Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.

The CRL4DCAF1 cullin-RING ubiquitin ligase is activated following a switch in oligomerization state.

The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.

Publisher Correction: DNA damage detection in nucleosomes involves DNA register shifting.

In this Article, in Fig. 1a, the 5' and 3' labels were reversed in the DNA sequence, and Fig. 4 was missing panel labels a-e. These errors have been corrected online.

DNA damage detection in nucleosomes involves DNA register shifting.

Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.

Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture.

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

Dynamic association of human Ebp1 with the ribosome.

Ribosomes are the macromolecular machines at the heart of protein synthesis; however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution. Ebp1 binds in the vicinity of the peptide exit tunnel on the 80S ribosome, and this binding is enhanced upon puromycin-mediated translational inhibition. The association of Ebp1 with the 80S ribosome centers around its interaction with ribosomal proteins eL19 and uL23 and the 28S rRNA. Further analysis of the Ebp1-ribosome complex suggests that Ebp1 can rotate around its insert domain, which may enable it to assume a wide range of conformations while maintaining its interaction with the ribosome. Structurally, Ebp1 shares homology with the methionine aminopeptidase 2 family of enzymes; therefore, this inherent flexibility may also be conserved.

Live Analysis and Reconstruction of Single-Particle Cryo-Electron Microscopy Data with CryoFLARE.

Efficient, reproducible and accountable single-particle cryo-electron microscopy structure determination is tedious and often impeded by the lack of a standardized procedure for data analysis and processing. To address this issue, we have developed the FMI Live Analysis and Reconstruction Engine (CryoFLARE). CryoFLARE is a modular open-source platform offering easy integration of new processing algorithms developed by the cryo-EM community. It provides a user-friendly interface that allows fast setup of standardized workflows, serving the need of pharmaceutical industry and academia alike who need to optimize throughput of their microscope. To consistently document how data is processed, CryoFLARE contains an integrated reporting facility to create reports. Live analysis and processing parallel to data acquisition are used to monitor and optimize data quality. Problems at the level of the sample preparation (heterogeneity, ice thickness, sparse particles, areas selected for acquisition, etc.) or misalignments of the microscope optics can quickly be detected and rectified before data collection is continued. Interfacing with automated data collection software for retrieval of acquisition metadata reduces user input needed for analysis, and with it minimizes potential sources of errors and workflow inconsistencies. Local and remote export support in Relion-compatible job and data format allows seamless integration into the refinement process. The support for nonlinear workflows and fine-grained scheduling for mixed workflows with separate CPU and GPU based calculation steps ensures optimal use of processing hardware. CryoFLARE's flexibility allows it to be used for all types of image acquisitions, ranging from sample screening to high-resolution data collection, and it offers a new alternative for setting up image processing workflows. It can be used without modifications of the hardware/software delivered by the microscope supplier. As it runs on a server in parallel to the hardware used for acquisition, it can easily be set up for remote display connections and fast control of the acquisition status.

Individual Defects in InAs/InGaAsSb/GaSb Nanowire Tunnel Field-Effect Transistors Operating below 60 mV/decade.

Tunneling field-effect transistors (TunnelFET), a leading steep-slope transistor candidate, is still plagued by defect response, and there is a large discrepancy between measured and simulated device performance. In this work, highly scaled InAs/InxGa1-xAsySb1-y/GaSb vertical nanowire TunnelFET with ability to operate well below 60 mV/decade at technically relevant currents are fabricated and characterized. The structure, composition, and strain is characterized using transmission electron microscopy with emphasis on the heterojunction. Using Technology Computer Aided Design (TCAD) simulations and Random Telegraph Signal (RTS) noise measurements, effects of different type of defects are studied. The study reveals that the bulk defects have the largest impact on the performance of these devices, although for these highly scaled devices interaction with even few oxide defects can have large impact on the performance. Understanding the contribution by individual defects, as outlined in this letter, is essential to verify the fundamental physics of device operation, and thus imperative for taking the III-V TunnelFETs to the next level.

Molecular driving forces defining lipid positions around aquaporin-0.

Lipid-protein interactions play pivotal roles in biological membranes. Electron crystallographic studies of the lens-specific water channel aquaporin-0 (AQP0) revealed atomistic views of such interactions, by providing high-resolution structures of annular lipids surrounding AQP0. It remained unclear, however, whether these lipid structures are representative of the positions of unconstrained lipids surrounding an individual protein, and what molecular determinants define the lipid positions around AQP0. We addressed these questions by using molecular dynamics simulations and crystallographic refinement, and calculated time-averaged densities of dimyristoyl-phosphatidylcholine lipids around AQP0. Our simulations demonstrate that, although the experimentally determined crystallographic lipid positions are constrained by the crystal packing, they appropriately describe the behavior of unconstrained lipids around an individual AQP0 tetramer, and thus likely represent physiologically relevant lipid positions.While the acyl chains were well localized, the lipid head groups were not. Furthermore, in silico mutations showed that electrostatic interactions do not play a major role attracting these phospholipids towards AQP0. Instead, the mobility of the protein crucially modulates the lipid localization and explains the difference in lipid density between extracellular and cytoplasmic leaflets. Moreover, our simulations support a general mechanism in which membrane proteins laterally diffuse accompanied by several layers of localized lipids, with the positions of the annular lipids being influenced the most by the protein surface. We conclude that the acyl chains rather than the head groups define the positions of dimyristoyl-phosphatidylcholine lipids around AQP0. Lipid localization is largely determined by the mobility of the protein surface, whereas hydrogen bonds play an important but secondary role.

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus.

The RNA-dependent RNA polymerase (RdRP) of nonsegmented negative-sense RNA viruses consists of a large catalytic protein (L) and a phosphoprotein cofactor (P). During infection, the RdRP replicates and transcribes the viral genome, which resides inside an oligomer of nucleocapsid protein (N-RNA). The classical view of P as a cofactor for L assigns a primary role of P as a bridge mediating the access of L to the RNA template, whereby its N-terminal domain (P(NTD)) binds L and its C-terminal domain (P(CTD)) binds N-RNA. Recent biochemical and structural studies of a prototype nonsegmented negative-sense RNA virus, vesicular stomatitis virus, suggest a role for P beyond that of a mere physical link: P induces a structural rearrangement in L and stimulates polymerase processivity. In this study, we investigated the critical requirements within P mediating the functional interaction with L to form a fully functional RdRP. We analyzed the correlation between the impact of P on the conformation of L and its activity in RNA synthesis and the consequences of these events on RdRP function. We identified three separable elements of the P(NTD) that are required for inducing the conformational rearrangement of L, stimulating polymerase processivity, and mediating transcription of the N-RNA. The functional interplay between these elements provides insight into the role of P as a dynamic player in the RNA synthesis machine, influencing essential aspects of polymerase structure and function.

[Awake fiberoptic intubation - gold standard for the anticipated difficult airway]. / Fiberoptische Wachintubation - Goldstandard für den erwartet schwierigen Atemweg.

Awake fiberoptic intubation seems to continually lose importance in recent years. Alternative options of airway management are coming more and more into the focus of clinical anaesthesia and are moreover advertised specifically for the difficult airway. The concern seems justified that this intubation technique - once the indisputed standard in the management of the anticipated difficult airway - is practised less and less so that especially younger colleagues can hardly gain routine practice. This article aims at counteracting this trend and particularly demonstrates a practical approach. Against this background the equipment is described and tips and tricks are given for execution. The main focus are safety aspects and the success factor of a good topical mucosal anaesthesia, which is of greater importance than a concomitant sedation.

A pipeline for comprehensive and automated processing of electron diffraction data in IPLT.

Electron crystallography of two-dimensional crystals allows the structural study of membrane proteins in their native environment, the lipid bilayer. Determining the structure of a membrane protein at near-atomic resolution by electron crystallography remains, however, a very labor-intense and time-consuming task. To simplify and accelerate the data processing aspect of electron crystallography, we implemented a pipeline for the processing of electron diffraction data using the Image Processing Library and Toolbox (IPLT), which provides a modular, flexible, integrated, and extendable cross-platform, open-source framework for image processing. The diffraction data processing pipeline is organized as several independent modules implemented in Python. The modules can be accessed either from a graphical user interface or through a command line interface, thus meeting the needs of both novice and expert users. The low-level image processing algorithms are implemented in C++ to achieve optimal processing performance, and their interface is exported to Python using a wrapper. For enhanced performance, the Python processing modules are complemented with a central data managing facility that provides a caching infrastructure. The validity of our data processing algorithms was verified by processing a set of aquaporin-0 diffraction patterns with the IPLT pipeline and comparing the resulting merged data set with that obtained by processing the same diffraction patterns with the classical set of MRC programs.

Molecular architecture of the vesicular stomatitis virus RNA polymerase.

Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.

Assembly of a functional Machupo virus polymerase complex.

Segmented negative-sense viruses of the family Arenaviridae encode a large polymerase (L) protein that contains all of the enzymatic activities required for RNA synthesis. These activities include an RNA-dependent RNA polymerase (RdRP) and an RNA endonuclease that cleaves capped primers from cellular mRNAs to prime transcription. Using purified catalytically active Machupo virus L, we provide a view of the overall architecture of this multifunctional polymerase and reconstitute complex formation with an RNA template in vitro. The L protein contains a central ring domain that is similar in appearance to the RdRP of dsRNA viruses and multiple accessory appendages that may be responsible for 5' cap formation. RNA template recognition by L requires a sequence-specific motif located at positions 2-5 in the 3' terminus of the viral genome. Moreover, L-RNA complex formation depends on single-stranded RNA, indicating that inter-termini dsRNA interactions must be partially broken for complex assembly to occur. Our results provide a model for arenavirus polymerase-template interactions and reveal the structural organization of a negative-strand RNA virus L protein.

Silicon nanowire Esaki diodes.

We report on the fabrication and characterization of silicon nanowire tunnel diodes. The silicon nanowires were grown on p-type Si substrates using Au-catalyzed vapor-liquid-solid growth and in situ n-type doping. Electrical measurements reveal Esaki diode characteristics with peak current densities of 3.6 kA/cm(2), peak-to-valley current ratios of up to 4.3, and reverse current densities of up to 300 kA/cm(2) at 0.5 V reverse bias. Strain-dependent current-voltage (I-V) measurements exhibit a decrease of the peak tunnel current with uniaxial tensile stress and an increase of 48% for 1.3 GPa compressive stress along the <111> growth direction, revealing the strain dependence of the Si band structure and thus the tunnel barrier. The contributions of phonons to the indirect tunneling process were probed by conductance measurements at 4.2 K. These measurements show phonon peaks at energies corresponding to the transverse acoustical and transverse optical phonons. In addition, the low-temperature conductance measurements were extended to higher biases to identify potential impurity states in the band gap. The results demonstrate that the most likely impurity, namely, Au from the catalyst particle, is not detectable, a finding that is also supported by the excellent device properties of the Esaki diodes reported here.

Family-centered delirium prevention and treatment using video calls: the FACE Delirium trial.

PURPOSE: In the FACE Delirium trial, we investigated the feasibility of a structured FAmily-CEntered delirium prevention and treatment during the corona pandemic. METHODS: Patients hospitalized in a German geriatric medicine department were included in this single-center, prospective, single-arm feasibility study. Their relatives received a short training on delirium and volunteers or paid staff members facilitated video calls. The primary endpoint was reached when contact between patients and their relatives occurred on ≥ 80% of treatment days, either via video call or visit. RESULTS: 38 patients were included (age 83.0 ± 5.9 years; 73.7% women). 76.3% reached the primary endpoint. Due to the pandemic, 99.3% of the contacts were video calls with a duration of 24.8 ± 16.3 min. CONCLUSION: Family-centered delirium prevention and treatment using video calls is feasible among hospitalized geriatric patients. Daily implementation in clinical practice poses challenges and requires motivated and qualified staff.

Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

In situ doping of catalyst-free InAs nanowires.

We report on in situ doping of InAs nanowires grown by metal-organic vapor-phase epitaxy without any catalyst particles. The effects of various dopant precursors (Si(2)H(6), H(2)S, DETe, CBr(4)) on the nanowire morphology and the axial and radial growth rates are investigated to select dopants that enable control of the conductivity in a broad range and that concomitantly lead to favorable nanowire growth. In addition, the resistivity of individual wires was measured for different gas-phase concentrations of the dopants selected, and the doping density and mobility were extracted. We find that by using Si(2)H(6) axially and radially uniform doping densities up to 7 × 10(19) cm(-3) can be obtained without affecting the morphology or growth rates. For sulfur-doped InAs nanowires, we find that the distribution coefficient depends on the growth conditions, making S doping more difficult to control than Si doping. Moreover, above a critical sulfur gas-phase concentration, compensation takes place, limiting the maximum doping level to 2 × 10(19) cm(-3). Finally, we extract the specific contact resistivity as a function of doping concentration for Ti and Ni contacts.

Trap-assisted tunneling in Si-InAs nanowire heterojunction tunnel diodes.

We report on the electrical characterization of one-sided p(+)-si/n-InAs nanowire heterojunction tunnel diodes to provide insight into the tunnel process occurring in this highly lattice mismatched material system. The lattice mismatch gives rise to dislocations at the interface as confirmed by electron microscopy. Despite this, a negative differential resistance with peak-to-valley current ratios of up to 2.4 at room temperature and with large current densities is observed, attesting to the very abrupt and high-quality interface. The presence of dislocations and other defects that increase the excess current is evident in the first and second derivative of the I-V characteristics as distinct peaks arising from trap-and phonon-assisted tunneling via the corresponding defect levels. We observe this assisted tunneling mainly in the forward direction and at low reverse bias but not at higher reverse biases because the band-to-band generation rates are peaked in the InAs, which is also confirmed by modeling. This indicates that most of the peaks are due to dislocations and defects in the immediate vicinity of the interface. Finally, we also demonstrate that these devices are very sensitive to electrical stress, in particular at room temperature, because of the extremely high electrical fields obtained at the abrupt junction even at low bias. The electrical stress induces additional defect levels in the band gap, which reduce the peak-to-valley current ratios.

Land use change in the Vietnamese Mekong Delta: New evidence from remote sensing.

This paper presents the first attempt to capture a comprehensive spatial view of land use change in the Vietnamese Mekong Delta (VMD) for a long period, i.e., from 2000 to 2020. It is aimed at monitoring holistically the land use change and flooding situation in the region, addressing the reasons for land use change, and assessing the impacts of land use change on hydraulic aspects and farmer livelihoods during the last 21 years. MODIS products, in particular, are used to study the dynamics of land use and floods after demonstrating high validation with statistical data and radar satellites, with R2 = 0.96 and R2 ≥ 0.97 for land use and flood maps, respectively. The results show that rice cultivation is the most dominant land use type, accounting for 40% to 46% of the delta area, while aquaculture accounts for 10% to 22%, respectively. The total rice cultivation area increased from 3764 thousand hectares (thous. ha) in 2001 to 4343 thous. ha in 2015 based on the intensive development of triple rice cropping in the upper zone, then decreased to 3963 thous. ha in 2020. In contrast, aquaculture areas are farmed mainly in the coastal area and remained relatively steady, increasing slightly from 619 thous. ha in 2001 to 856 thous. ha in 2020. The massive construction of dikes for triple rice cropping in the upper zone appears to cause a significant impact on the annual flooding regime. Land use policies have influenced the changes in land use patterns, flooding situations, and the livelihoods of local farmers.