Quantitative mapping of the in vivo O-GalNAc glycoproteome in mouse tissues identifies GalNAc-T2 O-glycosites in metabolic disorder.

The family of GalNAc-Ts (GalNAcpolypeptide:N-Acetylgalactosaminyl transferases) catalyzes the first committed step in the synthesis of O-glycans, which is an abundant and biologically important protein modification. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence a broad array of biological functions. How site-specific O-glycans regulate biology is unclear. Compiling in vivo O-glycosites would be an invaluable step in determining the function of site-specific O-glycans. We integrated chemical and enzymatic conditions that cleave O-glycosites, a higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation mass spectrometry (MS) workflow and software to study nine mouse tissues and whole blood. We identified 2,154 O-glycosites from 595 glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and shared functions as classified by Gene Ontology terms. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. Quantitative glycoproteomics and proteomics revealed a tissue-specific regulation of O-glycosites that the differential expression of Galnt isoenzymes in tissues partly contributes to. We examined the Galnt2-null mouse model, which phenocopies congenital disorder of glycosylation involving GALNT2 and revealed a network of glycoproteins that lack GalNAc-T2-specific O-glycans. The known direct and indirect functions of these glycoproteins appear consistent with the complex metabolic phenotypes observed in the Galnt2-null animals. Through this study and interrogation of databases and the literature, we have compiled an atlas of experimentally identified mouse O-glycosites consisting of 2,925 O-glycosites from 758 glycoproteins.

Loss of the glycosyltransferase Galnt11 affects vitamin D homeostasis and bone composition.

O-glycosylation is a conserved posttranslational modification that impacts many aspects of organismal viability and function. Recent studies examining the glycosyltransferase Galnt11 demonstrated that it glycosylates the endocytic receptor megalin in the kidneys, enabling proper binding and reabsorption of ligands, including vitamin D-binding protein (DBP). Galnt11-deficient mice were unable to properly reabsorb DBP from the urine. Vitamin D plays an essential role in mineral homeostasis and its deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. We therefore set out to examine the effects of the loss of Galnt11 on vitamin D homeostasis and bone composition. We found significantly decreased levels of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, consistent with decreased reabsorption of DBP. This was accompanied by a significant reduction in blood calcium levels and a physiologic increase in parathyroid hormone (PTH) in Galnt11-deficient mice. Bones in Galnt11-deficient mice were smaller and displayed a decrease in cortical bone accompanied by an increase in trabecular bone and an increase in a marker of bone formation, consistent with PTH-mediated effects on bone. These results support a unified model for the role of Galnt11 in bone and mineral homeostasis, wherein loss of Galnt11 leads to decreased reabsorption of DBP by megalin, resulting in a cascade of disrupted mineral and bone homeostasis including decreased circulating vitamin D and calcium levels, a physiological increase in PTH, an overall loss of cortical bone, and an increase in trabecular bone. Our study elucidates how defects in O-glycosylation can influence vitamin D and mineral homeostasis and the integrity of the skeletal system.

C. elegans screen identifies autophagy genes specific to multicellular organisms.

The molecular understanding of autophagy has originated almost exclusively from yeast genetic studies. Little is known about essential autophagy components specific to higher eukaryotes. Here we perform genetic screens in C. elegans and identify four metazoan-specific autophagy genes, named epg-2, -3, -4, and -5. Genetic analysis reveals that epg-2, -3, -4, and -5 define discrete genetic steps of the autophagy pathway. epg-2 encodes a coiled-coil protein that functions in specific autophagic cargo recognition. Mammalian homologs of EPG-3/VMP1, EPG-4/EI24, and EPG-5/mEPG5 are essential for starvation-induced autophagy. VMP1 regulates autophagosome formation by controlling the duration of omegasomes. EI24 and mEPG5 are required for formation of degradative autolysosomes. This study establishes C. elegans as a multicellular genetic model to delineate the autophagy pathway and provides mechanistic insights into the metazoan-specific autophagic process.

SEPA-1 mediates the specific recognition and degradation of P granule components by autophagy in C. elegans.

How autophagy, an evolutionarily conserved intracellular catabolic system for bulk degradation, selectively degrades protein aggregates is poorly understood. Here, we show that several maternally derived germ P granule components are selectively eliminated by autophagy in somatic cells during C. elegans embryogenesis. The activity of sepa-1 is required for the degradation of these P granule components and for their accumulation into aggregates, termed PGL granules, in autophagy mutants. SEPA-1 forms protein aggregates and is also a preferential target of autophagy. SEPA-1 directly binds to the P granule component PGL-3 and also to the autophagy protein LGG-1/Atg8. SEPA-1 aggregates consistently colocalize with PGL granules and with LGG-1 puncta. Thus, SEPA-1 functions as a bridging molecule in mediating the specific recognition and degradation of P granule components by autophagy. Our study reveals a mechanism for preferential degradation of protein aggregates by autophagy and emphasizes the physiological significance of selective autophagy during animal development.

GLRA2 gene mutations cause high myopia in humans and mice.

BACKGROUND: High myopia (HM) is a leading cause of blindness that has a strong genetic predisposition. However, its genetic and pathogenic mechanisms remain largely unknown. Thus, this study aims to determine the genetic profile of individuals from two large Chinese families with HM and 200 patients with familial/sporadic HM. We also explored the pathogenic mechanism of HM using HEK293 cells and a mouse model. METHODS: The participants underwent genome-wide linkage analysis and exome sequencing. Visual acuity, electroretinogram response, refractive error, optical parameters and retinal rod cell genesis were measured in knockout mice. Immunofluorescent staining, biotin-labelled membrane protein isolation and electrophysiological characterisation were conducted in cells transfected with overexpression plasmids. RESULTS: A novel HM locus on Xp22.2-p11.4 was identified. Variant c.539C>T (p.Pro180Leu) in GLRA2 gene was co-segregated with HM in the two families. Another variant, c.458G>A (p.Arg153Gln), was identified in a sporadic sample. The Glra2 knockout mice showed myopia-related phenotypes, decreased electroretinogram responses and impaired retinal rod cell genesis. Variants c.458G>A and c.539C>T altered the localisation of GlyRα2 on the cell membrane and decreased agonist sensitivity. CONCLUSION: GLRA2 was identified as a novel HM-causing gene. Its variants would cause HM through altered visual experience by impairing photoperception and visual transmission.

Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation.

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.

Dynamic expression of mucins and the genes controlling mucin-type O-glycosylation within the mouse respiratory system.

The COVID-19 global pandemic has underscored the need to understand how viruses and other pathogens are able to infect and replicate within the respiratory system. Recent studies have highlighted the role of highly O-glycosylated mucins in the protection of the respiratory system as well as how mucin-type O-glycosylation may be able to modify viral infectivity. Therefore, we set out to identify the specific genes controlling mucin-type O-glycosylation throughout the mouse respiratory system as well as determine how their expression and the expression of respiratory mucins is influenced by infection or injury. Here, we show that certain mucins and members of the Galnt family are abundantly expressed in specific respiratory tissues/cells and demonstrate unique patterns of O-glycosylation across diverse respiratory tissues. Moreover, we find that the expression of certain Galnts and mucins is altered during lung infection and injury in experimental mice challenged with infectious agents, toxins, and allergens. Finally, we examine gene expression changes of Galnts and mucins in a mouse model of SARS-CoV-2 infection. Our work provides foundational knowledge regarding the specific expression of Galnt enzyme family members and mucins throughout the respiratory system, and how their expression is altered upon lung infection and injury.

Association of Hearing Status and Cognition With Fall Among the Oldest-Old Chinese: A Nationally Representative Cohort Study.

OBJECTIVES: The oldest-old (aged ≥80 years) are the most rapidly growing population and age is related to hearing impairment (HI) and cognitive decline. We aimed to estimate the association between HI and fall, and the effect of different cognitive states on this association among the oldest-old Chinese population. DESIGN: A total of 6931 Chinese oldest-old were included in the 2018 cross-cohort from the Chinese Longitudinal Healthy Longevity Survey (CLHLS). The presence of HI was identified by using a dichotomized metric of self-reported hearing status. Cognitive function was evaluated by using the modified Mini-Mental State Examination (MMSE). Cognitive impairment was defined as the MMSE score below 24 points. Data on fall history were collected by questionnaires survey from the participants or their relatives. We studied the association of hearing status and cognitive function with fall by using multivariable logistic regressions, upon adjustment of sociodemographic characteristics, lifestyles, and health conditions. RESULTS: Our participants were aged 92 (range 80 to 117) on average, with 60.1% being women. In total, 39.1% of the participants had reported HI, 50.1% had cognitive impairment, and 26.2% had a history of falling. Participants with HI had a higher incidence of cognitive impairment (79.4%), as compared with their counterparts without HI (31.3%). Compared with those without HI, HI patients had a higher risk of falling after full adjustment for potential confounders (OR = 1.16 [95% confidence interval, CI, 1.01, 1.32], p = 0.031). In comparison with HI participants without cognitive impairment, HI patients with cognitive impairment had a higher fall risk (OR = 1.45 [95% CI = 1.23, 1.72], p < 0.001). CONCLUSIONS: Association of hearing status and cognition with fall was, for the first time, examined on the basis of a nationally-representative oldest-old Chinese population. Poor cognitive performance was common in individuals with HI, and those with HI and cognitive impairment further increased the risk of falling.

Study on the Effect of Pain Programmed Care Based on the Concept of Prehabilitation on the Recovery of Joint Function and WHOQOL-BREF Score in Elderly Patients after Total Hip Arthroplasty.

Objective: To assess the impact of pain-programmed care, utilizing the concept of prehabilitation, on the postoperative recovery of joint function and WHOQOL-BREF score in elderly patients following total hip arthroplasty. Methods: Ninety cases of elderly patients with total hip arthroplasty admitted to our hospital from January to December 2022 were selected as the observation sample, and the 90 elderly patients with total hip arthroplasty were divided into 45 control groups and 45 control groups by random number table method. The pain assessment, functional exercise compliance, hip joint function and quality of life of the two groups were compared after the intervention. Results: The nursing intervention led to a significant reduction in pain scores and improvement in quality of life for elderly patients undergoing total hip joint replacement. The observation group showed a greater reduction in resting pain scores (6.20 ± 0.63 vs. 3.78 ± 0.67, P < .05) and activity pain scores (8.78 ± 0.64 vs. 4.89 ± 0.68, P < .05) compared to the control group. Additionally, the observation group demonstrated significant improvements in physiology (55.73 ± 2.14 vs. 71.87 ± 21.59, P < .05), psychology (55.71 ± 2.13 vs. 72.60 ± 2.20, P < .05), social relations (55.73 ± 2.13 vs. 71.96 ± 1.57, P < .05), and environmental effect (55.60 ± 2.15 vs. 68.62 ± 1.51, P < .05) after care, whereas the control group exhibited lesser improvements in these areas (physiology: 55.60 ± 2.24 vs. 64.53±2.02, P < .05; psychology: 55.60 ± 2.22 vs. 66.33±1.99, P < .05; social relations: 55.82 ± 2.09 vs. 67.84 ± 1.73, P < .05; environmental effect: 55.89 ± 2.18 vs. 62.09 ± 51.49, P < .05). These findings demonstrate the significant impact of nursing intervention on pain reduction and improved quality of life for elderly patients undergoing total hip joint replacement. Conclusion: Pain programmed care based on the concept of prehabilitation for elderly patients undergoing total hip arthroplasty has a significant positive impact on pain control, compliance with functional exercise, recovery of hip function, and improvement of quality of life. These findings highlight the benefits of implementing pain management strategies and rehabilitation programs in the field of total hip arthroplasty and elderly care.

Insights into glucosinolate accumulation and metabolic pathways in Isatis indigotica Fort.

BACKGROUND: Glucosinolates (GSLs) play important roles in defending against exogenous damage and regulating physiological activities in plants. However, GSL accumulation patterns and molecular regulation mechanisms are largely unknown in Isatis indigotica Fort. RESULTS: Ten GSLs were identified in I. indigotica, and the dominant GSLs were epiprogoitrin (EPI) and indole-3-methyl GSL (I3M), followed by progoitrin (PRO) and gluconapin (GNA). The total GSL content was highest (over 20 µmol/g) in reproductive organs, lowest (less than 1.0 µmol/g) in mature organs, and medium in fresh leaves (2.6 µmol/g) and stems (1.5 µmol/g). In the seed germination process, the total GSL content decreased from 27.2 µmol/g (of seeds) to 2.7 µmol/g (on the 120th day) and then increased to 4.0 µmol/g (180th day). However, the content of indole GSL increased rapidly in the first week after germination and fluctuated between 1.13 µmol/g (28th day) and 2.82 µmol/g (150th day). Under the different elicitor treatments, the total GSL content increased significantly, ranging from 2.9-fold (mechanical damage, 3 h) to 10.7-fold (MeJA, 6 h). Moreover, 132 genes were involved in GSL metabolic pathways. Among them, no homologs of AtCYP79F2 and AtMAM3 were identified, leading to a distinctive GSL profile in I. indigotica. Furthermore, most genes involved in the GSL metabolic pathway were derived from tandem duplication, followed by dispersed duplication and segmental duplication. Purifying selection was observed, although some genes underwent relaxed selection. In addition, three tandem-arrayed GSL-OH genes showed different expression patterns, suggesting possible subfunctionalization during evolution. CONCLUSIONS: Ten different GSLs with their accumulation patterns and 132 genes involved in the GSL metabolic pathway were explored, which laid a foundation for the study of GSL metabolism and regulatory mechanisms in I. indigotica.

Boosting Fast Sodium Ion Storage by Synergistic Effect of Heterointerface Engineering and Nitrogen Doping Porous Carbon Nanofibers.

Heterointerface engineering with multiple electroactive and inactive supporting components is considered an efficient approach to enhance electrochemical performance for sodium-ion batteries (SIBs). Nevertheless, it is still a challenge to rationally design heterointerface engineering and understand the synergistic effect reaction mechanisms. In this paper, the two-phase heterointerface engineering (Sb2 S3 and FeS2 ) is well designed to incorporate into N-doped porous hollow carbon nanofibers (Sb-Fe-S@CNFs) by proper electrospinning design. The obtained Sb-Fe-S@CNFs are used as anode in SIBs to evaluate the electrochemical performance. It delivers a reversible capacity of 396 mA h g-1 after 2000 cycles at 1 A g-1 and exhibits an ultra-long high rate cycle life for 16 000 cycles at 10 A g-1 . The admirable electrochemical performance is mainly attributed to the following reasons: The porous carbon nanofibers serve as an accelerator of the electrons/ions and a buffer to alleviate volume expansion upon long cyclic performance. The abundant phase boundaries of Sb2 S3 /FeS2 exert low Na+ adsorption energy and greatly promote the charge transfer in the internal electric field calculated by first-principle density functional theory. Therefore, the as-prepared Sb-Fe-S@CNFs represents a promising candidate for an efficient anode electrode material in SIBs.

Handelin extends lifespan and healthspan of Caenorhabditis elegans by reducing ROS generation and improving motor function.

Aging and aging-related disorders contribute to formidable socioeconomic and healthcare challenges. Several promising small molecules have been identified to target conserved genetic pathways delaying aging to extend lifespan and healthspan in many organisms. We previously found that extract from an edible and medicinal plant Chrysanthemum indicum L. (C. indicum L.) protect skin from UVB-induced photoaging, partially by reducing reactive oxygen species (ROS) generation. Thus, we hypothesized that C. indicum L. and its biological active compound may extend lifespan and health span in vivo. We find that both water and ethanol extracts from C. indicum L. extended lifespan of Caenorhabditis elegans, with better biological effect on life extending for ethanol extracts. As one of the major biological active compounds, handelin extended lifespan of C. elegans too. RNA-seq analysis revealed overall gene expression change of C. elegans post stimulation of handelin focus on several antioxidative proteins. Handelin significantly reduced ROS level and maintained the number and morphology of mitochondria. Moreover, handelin improveed many C. elegans behaviors related to healthspan, including increased pharyngeal pumping and body movement. Muscle fiber imaging analyses revealed that handelin maintains muscle architecture by stabilizing myofilaments. In conclusion, our present study finds a novel compound handelin, from C. indicum L., which bring about biologically beneficial effects by mild stress response, termed as hormetin, that can extend both lifespan and healthspan in vivo on C. elegans. Further study on mammal animal model of natural aging or sarcopenia will verify the potential clinical value of handelin.

An Improved Self-Adaptive Differential Evolution with the Neighborhood Search Algorithm for Global Optimization of Bimetallic Clusters.

Global optimization of multicomponent cluster structures is considerably time-consuming due to the existence of a vast number of isomers. In this work, we proposed an improved self-adaptive differential evolution with the neighborhood search (SaNSDE) algorithm and applied it to the global optimization of bimetallic cluster structures. The cross operation was optimized, and an improved basin hopping module was introduced to enhance the searching efficiency of SaNSDE optimization. Taking (PtNi)N (N = 38 or 55) bimetallic clusters as examples, their structures were predicted by using this algorithm. The traditional SaNSDE algorithm was carried out for comparison with the improved SaNSDE algorithm. For all the optimized clusters, the excess energy and the second difference of the energy were calculated to examine their relative stabilities. Meanwhile, the bond order parameters were adopted to quantitatively characterize the cluster structures. The results reveal that the improved SaNSDE algorithm possessed significantly higher searching capability and faster convergence speed than the traditional SaNSDE algorithm. Furthermore, the lowest-energy configurations of (PtNi)38 clusters could be classified as the truncated octahedral and disordered structures. In contrast, all the optimal (PtNi)55 clusters were approximately icosahedral. Our work fully demonstrates the high efficiency of the improved algorithm and advances the development of global optimization algorithms and the structural prediction of multicomponent clusters.

Galnt11 regulates kidney function by glycosylating the endocytosis receptor megalin to modulate ligand binding.

Chronic kidney disease (CKD) affects more than 20 million Americans and ∼10% of the population worldwide. Genome-wide association studies (GWAS) of kidney functional decline have identified genes associated with CKD, but the precise mechanisms by which they influence kidney function remained largely unexplored. Here, we examine the role of 1 GWAS-identified gene by creating mice deficient for Galnt11, which encodes a member of the enzyme family that initiates protein O-glycosylation, an essential posttranslational modification known to influence protein function and stability. We find that Galnt11-deficient mice display low-molecular-weight proteinuria and have specific defects in proximal tubule-mediated resorption of vitamin D binding protein, α1-microglobulin, and retinol binding protein. Moreover, we identify the endocytic receptor megalin (LRP2) as a direct target of Galnt11 in vivo. Megalin in Galnt11-deficient mice displays reduced ligand binding and undergoes age-related loss within the kidney. Differential mass spectrometry revealed specific sites of Galnt11-mediated glycosylation within mouse kidney megalin/LRP2 that are known to be involved in ligand binding, suggesting that O-glycosylation directly influences the ability to bind ligands. In support of this, recombinant megalin containing these sites displayed reduced albumin binding in cells deficient for Galnt11 Our results provide insight into the association between GALNT11 and CKD, and identify a role for Galnt11 in proper kidney function.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome.

The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted.

Functional Proliferating Human Hepatocytes: In Vitro Hepatocyte Model for Drug Metabolism, Excretion, and Toxicity.

To develop a functional alternative hepatocyte model for primary human hepatocytes (PHHs) with proliferative property, essential drug metabolic, and transporter functions, proliferating human hepatocytes (ProliHHs) expanded from PHHs were fully characterized in vitro. Herein, ProliHHs generated from multiple PHHs donors could be expanded more than 200-fold within four passages and maintained their metabolic or transporter capacities partially. Furthermore, ProliHHs were able to regain the mature hepatic property after three-dimensional (3D) culture. Particularly, the downregulated mRNA expression and function of three major cytochrome P450 (P450) enzymes (CYP1A2, CYP2B6, and CYP3A4) in the proliferating process (ProliHHs-P) could be recovered by 3D culture. The metabolic variabilities across different PHHs donors could be inherited to their matured ProliHHs (ProliHHs-M). The intrinsic clearances of seven major P450 enzymes in ProliHHs-M correlated well (r = 0.87) with those in PHHs. Also, bile canaliculi structures could be observed in sandwich-cultured ProliHHs (SC-ProliHHs), and the biliary excretion index of four probe compounds [cholyl-lys-fluorescein, 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate (CDF), deuterium-labeled sodium taurocholate acid, and rosuvastatin] in SC-ProliHHs (>10%) were close to sandwich-cultured PHHs. More importantly, both ProliHHs-P and ProliHHs-M could be used to evaluate hepatotoxicity. Therefore, these findings demonstrated that the 3D and sandwich culture system could be used to recover the metabolic and transporter functions in ProliHHs for clearance prediction and cholestasis risk assessment, respectively. Together, ProliHHs could be a promising substitute for PHHs in drug metabolism, transport, and hepatotoxicity screening. SIGNIFICANCE STATEMENT: This report describes the study of drug metabolic capacities, efflux transporter functions, and toxicity assessments of proliferating human hepatocytes (ProliHHs). The metabolic variability in different primary human hepatocyte donors could be inherited by their matured ProliHHs derivatives. Also, ProliHHs could form canalicular networks in sandwich culture and display biliary excretion capacities. More importantly, both the proliferative and maturation statuses of ProliHHs could be used to evaluate hepatotoxicity. Together, ProliHHs were feasible to support drug candidate screening in hepatic metabolism, disposition, and toxicity.

Improved online LC-MS/MS identification of O-glycosites by EThcD fragmentation, chemoenzymatic reaction, and SPE enrichment.

O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites. O-glycosite mapping strategy via proteases, cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are enriched by SPE column, followed by release of N-glycans, collection of higher MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, and finally detected by LC-MS/MS using EThcD.

Arginine methylation modulates autophagic degradation of PGL granules in C. elegans.

The selective degradation of intracellular components by autophagy involves sequential interactions of the cargo with a receptor, which also binds the autophagosomal protein Atg8 and a scaffold protein. Here, we demonstrated that mutations in C. elegans epg-11, which encodes an arginine methyltransferase homologous to PRMT1, cause the defective removal of PGL-1 and PGL-3 (cargo)-SEPA-1 (receptor) complexes, known as PGL granules, from somatic cells during embryogenesis. Autophagic degradation of the PGL granule scaffold protein EPG-2 and other protein aggregates was unaffected in epg-11/prmt-1 mutants. Loss of epg-11/prmt-1 activity impairs the association of PGL granules with EPG-2 and LGG-1 puncta. EPG-11/PRMT-1 directly methylates arginines in the RGG domains of PGL-1 and PGL-3. Autophagic removal of PGL proteins is impaired when the methylated arginines are mutated. Our study reveals that posttranslational arginine methylation regulates the association of the cargo-receptor complex with the scaffold protein, providing a mechanism for modulating degradation efficiency in selective autophagy.

[Efficacy of multisensory training and rehabilitation in patients with balance disorders].

Objective: To analyze the clinical efficacy of multisensory training and rehabilitation treatment in patients with balance disorders. Methods: From January to December 2020, 95 patients with balance disorders in the Vertigo Center of Department of Otorhinolaryngology, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology were enrolled. All patients were treated according to the treatment guidelines of Chinese Medical Association or expert consensus. Those with poor recovery or unsatisfactory treatment results underwent multisensory training and rehabilitation for 1 month after a comprehensive evaluation. The scores of Visual Analogue Scale (VAS), Dizziness Handicap Inventory (DHI), Berg Balance Scale (BBS), and Activities-specific Balance Confidence Scale (ABC), Somatization Symptom Self-Rating Scale (SSS), 9-item Patient Health Questionnaire (PHQ9) and the 7-item Generalized Anxiety Disorder (GAD7) were evaluated and compared before and after the treatment. Results: Totally, 95 patients were enrolled. There were 34 males and 61 females, aged (44±14) years. VAS (2.1±1.1 vs 5.9±2.5, P<0.01), DHI (15.6±7.7 vs 33.1±13.2, P<0.01), SSS (1.5±0.6 vs 2.4±0.8, P<0.01), PHQ9 (6.0±2.7 vs 8.6±4.3, P=0.01) and GAD7 (5.2±2.6 vs 9.5±2.8, P<0.01) decreased after treatment, while BBS (53.4±10.0 vs 34.8±10.7, P<0.01) and ABC (89.6±8.0 vs 55.7±21.8, P<0.01) increased. Conclusion: Multisensory training and rehabilitation therapy can effectively enhance the vertigo control rate and balance ability in patients with balance disorders, reduce the risk of falling, promote their mental and psychological state, and thus improve the quality of life.

An electroanalytical platform for nereistoxin-related insecticide detection based on DNA conformational switching and exonuclease III assisted target recycling.

In this work, an electroanalytical platform for nereistoxin (NRT)-related insecticide detection is proposed on the basis of NRT induced DNA conformational switching and exonuclease III (Exo III) assisted target recycling. NRT-related insecticides were first hydrolyzed and converted into NRT with two thiol groups (-SH). Then, a cytosine-Ag+-cytosine (C-Ag+-C) mismatched base pair was adopted to induce a blunt-ended hairpin configuration of HP DNA. In the presence of converted NRT, it could take up Ag+ from HP DNA to change its conformation from a hairpin to single-stranded structure (HP ssDNA). Thereafter, the obtained HP ssDNA was further hybridized with an H1 hairpin probe on the electrode surface to trigger the Exo III cleavage process, releasing HP ssDNA for recycling leaving the G-quadruplex fragment of H1, which was used for hemin/G-quadruplex complex formation. The reversible redox reaction of Fe(iii)/Fe(ii) of hemin gave a remarkable electrochemical response for quantitative determination of the NRT-related insecticides. As an analytical model, a low detection limit of 3.9 ng L-1 and a wide linear range of 0.01-1500 µg L-1 with excellent selectivity were achieved for cartap detection. The proposed method also displayed great applicability for cartap detection in agricultural products.