Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

Structural basis for the tethered peptide activation of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1-6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7-12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical-bulge-ß-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved 'toggle switch' W6.53 and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49 and the P6.47/V6.47φφG6.50 motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gs coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gs coupling.

Structures of the glucocorticoid-bound adhesion receptor GPR97-Go complex.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

Label-Free Mapping of Multivalent Binding Pathways with Ligand-Receptor-Anchored Nanopores.

Understanding single-molecule multivalent ligand-receptor interactions is crucial for comprehending molecular recognition at biological interfaces. However, label-free identifications of these transient interactions during multistep binding processes remains challenging. Herein, we introduce a ligand-receptor-anchored nanopore that allows the protein to maintain structural flexibility and favorable orientations in native states, mapping dynamic multivalent interactions. Using a four-state Markov chain model, we clarify two concentration-dependent binding pathways for the Omicron spike protein (Omicron S) and soluble angiotensin-converting enzyme 2 (sACE2): sequential and concurrent. Real-time kinetic analysis at the single-monomeric subunit level reveals that three S1 monomers of Omicron S exhibit a consistent and robust binding affinity toward sACE2 (-13.1 ± 0.2 kcal/mol). These results highlight the enhanced infectivity of Omicron S compared to other homologous spike proteins (WT S and Delta S). Notably, the preceding binding of sACE2 to Omicron S facilitates the subsequent binding steps, which was previously obscured in bulk measurements. Our single-molecule studies resolve the controversy over the disparity between the measured spike protein binding affinity with sACE2 and the viral infectivity, offering valuable insights for drug design and therapies.

Prognostic value of GLIM-defined malnutrition in combination with hand-grip strength or gait speed for the prediction of postoperative outcomes in gastric cancer patients with cachexia.

BACKGROUND: Cancer cachexia is associated with impaired functional and nutritional status and worse clinical outcomes. Global Leadership Initiative in Malnutrition (GLIM) consensus recommended the application of GLIM criteria to diagnose malnutrition in patients with cachexia. However, few previous study has applied the GLIM criteria in patients with cancer cachexia. METHODS: From July 2014 to May 2019, patients who were diagnosed with cancer cachexia and underwent radical gastrectomy for gastric cancer were included in this study. Malnutrition was diagnosed using the GLIM criteria. Skeletal muscle index was measured using abdominal computed tomography (CT) images at the third lumbar vertebra (L3) level. Hand-grip strength and 6-meters gait speed were measured before surgery. RESULTS: A total of 356 patients with cancer cachexia were included in the present study, in which 269 (75.56%) were identified as having malnutrition based on the GLIM criteria. GLIM-defined malnutrition alone did not show significant association with short-term postoperative outcomes, including complications, costs or length of postoperative hospital stays. The combination of low hand-grip strength or low gait speed with GLIM-defined malnutrition led to a significant predictive value for these outcomes. Moreover, low hand-grip strength plus GLIM-defined malnutrition was independently associated with postoperative complications (OR 1.912, 95% CI 1.151-3.178, P = 0.012). GLIM-defined malnutrition was an independent predictive factor for worse OS (HR 2.310, 95% CI 1.421-3.754, P = 0.001) and DFS (HR 1.815, 95% CI 1.186-2.779, P = 0.006) after surgery. The addition of low hand-grip strength or low gait speed to GLIM-defined malnutrition did not increase its predictive value for survival. CONCLUSION: GLIM-defined malnutrition predicted worse long-term survival in gastric cancer patients with cachexia. Gait speed and hand-grip strength added prognostic value to GLIM-defined malnutrition for the prediction of short-term postoperative outcomes, which could be incorporated into preoperative assessment protocols in patients with cancer cachexia.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

HFM-Tracker: a cell tracking algorithm based on hybrid feature matching.

Cell migration is known to be a fundamental biological process, playing an essential role in development, homeostasis, and diseases. This paper introduces a cell tracking algorithm named HFM-Tracker (Hybrid Feature Matching Tracker) that automatically identifies cell migration behaviours in consecutive images. It combines Contour Attention (CA) and Adaptive Confusion Matrix (ACM) modules to accurately capture cell contours in each image and track the dynamic behaviors of migrating cells in the field of view. Cells are firstly located and identified via the CA module-based cell detection network, and then associated and tracked via a cell tracking algorithm employing a hybrid feature-matching strategy. This proposed HFM-Tracker exhibits superiorities in cell detection and tracking, achieving 75% in MOTA (Multiple Object Tracking Accuracy) and 65% in IDF1 (ID F1 score). It provides quantitative analysis of the cell morphology and migration features, which could further help in understanding the complicated and diverse cell migration processes.

Application of 3D computed tomography in emphysematous parenchyma patients scheduled for bronchoscopic lung volume reduction.

Bronchoscopic lung volume reduction (BLVR) is a feasible, safe, effective and minimally invasive technique to significantly improve the quality of life of advanced severe chronic obstructive pulmonary disease (COPD). In this study, three-dimensional computed tomography (3D-CT) automatic analysis software combined with pulmonary function test (PFT) was used to retrospectively evaluate the postoperative efficacy of BLVR patients. The purpose is to evaluate the improvement of lung function of local lung tissue after operation, maximize the benefits of patients, and facilitate BLVR in the treatment of patients with advanced COPD. All the reported cases of advanced COPD patients treated with BLVR with one-way valve were collected and analysed from 2017 to 2020. Three-dimensional-CT image analysis software system was used to analyse the distribution of low-density areas <950 Hounsfield units in both lungs pre- and post- BLVR. Meanwhile, all patients performed standard PFT pre- and post-operation for retrospective analysis. We reported six patients that underwent unilateral BLVR with 1 to 3 valves according to the range of emphysema. All patients showed a median increase in forced expiratory volume in 1 second (FEV1) of 34%, compared with baseline values. Hyperinflation was reduced by 16.6% (range, 4.9%-47.2%). The volumetric measurements showed a significant reduction in the treated lobe volume among these patients. Meanwhile, the targeted lobe volume changes were inversely correlated with change in FEV1/FEV1% in patients with heterogeneous emphysematous. We confirm that 3D-CT analysis can quantify the changes of lung volume, ventilation and perfusion, to accurately evaluate the distribution and improvement of emphysema and rely less on the observer.

Pore confined time-of-flight secondary ion electrochemical mass spectrometry.

Molecular structure conversion concomitant with mass transfer processes at the electrode-electrolyte interfaces plays a central role in energy electrochemistry. Mass spectrometry, as one of the most intuitive, sensitive techniques, provides the capability to collect transient intermediates and products and uncover reaction mechanisms and kinetics. In situ time-of-flight secondary ion electrochemical mass spectrometry with inherent high mass and spatiotemporal resolution has emerged as a promising strategy for investigating electrochemical processes at the electrode surface. This review illustrates the recent advancements in coupling time-of-flight secondary ion mass spectrometry and electrochemistry to visualize and quantify local dynamic electrochemical processes, identify solvated species distribution, and disclose hidden reaction pathways at the molecular level. Moreover, the key challenges in this field are further discussed to promote new applications and discoveries in operando studying the dynamic electrochemical interfaces of advanced energy systems.

Electrochemical Monitoring of Real-Time Vesicle Dynamics Induced by Tau in a Confined Nanopipette.

The microtubule-associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau's engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse-mimicking nanopipette to monitor vesicle dynamics induced by tau. A model vesicle of ~30 nm is confined within a lipid-modified nanopipette orifice with a comparable diameter to mimic the synaptic lipid environment. Both tau and phosphorylated tau (p-tau) present two-state dynamic behavior in this biomimetic system, showing typical ionic current oscillation, induced by lipid-tau interaction. The results indicate that p-tau has a stronger affinity to the lipid vesicles in the confined environment, blocking the vesicle movement to a higher degree. Taken together, this method bridges a gap for sensing synaptic vesicle dynamics in a confined lipid environment, mimicking vesicle movement near the synaptic membrane. These findings contribute to understanding how different types of tau protein regulate synaptic vesicle motility and to underlying its functional and pathological behaviours in disease.

Emerging Data Processing Methods for Single-Entity Electrochemistry.

Single-entity electrochemistry is a powerful tool that enables the study of electrochemical processes at interfaces and provides insights into the intrinsic chemical and structural heterogeneities of individual entities. Signal processing is a critical aspect of single-entity electrochemical measurements and can be used for data recognition, classification, and interpretation. In this review, we summarize the recent five-year advances in signal processing techniques for single-entity electrochemistry and highlight their importance in obtaining high-quality data and extracting effective features from electrochemical signals, which are generally applicable in single-entity electrochemistry. Moreover, we shed light on electrochemical noise analysis to obtain single-molecule frequency fingerprint spectra that can provide rich information about the ion networks at the interface. By incorporating advanced data analysis tools and artificial intelligence algorithms, single-entity electrochemical measurements would revolutionize the field of single-entity analysis, leading to new fundamental discoveries.

Nanopore Deciphering Single Digital Polymers Towards High-Density Data Storage.

Sequence-defined polymer is one of the most promising alternative media for high-density data storage. It could be used to alleviate the problem of insufficient storage capacity of conventional silicon-based devices for the explosively increasing data. To fulfil the goal of polymer data storage, suitable methods should be developed to accurately read and decode the information-containing polymers, especially for those composed by a combination of the natural and unnatural monomers. Nanopore-based approaches have become one of the most competitive analysis and sequencing techniques, which are expected to read both natural and synthetic polymers with single-molecule precision and monomeric resolution. Herein, this work emphasizes the advances being made in nanopore reading and decoding of information stored in the man-made polymers and DNA nanostructures, and discusses the challenges and opportunities towards the development and realization of high-density data storage.

Paper-based substrates for surface-enhanced Raman spectroscopy sensing.

Surface-enhanced Raman scattering (SERS) has been recognized as one of the most sensitive analytical methods by adsorbing the target of interest onto a plasmonic surface. Growing attention has been directed towards the fabrication of various substrates to broaden SERS applications. Among these, flexible SERS substrates, particularly paper-based ones, have gained popularity due to their easy-to-use features by full contact with the sample surface. Herein, we reviewed the latest advancements in flexible SERS substrates, with a focus on paper-based substrates. Firstly, it begins by introducing various methods for preparing paper-based substrates and highlights their advantages through several illustrative examples. Subsequently, we demonstrated the booming applications of these paper-based SERS substrates in abiotic and biological matrix detection, with particular emphasis on their potential application in clinical diagnosis. Finally, the prospects and challenges of paper-based SERS substrates in broader applications are discussed.

Observing Confined Local Oxygen-induced Reversible Thiol/Disulfide Cycle with a Protein Nanopore.

Disulfide bonds play an important role in thiol-based redox regulation. However, owing to the lack of analytical tools, little is known about how local O2 mediates the reversible thiol/disulfide cycle under protein confinement. In this study, a protein-nanopore inside a glove box is used to control local O2 for single-molecule reaction, as well as a single-molecule sensor for real-time monitoring of the reversible thiol/disulfide cycle. The results demonstrate that the local O2 molecules in protein nanopores could facilitate the redox cycle of disulfide formation and cleavage by promoting a higher fraction of effective reactant collisions owing to nanoconfinement. Further kinetic calculations indicate that the negatively charged residues near reactive sites facilitate proton-involved oxygen-induced disulfide cleavage under protein confinement. The unexpectedly strong oxidation ability of confined local O2 may play an essential role in cellular redox signaling and enzyme reactions.

In Situ Characterization of Oxygen Evolution Electrocatalysis of Silver Salt Oxide on a Wireless Nanopore Electrode.

Silver salt oxide shows superior oxidation ability for the applications of superconductivity, sterilization, and catalysis. However, due to the easy decomposition, the catalytic properties of silver salt oxide are difficult to characterize by conventional methods. Herein, we used a closed-type wireless nanopore electrode (CWNE) to in situ and real-time monitor the electrocatalytic performance of Ag7NO11 in the oxygen evolution reaction. The real-time current recording revealed that the deposited Ag7NO11 on the CWNE tip greatly enhanced the oxidative capacity of the electrode, resulting in water splitting. The statistical event analysis reveals the periodic O2 bubble formation and dissolution at the Ag7NO11 interface, which ensures the characterization of the oxygen evolution electrocatalytic process at the nanoscale. The calculated kcat and Markov chain modeling suggest the anisotropy of Ag7NO11 at a low voltage may lead to multiple catalytic rates. Therefore, our results demonstrate the powerful capability of CWNE in direct and in situ characterization of gas-liquid-solid catalytic reactions for unstable catalysts.

Confined Nanopipet as a Versatile Tool for Precise Single Cell Manipulation.

The precise manipulation of single cells plays a fundamental role for single cell measurement, which is crucial for understanding the diverse cellular mechanisms. Unusual single cell behavior could thus be identified by integrating with advanced analytical methods such as single cell omics, unraveling the intrinsic cellular heterogeneity hidden in ensemble measurements. Herein, this technical note reports a nanopipet-based versatile method for manipulation of an ultrasmall volume of liquid, which further enables the precise manipulation of single cells. Femtoliter volumes of cytoplasm were extracted from single living cells and analyzed by time-of-flight secondary ion mass spectrometry. Moreover, several kinds of exogenous components were injected simultaneously into a cell, offering a delicate tool for multi-imaging in single living cells.

Association of Long-term Ambient Fine Particulate Matter (PM2.5) and Incident CKD: A Prospective Cohort Study in China.

RATIONALE & OBJECTIVE: Increasing evidence has linked ambient fine particulate matter (ie, particulate matter no larger than 2.5 µm [PM2.5]) to chronic kidney disease (CKD), but their association has not been fully elucidated, especially in regions with high levels of PM2.5 pollution. This study aimed to investigate the long-term association of high PM2.5 exposure with incident CKD in mainland China. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 72,425 participants (age ≥18 years) without CKD were recruited from 121 counties in Hunan Province, China. EXPOSURE: Annual mean PM2.5 concentration at the residence of each participant derived from a long-term, full-coverage, high-resolution (1 × 1 km2), high-quality dataset of ground-level air pollutants in China. OUTCOMES: Incident CKD during the interval between the baseline examination of each participant (2005-2017) and the end of follow-up through 2018. ANALYTICAL APPROACH: Cox proportional hazards models were used to estimate the independent association of PM2.5 with incident CKD and the joint association of PM2.5 with temperature or humidity on the development of PM2.5-related CKD. Restricted cubic splines were used to model exposure-response relationships. RESULTS: Over a median follow-up of 3.79 (IQR, 2.03-5.48) years, a total of 2,188 participants with incident CKD were identified. PM2.5 exposure was associated with incident CKD with an adjusted hazard ratio of 1.71 (95% CI, 1.58-1.85) per 10-µg/m3 greater long-term exposure. Multiplicative interactions between PM2.5 and humidity or temperature on incident CKD were detected (all P < 0.001 for interaction), whereas an additive interaction was detected only for humidity (relative risk due to interaction, 3.59 [95% CI, 0.97-6.21]). LIMITATIONS: Lack of information on participants' activity patterns such as time spent outdoors. CONCLUSIONS: Greater long-term ambient PM2.5 pollution is associated with incident CKD in environments with high PM2.5 exposure. Ambient humidity has a potentially synergetic effect on the association of PM2.5 with the development of CKD. PLAIN-LANGUAGE SUMMARY: Exposure to a form of air pollution known as fine particulate matter (ie, particulate matter ≤2.5 µm [PM2.5]) has been linked to an increased risk of chronic kidney disease (CKD), but little is known about how PM2.5 affects CKD in regions with extremely high levels of PM2.5 pollution. This longitudinal cohort study in China investigates the effect of PM2.5 on the incidence of CKD and whether temperature or humidity interact with PM2.5. Our findings suggest that long-term exposure to high levels of ambient PM2.5 significantly increased the risk of CKD in mainland China, especially in terms of cumulative average PM2.5. The associations of PM2.5 and incident CKD were greater in high-humidity environments. These findings support the recommendation that reducing PM2.5 pollution should be a priority to decrease the burden of associated health risks, including CKD.

Antagonism of histamine H3 receptor promotes angiogenesis following focal cerebral ischemia.

Our previous study showed that H3 receptor antagonists reduced neuronal apoptosis and cerebral infarction in the acute stage after cerebral ischemia, but through an action independent of activation of histaminergic neurons. Because enhanced angiogenesis facilitates neurogenesis and neurological recovery after ischemic stroke, we herein investigated whether antagonism of H3R promoted angiogenesis after brain ischemia. Photothrombotic stroke was induced in mice. We showed that administration of H3R antagonist thioperamide (THIO, 10 mg·kg-1·d-1, i.p., from D1 after cerebral ischemia) significantly improved angiogenesis assessed on D14, and attenuated neurological defects on D28 after cerebral ischemia. Compared with wild-type mice, Hrh3-/- mice displayed more blood vessels in the ischemic boundary zone on D14, and THIO administration did not promote angiogenesis in these knockout mice. THIO-promoted angiogenesis in mice was reversed by i.c.v. injection of H3R agonist immepip, but not by H1 and H2 receptor antagonists, histidine decarboxylase inhibitor α-fluoromethylhistidine, or histidine decarboxylase gene knockout (HDC-/-), suggesting that THIO-promoted angiogenesis was independent of activation of histaminergic neurons. In vascular endothelial cells (bEnd.3), THIO (10-9-10-7 M) dose-dependently facilitated cell migration and tube formation after oxygen glucose deprivation (OGD), and H3R knockdown caused similar effects. We further revealed that H3R antagonism reduced the interaction between H3R and Annexin A2, while knockdown of Annexin A2 abrogated THIO-promoted angiogenesis in bEnd.3 cells after OGD. Annexin A2-overexpressing mice displayed more blood vessels in the ischemic boundary zone, which was reversed by i.c.v. injection of immepip. In conclusion, this study demonstrates that H3R antagonism promotes angiogenesis after cerebral ischemia, which is independent of activation of histaminergic neurons, but related to the H3R on vascular endothelial cells and its interaction with Annexin A2. Thus, H3R antagonists might be promising drug candidates to improve angiogenesis and neurological recovery after ischemic stroke.

Roles of C/EBP-homologous protein and histone H3 lysine 4 methylation in arsenic-induced mitochondrial apoptosis in hepatocytes.

C/EBP-homologous protein (CHOP) and histone H3 lysine 4 (H3K4) methylation have been verified to be correlated with apoptosis, whereas their biological function in arsenic-induced hepatocyte apoptosis through the mitochondrial pathway is still unclear. This study aimed to explore the specific regulatory mechanism of CHOP and H3K4me1/2 in arsenic-induced mitochondrial apoptosis in hepatocytes. Apoptosis and proliferation results showed arsenic promoted apoptosis and inhibited cell growth in BRL-3A cells. Meanwhile, arsenic treatment significantly upregulated the 78-kDa glucose-regulated protein (GRP78), CHOP, su(var)-3-9,enhancer-of-zeste,trithorax (SET) domain containing 7/9 (SET7/9), H3K4me1/2, BIM and BAX expression, while markedly downregulated lysine-specific histone demethylase 1 (LSD1) and BCL2 expression. After down-regulating CHOP, LSD1, and (su(var)-3-9,enhancer-of-zeste,trithorax) domain-containing protein 7/9 (SET7/9) in BRL-3A cells by siRNA, silencing CHOP and SET7/9 notably attenuated the pro-apoptotic and anti-proliferative effects of arsenic treatment on BRL-3A cells, which was reversed after inhibiting LSD1. In addition, our results suggested that knockdown of CHOP altered the expression of mitochondrial-associated proteins BCL2 and BIM, whereas knockdown of LSD1 and SET7/8 regulated the level of H3K4me1/2 modification and BAX protein. Coupled with chromatin immunoprecipitation results, we found that the level of CHOP in the promoter regions of BCL2 and BIM was significantly increased in BRL-3A cells exposed to 30 µmol/L NaAsO2 for 24 h, whereas the levels of H3K4me1/2 in the promoter regions of BAX were unchanged. Collectively, these data indicated that arsenic triggered the mitochondrial pathway to induce hepatocyte apoptosis by up-regulating the levels of CHOP and H3K4me1/2.

Identification of rare variants causing urea cycle disorders: A clinical, genetic, and biophysical study.

Urea cycle disorders (UCDs) are a group of rare metabolic conditions characterized by hyperammonemia and a broad spectrum of phenotypic severity. They are caused by the congenital deficiency in the eight biomolecules involved in urea cycle. In the present study, five cases of UCD were recruited and submitted to a series of clinical, biochemical, and genetic analysis with a combination of high throughput techniques. Moreover, in silico analysis was conducted on the identified missense genetic variants. Various clinical and biochemical indications (including profiles of amino acids and urinary orotic acids) of UCD were manifested by the five probands. Sequence analysis revealed nine diagnostic variants, including three novel ones, which caused Argininosuccinic aciduria (ASA) in one case, Carbamoyl phosphate synthetase 1deficiency (CPS1D) in two cases, Ornithine transcarbamylase deficiency (OTCD) in one case, and Citrin deficiency in 1case. Results of in silico biophysical analysis strongly suggested the pathogenicity of each the five missense variants and provided insight into their intramolecular impacts. In conclusion, this study expanded the genetic variation spectrum of UCD, gave solid evidence for counselling to the affected families, and should facilitate the functional study on the proteins in urea cycle.