Large-scale multiplexed mosaic CRISPR perturbation in the whole organism.

Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.

The kpc-1 3'UTR facilitates dendritic transport and translation efficiency of mRNAs for dendrite arborization of a mechanosensory neuron important for male courtship.

A recently reported Schizophrenia-associated genetic variant in the 3'UTR of the human furin gene, a homolog of C. elegans kpc-1, highlights an important role of the furin 3'UTR in neuronal development. We isolate three kpc-1 mutants that display abnormal dendrite arborization in PVD neurons and defective male mating behaviors. We show that the kpc-1 3'UTR participates in dendrite branching and self-avoidance. The kpc-1 3'UTR facilitates mRNA localization to branching points and contact points between sibling dendrites and promotes translation efficiency. A predicted secondary structural motif in the kpc-1 3'UTR is required for dendrite self-avoidance. Animals with over-expression of DMA-1, a PVD dendrite receptor, exhibit similar dendrite branching and self-avoidance defects that are suppressed with kpc-1 over-expression. Our results support a model in which KPC-1 proteins are synthesized at branching points and contact points to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance of a mechanosensory neuron important for male courtship.

The codriven assembly of molecular metalla-links ([Formula: see text], [Formula: see text]) and metalla-knots ([Formula: see text], [Formula: see text]) via coordination and noncovalent interactions.

Although mechanically interlocked molecules (MIMs) display unique properties and functions associated with their intricate connectivity, limited assembly strategies are available for their synthesis. Herein, we presented a synergistic assembly strategy based on coordination and noncovalent interactions (π-π stacking and CH⋯π interactions) to selectively synthesize molecular closed three-link chains ([Formula: see text] links), highly entangled figure-eight knots ([Formula: see text] knots), trefoil knot ([Formula: see text] knot), and Borromean ring ([Formula: see text] link). [Formula: see text] links can be created by the strategic assembly of nonlinear multicurved ligands incorporating a furan or phenyl group with the long binuclear half-sandwich organometallic Cp*RhIII (Cp* = η5-pentamethylcyclopentadienyl) clip. However, utilizing much shorter binuclear Cp*RhIII units for union with the 2,6-naphthyl-containing ligand led to a [Formula: see text] knot because of the increased π-π stacking interactions between four consecutive stacked layers and CH⋯π interactions. Weakening such π-π stacking interactions resulted in a [Formula: see text] knot. The universality of this synergistic assembly strategy for building [Formula: see text] knots was verified by utilizing a 1,5-naphthyl-containing ligand. Quantitative conversion between the [Formula: see text] knot and the simple macrocycle species was accomplished by adjusting the concentrations monitored by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Furthermore, increasing the stiff π-conjugated area of the binuclear unit afforded molecular Borromean ring, and this topology is a topological isomer of the [Formula: see text] link. These artificial metalla-links and metalla-knots were confirmed by single-crystal X-ray diffraction, NMR and ESI-MS. The results offer a potent strategy for building higher-order MIMs and emphasize the critical role that noncovalent interactions play in creating sophisticated topologies.

Two intrinsic timing mechanisms set start and end times for dendritic arborization of a nociceptive neuron.

Choreographic dendritic arborization takes place within a defined time frame, but the timing mechanism is currently not known. Here, we report that the precisely timed lin-4-lin-14 regulatory circuit triggers an initial dendritic growth activity, whereas the precisely timed lin-28-let-7-lin-41 regulatory circuit signals a subsequent developmental decline in dendritic growth ability, hence restricting dendritic arborization within a set time frame. Loss-of-function mutations in the lin-4 microRNA gene cause limited dendritic outgrowth, whereas loss-of-function mutations in its direct target, the lin-14 transcription factor gene, cause precocious and excessive outgrowth. In contrast, loss-of-function mutations in the let-7 microRNA gene prevent a developmental decline in dendritic growth ability, whereas loss-of-function mutations in its direct target, the lin-41 tripartite motif protein gene, cause further decline. lin-4 and let-7 regulatory circuits are expressed in the right place at the right time to set start and end times for dendritic arborization. Replacing the lin-4 upstream cis-regulatory sequence at the lin-4 locus with a late-onset let-7 upstream cis-regulatory sequence delays dendrite arborization, whereas replacing the let-7 upstream cis-regulatory sequence at the let-7 locus with an early-onset lin-4 upstream cis-regulatory sequence causes a precocious decline in dendritic growth ability. Our results indicate that the lin-4-lin-14 and the lin-28-let-7-lin-41 regulatory circuits control the timing of dendrite arborization through antagonistic regulation of the DMA-1 receptor level on dendrites. The LIN-14 transcription factor likely directly represses dma-1 gene expression through a transcriptional means, whereas the LIN-41 tripartite motif protein likely indirectly promotes dma-1 gene expression through a posttranscriptional means.

Threshold of hyperglycaemia associated with mortality in critically ill patients: a multicentre, prospective, observational study using continuous glucose monitoring.

AIMS/HYPOTHESIS: Continuous glucose monitoring (CGM) provides comprehensive information on the exposure to dysglycaemia. This study aimed to investigate the threshold of hyperglycaemia related to mortality risk in critically ill patients using CGM technology. METHODS: A total of 293 adult critically ill patients admitted to intensive care units of five medical centres were prospectively included between May 2020 and November 2021. Participants wore intermittently scanned CGM for a median of 12.0 days. The relationships between different predefined time above ranges (TARs), with the thresholds of hyperglycaemia ranging from 7.8 to 13.9 mmol/l (140-250 mg/dl), and in-hospital mortality risk were assessed by multivariate Cox proportional regression analysis. Time in ranges (TIRs) of 3.9 mmol/l (70 mg/dl) to the predefined hyperglycaemic thresholds were also assessed. RESULTS: Overall, 66 (22.5%) in-hospital deaths were identified. Only TARs with a threshold of 10.5 mmol/l (190 mg/dl) or above were significantly associated with the risk of in-hospital mortality, after adjustment for covariates. Furthermore, as the thresholds for TAR increased from 10.5 mmol/l to 13.9 mmol/l (190 mg/dl to 250 mg/dl), the hazards of in-hospital mortality increased incrementally with every 10% increase in TARs. Similar results were observed concerning the associations between TIRs with various upper thresholds and in-hospital mortality risk. For per absolute 10% decrease in TIR 3.9-10.5 mmol/l (70-190 mg/dl), the risk of in-hospital mortality was increased by 12.1% (HR 1.121 [95% CI 1.003, 1.253]). CONCLUSIONS/INTERPRETATION: A glucose level exceeding 10.5 mmol/l (190 mg/dl) was significantly associated with higher risk of in-hospital mortality in critically ill patients.

The Topological Transformation of Trefoil Knots to Solomon Links via Diels-Alder Click Reaction.

The quest for more efficient, user-friendly, and less wasteful topological transformations remains a significant challenge in the realm of postassembly modifications. In this article, high yields of two molecular trefoil knots (Rh-1, Ir-1) were obtained using ligand 3,6-bis(3-(pyridin-4-yl)phenyl)-1,2,4,5-tetrazine (L1) with reactive tetrazine units and binuclear half-sandwich organometallic units [Cp*2M2(µ-TPPHZ)(OTf)2](OTf)2 (Rh-B, M = RhIII; Ir-B, M = IrIII). 2,5-Norbornadiene was used as an inducer of the Diels-Alder click reaction to modulate rapidly and efficiently the transformation of Trefoil knots to Solomon links. However, the key to achieving this topological structural change is the subtle increase in site steric of the pyridazine fragments (L2), which allows the molecular structures to spread and bend in three-dimensional space, as confirmed by single-crystal X-ray diffraction, ESI-TOF/MS, elementary analysis and detailed solution-state NMR techniques.

Two-sample Mendelian randomization analysis reveals causal relationships between blood lipids and venous thromboembolism.

Venous Thromboembolism (VTE) is a complex disease that can be classified into two subtypes: Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE). Previous observational studies have shown associations between lipids and VTE, but causality remains unclear. Hence, by utilizing 241 lipid-related traits as exposures and data from the FinnGen consortium on VTE, DVT, and PE as outcomes, we conducted two-sample Mendelian randomization (MR) analysis to investigate causal relationships between lipids and VTE, DVT and PE. The MR results identified that fatty acid (FA) unsaturation traits (Ratio of bis-allylic bonds to double bonds in lipids, and Ratio of bis-allylic bonds to total fatty acids in lipids) were associated with VTE (OR [95% CI]: 1.21 [1.15-1.27]; 1.21 [1.13-1.30]), DVT (OR [95%CI]: 1.24 [1.16-1.33]; 1.26 [1.16-1.36]) and PE (OR [95%CI]: 1.18 [1.08-1.29]; 1.18 [1.09-1.27]). Phosphatidylcholines exhibit potential causal effects on VTE and PE. Phosphatidylcholine acyl-alkyl C40:4 (PC ae C40:4) was negatively associated with VTE (OR [95% CI]: 0.79 [0.73-0.86]), while phosphatidylcholine diacyl C42:6 (PC aa C42:6) and phosphatidylcholine acyl-alkyl C36:4 (PC ae C36:4) were positively associated with PE (OR [95%CI]: 1.44 [1.20-1.72]; 1.22 [1.10-1.35]). Additionally, we found that medium LDL had a protective effect on VTE. Our study indicates that higher FA unsaturation may increase the risk of VTE, DVT, and PE. Different types of phosphatidylcholine have either promotive or inhibitory effects on VTE and PE, contributing to a better understanding of the risk factors for VTE.

Small Scale, Big Impact: Nanotechnology-Enhanced Drug Delivery for Brain Diseases.

Central nervous system (CNS) diseases, ranging from brain cancers to neurodegenerative disorders like dementia and acute conditions such as strokes, have been heavily burdening healthcare and have a direct impact on patient quality of life. A significant hurdle in developing effective treatments is the presence of the blood-brain barrier (BBB), a highly selective barrier that prevents most drugs from reaching the brain. The tight junctions and adherens junctions between the endothelial cells and various receptors expressed on the cells make the BBB form a nonfenestrated and highly selective structure that is crucial for brain homeostasis but complicates drug delivery. Nanotechnology offers a novel pathway to circumvent this barrier, with nanoparticles engineered to ferry drugs across the BBB, protect drugs from degradation, and deliver medications to the designated area. After years of development, nanoparticle optimization, including sizes, shapes, surface modifications, and targeting ligands, can enable nanomaterials tailored to specific brain drug delivery settings. Moreover, smart nano drug delivery systems can respond to endogenous and exogenous stimuli that control subsequent drug release. Here, we address the importance of the BBB in brain disease treatment, summarize different delivery routes for brain drug delivery, discuss the cutting-edge nanotechnology-based strategies for brain drug delivery, and further offer valuable insights into how these innovations in nanoparticle technology could revolutionize the treatment of CNS diseases, presenting a promising avenue for noninvasive, targeted therapeutic interventions.

Deep Learning-Assisted Two-Dimensional Transperineal Ultrasound for Analyzing Bladder Neck Motion in Women With Stress Urinary Incontinence.

BACKGROUND: No universally recognized transperineal ultrasound parameters are available for evaluating stress urinary incontinence. The information captured by commonly used perineal ultrasound parameters is limited and insufficient for a comprehensive assessment of stress urinary incontinence. Although bladder neck motion plays a major role in stress urinary incontinence, objective and visual methods to evaluate its impact on stress urinary incontinence remain lacking. OBJECTIVE: To use a deep learning-based system to evaluate bladder neck motion using two-dimensional transperineal ultrasound videos, exploring motion parameters for diagnosing and evaluating stress urinary incontinence. We hypothesized that bladder neck motion parameters are associated with stress urinary incontinence and are useful for stress urinary incontinence diagnosis and evaluation. STUDY DESIGN: This retrospective study including 217 women involved the following parameters: maximum and average speeds of bladder neck descent, ß angle, urethral rotation angle, and duration of the Valsalva maneuver. The fitted curves were derived to visualize bladder neck motion trajectories. Comparative analyses were conducted to assess these parameters between stress urinary incontinence and control groups. Logistic regression and receiver operating characteristic curve analyses were employed to evaluate the diagnostic performance of each motion parameter and their combinations for stress urinary incontinence. RESULTS: Overall, 173 women were enrolled in this study (82, stress urinary incontinence group; 91, control group). No significant differences were observed in the maximum and average speeds of bladder neck descent and in the speed variance of bladder neck descent. The maximum and average speed of the ß and urethral rotation angles were faster in the stress urinary incontinence group than in the control group (151.2 vs 109.0 mm/s, P=0.001; 6.0 vs 3.1 mm/s, P <0.001; 105.5 vs 69.6 mm/s, P <0.001; 10.1 vs 7.9 mm/s, P=0.011, respectively). The speed variance of the ß and urethral rotation angles were higher in the stress urinary incontinence group (844.8 vs 336.4, P <0.001; 347.6 vs 131.1, P <0.001, respectively). The combination of the average speed of the ß angle, maximum speed of the urethral rotation angle, and duration of the Valsalva maneuver demonstrated a strong diagnostic performance (area under the curve, 0.87). When 0.481*ß anglea + 0.013*URAm + 0.483*Dval = 7.405, the diagnostic sensitivity was 70% and specificity was 92%, highlighting the significant role of bladder neck motion in stress urinary incontinence, particularly changes in the speed of the ß and urethral rotation angles. CONCLUSIONS: A system utilizing deep learning can describe the motion of the bladder neck in women with stress urinary incontinence during the Valsalva maneuver, making it possible to visualize and quantify bladder neck motion on transperineal ultrasound. The speeds of the ß and urethral rotation angles and duration of the Valsalva maneuver were relatively reliable diagnostic parameters.

External light-dark cycle shapes gut microbiota through intrinsically photosensitive retinal ganglion cells.

Gut microbiota are involved in many physiological functions such as metabolism, brain development, and neurodegenerative diseases. Many microbes in the digestive tract do not maintain a constant level of their relative abundance but show daily oscillations under normal conditions. Recent evidence indicates that chronic jetlag, constant darkness, or deletion of the circadian core gene can alter the composition of gut microbiota and dampen the daily oscillation of gut microbes. However, the neuronal circuit responsible for modulating gut microbiota remained unclear. Using genetic mouse models and 16s rRNA metagenomic analysis, we find that light-dark cycle information transmitted by the intrinsically photosensitive retinal ganglion cells (ipRGCs) is essential for daily oscillations of gut microbes under temporal restricted high-fat diet conditions. Furthermore, aberrant light exposure such as dim light at night (dLAN) can alter the composition, relative abundance, and daily oscillations of gut microbiota. Together, our results indicate that external light-dark cycle information can modulate gut microbiota in the direction from the brain to the gut via the sensory system.

Further structural optimization and SAR study of sungsanpin derivatives as cell-invasion inhibitors.

Metastasis is one of the major causes of death in patients with cancer, and cell invasion plays a fundamental part in this process. Because of the absence of efficacious treatments, caring for these patients is challenging. Recently, we optimized the structure of the naturally occurring lasso peptide sungsanpin. We identified two peptides, octapeptide S3 and cyclic peptide S4, which inhibited invasion into A549 cells effectively. We undertook an alanine scan of S3 to explore the structure-activity relationship. The linear octapeptide S3-4 and cyclic peptide S4-1 exhibited improved inhibition of invasion into A549 cells. We modified S3-4 to obtain S3-4K, which displayed much higher inhibitory activity against invasion into A549 cells than S3-4. Of all peptides tested, S4-1 upregulated significantly mRNA of tissue inhibitor matrix metalloproteinase TIMP-1 and TIMP-2.

Factors Influencing Plasma Concentrations of Valproic Acid in Pediatric Patients With Epilepsy and the Clinical Significance of CYP2C9 Genotypes in Personalized Valproic Acid Therapy.

BACKGROUND: The aim of this study was to investigate the factors affecting plasma valproic acid (VPA) concentration in pediatric patients with epilepsy and the clinical significance of CYP2C9 gene polymorphisms in personalized dosing using therapeutic drug monitoring and pharmacogenetic testing. METHODS: The medical records of children with epilepsy who underwent therapeutic drug monitoring at our institution between July 2022 and July 2023 and met the inclusion criteria were reviewed. Statistical analysis was performed to determine whether age, sex, blood ammonia, liver function, kidney function, and other characteristics affected the concentration-to-dose ratio of VPA (CDRV) in these patients. To investigate the effect of CYP2C9 polymorphisms on CDRV, DNA samples were collected from patients and the CYP2C9 genotypes were identified using real-time quantitative PCR. RESULTS: The mean age of 208 pediatric patients with epilepsy was 5.50 ± 3.50 years. Among these patients, 182 had the CYP2C9 *1/*1 genotype, with a mean CDRV (mcg.kg/mL.mg) of 2.64 ± 1.46, 24 had the CYP2C9 *1/*3 genotype, with a mean CDRV of 3.28 ± 1.74, and 2 had the CYP2C9 *3/*3 genotype, with a mean CDRV of 6.46 ± 3.33. There were statistical differences among these 3 genotypes ( P < 0.05). The CDRV in these patients were significantly influenced by age, aspartate aminotransferase, total bilirubin, direct bilirubin, globulin, albumin/globulin ratio, prealbumin, creatinine, and CYP2C9 polymorphisms. In addition, multivariate linear regression analysis identified total bilirubin, direct bilirubin, and CYP2C9 polymorphisms as independent risk factors for high CDRV. CONCLUSIONS: Liver problems and mutations in the CYP2C9 gene increase VPA levels. This underscores the importance of considering these factors when prescribing VPA to children with epilepsy, thereby enhancing the safety and efficacy of the therapy.

Controlled Assembly of a Dawson-like Antimoniotungstate-Supported Trefoil-type Trimer with Good Proton Conductivity Performance.

With the excellent properties of POM in the field of proton conductivity, the preparation of POM-based proton-conductive materials has burst into life. Herein, an unprecedented Sb-templated all-inorganic trimer Na8H18.64[(SbW14O52)3(Sb2W6.12Ru5.88O18)]·85H2O (1), which is based on tetravacant Dawson-like [SbW14O52]17- blocks and exhibits a trefoil type with D3 symmetry, has been successfully designed and synthesized by the assembly of simple materials with a one-pot hydrothermal method under acidic conditions. Also, compound 1 is systematically characterized by single-crystal X-ray diffraction, PXRD, ESI-MS, IR spectroscopy, UV-vis, elemental analysis, and TGA. Crystal structure data analysis demonstrates that compound 1 is constructed by a hexagonal prismatic heterometallic {Sb2W6.12Ru5.88O18} core and three equivalent {SbW14} units bridged through µ2-O atoms in periphery. Subsequently, further property experiments show that compound 1 exhibits high proton conductivity with a conductivity value (σ) of 3.07 × 10-2 S cm-1 at 75 °C and 80% relative humidity (RH). The activation energy of compound 1 evaluated by the Arrhenius plots is 0.22 eV, which indicates that the Grotthuss mechanism is dominant during the process of proton transfer.

Design, synthesis, evaluation and optimization of novel azole analogues as potent antifungal agents.

In order to develop antifungal drugs, a series of novel azole analogues were designed and synthesized based on our previous work. Most of the target compounds had broad-spectrum antifungal activity, which showed excellent to moderate inhibitory activity against the tested strains, except A. fum 0504656. Among these, compounds B3, B7, B8, B11, B12 and E9 showed excellent activity against C. alb Y0109 and C. alb SC5314 (with the MIC80: 0.0156 ug/mL). In addition, compound B3 showed the best inhibitory activity against fluconazole-resistant strains C. alb 901 and C. alb 904, and had low toxicity against NIH/3T3 cells at the effective MIC range against fungi. Structure-activity relationship and docking studies of the derivatives suggest that the presence of the 2-fluoro-4-hydroxyphenyl and 1,2,3-triazole group enhance the antifungal activity of the compounds, which may be related to the interaction of the key groups with the amino acids surrounding the target enzyme.

Health-related quality of life in Chinese SLE patients: evidence from 1568 SLE patients and 2610 healthy controls.

OBJECTIVE: To investigate the effects of systemic lupus erythematosus (SLE) on health-related quality of life (HRQOL), the relationship between disease activity and HRQOL, and potential factors affecting HRQOL in Chinese SLE patients. METHODS: This study recruited 1568 patients and 2610 controls to explore the effects of SLE on HRQOL. The association between disease activity and HRQOL, and the influencing factors of HRQOL were determined in 1568 patients. Then, we prospectively followed 1096 patients to explore the association between reduced disease activity and improved HRQOL, and the influencing factors of improved HRQOL. The Short-Form 36 (SF-36) and SLE disease activity index (SLEDAI) were used to evaluate HRQOL and disease activity. RESULTS: Chinese SLE patients had lower HRQOL than controls in all domains (P < 0.001), especially in role-physical (RP) and role-emotional (RE). Compared with SLE patients from outside China, the HRQOL of Chinese patients appeared to be higher in mental component summary (MCS) but lower in RP and RE. SLEDAI was negatively correlated with HRQOL, which was validated using the results of a follow-up study, where SLEDAI reduction was positively associated with HRQOL improvements (P < 0.05). Furthermore, personality, life nervous and experiences of adverse life events may influence HRQOL and HRQOL improvements. CONCLUSION: SLE significantly affected the HRQOL of Chinese patients, especially in RP and RE. Disease activity was negatively correlated with HRQOL. We also found for the first time some factors affecting HRQOL, which can be regarded as the basis for improving the HRQOL of SLE patients.

Characteristics of the time processing of adults' strongest sustained attentional bias toward neutral infant faces.

Previous studies have shown that adults exhibit the strongest attentional bias toward neutral infant faces when viewing faces with different expressions at different attentional processing stages due to different stimulus presentation times. However, it is not clear how the characteristics of the temporal processing associated with the strongest effect change over time. Thus, we combined a free-viewing task with eye-tracking technology to measure adults' attentional bias toward infant and adult faces with happy, neutral, and sad expressions of the same face. The results of the analysis of the total time course indicated that the strongest effect occurred during the strategic processing stage. However, the results of the analysis of the split time course revealed that sad infant faces first elicited adults' attentional bias at 0 to 500 ms, whereas the strongest effect of attentional bias toward neutral infant faces was observed at 1000 to 3000 ms, peaking at 1500 to 2000 ms. In addition, women and men had no differences in their responses to different expressions. In summary, this study provides further evidence that adults' attentional bias toward infant faces across stages of attention processing is modulated by expressions. Specifically, during automatic processing adults' attentional bias was directed toward sad infant faces, followed by a shift to the processing of neutral infant faces during strategic processing, which ultimately resulted in the strongest effect. These findings highlight that this strongest effect is dynamic and associated with a specific time window in the strategic process.

Synergizing Steric Hindrance and Stacking Interactions to Facilitate the Controlled Assembly of Multiple 41 Metalla-Knots and Pseudo-Solomon Links.

In this work, a noncoplanar terphenyl served as building block to synthesize a novel 3,3'-substituted bipyridyl ligand (L1) which further reacted with binuclear half-sandwich units A/B, giving rise to two aesthetical 41 metalla-knots in high yields via coordination-driven self-assembly strategy. Furthermore, given the inherent compactness of the 41 metalla-knots, it creates favorable conditions for the emergence of steric repulsion. We focused on progressively introducing nitrogen atoms featuring lone pair electrons (LPEs) into ligand L1 to manipulate the balance of H···H/LPEs···LPEs steric repulsion during the assembly process, ultimately achieving controlled assembly from 41 metalla-knots to the pseudo-Solomon link and then to molecular tweezer-like assembly facilitated by stacking interactions. All the assemblies were well characterized by solution-state NMR techniques, ESI-TOF/MS, and single-crystal X-ray diffraction. The evolutionary process of topological architectures is equivalent to visualizing the synergistic effect of steric hindrance and stacking interactions on structural assembly, providing a new avenue for achieving the controlled synthesis of different topologies.

Functional polymorphisms of the TET1 gene increase the risk of neuroblastoma in Chinese children.

Common genetic mutations are absent in neuroblastoma, one of the most common childhood tumours. As a demethylase of 5-methylcytosine (m5C) modification, TET1 plays an important role in tumourigenesis and differentiation. However, the association between TET1 gene polymorphisms and susceptibility to neuroblastoma has not been reported. Three TET1 gene polymorphisms (rs16925541 A > G, rs3998860 G > A and rs12781492 A > C) in 402 Chinese patients with neuroblastoma and 473 cancer-free controls were assessed using TaqMan. Multivariate logistic regression analysis was used to evaluate the association between TET1 gene polymorphisms and susceptibility to neuroblastoma. The GTEx database was used to analyse the impact of these polymorphisms on peripheral gene expression. The relationship between gene expression and prognosis was analysed using Kaplan-Meier analysis with the R2 platform. We found that both rs3998860 G > A and rs12781492 A > C were significantly associated with increased neuroblastoma risk. Stratified analysis further showed that rs3998860 G > A and rs12781492 A > C significantly increased neuroblastoma risk in certain subgroups. In the combined risk genotype model, 1-3 risk genotypes significantly increased risk of neuroblastoma compared with the 0 risk genotype. rs3998860 G > A and rs12781492 A > C were significantly associated with increased STOX1 mRNA expression in adrenal and whole blood, and high expression of STOX1 mRNA in adrenal and whole blood was significantly associated with worse prognosis. In summary, TET1 gene polymorphisms are significantly associated with increased neuroblastoma risk; further research is required for the potential mechanism and therapeutic prospects in neuroblastoma.

Configuration-Specific Antibody for Bacterial Heptosylation: An Antiadhesion Therapeutic Strategy.

Alternative antibacterial therapies refractory to existing mechanisms of antibiotic resistance are urgently needed. One such attractive therapy is to inhibit bacterial adhesion and colonization. Ser O-heptosylation (Ser O-Hep) on autotransporters of Gram-negative bacteria is a novel glycosylation and has been proven to be essential for bacterial colonization. Herein, we chemically synthesized glycopeptides containing this atypical glycan structure and an absolute C6 configuration through the assembly of Ser O-Hep building blocks. Using glycopeptides as haptens, we generated first-in-class poly- and monoclonal antibodies, termed Anti-SerHep1a and Anti-SerHep1b, that stereoselectively recognize Ser O-heptosylation (d/l-glycero) with high specificity in vitro and in vivo. Importantly, these antibodies effectively blocked diffusely adhering Escherichia coli 2787 adhesion to HeLa cells and in mice in a dose- and Ser O-Hep-dependent manner. Together, these antibodies represent not only useful tools for the discovery of unknown serine O-heptosylated proteins bearing various C6 chiral centers but also a novel class of antiadhesion therapeutic agents for the treatment of bacterial infection.

Nanoarray Enabled Size-Dependent Isolation and Proteomics Profiling of Small Extracellular Vesicle Subpopulations toward Accurate Cancer Diagnosis and Prognosis.

Small extracellular vesicles (sEVs) have emerged as noninvasive biomarkers in liquid biopsy due to their significant function in pathology and physiology. However, the phenotypic heterogeneity of sEVs presents a significant challenge to their study and has significant implications for their applications in liquid biopsies. In this study, anodic aluminum oxide films with different pore sizes (AAO nanoarray) were introduced to enable size-based isolation and downstream proteomics profiling of sEV subpopulations. The adjustable pore size and abundant Al3+ on the framework of AAOs allowed size-dependent isolation of sEV subpopulations through nanoconfined effects and Lewis acid-base interaction between AAOs and sEVs. Benefiting from the strong concerted effect, the simple AAO nanoarray enabled specific isolation of three sEV subpopulations, termed "50", "90", and "150 nm" groups, from 10 µL of complex biological samples within 10 min with high capture efficiencies and purities. Moreover, the nanopores of AAOs also acted as nanoreactors for comprehensive proteomic profiling of the captured sEV subpopulations to reveal their heterogeneity. The AAO nanoarray was first investigated on sEVs from a cell culture medium, where sEV subpopulations could be clearly distinguished, and three traditional sEV-specific proteins (CD81, CD9, and FLOT1) could be identified by proteomic analysis. A total of 3946, 3951, and 3940 proteins were identified from 50, 90, and 150 nm sEV subpopulations, respectively, which is almost twice the number compared to those obtained from the conventional approach. The concept was further applied to complex real-case sample analysis from prostate cancer patients. Machine learning and gene ontology (GO) information analysis of the identified proteins indicate that different-sized sEV subpopulations contain unique protein cargos and have distinct cellular components and molecular functions. Further receiver operating characteristic curve (ROC) analysis of the top five differential proteins from the three sEV subpopulations demonstrated the high accuracy of the proposed approach toward prostate cancer diagnosis (AUC > 0.99). More importantly, several proteins involved in focal adhesion and antigen processing and presentation pathways were found to be upregulated in prostate cancer patients, which may serve as potential biomarkers of prostate cancer. These results suggest that the sEV subpopulation-based AAO nanoarray is of great value in facilitating the early diagnosis and prognosis of cancer and opens a new avenue for sEVs in liquid biopsy.