Genome-wide CRISPR screenings identified SMCHD1 as a host-restricting factor for AAV transduction.

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy.

ALS-linked C9orf72-SMCR8 complex is a negative regulator of primary ciliogenesis.

Massive GGGGCC (G4C2) repeat expansion in C9orf72 and the resulting loss of C9orf72 function are the key features of ~50% of inherited amyotrophic lateral sclerosis and frontotemporal dementia cases. However, the biological function of C9orf72 remains unclear. We previously found that C9orf72 can form a stable GTPase activating protein (GAP) complex with SMCR8 (Smith-Magenis chromosome region 8). Herein, we report that the C9orf72-SMCR8 complex is a major negative regulator of primary ciliogenesis, abnormalities in which lead to ciliopathies. Mechanistically, the C9orf72-SMCR8 complex suppresses the primary cilium as a RAB8A GAP. Moreover, based on biochemical analysis, we found that C9orf72 is the RAB8A binding subunit and that SMCR8 is the GAP subunit in the complex. We further found that the C9orf72-SMCR8 complex suppressed the primary cilium in multiple tissues from mice, including but not limited to the brain, kidney, and spleen. Importantly, cells with C9orf72 or SMCR8 knocked out were more sensitive to hedgehog signaling. These results reveal the unexpected impact of C9orf72 on primary ciliogenesis and elucidate the pathogenesis of diseases caused by the loss of C9orf72 function.

MYC-Targeting Inhibitors Generated from a Stereodiversified Bicyclic Peptide Library.

Here, we present the second generation of our bicyclic peptide library (NTB), featuring a stereodiversified structure and a simplified construction strategy. We utilized a tandem ring-opening metathesis and ring-closing metathesis reaction (ROM-RCM) to cyclize the linear peptide library in a single step, representing the first reported instance of this reaction being applied to the preparation of macrocyclic peptides. Moreover, the resulting bicyclic peptide can be easily linearized for MS/MS sequencing with a one-step deallylation process. We employed this library to screen against the E363-R378 epitope of MYC and identified several MYC-targeting bicyclic peptides. Subsequent in vitro cell studies demonstrated that one candidate, NT-B2R, effectively suppressed MYC transcription activities and cell proliferation.

CRISPR-GRANT: a cross-platform graphical analysis tool for high-throughput CRISPR-based genome editing evaluation.

BACKGROUD: CRISPR/Cas is an efficient genome editing system that has been widely used for functional genetic studies and exhibits high potential in biomedical translational applications. Indel analysis has thus become one of the most common practices in the lab to evaluate DNA editing events generated by CRISPR/Cas. Several indel analysis tools have been reported, however, it is often required that users have certain bioinformatics training and basic command-line processing capability. RESULTS: Here, we developed CRISPR-GRANT, a stand-alone graphical CRISPR indel analysis tool, which could be easily installed for multi-platforms, including Linux, Windows, and macOS. CRISPR-GRANT offered a straightforward GUI by simple click-and-run for genome editing analysis of single or pooled amplicons and one-step analysis for whole-genome sequencing without the need of data pre-processing, making it ideal for novice lab scientists. Moreover, it also exhibited shorter run-time compared with tools currently available. CONCLUSION: Therefore, CRISPR-GRANT is a valuable addition to the current CRISPR toolkits that significantly lower the barrier for wet-lab researchers to conduct indel analysis from large NGS datasets. CRISPR-GRANT binaries are freely available for Linux (above Ubuntu 16.04), macOS (above High Sierra 10.13) and Windows (above Windows 7) at https://github.com/fuhuancheng/CRISPR-GRANT . CRISPR-GRANT source code is licensed under the GPLv3 license and free to download and use.

The long noncoding RNA ROCKI regulates inflammatory gene expression.

Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. ROCKI interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the MARCKS promoter. In turn, ROCKI-APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing MARCKS transcription and subsequent Ca2+ signaling and inflammatory gene expression. Finally, genetic variants affecting ROCKI expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of cis-acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation.

Non-Mass Spectrometric Targeted Single-Cell Metabolomics.

Metabolic assays serve as pivotal tools in biomedical research, offering keen insights into cellular physiological and pathological states. While mass spectrometry (MS)-based metabolomics remains the gold standard for comprehensive, multiplexed analyses of cellular metabolites, innovative technologies are now emerging for the targeted, quantitative scrutiny of metabolites and metabolic pathways at the single-cell level. In this review, we elucidate an array of these advanced methodologies, spanning synthetic and surface chemistry techniques, imaging-based methods, and electrochemical approaches. We summarize the rationale, design principles, and practical applications for each method, and underscore the synergistic benefits of integrating single-cell metabolomics (scMet) with other single-cell omics technologies. Concluding, we identify prevailing challenges in the targeted scMet arena and offer a forward-looking commentary on future avenues and opportunities in this rapidly evolving field.

A noncanonical role of NOD-like receptor NLRP14 in PGCLC differentiation and spermatogenesis.

NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonad-specific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14-HSPA2-BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development.

Cryo-EM structure of C9ORF72-SMCR8-WDR41 reveals the role as a GAP for Rab8a and Rab11a.

A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryogenic electron microscopy (cryo-EM) structure of the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 Å. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP activity of the C9ORF72-SMCR8 complex.

Performance Enhancement of INS and UWB Fusion Positioning Method Based on Two-Level Error Model.

In GNSS-denied environments, especially when losing measurement sensor data, inertial navigation system (INS) accuracy is critical to the precise positioning of vehicles, and an accurate INS error compensation model is the most effective way to improve INS accuracy. To this end, a two-level error model is proposed, which comprehensively utilizes the mechanism error model and propagation error model. Based on this model, the INS and ultra-wideband (UWB) fusion positioning method is derived relying on the extended Kalman filter (EKF) method. To further improve accuracy, the data prefiltering algorithm of the wavelet shrinkage method based on Stein's unbiased risk estimate-Shrink (SURE-Shrink) threshold is summarized for raw inertial measurement unit (IMU) data. The experimental results show that by employing the SURE-Shrink wavelet denoising method, positioning accuracy is improved by 76.6%; by applying the two-level error model, the accuracy is further improved by 84.3%. More importantly, at the point when the vehicle motion state changes, adopting the two-level error model can provide higher computational stability and less fluctuation in trajectory curves.

IMU/UWB Fusion Method Using a Complementary Filter and a Kalman Filter for Hybrid Upper Limb Motion Estimation.

Motion capture systems have enormously benefited the research into human-computer interaction in the aerospace field. Given the high cost and susceptibility to lighting conditions of optical motion capture systems, as well as considering the drift in IMU sensors, this paper utilizes a fusion approach with low-cost wearable sensors for hybrid upper limb motion tracking. We propose a novel algorithm that combines the fourth-order Runge-Kutta (RK4) Madgwick complementary orientation filter and the Kalman filter for motion estimation through the data fusion of an inertial measurement unit (IMU) and an ultrawideband (UWB). The Madgwick RK4 orientation filter is used to compensate gyroscope drift through the optimal fusion of a magnetic, angular rate, and gravity (MARG) system, without requiring knowledge of noise distribution for implementation. Then, considering the error distribution provided by the UWB system, we employ a Kalman filter to estimate and fuse the UWB measurements to further reduce the drift error. Adopting the cube distribution of four anchors, the drift-free position obtained by the UWB localization Kalman filter is used to fuse the position calculated by IMU. The proposed algorithm has been tested by various movements and has demonstrated an average decrease in the RMSE of 1.2 cm from the IMU method to IMU/UWB fusion method. The experimental results represent the high feasibility and stability of our proposed algorithm for accurately tracking the movements of human upper limbs.

Effects of Coverlys TF150® on the Photosynthetic Characteristics of Grape.

Grape rain-shelter cultivation is a widely employed practice in China. At present, the most commonly used rain shelter film materials are polyvinyl chloride (PVC), polyethylene (PE), ethylene-vinyl acetate copolymer (EVA), and polyolefin (PO). Coverlys TF150® is a woven fabric with an internal antifoggy PE coating that has not yet been popularized as a rain shelter film for grapes in China. To investigate the effects of Coverlys TF150® on grapes, we measured the microdomain environment, leaf development, and photosynthetic characteristics of 'Miguang' (Vitis vinifera × V. labrusca) under rain-shelter cultivation and performed transcriptome analysis. The results showed that Coverlys TF150® significantly reduced (p < 0.05) the light intensity, temperature, and humidity compared with PO film, increased the chlorophyll content and leaf thickness (particularly palisade tissue thickness), and increased stomatal density and stomatal opening from 10:00 to 14:00. Coverlys TF150® was observed to improve the maximum efficiency of photosystem II (Fv/Fm), photochemical quenching (qP), the electron transfer rate (ETR), and the actual photochemical efficiency (ΦPSII) from 10:00 to 14:00. Moreover, the net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr) of grape leaves significantly increased (p < 0.05) from 10:00 to 14:00. RNA-Seq analysis of the grape leaves at 8:00, 10:00, and 12:00 revealed 1388, 1562, and 1436 differential genes at these points in time, respectively. KEGG enrichment analysis showed the occurrence of protein processing in the endoplasmic reticulum. Plant hormone signal transduction and plant-pathogen interaction were identified as the metabolic pathways with the highest differential gene expression enrichment. The psbA encoding D1 protein was significantly up-regulated in both CO10vsPO10 and CO12vsPO12, while the sHSPs family genes were significantly down-regulated in all time periods, and thus may play an important role in the maintenance of the photosystem II (PSII) activity in grape leaves under Coverlys TF150®. Compared with PO film, the PSI-related gene psaB was up-regulated, indicating the ability of Coverlys TF150® to better maintain PSI activity. Compared with PO film, the abolic acid receptacle-associated gene PYL1 was down-regulated at all time periods under the Coverlys TF150® treatment, while PP2C47 was significantly up-regulated in CO10vsPO10 and CO12vsPO12, inducing stomatal closure. The results reveal that Coverlys TF150® alleviates the stress of high temperature and strong light compared with PO film, improves the photosynthetic capacity of grape leaves, and reduces the midday depression of photosynthesis.

Hydroxyl-Rich Hydrophilic Endocytosis-Promoting Peptide with No Positive Charge.

Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.

An evolutionarily conserved long noncoding RNA TUNA controls pluripotency and neural lineage commitment.

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.

The crystal structure of Atg18 reveals a new binding site for Atg2 in Saccharomyces cerevisiae.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved catabolic eukaryotic pathway that is critical for stress responses and homeostasis. Atg18, one of the core proteins involved in autophagy, belongs to the PROPPIN family and is composed of seven WD40 repeats. Together with Atg2, Atg18 participates in the elongation of phagophores and the recycling of Atg9 in yeast. Despite extensive studies on the PROPPIN family, the structure of Atg18 from Saccharomyces cerevisiae has not been determined. Here, we report the structure of ScAtg18 at a resolution of 2.8 Å. Based on bioinformatics and structural analysis, we found that the 7AB loop of ScAtg18 is extended in Atg18, in comparison to other members of the PROPPIN family. Genetic analysis revealed that the 7AB loop of ScAtg18 is required for autophagy. Biochemical and biophysical experiments indicated that the 7AB loop of ScAtg18 is critical for interaction with ScAtg2 and the recruitment of ScAtg2 to the autophagy-initiating site. Collectively, our results show that the 7AB loop of ScAtg18 is a new binding site for Atg2 and is of functional importance to autophagy.

Constrained ESKF for UAV Positioning in Indoor Corridor Environment Based on IMU and WiFi.

The indoor autonomous navigation of unmanned aerial vehicles (UAVs) is the current research hotspot. Unlike the outdoor broad environment, the indoor environment is unknown and complicated. Global Navigation Satellite System (GNSS) signals are easily blocked and reflected because of complex indoor spatial features, which make it impossible to achieve positioning and navigation indoors relying on GNSS. This article proposes a set of indoor corridor environment positioning methods based on the integration of WiFi and IMU. The zone partition-based Weighted K Nearest Neighbors (WKNN) algorithm is used to achieve higher WiFi-based positioning accuracy. On the basis of the Error-State Kalman Filter (ESKF) algorithm, WiFi-based and IMU-based methods are fused together and realize higher positioning accuracy. The probability-based optimization method is used for further accuracy improvement. After data fusion, the positioning accuracy increased by 51.09% compared to the IMU-based algorithm and by 66.16% compared to the WiFi-based algorithm. After optimization, the positioning accuracy increased by 20.9% compared to the ESKF-based data fusion algorithm. All of the above results prove that methods based on WiFi and IMU (low-cost sensors) are very capable of obtaining high indoor positioning accuracy.

Single-Cell Profiling of Fatty Acid Uptake Using Surface-Immobilized Dendrimers.

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

Digitonin-facilitated delivery of imaging probes enables single-cell analysis of AKT signalling activities in suspension cells.

Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.

A cyclic peptide antenna ligand for enhancing terbium luminescence.

We present here a cyclic peptide ligand, cy(WQETR), that binds to the terbium ion (Tb3+) and enhances Tb3+ luminescence intensity through the antenna effect. This peptide was identified through screening a cyclic peptide library against Tb3+ with an apparent EC50 of 540 µM. The tryptophan residue from the peptide directly interacts with the Tb3+ ion, which provides access to a low-lying triplet excited state of the tryptophan. Direct excitation of this triplet state enables energy transfer to the Tb3+ ion and enhances Tb3+ luminescence intensity by 150 fold. We further showcase the application of this cy(WQETR)-Tb3+ system by demonstrating the detection of tromethamine with a detection limit of 0.5 mM.

Photo-Click-Facilitated Screening Platform for the Development of Hetero-Bivalent Agents with High Potency.

A novel photo-click-based platform has been developed for rapid screening and affinity optimization of heterobivalent agents. This method allows for the efficient selection of high-affinity dual receptor-targeting agents via streamlining tedious organic synthesis and biological evaluation procedures required by traditional approaches. The high-avidity heterobivalent agents targeting both integrin αvß3 and urokinase-type plasminogen activator receptors have been developed using this photo-click-facilitated screening platform. The affinity screening results were further validated by traditional in vitro and in vivo evaluation techniques, reaffirming the reliability of the method. The convenience, rapidity, universality, and robustness of the screening platform, discussed in this report, can greatly facilitate the development of new heterobivalent agents for research and/or clinical applications.

Fluorescence imaging-based methods for single-cell protein analysis.

The quantity and activity of proteins in many biological systems exhibit prominent heterogeneities. Single-cell analytical methods can resolve subpopulations and dissect their unique signatures from heterogeneous samples, enabling a clarifying view of the biological process. Over the last 5 years, technologies for single-cell protein analysis have significantly advanced. In this article, we highlight a branch of those technology developments involving fluorescence-based approaches, with a focus on the methods that increase the ability to multiplex and enable dynamic measurements. We also analyze the limitations of these techniques and discuss current challenges in the field, with the hope that more transformative platforms can soon emerge.