Fully digital workflow of occlusal reconstruction treatment in a patient with congenital dentition defects.

OBJECTIVE: Occlusal reconstruction is a critical intervention for patients with dental hard tissue defects, temporomandibular joint (TMJ) disorders, and jaw position abnormalities. Clinical efficiency and outcomes of these procedures have improved with advances in digital technologies. This case report aims to illustrate a comprehensive digital workflow for occlusal reconstruction in a patient with congenital dentition defects, emphasizing the application of digital technologies to enhance treatment outcomes. CLINICAL CONSIDERATIONS: A 28-year-old woman with previously installed porcelain-fused-to-metal bridge restorations presented with a fractured prosthesis and TMJ symptoms. A multidisciplinary approach was adopted involving the use of digital facebow, intraoral scanners, digital smile design, and CAD/CAM technologies. The process included the extraction of defective restorations, temporary restorations to refine jaw position, and final permanent restorations. The digital workflow facilitated precise diagnostics and treatment, culminating in the successful installation of permanent restorations. Regular follow-ups at one- and three-months post-treatment confirmed stable occlusal function and high patient satisfaction. CONCLUSIONS: This case report showcases the potential of multiple digital technologies to streamline complex dental treatments and achieve high-quality results. CLINICAL SIGNIFICANCE: The integration of digital technologies in occlusal reconstruction treatments offers significant benefits in terms of precision, patient comfort, and esthetic outcomes.

Soil moisture decline in China's monsoon loess critical zone: More a result of land-use conversion than climate change.

Soil moisture (SM) is essential for sustaining services from Earth's critical zone, a thin-living skin spanning from the canopy to groundwater. In the Anthropocene epoch, intensive afforestation has remarkably contributed to global greening and certain service improvements, often at the cost of reduced SM. However, attributing the response of SM in deep soil to such human activities is a great challenge because of the scarcity of long-term observations. Here, we present a 37 y (1985 to 2021) analysis of SM dynamics at two scales across China's monsoon loess critical zone. Site-scale data indicate that land-use conversion from arable cropland to forest/grassland caused an 18% increase in SM deficit over 0 to 18 m depth (P < 0.01). Importantly, this SM deficit intensified over time, despite limited climate change influence. Across the Loess Plateau, SM storage in 0 to 10 m layer exhibited a significant decreasing trend from 1985 to 2021, with a turning point in 1999 when starting afforestation. Compared with SM storage before 1999, the relative contributions of climate change and afforestation to SM decline after 1999 were -8% and 108%, respectively. This emphasizes the pronounced impacts of intensifying land-use conversions as the principal catalyst of SM decline. Such a decline shifts 18% of total area into an at-risk status, mainly in the semiarid region, thereby threatening SM security. To mitigate this risk, future land management policies should acknowledge the crucial role of intensifying land-use conversions and their interplay with climate change. This is imperative to ensure SM security and sustain critical zone services.

LINC00909 up-regulates pluripotency factors and promotes cancer stemness and metastasis in pancreatic ductal adenocarcinoma by targeting SMAD4.

BACKGROUND: Pancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis. METHODS: The expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People's Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied. RESULTS: LINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models. CONCLUSION: We demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.

Medical service quality evaluation based on LDA and sentiment analysis: Examples of seven chronic diseases.

Objective: In this article, we investigate how chronic noncommunicable disease (CND) patients evaluate the medical service, and what obstacles exist in this process, which is useful for hospitals to improve efficiency and enhance patient satisfaction. Methods: Based on the total number of CND patients in China, 7 CNDs were selected as the evaluation objects, and then selected the Haodaifu website as the data source, crawled 15,682 medical service reviews, then the 9 themes were analyzed by the LDA theme model. The evaluation index system of six indicators was constructed based on quality management theory. The binary long short-term memory model was used to analyze the sentiment, and the entropy-valued, TOPSIS and gray correlation model was implemented for medical service quality evaluation; the barrier model was used to find out the key factors limiting medical services. Results: (a) Hypertension was rated at a good level in the degree of gray correlation closeness, bronchitis was rated at a low level and the rest were at an intermediate level. (b) The first two overall barriers were the hospitalization process and registration services which occupy about 30%, respectively. This implies that hospitals should focus on providing registration services and inpatient settings in the future. Conclusion: To promote hospitals to provide better services for patients with CNDs and improve patient satisfaction with medical care. And it is necessary to optimize medical services fundamentally by optimizing the inpatient process and improving the registration process to improve efficiency.

Synthesis of a novel monomer "DDTU-IDI" for the development of low-shrinkage dental resin composites.

OBJECTIVE: The current dental resin composites often suffer from polymerization shrinkage, which can lead to microleakage and potentially result in recurring tooth decay. This study presents the synthesis of a novel monomer, (3,9-diethyl-1,5,7,11-tetraoxaspiro[5,5]undecane-3,9-diyl)bis(methylene) bis((2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)carbamate) (DDTU-IDI), and evaluates its effect in the formulation of low-shrinkage dental resin composites. METHODS: DDTU-IDI was synthesized through a two-step reaction route, with the initial synthesis of the required raw material monomer 3,9-diethyl-3,9-dihydroxymethyl-1,5,7,11-tetraoxaspiro-[5,5] undecane (DDTU). The structures were confirmed using Fourier-transform infrared (FT-IR) spectroscopy and hydrogen nuclear magnetic resonance (1HNMR) spectroscopy. Subsequently, DDTU-IDI was incorporated into Bis-GMA-based composites at varying weight percentages (5, 10, 15, and 20 wt%). The polymerization reaction, degree of conversion, polymerization shrinkage, mechanical properties, physicochemical properties and biocompatibility of the low-shrinkage composites were thoroughly evaluated. Furthermore, the mechanical properties were assessed after a thermal cycling test with 10,000 cycles to determine the stability. RESULTS: The addition of DDTU-IDI at 10, 15, and 20 wt% significantly reduced the polymerization volumetric shrinkage of the experimental resin composites, without compromising the degree of conversion, mechanical and physicochemical properties. Remarkably, at a monomer content of 20 wt%, the polymerization shrinkage was reduced to 1.83 ± 0.53%. Composites containing 10, 15, and 20 wt% DDTU-IDI exhibited lower water sorption and higher contact angle. Following thermal cycling, the composites exhibited no significant decrease in mechanical properties, except for the flexural properties. SIGNIFICANCE: DDTU-IDI has favorable potential as a component which could produce volume expansion and increase rigidity in the development of low-shrinkage dental resin composites. The development of low-shrinkage composites containing DDTU-IDI appears to be a promising strategy for reducing polymerization shrinkage, thereby potentially enhancing the longevity of dental restorations.

Machine learning assisted biosensing technology: An emerging powerful tool for improving the intelligence of food safety detection.

Recently, the application of biosensors in food safety assessment has gained considerable research attention. Nevertheless, the evaluation of biosensors' sensitivity, accuracy, and efficiency is still ongoing. The advent of machine learning has enhanced the application of biosensors in food security assessment, yielding improved results. Machine learning has been preliminarily applied in combination with different biosensors in food safety assessment, with positive results. This review offers a comprehensive summary of the diverse machine learning methods employed in biosensors for food safety. Initially, the primary machine learning methods were outlined, and the integrated application of biosensors and machine learning in food safety was thoroughly examined. Lastly, the challenges and limitations of machine learning and biosensors in the realm of food safety were underscored, and potential solutions were explored. The review's findings demonstrated that algorithms grounded in machine learning can aid in the early detection of food safety issues. Furthermore, preliminary research suggests that biosensors could be optimized through machine learning for real-time, multifaceted analyses of food safety variables and their interactions. The potential of machine learning and biosensors in real-time monitoring of food quality has been discussed.

Programmable DNA tweezers-SDA for ultra-sensitive signal amplification fluorescence sensing strategy.

BACKGROUND: DNA tweezers, classified as DNA nanomachines, have gained prominence as multifunctional biosensors due to their advantages, including a straightforward structure, response mechanism, and high programmability. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. Some small molecules, such as mycotoxins, often require more sensitive detection due to their extremely high toxicity. Therefore, more effective signal amplification strategies are needed to further enhance the sensitivity of DNA tweezers in biosensing. RESULTS: We designed programmable DNA tweezers that detect small-molecule mycotoxins and miRNAs through simple sequence substitution. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. We introduced the Strand Displacement Amplification (SDA) technique to address this limitation, proposing a strategy of novel programmable DNA tweezers-SDA ultrasensitive signal amplification fluorescence sensing. We specifically investigate the effectiveness of this approach concerning signal amplification for two critical mycotoxins: aflatoxin B1 (AFB1) and zearalenone (ZEN). Results indicate that the detection ranges of AFB1 and ZEN via this strategy were 1-10,000 pg mL -1 and 10-100,000 pg mL -1, respectively, with corresponding detection limits of 0.933 pg mL -1 and 1.07 pg mL -1. Compared with the DNA tweezers direct detection method for mycotoxins, the newly constructed programmable DNA tweezers-SDA fluorescence sensing strategy achieved a remarkable 104-fold increase in the detection sensitivity for AFB1 and ZEN. SIGNIFICANCE: The constructed programmable DNA tweezers-SDA ultrasensitive signal-amplified fluorescence sensing strategy exhibits excellent detection performance for mycotoxins. The superb versatility of this strategy allows the developed method to be easily used for detecting other analytes by simply replacing the aptamer and cDNA, which has incredible potential in various fields such as food safety screening, clinical diagnostics, and environmental analysis.

Transport and inhibition mechanism for VMAT2-mediated synaptic vesicle loading of monoamines.

Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington's Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.

Molecular basis of the inositol deacylase PGAP1 involved in quality control of GPI-AP biogenesis.

The secretion and quality control of glycosylphosphatidylinositol-anchored proteins (GPI-APs) necessitates post-attachment remodeling initiated by the evolutionarily conserved PGAP1, which deacylates the inositol in nascent GPI-APs. Impairment of PGAP1 activity leads to developmental diseases in humans and fatality and infertility in animals. Here, we present three PGAP1 structures (2.66-2.84 Å), revealing its 10-transmembrane architecture and product-enzyme interaction details. PGAP1 holds GPI-AP acyl chains in an optimally organized, guitar-shaped cavity with apparent energetic penalties from hydrophobic-hydrophilic mismatches. However, abundant glycan-mediated interactions in the lumen counterbalance these repulsions, likely conferring substrate fidelity and preventing off-target hydrolysis of bulk membrane lipids. Structural and biochemical analyses uncover a serine hydrolase-type catalysis with atypical features and imply mechanisms for substrate entrance and product release involving a drawing compass movement of GPI-APs. Our findings advance the mechanistic understanding of GPI-AP remodeling.

Regulation of regulated cell death by extracellular vesicles in acute kidney injury and chronic kidney disease.

The imbalance between proliferation and death of kidney resident cells is a crucial factor in the development of acute or chronic renal dysfunction. Acute kidney injury (AKI) is often associated with the rapid loss of tubular epithelial cells (TECs). Sustained injury leads to the loss of glomerular endothelial cells (GECs) and podocytes, which is a key mechanism in the pathogenesis of glomerular diseases. This irreversible damage resulting from progressive cell loss eventually leads to deterioration of renal function characterized by glomerular compensatory hypertrophy, tubular degeneration, and renal fibrosis. Regulated cell death (RCD), which involves a cascade of gene expression events with tight structures, plays a certain role in regulating kidney health by determining the fate of kidney resident cells. Under pathological conditions, cells in the nephron have been demonstrated to constitutively release extracellular vesicles (EVs) which act as messengers that specifically interact with recipient cells to regulate their cell death process. For therapeutic intervention, exogenous EVs have exhibited great potential for the prevention and treatment of kidney disease by modulating RCD, with enhanced effects through engineering modification. Based on the functional role of EVs, this review comprehensively explores the regulation of RCD by EVs in AKI and chronic kidney disease (CKD), with emphasis on pathogenesis and therapeutic intervention.

MOF-on-MOF heterostructure boosting AIE sensing and triggered structural collapse for histamine detection.

We explored a novel preparation method for MOF-on-MOF heterostructured material (Zn-BTEC@ZIF-8). This prepared heterostructured material acts as a container, capable of adsorbing tetracycline hydrochloride molecules into its backbone through hydrogen bonding and π-π interactions. This phenomenon triggers an aggregation induced emission (AIE) effect, leading to the formation of luminescent bodies. The coordination between histamine and MOF was found to collapse the originally stabilized MOF-on-MOF structure. This collapse causes the splitting of the initially stabilized MOF-on-MOF structure from the aggregated state into fragments, resulting in the quenching of fluorescence in the fluorophore. Remarkably, the fluorescence quenching efficiency of this composite surpasses that of single-layer metal-organic framework (MOF) zeolitic imidazolate framework-8 (ZIF-8) or zinc-based MOF of pyromellitic acid (Zn-BTEC), enabling more sensitive detection of histamine. In this investigation, we constructed a label-free fluorescent sensor specifically designed for the detection of histamine, capitalizing on the AIE effect inherent in MOF-on-MOF architecture and the presence of tetracycline hydrochloride (Tet). The sensor demonstrates a rapid, straightforward, and stable response, allowing for histamine detection within 20 min. Notably, the sensor covers a detection range of 2-400 mg L-1, achieving a low detection limit of 1.458 mg L-1 The practical application of this sensor for quantitative detection of histamine in river water and various fish species exhibited robust performance, ensuring reliability and accuracy in real samples. Its potential application in food safety and environmental monitoring is evident, making it a valuable tool for addressing histamine-related challenges in these domains.

Editorial: Lifespan Connectome Gradients for a Road to Mental Health.

The connectome, generally defined as a comprehensive map of the structural and/or functional connections of a complete set of neural elements, has been recognized to condense the mechanistic architecture of the brain and capture meaningful individual differences. Increasing efforts are being invested in exploring the associations between connectomes and behavior/symptoms to piece together guidelines from a systems/cognitive neuroscience perspective for reforming mental health care. This editorial sketches a road to mental health with lifespan connectome gradients (LCG), highlighting unique perspectives, prospects, and priorities.

A turn-on fluorescent probe for detecting and bioimaging of HOCl in inflammatory and liver disease models.

Hypochlorous acid (HOCl) is a common reactive oxygen species (ROS) associated with the development of liver, tumor, inflammatory, and other diseases. In this work, the turn-on fluorescent probe named (WZ-HOCl) with a naphthalimide structure was designed and synthesized to detect endogenous HOCl in disease models. WZ-HOCl can achieve a fast response to HOCl with good linearity in the range of 0-45 µM (LOD = 147 nM). The application of WZ-HOCl in bioimaging was investigated by constructing a series of cellular disease models, and the results showed that WZ-HOCl could sensitively detect endogenous HOCl in inflammatory and liver disease models. It can also be used to differentiate between hepatocytes and hepatoma cells. WZ-HOCl will provide new methods and ideas for fluorescent probes in detecting drug-induced liver injury, alcoholic and non-alcoholic steatohepatitis, and some inflammation-related diseases.

Treatment of periorbital aging with negative pressure fractional microneedle radiofrequency: A self-controlled clinical trial.

BACKGROUND: Several treatment modalities are used for the treatment of periorbital rejuvenation with variable results. Recent studies showed that fractional radiofrequency may be an effective treatment modality for periorbital aging. This study aims to determine the efficacy and safety of negative pressure fractional microneedle radiofrequency (NPFMR) as a treatment for periorbital aging. METHODS: Twenty-five patients with periorbital aging were involved in this study. They were treated two times with an interval of 1 month. The patients were evaluated before treatment and 1, 3, and 6 months after the final treatment. RESULTS: The research findings suggest that periorbital wrinkles of the patients were significantly improved by VISIA system (p < 0.05). Physiological indicators detected by MPA10 system showed that compared with before treatment, the hydration increased (p < 0.05) and trans epidermal water loss (TEWL) decreased (p < 0.05) at 3 and 6 months after treatment. The glossiness increased at 1 month after treatment compared to pre-treatment (p < 0.05) and returned to the baseline level at 3 and 6 months after treatment. There was no significant change in melanin content (p > 0.05). Periorbital dermal thickness of the patients significantly increased at 1, 3, and 6 months after treatment according to skin ultrasound (p < 0.05). A periorbital skin biopsy revealed that the collagen fibers in the dermis were significantly thicker and more orderly after treatment, and the expression of type I collagen fibers and elastic fibers was increased compared with that before treatment. One patient developed post-inflammatory hyperpigmentation (PIH) at 1 month after the first treatment, which improved after active treatment. No other adverse reactions were observed. CONCLUSIONS: NPMFR could be an effective and safe treatment modality for the treatment of periorbital aging.

FeNbO4 nanochains with a five-electron transfer reaction toward high capacity and fast Li storage.

High capacity and outstanding rate performance of the FeNbO4 nanochain anode with both intercalation and conversion reactions for lithium-ion batteries are demonstrated. The unique one-dimensional structure and intercalation pseudocapacitive behavior of FeNbO4 accelerate the reaction kinetics. In situ X-ray diffractometer measurement confirms a five-electron transfer mechanism for Li storage.

Branched Copolymers with Tunable Clusteroluminescence in High Quantum Yield.

A novel type of fluorescence without large conjugated structures called clusteroluminescence (CL) has attracted a great deal of attention in recent years. Despite its many advantages, the emerging CL still encounters difficulties of low quantum yield (QY) and preliminary mechanisms. In this work, the branched structure was introduced into poly(maleic anhydride-alt-vinyl acetate) by chain transfer monomer. The emission wavelength of the branched copolymers is red-shifted with the increase of branching degree, and the absolute QY of solids can reach up to 29.87%. Further characterizations reveal that the branched structure can improve the flexibility of polymer chains, thereby promoting the intrachain interactions of subgroups. Furthermore, in the case of branched anhydride copolymers, the equilibrium between intrachain interactions and nonradiative transitions holds a crucial significance in determining the QY. This endeavor not only offers new insights into the mechanism of CL but also presents a novel approach to surmount the low QY of anhydride copolymers, thus broadening the horizons of CLgens to unexplored domains.

Establishment and Clinical Application in Stroke of a Serum Copeptin Time-Resolved Fluorescence Immunoassay.

The serum biomarker copeptin, an innovative and stable substitute biomarker of vasopressin, is associated with stroke. Therefore, establishing a highly sensitive time-resolved fluorescence immunoassay for copeptin (copeptin-TRFIA) is helpful to measure stroke and evaluate its value in clinical applications. Double antibody sandwich was used to establish copeptin-TRFIA. The established method was then assessed. Two coated and Eu3+-labeled copeptin monoclonal specific antibodies targeting different antigen epitopes were employed. The serum fluorescence counts of patients with stroke and healthy volunteers were detected by using the well-established copeptin-TRFIA. Serum copeptin levels were measured and analyzed statistically. The actual measurement linearity range of the proposed method was 0.13-44.66 ng/mL. Copeptin-TRFIA had the inter-assay coefficient of variation (CV) of 6.49%-9.08% and the intra-assay CV of 4.75%-7.77%. Patients with cerebral infarction (CI) and intracerebral hemorrhage (ICH) had significantly higher serum copeptin levels than healthy subjects. Copeptin concentrations in the serum of patients with stroke were significantly correlated with the scores of the National Institute for Healthy Stroke Scale (NIHSS) and modified Rankin Scale (mRS). A highly sensitive copeptin-TRFIA was successfully established. Serum copeptin has a certain value in the clinical diagnosis and prognosis of stroke.

Application and limitations of configuration factor (C-factor) in stress analysis of dental restorations.

OBJECTIVE: The configuration factor (C-factor) is an index used to evaluate the relationship between cavity configuration and the development of polymerization shrinkage stress in dental restorations. Although C-factor has been widely researched, its correlation with stress analysis in dental restorations remains controversial. This review aims to discuss the application and limitations of C-factor and define the restricted conditions under which the C-factor "rule of thumb" is applicable. METHODS: A thorough literature review was conducted on the application and limitations of C-factor in stress analysis of dental restorations. This was principally based on MEDLINE/PubMed and Web of Science databases and a review of the relevant studies and publications in scientific papers in international peer-reviewed journals for the specific topic of C-factor and polymerization shrinkage. RESULTS: The C-factor alone cannot provide an accurate prediction of the shrinkage stress of restorations and the mechanical behavior of material-tooth interfaces. C-factor is only applicable under one condition not typically seen in clinical practice: low, near-rigid compliance. SIGNIFICANCE: Conditions for the application of C-factor have been explicitly defined. A more accurate and precise understanding and utilization of the C-factor is of benefit as it contributes to better understanding of polymerization shrinkage behavior of restorations.

Structures of liganded glycosylphosphatidylinositol transamidase illuminate GPI-AP biogenesis.

Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a signal peptide region that lacks consensus sequence and replaces it with GPI via a transamidation reaction. How GPI-T maintains broad specificity while preventing unintentional cleavage is unclear. Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity. In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements. Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft. GPI-T thus integrates various weak specificity regions to form strong selectivity and prevent accidental activation. These findings provide important mechanistic insights into GPI-anchored protein biogenesis.