: This study analyzed the data of 115 patients who underwent EUS-guided debridement using LAMS from 2011 to 2015. Pre-intervention CT or MRI was used to analyze the total volume of WON, percentage of debris, multilocularity, and density. Success measures included technical success, the number of endoscopic sessions, the requirement of percutaneous drainage, long-term success, and recurrence. RESULTS: The primary cause of pancreatitis was gallstones (50.4%), followed by alcohol (27.8%), hypertriglyceridemia (11.3%), idiopathic (8.7%), and autoimmune (1.7%). The mean WON size was 674 mL. All patients underwent endoscopic necrosectomy, averaging 3.1 sessions. Stent placement was successful in 96.5% of cases. Procedural complications were observed in 13 patients (11.3%) and 6 patients (5.2%) who needed additional percutaneous drainage. No patients reported recurrent WON posttreatment. Univariate analysis indicated a significant correlation between debris percentage and the need for additional drainage and long-term success (p < 0.001). The number of endoscopic sessions correlated significantly with debris percentage (p < 0.001). CONCLUSION: Pre-procedural imaging, particularly debris percentage within WON, significantly predicts the number of endoscopic sessions, the need for further percutaneous drainage, and overall long-term success.

A Subpopulation of Microglia Generated in the Adult Mouse Brain Originates from Prominin-1-Expressing Progenitors.

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro To determine whether Prom1-expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion.SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

Microglial depletion abolishes ischemic preconditioning in white matter.

Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC+ ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring.

Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences.

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.

PROSS 2: a new server for the design of stable and highly expressed protein variants.

SUMMARY: Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence analysis, error handling and automated analysis of the quality of the sequence alignment that is used in design calculations. AVAILABILITY AND IMPLEMENTATION: PROSS2 is freely available for academic use at https://pross.weizmann.ac.il.

Distressed Communities Index in Patients Undergoing Transcatheter Aortic Valve Implantation in an Affluent County in New York.

BACKGROUND: The clinical impact of the distressed communities index (DCI), a composite measure of economic well-being based on the U.S. zip code, is becoming increasingly recognized. Ranging from 0 (prosperous) to 100 (distressed), DCI's association with cardiovascular outcomes remains unknown. We aimed to study the association of the DCI with presentation and outcomes in adults with severe symptomatic aortic stenosis (AS) undergoing transcatheter aortic valve intervention (TAVR) in an affluent county in New York. METHODS: The study population included 286 patients with severe symptomatic AS or degeneration of a bioprosthetic valve who underwent TAVR with a newer generation transcatheter heart valve (THV) from December 2015 to June 2018 at an academic tertiary medical center. DCI for each patient was derived from their primary residence zip code. Patients were classified into DCI deciles and then categorized into 4 groups. The primary and secondary outcomes of interest were 30-day, 1-year, and 3-year mortality, respectively. RESULTS: Among 286 patients studied, 26%, 28%, 28%, and 18% were categorized into DCI groups 1-4, respectively (DCI <10: n = 73; DCI 10-20: n = 81; DCI 20-30: n = 80; DCI >30: n = 52). Patients in group 4 were younger with worse kidney function compared to patients in groups 1 and 2. They also had smaller aortic annuli and were more likely to receive a smaller THV. No significant difference in hospital length of stay or distribution of in-hospital, 30-day, 1-year, and 3-year mortality was demonstrated. CONCLUSIONS: While the DCI was associated with differences in the clinical and anatomic profile, it was not associated with differences in clinical outcomes in this prospective observational study of adults undergoing TAVR suggesting that access to care is the likely discriminator.

The role of microglia in ischemic preconditioning.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic stimulus confers protection against a subsequent prolonged ischemic event. Initially thought to be due to mechanistic changes in neurons, our understanding of IPC has evolved to encompass a global reprogramming of the Central Nervous System (CNS) after transient ischemia/reperfusion that requires innate immune signaling pathways including Toll-like receptors (TLRs) and Type I interferons. Microglia are the CNS resident neuroimmune cells that express these key innate immune receptors. Studies suggest that microglia are required for IPC-mediated neuronal and axonal protection. Multiple paradigms targeting TLRs have converged on a distinctive Type I interferon response in microglia that is critical for preconditioning-mediated protection against ischemia. These pathways can be targeted through administration of TLR agonists, cytokines including interferon-ß, and pharmaceutical agents that induce preconditioning through cross-tolerance mechanisms. Transcriptomic analyses and single cell RNA studies point to specific gene expression signatures in microglia that functionally shift these mutable cells to an immunomodulatory or protective phenotype. Although there are technological challenges and gaps in knowledge to overcome, the targeting of specific molecular signaling pathways in microglia is a promising direction for development of novel and effective pharmacotherapies for stroke. Studies on preconditioning in animal models, including nonhuman primates, show promise as prophylactic preconditioning treatments for selected at risk patient populations. In addition, our growing understanding of the mechanisms of IPC-mediated protection is identifying novel cellular and molecular targets for therapeutic interventions that could apply broadly to both acute stroke and chronic vascular cognitive impairment patients.

Ischemic preconditioning induces cortical microglial proliferation and a transcriptomic program of robust cell cycle activation.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a subthreshold ischemic insult applied to the brain reduces damage caused by a subsequent more severe ischemic episode. Identifying key molecular and cellular mediators of IPC will provide critical information needed to develop novel therapies for stroke. Here we report that the transcriptomic response of acutely isolated preconditioned cortical microglia is dominated by marked upregulation of genes involved in cell cycle activation and cellular proliferation. Notably, this transcriptional response occurs in the absence of cortical infarction. We employed ex vivo flow cytometry, immunofluorescent microscopy, and quantitative stereology methods on brain tissue to evaluate microglia proliferation following IPC. Using cellular colocalization of microglial (Iba1) and proliferation (Ki67 and BrdU) markers, we observed a localized increase in the number of microglia and proliferating microglia within the preconditioned hemicortex at 72, but not 24, hours post-IPC. Our quantification demonstrated that the IPC-induced increase in total microglia was due entirely to proliferation. Furthermore, microglia in the preconditioned hemisphere had altered morphology and increased soma volumes, indicative of an activated phenotype. Using transgenic mouse models with either fractalkine receptor (CX3CR1)-haploinsufficiency or systemic type I interferon signaling loss, we determined that microglial proliferation after IPC is dependent on fractalkine signaling but independent of type I interferon signaling. These findings suggest there are multiple distinct targetable signaling pathways in microglia, including CX3CR1-dependent proliferation that may be involved in IPC-mediated protection.

Ultralong Spin-Coherence Times for Rubidium Atoms in Solid Parahydrogen via Dynamical Decoupling.

Coherence time is an essential parameter for quantum sensing, quantum information, and quantum computation. In this work, we demonstrate electron spin coherence times as long as 0.1 s for an ensemble of rubidium atoms trapped in a solid parahydrogen matrix. We explore the underlying physics limiting the coherence time. The properties of these matrix isolated atoms are very promising for future applications, including quantum sensing of nuclear spins. If combined with efficient single-atom readout, this would enable NMR and magnetic resonance imaging of single molecules cotrapped with alkali-metal atom quantum sensors within a parahydrogen matrix.

A lipophilicity-based energy function for membrane-protein modelling and design.

Membrane-protein design is an exciting and increasingly successful research area which has led to landmarks including the design of stable and accurate membrane-integral proteins based on coiled-coil motifs. Design of topologically more complex proteins, such as most receptors, channels, and transporters, however, demands an energy function that balances contributions from intra-protein contacts and protein-membrane interactions. Recent advances in water-soluble all-atom energy functions have increased the accuracy in structure-prediction benchmarks. The plasma membrane, however, imposes different physical constraints on protein solvation. To understand these constraints, we recently developed a high-throughput experimental screen, called dsTßL, and inferred apparent insertion energies for each amino acid at dozens of positions across the bacterial plasma membrane. Here, we express these profiles as lipophilicity energy terms in Rosetta and demonstrate that the new energy function outperforms previous ones in modelling and design benchmarks. Rosetta ab initio simulations starting from an extended chain recapitulate two-thirds of the experimentally determined structures of membrane-spanning homo-oligomers with <2.5Å root-mean-square deviation within the top-predicted five models (available online: http://tmhop.weizmann.ac.il). Furthermore, in two sequence-design benchmarks, the energy function improves discrimination of stabilizing point mutations and recapitulates natural membrane-protein sequences of known structure, thereby recommending this new energy function for membrane-protein modelling and design.

Endovascular Repair of the Ascending Aorta for an Anastomotic Saphenous Vein Graft Aneurysm.

The ascending aorta is the final segment of the aorta to be explored with endovascular stent grafts. With a patient population of increasingly advanced age and disease, there are situations where traditional open repair for ascending aneurysms or dissections may be prohibitive. However, the ascending aorta has multiple hostile characteristics that make endovascular treatment challenging. There is also a lack of approved specialized devices in the United States for this aortic territory. We demonstrate the feasibility of adapting an abdominal aortic graft to the ascending aorta for the treatment of a saphenous vein graft aneurysm with a discussion of the technical considerations for the operation.

Searching for Ultralight Dark Matter with Optical Cavities.

We discuss the use of optical cavities as tools to search for dark matter (DM) composed of virialized ultralight fields (VULFs). Such fields could lead to oscillating fundamental constants, resulting in oscillations of the length of rigid bodies. We propose searching for these effects via differential strain measurement of rigid and suspended-mirror cavities. We estimate that more than 2 orders of magnitude of unexplored phase space for VULF DM couplings can be probed at VULF Compton frequencies in the audible range of 0.1-10 kHz.

Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology.

The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTßL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTßL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is -6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il).

Ischemia/Reperfusion Induces Interferon-Stimulated Gene Expression in Microglia.

Innate immune signaling is important in the pathophysiology of ischemia/reperfusion (stroke)-induced injury and recovery. Several lines of evidence support a central role for microglia in these processes. Recent work has identified Toll-like receptors (TLRs) and type I interferon (IFN) signaling in both ischemia/reperfusion-induced brain injury and ischemic preconditioning-mediated neuroprotection. To determine the effects of "ischemia/reperfusion-like" conditions on microglia, we performed genomic analyses on wild-type (WT) and TLR4-/- cultured microglia after sequential exposure to hypoxia/hypoglycemia and normoxia/normoglycemia (H/H-N/N). We observed increased expression of type 1 IFN-stimulated genes (ISGs) as the predominant transcriptomal feature of H/H-N/N-exposed WT, but not TLR4-/-, microglia. Microarray analysis on ex vivo sorted microglia from ipsilateral male mouse cortex after a transient in vivo ischemic pulse also demonstrated robust expression of ISGs. Type 1 IFNs, including the IFN-αs and IFN-ß, activate the interferon-α/ß receptor (IFNAR) complex. We confirmed both in vitro H/H-N/N- and in vivo ischemia/reperfusion-induced microglial ISG responses by quantitative real-time PCR and demonstrated that both were dependent on IFNAR1. We characterized the effects of hypoxia/hypoglycemia on phosphorylation of signal transducer and activator of transcription 1 (STAT1), release of type 1 IFNs, and surface expression of IFNAR1 in microglia. We demonstrated that IFN-ß induces dose-dependent secretion of ISG chemokines in cultured microglia and robust ISG expression in microglia both in vitro and in vivo Finally, we demonstrated that the microglial ISG chemokine responses to TLR4 agonists were dependent on TLR4 and IFNAR1. Together, these data suggest novel ischemia/reperfusion-induced pathways for both TLR4-dependent and -independent, IFNAR1-dependent, type 1 IFN signaling in microglia.SIGNIFICANCE STATEMENT Stroke is the fifth leading cause of death in the United States and is a leading cause of serious long-term disability worldwide. Innate immune responses are critical in stroke pathophysiology, and microglia are key cellular effectors in the CNS response to ischemia/reperfusion. Using a transcriptional analysis approach, we identified a robust interferon (IFN)-stimulated gene response within microglia exposed to ischemia/reperfusion in both in vitro and in vivo experimental paradigms. Using a number of complementary techniques, we have demonstrated that these responses are dependent on innate immune signaling components including Toll-like receptor-4 and type I IFNs. We have also elucidated several novel ischemia/reperfusion-induced microglial signaling mechanisms.

Cold Anisotropically Interacting van der Waals Molecule: TiHe.

We have used laser ablation and helium buffer-gas cooling to produce titanium-helium van der Waals molecules at cryogenic temperatures. The molecules were detected through laser-induced fluorescence spectroscopy. Ground-state Ti(a^{3}F_{2})-He binding energies were determined for the ground and first rotationally excited states from studying equilibrium thermodynamic properties, and found to agree well with theoretical calculations based on newly calculated ab initio Ti-He interaction potentials, opening up novel possibilities for studying the formation, dynamics, and nonuniversal chemistry of van der Waals clusters at low temperatures.