The IRE1ß-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells.

Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1ß) with the luminal domain of IRE1ß deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1ß/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1ß/α chimera. In vitro, AGR2 actively de-stabilised the IRE1ß luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1ß-AGR2 couple suggest that active repression of IRE1ß by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.

Disease-specific tau filaments assemble via polymorphic intermediates.

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, ß-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

Domestication through clandestine cultivation constrained genetic diversity in magic mushrooms relative to naturalized populations.

Fungi that are edible or fermentative were domesticated through selective cultivation of their desired traits. Domestication is often associated with inbreeding or selfing, which may fix traits other than those under selection, and causes an overall decrease in heterozygosity. A hallucinogenic mushroom, Psilocybe cubensis, was domesticated from its niche in livestock dung for production of psilocybin. It has caused accidental poisonings since the 1940s in Australia, which is a population hypothesized to be introduced from an unknown center of origin. We sequenced genomes of 38 isolates from Australia and compared them with 86 genomes of commercially available cultivars to determine (1) whether P. cubensis was introduced to Australia, and (2) how domestication has impacted commercial cultivars. Our analyses of genome-wide SNPs and single-copy orthologs showed that the Australian population is naturalized, having recovered its effective population size after a bottleneck when it was introduced, and it has maintained relatively high genetic diversity based on measures of nucleotide and allelic diversity. In contrast, domesticated cultivars generally have low effective population sizes and hallmarks of selfing and clonal propagation, including low genetic diversity, low heterozygosity, high linkage disequilibrium, and low allelic diversity of mating-compatibility genes. Analyses of kinship show that most cultivars are founded from related populations. Alleles in the psilocybin gene cluster are identical across most cultivars of P. cubensis with low diversity across coding sequence; however, unique allelic diversity in Australia and some cultivars may translate to differences in biosynthesis of psilocybin and its analogs.

Rapid single-photon color imaging of moving objects.

This paper outlines an experimental demonstration of a Bayesian image reconstruction approach to achieve rapid single-photon color imaging of moving objects. The capacity to extract the color of objects is important in a variety of target identification and computer vision applications. Nonetheless, it remains challenging to achieve high-speed color imaging of moving objects in low-photon flux environments. The low-photon regime presents particular challenges for efficient spectral separation and identification, while unsupervised image reconstruction algorithms are often slow and computationally expensive. In this paper, we address both of these difficulties using a combination of hardware and computational solutions. We demonstrate color imaging using a Single-Photon Avalanche Diode (SPAD) detector array for rapid, low-light-level data acquisition, with an integrated color filter array (CFA) for efficient spectral unmixing. High-speed image reconstruction is achieved using a bespoke Bayesian algorithm to produce high-fidelity color videos. The analysis is conducted first on simulated data allowing different pixel formats and photon flux scenarios to be investigated. Experiments are then performed using a plasmonic metasurface-based CFA, integrated with a 64 × 64 pixel format SPAD array. Passive imaging is conducted using white-light illumination of multi-colored, moving targets. Intensity information is recorded in a series of 2D photon-counting SPAD frames, from which accurate color information is extracted using the fast Bayesian method introduced herein. The per-frame reconstruction rate proves to be hundreds of times faster than the previous computational method. Furthermore, this approach yields additional information in the form of uncertainty measures, which can be used to assist with imaging system optimization and decision-making in real-world applications. The techniques demonstrated point the way towards rapid video-rate single-photon color imaging. The developed Bayesian algorithm, along with more advanced SPAD technology and utilization of time-correlated single-photon counting (TCSPC) will permit live 3D, color videography in extremely low-photon flux environments.

3D target detection and spectral classification for single-photon LiDAR data.

3D single-photon LiDAR imaging has an important role in many applications. However, full deployment of this modality will require the analysis of low signal to noise ratio target returns and very high volume of data. This is particularly evident when imaging through obscurants or in high ambient background light conditions. This paper proposes a multiscale approach for 3D surface detection from the photon timing histogram to permit a significant reduction in data volume. The resulting surfaces are background-free and can be used to infer depth and reflectivity information about the target. We demonstrate this by proposing a hierarchical Bayesian model for 3D reconstruction and spectral classification of multispectral single-photon LiDAR data. The reconstruction method promotes spatial correlation between point-cloud estimates and uses a coordinate gradient descent algorithm for parameter estimation. Results on simulated and real data show the benefits of the proposed target detection and reconstruction approaches when compared to state-of-the-art processing algorithms.

Robust real-time imaging through flexible multimode fibers.

Conventional endoscopes comprise a bundle of optical fibers, associating one fiber for each pixel in the image. In principle, this can be reduced to a single multimode optical fiber (MMF), the width of a human hair, with one fiber spatial-mode per image pixel. However, images transmitted through a MMF emerge as unrecognizable speckle patterns due to dispersion and coupling between the spatial modes of the fiber. Furthermore, speckle patterns change as the fiber undergoes bending, making the use of MMFs in flexible imaging applications even more complicated. In this paper, we propose a real-time imaging system using flexible MMFs, but which is robust to bending. Our approach does not require access or feedback signal from the distal end of the fiber during imaging. We leverage a variational autoencoder to reconstruct and classify images from the speckles and show that these images can still be recovered when the bend configuration of the fiber is changed to one that was not part of the training set. We utilize a MMF 300 mm long with a 62.5 µm core for imaging [Formula: see text] cm objects placed approximately at 20 cm from the fiber and the system can deal with a change in fiber bend of 50[Formula: see text] and range of movement of 8 cm.

Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere.

The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-ANuc). This revealed a central CENP-ANuc with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-ANuc can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Submerged single-photon LiDAR imaging sensor used for real-time 3D scene reconstruction in scattering underwater environments.

We demonstrate a fully submerged underwater LiDAR transceiver system based on single-photon detection technologies. The LiDAR imaging system used a silicon single-photon avalanche diode (SPAD) detector array fabricated in complementary metal-oxide semiconductor (CMOS) technology to measure photon time-of-flight using picosecond resolution time-correlated single-photon counting. The SPAD detector array was directly interfaced to a Graphics Processing Unit (GPU) for real-time image reconstruction capability. Experiments were performed with the transceiver system and target objects immersed in a water tank at a depth of 1.8 meters, with the targets placed at a stand-off distance of approximately 3 meters. The transceiver used a picosecond pulsed laser source with a central wavelength of 532 nm, operating at a repetition rate of 20 MHz and average optical power of up to 52 mW, dependent on scattering conditions. Three-dimensional imaging was demonstrated by implementing a joint surface detection and distance estimation algorithm for real-time processing and visualization, which achieved images of stationary targets with up to 7.5 attenuation lengths between the transceiver and the target. The average processing time per frame was approximately 33 ms, allowing real-time three-dimensional video demonstrations of moving targets at ten frames per second at up to 5.5 attenuation lengths between transceiver and target.

Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly.

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC comprises Mad2:Cdc20:BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O-Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires O-Mad2 to C-Mad2 conversion and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. Here, we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C-terminal domain of Mad1 (Mad1CTD). We additionally identify flexibility of Mad1:Mad2 that suggests how the Cdc20:Mad1CTD interaction brings the Mad2-interacting motif (MIM) of Cdc20 near O-Mad2. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination.

DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.

Fast Scalable Image Restoration Using Total Variation Priors and Expectation Propagation.

This paper presents a scalable approximate Bayesian method for image restoration using Total Variation (TV) priors, with the ability to offer uncertainty quantification. In contrast to most optimization methods based on maximum a posteriori estimation, we use the Expectation Propagation (EP) framework to approximate minimum mean squared error (MMSE) estimates and marginal (pixel-wise) variances, without resorting to Monte Carlo sampling. For the classical anisotropic TV-based prior, we also propose an iterative scheme to automatically adjust the regularization parameter via Expectation Maximization (EM). Using Gaussian approximating densities with diagonal covariance matrices, the resulting method allows highly parallelizable steps and can scale to large images for denoising, deconvolution, and compressive sensing (CS) problems. The simulation results illustrate that such EP methods can provide a posteriori estimates on par with those obtained via sampling methods but at a fraction of the computational cost. Moreover, EP does not exhibit strong underestimation of posteriori variances, in contrast to variational Bayes alternatives.

Bacterial divisome protein FtsA forms curved antiparallel double filaments when binding to FtsN.

During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA-FtsN interaction site in the IA-IC interdomain cleft of FtsA using X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the actin-like protein of the elongasome. We propose that curved antiparallel FtsA double filaments together with treadmilling FtsZ filaments organize septal peptidoglycan synthesis in the division plane.

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome.

Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.

Bayesian Activity Estimation and Uncertainty Quantification of Spent Nuclear Fuel Using Passive Gamma Emission Tomography.

In this paper, we address the problem of activity estimation in passive gamma emission tomography (PGET) of spent nuclear fuel. Two different noise models are considered and compared, namely, the isotropic Gaussian and the Poisson noise models. The problem is formulated within a Bayesian framework as a linear inverse problem and prior distributions are assigned to the unknown model parameters. In particular, a Bernoulli-truncated Gaussian prior model is considered to promote sparse pin configurations. A Markov chain Monte Carlo (MCMC) method, based on a split and augmented Gibbs sampler, is then used to sample the posterior distribution of the unknown parameters. The proposed algorithm is first validated by simulations conducted using synthetic data, generated using the nominal models. We then consider more realistic data simulated using a bespoke simulator, whose forward model is non-linear and not available analytically. In that case, the linear models used are mis-specified and we analyse their robustness for activity estimation. The results demonstrate superior performance of the proposed approach in estimating the pin activities in different assembly patterns, in addition to being able to quantify their uncertainty measures, in comparison with existing methods.

Whole genome sequencing of colonies derived from cannabis flowers and the impact of media selection on benchmarking total yeast and mold detection tools.

BACKGROUND: Cannabis products are subjected to microbial testing for human pathogenic fungi and bacteria. These testing requirements often rely on non-specific colony forming unit (CFU/g) specifications without clarity on which medium, selection or growth times are required. We performed whole genome sequencing to assess the specificity of colony forming units (CFU) derived from three different plating media: Potato Dextrose Agar (PDA), PDA with chloramphenicol and Dichloran Rose Bengal with chloramphenicol (DRBC). METHODS: Colonies were isolated from each medium type and their whole genomes sequenced to identify the diversity of microbes present on each medium selection. Fungal Internal Transcribed Spacer (ITS3) and Bacterial 16S RNA(16S) quantitative polymerase chain reactions (qPCR) were performed, to correlate these CFUs with fungi- and bacterial- specific qPCR. RESULTS: Each plating medium displayed a ten-fold difference in CFU counts. PDA with chloramphenicol showed the highest diversity and the highest concordance with whole genome sequencing. According to ITS3 and 16S qPCR confirmed with whole genome sequencing, DRBC under counted yeast and mold while PDA without chloramphenicol over counted CFUs due to bacterial growth without selection. CONCLUSIONS: Colony Forming Unit regulations lack specificity. Each medium produces significant differences in CFU counts. These are further dependent on subjective interpretation, failure to culture most microbes, and poor selection between bacteria and fungi. Given the most human pathogenic microbes found on cannabis are endophytes which culture fails to detect, molecular methods offer a solution to this long-standing quantification problem in the cannabis testing field.

Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace.

The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.

A draft reference assembly of the Psilocybe cubensis genome.

We describe the use of high-fidelity single molecule sequencing to assemble the genome of the psychoactive Psilocybe cubensis mushroom. The genome is 46.6Mb, 46% GC, and in 32 contigs with an N50 of 3.3Mb. The BUSCO completeness scores are 97.6% with 1.2% duplicates. The Psilocybin synthesis cluster exists in a single 3.2Mb contig. The dataset is available from NCBI BioProject with accessions PRJNA687911 and PRJNA700437.

Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1.

During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.