Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration.

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.

Structural architecture of the human NALCN channelosome.

Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1-8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9-12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.

Theranostic cells: emerging clinical applications of synthetic biology.

Synthetic biology seeks to redesign biological systems to perform novel functions in a predictable manner. Recent advances in bacterial and mammalian cell engineering include the development of cells that function in biological samples or within the body as minimally invasive diagnostics or theranostics for the real-time regulation of complex diseased states. Ex vivo and in vivo cell-based biosensors and therapeutics have been developed to target a wide range of diseases including cancer, microbiome dysbiosis and autoimmune and metabolic diseases. While probiotic therapies have advanced to clinical trials, chimeric antigen receptor (CAR) T cell therapies have received regulatory approval, exemplifying the clinical potential of cellular therapies. This Review discusses preclinical and clinical applications of bacterial and mammalian sensing and drug delivery platforms as well as the underlying biological designs that could enable new classes of cell diagnostics and therapeutics. Additionally, we describe challenges that must be overcome for more rapid and safer clinical use of engineered systems.

Beyond the Four Bases: A Home Run for Synthetic Epigenetic Control?

Park et al. (2019) create a synthetic self-propagating adenine methylation system for epigenetic control in human cells. Targeting adenine allows their modular system to act orthogonally to most epigenetic processes, thereby opening the door for novel methods of controlling gene expression.

Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster.

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins.

SMOC-1 interacts with both BMP and glypican to regulate BMP signaling in C. elegans.

Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium-binding (EC) domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. In this study, we used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. We showed that CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. We provide both in vitro biochemical and in vivo functional evidence demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. We further showed that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. Our work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling.

The contributions of rare inherited and polygenic risk to ASD in multiplex families.

Autism spectrum disorder (ASD) has a complex genetic architecture involving contributions from both de novo and inherited variation. Few studies have been designed to address the role of rare inherited variation or its interaction with common polygenic risk in ASD. Here, we performed whole-genome sequencing of the largest cohort of multiplex families to date, consisting of 4,551 individuals in 1,004 families having two or more autistic children. Using this study design, we identify seven previously unrecognized ASD risk genes supported by a majority of rare inherited variants, finding support for a total of 74 genes in our cohort and a total of 152 genes after combined analysis with other studies. Autistic children from multiplex families demonstrate an increased burden of rare inherited protein-truncating variants in known ASD risk genes. We also find that ASD polygenic score (PGS) is overtransmitted from nonautistic parents to autistic children who also harbor rare inherited variants, consistent with combinatorial effects in the offspring, which may explain the reduced penetrance of these rare variants in parents. We also observe that in addition to social dysfunction, language delay is associated with ASD PGS overtransmission. These results are consistent with an additive complex genetic risk architecture of ASD involving rare and common variation and further suggest that language delay is a core biological feature of ASD.

Exome-wide association study to identify rare variants influencing COVID-19 outcomes: Results from the Host Genetics Initiative.

Host genetics is a key determinant of COVID-19 outcomes. Previously, the COVID-19 Host Genetics Initiative genome-wide association study used common variants to identify multiple loci associated with COVID-19 outcomes. However, variants with the largest impact on COVID-19 outcomes are expected to be rare in the population. Hence, studying rare variants may provide additional insights into disease susceptibility and pathogenesis, thereby informing therapeutics development. Here, we combined whole-exome and whole-genome sequencing from 21 cohorts across 12 countries and performed rare variant exome-wide burden analyses for COVID-19 outcomes. In an analysis of 5,085 severe disease cases and 571,737 controls, we observed that carrying a rare deleterious variant in the SARS-CoV-2 sensor toll-like receptor TLR7 (on chromosome X) was associated with a 5.3-fold increase in severe disease (95% CI: 2.75-10.05, p = 5.41x10-7). This association was consistent across sexes. These results further support TLR7 as a genetic determinant of severe disease and suggest that larger studies on rare variants influencing COVID-19 outcomes could provide additional insights.

Absence of Neutropenia in Patients With Early Exon Nonsense Mutations in ELANE: Clinical Evidence to Support Gene Therapy Approaches for Severe Congenital Neutropenia.

Severe congenital neutropenia is an inherited bone marrow failure disorder characterized by profoundly low neutrophil counts and promyelocytic maturation arrest in bone marrow. Severe congenital neutropenia is most often caused by heterozygous ELANE mutations. In vitro and mouse xenograft studies using CRISPR/Cas9 have shown that introduction of frameshift/nonsense mutations in mutant ELANE may restore neutrophil counts, providing a model for gene therapy. Here, we present 2 children with inherited nonsense mutations in ELANE analogous to those proposed for gene therapy. Their normal peripheral blood neutrophil counts provide support for this approach through human "experiments of nature."

Defining the distance between diseases using SNOMED CT embeddings.

Characterizing disease relationships is essential to biomedical research to understand disease etiology and improve clinical decision-making. Measurements of distance between disease pairs enable valuable research tasks, such as subgrouping patients and identifying common time courses of disease onset. Distance metrics developed in prior work focused on smaller, targeted disease sets. Distance metrics covering all diseases have not yet been defined, which limits the applications to a broader disease spectrum. Our current study defines disease distances for all disease pairs within the International Classification of Diseases, version 10 (ICD-10), the diagnostic classification system universally used in electronic health records. Our proposed distance is computed based on a biomedical ontology, SNOMED CT (Systemized Nomenclature of Medicine, Clinical Terms), which can also be viewed as a structured knowledge graph. We compared the knowledge graph-based metric to three other distance metrics based on the hierarchical structure of ICD, clinical comorbidity, and genetic correlation, to evaluate how each may capture similar or unique aspects of disease relationships. We show that our knowledge graph-based distance metric captures known phenotypic, clinical, and molecular characteristics at a finer granularity than the other three. With the continued growth of using electronic health records data for research, we believe that our distance metric will play an important role in subgrouping patients for precision health, and enabling individualized disease prevention and treatments.

Protein and Peptide Nanocluster Vaccines.

Recombinant protein- and peptide-based vaccines can deliver large amounts of specific antigens for tailored immune responses. One class of these are protein and peptide nanoclusters (PNCs), which are made entirely from the crosslinked antigen. PNCs leverage the inherent immunogenicity of nanoparticulate antigens while minimizing the use of excipients normally used to create them. In this chapter, we discuss PNC fabrication methods, immunostimulatory properties of nanoclusters observed in vitro and in vivo, and protective benefits of PNC vaccines against influenza and cancer mouse models. We conclude with an outlook on future studies of PNCs and PNC design strategies, as well as their use in future vaccine formulations.

Enhancing Synchronization by Optimal Correlated Noise.

From the flashes of fireflies to Josephson junctions and power infrastructure, networks of coupled phase oscillators provide a powerful framework to describe synchronization phenomena in many natural and engineered systems. Most real-world networks are under the influence of noisy, random inputs, potentially inhibiting synchronization. While noise is unavoidable, here we show that there exist optimal noise patterns which minimize desynchronizing effects and even enhance order. Specifically, using analytical arguments we show that in the case of a two-oscillator model, there exists a sharp transition from a regime where the optimal synchrony-enhancing noise is perfectly anticorrelated, to one where the optimal noise is correlated. More generally, we then use numerical optimization methods to demonstrate that there exist anticorrelated noise patterns that optimally enhance synchronization in large complex oscillator networks. Our results may have implications in networks such as power grids and neuronal networks, which are subject to significant amounts of correlated input noise.

Heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice.

Influenza is a persistent threat to public health. Here we report that double-layered peptide nanoparticles induced robust specific immunity and protected mice against heterosubtypic influenza A virus challenges. We fabricated the nanoparticles by desolvating a composite peptide of tandem copies of nucleoprotein epitopes into nanoparticles as cores and cross-linking another composite peptide of four tandem copies of influenza matrix protein 2 ectodomain epitopes to the core surfaces as a coating. Delivering the nanoparticles via dissolvable microneedle patch-based skin vaccination further enhanced the induced immunity. These peptide-only, layered nanoparticles demonstrated a strong antigen depot effect and migrated into spleens and draining (inguinal) lymph nodes for an extended period compared with soluble antigens. This increased antigen-presentation time correlated with the stronger immune responses in the nanoparticle-immunized group. The protection conferred by nanoparticle immunization was transferable by passive immune serum transfusion and depended partially on a functional IgG receptor FcγRIV. Using a conditional cell depletion, we found that CD8+ T cells were involved in the protection. The immunological potency and stability of the layered peptide nanoparticles indicate applications for other peptide-based vaccines and peptide drug delivery.

APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress.

The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3'-5' exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.

Initial skin cancer screening for solid organ transplant recipients in the United States: Delphi method development of expert consensus guidelines.

Skin cancer is the most common malignancy affecting solid organ transplant recipients (SOTR), and SOTR experience increased skin cancer-associated morbidity and mortality. There are no formal multidisciplinary guidelines for skin cancer screening after transplant, and current practices are widely variable. We conducted three rounds of Delphi method surveys with a panel of 84 U.S. dermatologists and transplant physicians to establish skin cancer screening recommendations for SOTR. The transplant team should risk stratify SOTR for screening, and dermatologists should perform skin cancer screening by full-body skin examination. SOTR with a history of skin cancer should continue regular follow-up with dermatology for skin cancer surveillance. High-risk transplant patients include thoracic organ recipients, SOTR age 50 and above, and male SOTR. High-risk Caucasian patients should be screened within 2 years after transplant, all Caucasian, Asian, Hispanic, and high-risk African American patients should be screened within 5 years after transplant. No consensus was reached regarding screening for low-risk African American SOTR. We propose a standardized approach to skin cancer screening in SOTR based on multidisciplinary expert consensus. These guidelines prioritize and emphasize the need for screening for SOTR at greatest risk for skin cancer.

Outcomes of Adjuvant Radiotherapy Following Negative Surgical Margins for Cutaneous Squamous Cell Carcinoma.

BACKGROUND: The role of adjuvant radiotherapy (ART) for cutaneous squamous cell carcinoma (cSCC) following negative surgical margins is unclear. OBJECTIVE: To retrospectively examine the clinical outcomes in a cohort of patients with cSCC who completed ART after Mohs micrographic surgery or wide local excision with negative margins. METHODS AND MATERIALS: After the institutional review board approval, a retrospective review was conducted of all patients with cSCC treated in the Mayo Clinic Department of Radiation Oncology from March 10, 1998, through April 26, 2013. Inclusion criteria were age >18 years, resection with negative histologic surgical margins, and completion of ART. RESULTS: Thirty-two patients met the inclusion criteria: 15 patients died, 12 without evidence of disease related to cSCC. Three patients developed recurrent disease, all with poorly differentiated cSCC, > 2 cm in clinical diameter, perineural invasion, and Brigham and Women's (BWH) stage T2b/T3; 2 of 3 patients were immunosuppressed; and 2 of 3 patients died of cSCC-related causes. CONCLUSION: These data suggest that the combination of surgical resection and ART is a reasonable option for Brigham and Women's T2b/T3 tumors.

Evaluating the catalytic importance of a conserved Glu97 residue in triosephosphate isomerase.

Investigating enzyme activity is central to our understanding of biological function, and the design of biocatalysts continues to find applications in synthesis. While a role for active site residues can be proposed based on structure and mechanism, our understanding of the catalytic importance for residues surrounding the active site is less well understood. In triosephosphate isomerase (TIM), Glu97 is situated adjacent to the active site and is found in essentially all sequences. Prior studies reported mutation of Glu97 to Asp and Gln in TIM from Plasmodium falciparum (PfTIM) led to a 100- and 4000-fold decrease in activity, respectively, while the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity. To investigate further the question of how mutations in essentially superimposable structures give different effects, we mutated E97 in TIM from Trypanosoma brucei brucei (TbbTIM), Saccharomyces cerevisiae (yTIM), and human (hTIM). The E97D, E97A, and E97Q mutations led to a ∼three-tenfold decrease in activity, a modest effect compared to the 102-103-fold effect in PfTIM. CD and fluorescence studies showed the overall structures for the mutants were essentially unchanged. Structural analysis shows that several residues surrounding E97 differ between PfTIM and TIM from the other organisms, and rearrangements or mispositioning of residues in PfTIM may lead to the different rate effects. The results illustrate the interplay of active site and surrounding residues in affecting catalysis and highlight that understanding of the role of residues surrounding the active site may aid in the incorporation of favorable or avoidance of unfavorable interactions when designing enzymes.

Hybrid, metal oxide-peptide amphiphile micelles for molecular magnetic resonance imaging of atherosclerosis.

BACKGROUND: Atherosclerosis, a major source of cardiovascular disease, is asymptomatic for decades until the activation of thrombosis and the rupture of enlarged plaques, resulting in acute coronary syndromes and sudden cardiac arrest. Magnetic resonance imaging (MRI) is a noninvasive nuclear imaging technique to assess the degree of atherosclerotic plaque with high spatial resolution and excellent soft tissue contrast. However, MRI lacks sensitivity for preventive medicine, which limits the ability to observe the onset of vulnerable plaques. In this study, we engineered hybrid metal oxide-peptide amphiphile micelles (HMO-Ms) that combine an inorganic, magnetic iron oxide or manganese oxide inner core with organic, fibrin-targeting peptide amphiphiles, consisting of the sequence CREKA, for potential MRI imaging of thrombosis on atherosclerotic plaques. RESULTS: Hybrid metal oxide-peptide amphiphile micelles, consisting of an iron oxide (Fe-Ms) or manganese oxide (Mn-Ms) core with CREKA peptides, were self-assembled into 20-30 nm spherical nanoparticles, as confirmed by dynamic light scattering and transmission electron microscopy. These hybrid nanoparticles were found to be biocompatible with human aortic endothelial cells in vitro, and HMO-Ms bound to human clots three to five times more efficiently than its non-targeted counterparts. Relaxivity studies showed ultra-high r2 value of 457 mM-1 s-1 and r1 value of 0.48 mM-1 s-1 for Fe-Ms and Mn-Ms, respectively. In vitro, MR imaging studies demonstrated the targeting capability of CREKA-functionalized hybrid nanoparticles with twofold enhancement of MR signals. CONCLUSION: This novel hybrid class of MR agents has potential as a non-invasive imaging method that specifically detects thrombosis during the pathogenesis of atherosclerosis.

Optimizing Effect Sizes With Imaging Enrichment and Outcome Choices for Mild Alzheimer Disease Clinical Trials.

Recent clinical trials in mild Alzheimer disease (AD) have enriched for amyloid-specific positron emission tomography (PET) imaging and used extended versions of the AD Assessment Scale-Cognitive Subscale (ADAS-Cog) in an effort to increase the sensitivity to detect treatment effects. We used data from mild AD participants in the AD Neuroimaging Initiative to model trial effect sizes for 12- and 24-month trials using 3 versions of the ADAS-Cog and increased standardized uptake value ratio (SUVR) cutoffs for amyloid imaging inclusion criteria. For 12-month trials, extended ADAS-Cog versions improved effect sizes. The ADAS-Cog11 elicited larger effect sizes when enriching for SUVR 1.1 only, whereas the ADAS-Cog12 and ADAS-Cog13 were associated with larger effect sizes with higher SUVR thresholds. For 24-month trials, extended ADAS-Cog versions increased effect sizes for trials not enriched for amyloid and trials enriched for SUVR 1.1. Only enriching for higher SUVR thresholds (1.3 and 1.4, not 1.1) increased trial power. We conclude that extended versions of the ADAS-Cog improve mild AD trial effect sizes for both 12- and 24-month long studies, whereas amyloid imaging criteria may be most valuable for 12-month trials.

Coated protein nanoclusters from influenza H7N9 HA are highly immunogenic and induce robust protective immunity.

Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines.