Structural basis of the recognition of adeno-associated virus by the neurological system-related receptor carbonic anhydrase IV.

Carbonic anhydrase IV (Car4) is a newly identified receptor that allows adeno-associated virus (AAV) 9P31 to cross the blood-brain barrier and achieve efficient infection in the central nervous system (CNS) in mouse models. However, the molecular mechanism by which engineered AAV capsids with 7-mer insertion in the variable region (VR) VIII recognize these novel cellular receptors is unknown. Here we report the cryo-EM structures of AAV9P31 and its complex with Mus musculus Car4 at atomic resolution by utilizing the block-based reconstruction (BBR) method. The structures demonstrated that Car4 binds to the protrusions at 3-fold axes of the capsid. The inserted 7-mer extends into a hydrophobic region near the catalytic center of Car4 to form stable interactions. Mutagenesis studies also identified the key residues in Car4 responsible for the AAV9P31 interaction. These findings provide new insights into the novel receptor recognition mechanism of AAV generated by directed evolution and highlight the application of the BBR method to studying the virus-receptor molecular mechanism.

Structural basis for pH gating of the two-pore domain K+ channel TASK2.

TASK2 (also known as KCNK5) channels generate pH-gated leak-type K+ currents to control cellular electrical excitability1-3. TASK2 is involved in the regulation of breathing by chemosensory neurons of the retrotrapezoid nucleus in the brainstem4-6 and pH homeostasis by kidney proximal tubule cells7,8. These roles depend on channel activation by intracellular and extracellular alkalization3,8,9, but the mechanistic basis for TASK2 gating by pH is unknown. Here we present cryo-electron microscopy structures of Mus musculus TASK2 in lipid nanodiscs in open and closed conformations. We identify two gates, distinct from previously observed K+ channel gates, controlled by stimuli on either side of the membrane. Intracellular gating involves lysine protonation on inner helices and the formation of a protein seal between the cytoplasm and the channel. Extracellular gating involves arginine protonation on the channel surface and correlated conformational changes that displace the K+-selectivity filter to render it nonconductive. These results explain how internal and external protons control intracellular and selectivity filter gates to modulate TASK2 activity.

Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase.

O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential human glycosyltransferase that adds O-GlcNAc modifications to numerous proteins. However, little is known about the mechanism with which OGT recognizes various protein substrates. Here we report on GlcNAc electrophilic probes (GEPs) to expedite the characterization of OGT-substrate recognition. Data from mass spectrometry, X-ray crystallization, and biochemical and radiolabeled kinetic assays support the application of GEPs to rapidly report the impacts of OGT mutations on protein substrate or sugar binding and to discover OGT residues crucial for protein recognition. Interestingly, we found that the same residues on the inner surface of the N-terminal domain contribute to OGT interactions with different protein substrates. By tuning reaction conditions, a GEP enables crosslinking of OGT with acceptor substrates in situ, affording a unique method to discover genuine substrates that weakly or transiently interact with OGT. Hence, GEPs provide new strategies to dissect OGT-substrate binding and recognition.

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation.

UNLABELLED: Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE: Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection.

Molecular basis for the formation of ribonucleoprotein complex of Crimean-Congo hemorrhagic fever virus.

Negative-sense single-strand RNA (-ssRNA) viruses comprise a large family of pathogens that cause severe human infectious diseases. All -ssRNA viruses encode a nucleocapsid protein (NP) to encapsidate the viral genome, which, together with polymerase, forms a ribonucleoprotein complex (RNP) that is packaged into virions and acts as the template for viral replication and transcription. In our previous work, we solved the monomeric structure of NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV), which belongs to the Nairovirus genus within the Bunyaviridae family, and revealed its unusual endonuclease activity. However, the mechanism of CCHFV RNP formation remains unclear, due to the difficulty in reconstructing the oligomeric CCHFV NP-RNA complex. Here, we identified and isolated the oligomeric CCHFV NP-RNA complex that formed in expression cells. Sequencing of RNA extracted from the complex revealed sequence specificity and suggested a potential encapsidation signal facilitating the association between NP and viral genome. A cryo-EM reconstruction revealed the ring-shaped architecture of the CCHFV NP-RNA oligomer, thus defining the interaction between the head and stalk domains that results in NP multimerization. This structure also suggested a modified gating mechanism for viral genome encapsidation, in which both the head and stalk domains participate in RNA binding. This work provides insight into the distinct mechanism underlying CCHFV RNP formation compared to other -ssRNA viruses.

Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation.

Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus, is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP-RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ring-shaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization was observed for the recombinant BUNV NP-RNA complex, which was similar to the Rift Valley fever virus NP-RNA complex. This result suggested that the flexibility of one NP protomer with adjacent protomers underlies the BUNV ribonucleoprotein complex (RNP) formation. Electron microscopy revealed that the monomer-sized NP-RNA complex was the building block of the natural BUNV RNP. Combined with previous results indicating that mutagenesis of the interprotomer or protein-RNA interface affects BUNV replication, our structure provides a great potential for understanding the mechanism underlying negative-sense single-stranded RNA RNP formation and enables the development of antiviral therapies targeting BUNV RNP formation.

Using gait videos to automatically assess anxiety.

Background: In recent years, the number of people with anxiety disorders has increased worldwide. Methods for identifying anxiety through objective clues are not yet mature, and the reliability and validity of existing modeling methods have not been tested. The objective of this paper is to propose an automatic anxiety assessment model with good reliability and validity. Methods: This study collected 2D gait videos and Generalized Anxiety Disorder (GAD-7) scale data from 150 participants. We extracted static and dynamic time-domain features and frequency-domain features from the gait videos and used various machine learning approaches to build anxiety assessment models. We evaluated the reliability and validity of the models by comparing the influence of factors such as the frequency-domain feature construction method, training data size, time-frequency features, gender, and odd and even frame data on the model. Results: The results show that the number of wavelet decomposition layers has a significant impact on the frequency-domain feature modeling, while the size of the gait training data has little impact on the modeling effect. In this study, the time-frequency features contributed to the modeling, with the dynamic features contributing more than the static features. Our model predicts anxiety significantly better in women than in men (r Male = 0.666, r Female = 0.763, p < 0.001). The best correlation coefficient between the model prediction scores and scale scores for all participants is 0.725 (p < 0.001). The correlation coefficient between the model prediction scores for odd and even frame data is 0.801~0.883 (p < 0.001). Conclusion: This study shows that anxiety assessment based on 2D gait video modeling is reliable and effective. Moreover, we provide a basis for the development of a real-time, convenient and non-invasive automatic anxiety assessment method.

Volatile Profiling and Transcriptome Sequencing Provide Insights into the Biosynthesis of α-Pinene and ß-Pinene in Liquidambar formosana Hance Leaves.

Liquidambar formosana Hance is a pinene-rich deciduous plant species in the Altingiaceae family that is used as a medicinal plant in China. However, the regulatory mechanisms underlying α-pinene and ß-pinene biosynthesis in L. formosana leaves remain unknown. Here, a joint analysis of the volatile compounds and transcriptomes of L. formosana leaves was performed to comprehensively explore the terpene synthase (TPS) that may participate in α-pinene and ß-pinene biosynthesis. Headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) jointly detected volatile L. formosana leaves. Trees with high and low levels of both α-pinene and ß-pinene were defined as the H group and L group, respectively. RNA sequencing data revealed that DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase), HDS [(E)-4-hydroxy-3-methylbut-2-eny-l-diphosphate synthase], and TPS may be the major regulators of monoterpenoid biosynthesis. We identified three TPSs (LfTPS1, LfTPS2, and LfTPS3), which are highly homologous to α-pinene and ß-pinene synthases of other species in phylogenetic analysis. Four TPS genes (LfTPS1, LfTPS2, LfTPS4, LfTPS5) may be critically involved in the biosynthesis and regulation of α-pinene and ß-pinene in L. formosana. Bioinformatic and transcriptomic results were verified using quantitative real-time PCR. We identified LfTPS1, LfTPS2 as candidate genes for α-pinene and ß-pinene biosynthesis that significantly improve the yield of beneficial terpenoids.

Reliability and validity analysis of personality assessment model based on gait video.

Personality affects an individual's academic achievements, occupational tendencies, marriage quality and physical health, so more convenient and objective personality assessment methods are needed. Gait is a natural, stable, and easy-to-observe body movement that is closely related to personality. The purpose of this paper is to propose a personality assessment model based on gait video and evaluate the reliability and validity of the multidimensional model. This study recruited 152 participants and used cameras to record their gait videos. Each participant completed a 44-item Big Five Inventory (BFI-44) assessment. We constructed diverse static and dynamic time-frequency features based on gait skeleton coordinates, interframe differences, distances between joints, angles between joints, and wavelet decomposition coefficient arrays. We established multidimensional personality trait assessment models through machine learning algorithms and evaluated the criterion validity, split-half reliability, convergent validity, and discriminant validity of these models. The results showed that the reliability and validity of the Gaussian process regression (GPR) and linear regression (LR) models were best. The mean values of their criterion validity were 0.478 and 0.508, respectively, and the mean values of their split-half reliability were all greater than 0.8. In the formed multitrait-multimethod matrix, these methods also had higher convergent and discriminative validity. The proposed approach shows that gait video can be effectively used to evaluate personality traits, providing a new idea for the formation of convenient and non-invasive personality assessment methods.

Comparing the Effectiveness of Brain Structural Imaging, Resting-state fMRI, and Naturalistic fMRI in Recognizing Social Anxiety Disorder in Children and Adolescents.

Social anxiety disorder (SAD) is a common anxiety disorder in childhood and adolescence. Studies on SAD in adults have reported both structural and functional aberrancies of the brain at the group level. However, evidence has shown differences in anxiety-related brain abnormalities between adolescents and adults. Since children and adolescents can afford limited scan time, optimizing the scan tasks is essential for SAD research in children and adolescents. Thus, we need to address whether brain structure, resting-state fMRI, and naturalistic imaging enable individualized identification of SAD in children and adolescents, which measurement is more effective, and whether pooling multi-modal features can improve the identification of SAD. We comprehensively addressed these questions by building machine learning models based on parcel-wise brain features. We found that naturalistic fMRI yielded higher classification accuracy (69.17%) than the other modalities and the classification performance showed dependence on the contents of the movie. The classification models also identified contributing brain regions, some of which exhibited correlations with the symptoms scores of SAD. However, pooling brain features from the three modalities did not help enhance the classification accuracy. These results support the application of carefully designed naturalistic imaging in recognizing children and adolescents at risk of SAD.

Complete genome of Pseudomonas mendocina NK-01, which synthesizes medium-chain-length polyhydroxyalkanoates and alginate oligosaccharides.

Pseudomonas mendocina NK-01 can synthesize medium-chain-length polyhydroxyalkanoate (PHA(MCL)) and alginate oligosaccharides (AO) simultaneously from glucose under conditions of limited nitrogen. Here, we report the complete sequence of the 5.4-Mbp genome of Pseudomonas mendocina NK-01, which was isolated from farmland soil in Tianjin, China.

Phylogenetic diversity and metabolic potential of activated sludge microbial communities in full-scale wastewater treatment plants.

The activated sludge process is an essential process for treating domestic and industrial wastewaters in most wastewater treatment plants (WWTPs). This process consists of a mixture of general and special microorganisms in a form of a complex enrichment population. Thus, the exploration of activated sludge microbial communities is crucial to improve the performance of activated sludge process. In this study, we investigated the phylogenetic diversity and metabolic potential of activated sludge microbial communities in full-scale WWTPs. Four 16S rRNA gene clone libraries were constructed from activated sludge samples. In all samples, Proteobacteria was the most abundant phylogenetic group, followed by Bacteroidetes and Firmicutes. The dominance of Proteobacteria was further demonstrated by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP). Some specific genera, e.g., Nitrosomonas, Thauera, and Dechloromonas, which significantly correlate with the functions and performance of wastewater treatment, were abundant in all samples. A large number of unclassified sequences were found in the library, suggesting that a wide variety of novel species may inhabit complex activated sludge communities. The structures of the bacterial community did not differ significantly among samples. All samples utilized the vast majority of 31 carbon sources of an EcoPlate (Biolog), suggesting that activated sludge microbial communities possess high metabolic potential and equivalent functions required for wastewater treatment.

Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization.

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

Physical basis for distinct basal and mechanically gated activity of the human K+ channel TRAAK.

TRAAK is a mechanosensitive two-pore domain K+ (K2P) channel localized to nodes of Ranvier in myelinated neurons. TRAAK deletion in mice results in mechanical and thermal allodynia, and gain-of-function mutations cause the human neurodevelopmental disorder FHEIG. TRAAK displays basal and stimulus-gated activities typical of K2Ps, but the mechanistic and structural differences between these modes are unknown. Here, we demonstrate that basal and mechanically gated openings are distinguished by their conductance, kinetics, and structure. Basal openings are low conductance, short duration, and due to a conductive channel conformation with the interior cavity exposed to the surrounding membrane. Mechanically gated openings are high conductance, long duration, and due to a channel conformation in which the interior cavity is sealed to the surrounding membrane. Our results explain how dual modes of activity are produced by a single ion channel and provide a basis for the development of state-selective pharmacology with the potential to treat disease.

Identifying Psychological Symptoms Based on Facial Movements.

Background: Many methods have been proposed to automatically identify the presence of mental illness, but these have mostly focused on one specific mental illness. In some non-professional scenarios, it would be more helpful to understand an individual's mental health status from all perspectives. Methods: We recruited 100 participants. Their multi-dimensional psychological symptoms of mental health were evaluated using the Symptom Checklist 90 (SCL-90) and their facial movements under neutral stimulation were recorded using Microsoft Kinect. We extracted the time-series characteristics of the key points as the input, and the subscale scores of the SCL-90 as the output to build facial prediction models. Finally, the convergent validity, discriminant validity, criterion validity, and the split-half reliability were respectively assessed using a multitrait-multimethod matrix and correlation coefficients. Results: The correlation coefficients between the predicted values and actual scores were 0.26 and 0.42 (P < 0.01), which indicated good criterion validity. All models except depression had high convergent validity but low discriminant validity. Results also indicated good levels of split-half reliability for each model [from 0.516 (hostility) to 0.817 (interpersonal sensitivity)] (P < 0.001). Conclusion: The validity and reliability of facial prediction models were confirmed for the measurement of mental health based on the SCL-90. Our research demonstrated that fine-grained aspects of mental health can be identified from the face, and provided a feasible evaluation method for multi-dimensional prediction models.

See your mental state from your walk: Recognizing anxiety and depression through Kinect-recorded gait data.

As the challenge of mental health problems such as anxiety and depression increasing today, more convenient, objective, real-time assessing techniques of mental state are in need. The Microsoft Kinect camera is a possible option for contactlessly capturing human gait, which could reflect the walkers' mental state. So we tried to propose a novel method for monitoring individual's anxiety and depression based on the Kinect-recorded gait pattern. In this study, after finishing the 7-item Generalized Anxiety Disorder Scale (GAD-7) and the 9-item Patient Health Questionnaire (PHQ-9), 179 participants were required to walked on the footpath naturally while shot by the Kinect cameras. Fast Fourier Transforms (FFT) were conducted to extract features from the Kinect-captured gait data after preprocessing, and different machine learning algorithms were used to train the regression models recognizing anxiety and depression levels, and the classification models detecting the cases with specific depressive symptoms. The predictive accuracies of the regression models achieved medium to large level: The correlation coefficient between predicted and questionnaire scores reached 0.51 on anxiety (by epsilon-Support Vector Regression, e-SVR) and 0.51 on depression (by Gaussian Processes, GP). The predictive accuracies could be even higher, 0.74 on anxiety (by GP) and 0.64 on depression (by GP), while training and testing the models on the female sample. The classification models also showed effectiveness on detecting the cases with some symptoms. These results demonstrate the possibility to recognize individual's questionnaire measured anxiety/depression levels and some depressive symptoms based on Kinect-recorded gait data through machine learning method. This approach shows the potential to develop non-intrusive, low-cost methods for monitoring individuals' mental health in real time.

Structural insights into the substrate binding adaptability and specificity of human O-GlcNAcase.

The O-linked ß-N-acetyl glucosamine (O-GlcNAc) modification dynamically regulates the functions of numerous proteins. A single human enzyme O-linked ß-N-acetyl glucosaminase (O-GlcNAcase or OGA) hydrolyzes this modification. To date, it remains largely unknown how OGA recognizes various substrates. Here we report the structures of OGA in complex with each of four distinct glycopeptide substrates that contain a single O-GlcNAc modification on a serine or threonine residue. Intriguingly, these glycopeptides bind in a bidirectional yet conserved conformation within the substrate-binding cleft of OGA. This study provides fundamental insights into a general principle that confers the substrate binding adaptability and specificity to OGA in O-GlcNAc regulation.O-linked ß-N-acetyl glucosamine (O-GlcNAc) is an important protein modification that is hydrolyzed by O-GlcNAcase (OGA). Here the authors give insights into OGA substrate recognition by presenting four human OGA structures complexed with glycopeptide substrates containing a single O-GlcNAc modification on either a serine or threonine.

Structures of human O-GlcNAcase and its complexes reveal a new substrate recognition mode.

Human O-GlcNAcase (hOGA) is the unique enzyme responsible for the hydrolysis of the O-linked ß-N-acetyl glucosamine (O-GlcNAc) modification, an essential protein glycosylation event that modulates the function of numerous cellular proteins in response to nutrients and stress. Here we report crystal structures of a truncated hOGA, which comprises the catalytic and stalk domains, in apo form, in complex with an inhibitor, and in complex with a glycopeptide substrate. We found that hOGA forms an unusual arm-in-arm homodimer in which the catalytic domain of one monomer is covered by the stalk domain of the sister monomer to create a substrate-binding cleft. Notably, the residues on the cleft surface afford extensive interactions with the peptide substrate in a recognition mode that is distinct from that of its bacterial homologs. These structures represent the first model of eukaryotic enzymes in the glycoside hydrolase 84 (GH84) family and provide a crucial starting point for understanding the substrate specificity of hOGA, which regulates a broad range of biological and pathological processes.

Erratum: PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis.

This corrects the article DOI: 10.1038/ncb3630.

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis.

Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP3Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP3R-dependent mitochondrial functions.