OBJECTIVE: A recent genome-wide association study (GWAS) reported a significant genetic association between rs34330 of cyclin-dependent kinase inhibitor 1B (CDKN1B) and risk of systemic lupus erythematosus (SLE) in Han Chinese. This study aims to validate the reported association and elucidate the biochemical mechanisms underlying the variant's effect.

METHODS: We performed allelic association with SLE followed by meta-analysis across 11 independent cohorts (n=28,872). We applied in silico bioinformatics and experimental validation in SLE-relevant cell lines to determine the functional consequences of rs34330.

RESULTS: We replicated genetic association between SLE and rs34330 (P =5.29x10 , OR (95% CI)=0.84 (0.81-0.87)). Follow-up bioinformatics and eQTL analysis suggest that rs34330 is located in active chromatin and potentially regulates several target genes. Using luciferase and ChIP-qPCR, we demonstrated substantial allele-specific promoter and enhancer activity, and allele-specific binding of three histone marks (H3K27ac, H3K4me3, H3K4me1), RNA pol II, CTCF, and a critical immune transcription factor (IRF-1). Chromosome conformation capture (3C) detected long-range chromatin interactions between rs34330 and the promoters of neighboring genes APOLD1 and DDX47, and effects on CDKN1B and the other target genes were directly validated by CRISPR-based genome editing. Finally, CRISPR-dCas9-based epigenetic activation/silencing confirmed these results. Gene-edited cell lines also showed higher levels of proliferation and apoptosis.

CONCLUSION: Collectively, these findings suggest a mechanism whereby the rs34330 risk allele (C) influences the presence of histone marks, RNA pol II, and the IRF-1 transcription factor to regulate expression of several target genes linked to proliferation and apoptosis, which potentially underlie the association of rs34330 with SLE.

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.

We aim to improve segmentation through the use of machine learning tools during region agglomeration. We propose an active learning approach for performing hierarchical agglomerative segmentation from superpixels. Our method combines multiple features at all scales of the agglomerative process, works for data with an arbitrary number of dimensions, and scales to very large datasets. We advocate the use of variation of information to measure segmentation accuracy, particularly in 3D electron microscopy (EM) images of neural tissue, and using this metric demonstrate an improvement over competing algorithms in EM and natural images.

Insects constitute the most species-rich radiation of metazoa, a success that is due to the evolution of active flight. Unlike pterosaurs, birds and bats, the wings of insects did not evolve from legs, but are novel structures that are attached to the body via a biomechanically complex hinge that transforms tiny, high-frequency oscillations of specialized power muscles into the sweeping back-and-forth motion of the wings. The hinge consists of a system of tiny, hardened structures called sclerites that are interconnected to one another via flexible joints and regulated by the activity of specialized control muscles. Here we imaged the activity of these muscles in a fly using a genetically encoded calcium indicator, while simultaneously tracking the three-dimensional motion of the wings with high-speed cameras. Using machine learning, we created a convolutional neural network that accurately predicts wing motion from the activity of the steering muscles, and an encoder-decoder that predicts the role of the individual sclerites on wing motion. By replaying patterns of wing motion on a dynamically scaled robotic fly, we quantified the effects of steering muscle activity on aerodynamic forces. A physics-based simulation incorporating our hinge model generates flight manoeuvres that are remarkably similar to those of free-flying flies. This integrative, multi-disciplinary approach reveals the mechanical control logic of the insect wing hinge, arguably among the most sophisticated and evolutionarily important skeletal structures in the natural world.

Recent developments in machine vision methods for automatic, quantitative analysis of social behavior have immensely improved both the scale and level of resolution with which we can dissect interactions between members of the same species. In this paper, we review these methods, with a particular focus on how biologists can apply them to their own work. We discuss several components of machine vision-based analyses: methods to record high-quality video for automated analyses, video-based tracking algorithms for estimating the positions of interacting animals, and machine learning methods for recognizing patterns of interactions. These methods are extremely general in their applicability, and we review a subset of successful applications of them to biological questions in several model systems with very different types of social behaviors.

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.

A 60-year-old man diagnosed with macular telangiectasia type 1 (MacTel 1) was treated for 3 years with monthly aflibercept (Eylea; Regeneron, Tarrytown, NY) and serially imaged with spectral-domain optical coherence tomography. When administered monthly, aflibercept appeared to have a beneficial effect on macular edema secondary to MacTel 1. Visual acuity preservation despite minimal chronic macular edema could be attributed to the lack of significant photoreceptor disruption.

The precise neural mechanisms within the brain that contribute to the remarkable lifetime persistence of memory remain unknown. Existing techniques to record neurons in animals are either unsuitable for longitudinal recording from the same cells or make it difficult for animals to express their full naturalistic behavioral repertoire. We present a magnetic voluntary head-fixation system that provides stable optical access to the brain during complex behavior. Compared to previous systems that used mechanical restraint, there are no moving parts and animals can engage and disengage entirely at will. This system is failsafe, easy for animals to use and reliable enough to allow long-term experiments to be routinely performed. Together with a novel two-photon fluorescence collection scheme that increases two-photon signal and a transgenic rat line that stably expresses the calcium sensor GCaMP6f in dorsal CA1, we are able to track and record activity from the same hippocampal neurons, during behavior, over a large fraction of animals’ lives.

PURPOSE: To develop switchable and tunable labels with high contrast ratio for MRI using magnetocaloric materials that have sharp first-order magnetic phase transitions at physiological temperatures and typical MRI magnetic field strengths.

METHODS: A prototypical magnetocaloric material iron-rhodium (FeRh) was prepared by melt mixing, high-temperature annealing, and ice-water quenching. Temperature- and magnetic field-dependent magnetization measurements of wire-cut FeRh samples were performed on a vibrating sample magnetometer. Temperature-dependent MRI of FeRh samples was performed on a 4.7T MRI.

RESULTS: Temperature-dependent MRI clearly demonstrated image contrast changes due to the sharp magnetic state transition of the FeRh samples in the MRI magnetic field (4.7T) and at a physiologically relevant temperature (~37°C).

CONCLUSION: A magnetocaloric material, FeRh, was demonstrated to act as a high contrast ratio switchable MRI contrast agent due to its sharp first-order magnetic phase transition in the DC magnetic field of MRI and at physiologically relevant temperatures. A wide range of magnetocaloric materials are available that can be tuned by materials science techniques to optimize their response under MRI-appropriate conditions and be controllably switched in situ with temperature, magnetic field, or a combination of both.

Many animals rely on a representation of head direction for flexible, goal-directed navigation. In insects, a compass-like head direction representation is maintained in a conserved brain region called the central complex. This head direction representation is updated by self-motion information and by tethering to sensory cues in the surroundings through a plasticity mechanism. However, under natural settings, some of these sensory cues may temporarily disappear—for example, when clouds hide the sun—and prominent landmarks at different distances from the insect may move across the animal's field of view during translation, creating potential conflicts for a neural compass. We used two-photon calcium imaging in head-fixed Drosophila behaving in virtual reality to monitor the fly's compass during navigation in immersive naturalistic environments with approachable local landmarks. We found that the fly's compass remains stable even in these settings by tethering to available global cues, likely preserving the animal's ability to perform compass-driven behaviors such as maintaining a constant heading.