Enhancement of extraction efficiency and functional properties of chickpea protein isolate using pulsed electric field combined with ultrasound treatment.

Chickpea protein isolate (CPI) is a promising dietary protein with the advantages of low allergenicity, easy digestion and balanced composition of essential amino acids. However, due to the thick skin of chickpeas, the extraction of CPI is challenging, resulting in lower efficiency of the alkaline extraction-isoelectric precipitation (AE-IEP) method. Therefore, the present study investigated the effect of pulsed electric field combined with ultrasound (PEF-US) treatment on the extraction efficiency of CPI and the functional properties was characterized. Parameter optimization was carried out using response surface methodology (RSM), with the following optimized conditions: pulse duration of 87 s, electric field intensity of 0.9 kV/cm, ultrasonic time of 15 min, and ultrasonic power of 325 W. Under the optimized conditions, the yield of CPI after combined (PEF-US) treatment was 13.52 ± 0.13 %, which was a 47.28 % improvement over the AE-IEP method. This yield was better than that obtained with either individual PEF or US treatment. Additionally, the functional properties (solubility, emulsification, and foaming) of CPI were significantly enhanced compared to AE-IEP. However, the stability of emulsification and foaming did not show significant differences among the four methods. The PEF-US method efficiently extracts CPI with excellent functional properties, enabling the production of proteins as desired functional additives in the food industry.

Comparison of objective visual quality between SMILE and FS-LASIK in moderate-to-high myopia.

Purpose: This study aims to compare the changes in the corneal wavefront aberrations and the objective visual quality resulting from two types of eye surgery-small incision lenticule extraction (SMILE) and femtosecond laser-assisted in situ keratomileusis (FS-LASIK)-in patients with moderate-to-high myopia. Methods: A prospective analysis was performed on 98 eyes of 51 patients who underwent SMILE. Additionally, 88 eyes of 45 patients who underwent FS-LASIK were analyzed. All patients underwent ocular examination preoperatively and at 1 day, 1 week, 1 month, and 3 months postoperatively. Corneal aberrations and objective visual quality were measured using the Optical Quality Analysis System II (OQAS II) and Optical Path Difference Scan III (OPD-Scan III). Results: At postoperative 1 day and 1 week, there was a statistically significant difference in uncorrected distance visual acuity (UDVA) between SMILE and FS-LASIK (P < 0.05). Postoperative spherical (S), cylinder (C) and spherical equivalent refraction (SE) were similar between the two groups (P > 0.05). In both groups, the absolute magnitude of total higher-aberrations (tHOA), piston, vertical tilt, vertical coma, and spherical aberration (SA) increased after surgery compared to preoperative values (P < 0.05). There was no significant difference in Δhorizontal tHOA, Δhorizontal tilt, Δhorizontal coma, and Δhorizontal trefoil between the two groups (P > 0.05), and the FS-LASIK had higher Δvertical trefoil and ΔSA (P < 0.05) but lower Δpiston, Δvertical tilt, and Δvertical coma than the SMILE group (P < 0.05). There was a rise in objective scattering index (OSI) and a decline in both modulation transfer function (MTF) cutoff and Strehl ratio (SR) after surgery compared to preoperative values in both groups (P < 0.05). There was a statistically significant difference in the OSI at 1 day and 3 months between the two groups (P < 0.05). Postoperative MTF cutoff and SR were similar between the two groups (P > 0.05). Postoperative OSI was positively correlated with corneal tHOA (0.261 ≤ R ≤ 0.483, P < 0.05) and was negatively correlated with vertical tilt and vertical coma (-0.315 ≤ R ≤ -0.209, P < 0.05) in both groups. Conclusion: While both SMILE and FS-LASIK can effectively correct moderate-to-high myopia, there is an increase in corneal aberrations and a postoperative delay in objective visual quality. The cornea may require a longer recovery period in the SMILE. OPD-Scan III combined with OQAS II is a useful supplementary inspection for assessing the optical quality following refractive surgery.

Manganese-arginine nanozymes as oxidase mimics for smartphone-based intelligent detection of 4'-O-methylpyridoxine.

By self-assembly of MnCl2 and arginine under alkaline conditions, ultra-small MnArg nanoparticles were successfully constructed as oxidase (OXD) mimics for intelligent detection of the Ginkgo toxin 4'-O-methylpyridoxal (MPN). The obtained MnArg nanozymes possessed excellent OXD-like activity and thermal stability. Based on the inhibitory effect of MPN for the catalytic activity of MnArg, this system was utilized for the colorimetric sensing of MPN with a low limit of detection (LOD) of 2.16 µg mL-1. The detection  system exhibited good selectivity against other potential interferents. FTIR data showed that the presence of MPN binds with MnArg and shields the active sites, thereby interfering with the oxidase-like activity. Combined with a smartphone and the ColorMax software, this nanozyme-based intelligent detection platform could effectively detect MPN with a LOD of 2.1 µg mL-1. Our MnArg nanozyme-based system was applied to detect real ginkgo nut samples with recoveries of 92.4-108.7%, and the relative standard deviations were less than 0.7%. This work may promote the development of novel nanozymes and expand their applications in the field of food safety detection.

Unleash the Power of State Space Model for Whole Slide Image with Local Aware Scanning and Importance Resampling.

Whole slide image (WSI) analysis is gaining prominence within the medical imaging field. However, previous methods often fall short of efficiently processing entire WSIs due to their gigapixel size. Inspired by recent developments in state space models, this paper introduces a new Pathology Mamba (PAM) for more accurate and robust WSI analysis. PAM includes three carefully designed components to tackle the challenges of enormous image size, the utilization of local and hierarchical information, and the mismatch between the feature distributions of training and testing during WSI analysis. Specifically, we design a Bi-directional Mamba Encoder to process the extensive patches present in WSIs effectively and efficiently, which can handle large-scale pathological images while achieving high performance and accuracy. To further harness the local information and inherent hierarchical structure of WSI, we introduce a novel Local-aware Scanning module, which employs a local-aware mechanism alongside hierarchical scanning to adeptly capture both the local information and the overarching structure within WSIs. Moreover, to alleviate the patch feature distribution misalignment between training and testing, we propose a Test-time Importance Resampling module to conduct testing patch resampling to ensure consistency of feature distribution between the training and testing phases, and thus enhance model prediction. Extensive evaluation on nine WSI datasets with cancer subtyping and survival prediction tasks demonstrates that PAM outperforms current state-of-the-art methods and also its enhanced capability in modeling discriminative areas within WSIs. The source code is available at https://github.com/HKU-MedAI/PAM.

Effectiveness and safety of canakinumab in cryopyrin-associated periodic syndrome: a retrospective study in China.

OBJECTIVE: Cryopyrin-associated periodic syndrome (CAPS) is characterized by excessive IL-1ß release resulting in systemic and organ inflammation. As an anti-IL-1 agent, canakinumab has been approved with all CAPS phenotypes in USA and European countries. However, the use of canakinumab in CAPS in Chinese patients was rarely reported. In this study, we aimed to assess the effectiveness and safety of canakinumab in Chinese patients with CAPS. METHODS: Patients with CAPS treated with canakinumab were included. Clinical data were collected retrospectively from medical records. Treatment response was evaluated by CAPS disease activity score, C-reactive protein (CRP), and/or serum amyloid A (SAA) levels. Data was analyzed at canakinumab initiation, at months 1, 3, 6, 9, and 12, or the last follow-up. RESULTS: A total of 10 CAPS patients were included. 40% of patients were males, the median age at disease onset was 2.5 (2.5, 6) days and the median duration of follow-up while on canakinumab was 22.5 (8.5, 27.5) months. 80% (8/10) of CAPS patients presented with moderate-severe disease activity before the canakinumab treatment. 30% (3/10) of patients required canakinumab dose increase to control disease activity. After treatments, 60% (6/10) of CAPS patients achieved complete remission without relapse and the rest showed minimal disease activity. Clinical symptoms such as fever and rash were improved significantly in most patients (80%). Although abnormal imaging in brain MRI remained in over half of those patients, neurological manifestations were all relieved. 60% (6/10) of patients received prednisone before starting canakinumab therapy and five of them discontinued prednisone later. The most common adverse event was infection (40%). No serious adverse events occurred during the treatment of canakinumab. CONCLUSIONS: Canakinumab may be effective and tolerable for Chinese CAPS patients, helping to reduce the dosage of corticosteroids. However, additional trials on large samples are required to further evaluate its efficacy and safety in China.

The imaging manifestations of persistent truncus arteriosus in children.

Persistent truncus arteriosus (PTA) is a rare congenital heart malformation. A 4-year-old girl was diagnosed with type I PTA through echocardiography and confirmed by cardiac CTA.

A retrospective comparative study on the treatment of non-metastatic pancreatic cancer using high-intensity focused ultrasound versus radical surgery.

OBJECTIVE: To compare the efficacy and safety of high-intensity focused ultrasound (HIFU) and radical surgery for non-metastatic pancreatic cancer (PC). MATERIALS AND METHODS: We retrospectively analyzed 89 stage I/II/III PC patients who underwent HIFU (n = 43) or surgery (n = 46) at the Third Xiangya Hospital from January 2020 to December 2021. Pain relief, Karnofsky Performance Scale (KPS), overall survival (OS), treatment-related complications and risk factors for OS were assessed. RESULTS: There was no significant difference in the pain relief rate at 30 days post-treatment between the two groups. However, compared with the surgery group, the HIFU group showed significantly lower post-treatment VAS scores (p = 0.019). In the surgery group, the KPS at 30 days post-treatment was lower than pretreatment KPS (70 vs 80; p = 0.015). This relationship was reversed in the HIFU group (80 vs 70; p = 0.024). Median OS favored surgery over HIFU (23 vs 10 months; p < 0.001), with a higher 1-year OS rate (69.57% vs 32.6%; p < 0.001). However, there was no significant difference in OS between the two groups for stage III patients (p = 0.177). Complications rated ≥ grade III were 2.33% in the HIFU group and 32.6% in the surgery group. Multivariate analyses showed that age, KPS, and treatment methods were independent prognostic factors for OS. CONCLUSION: HIFU demonstrates advantages over surgery in terms of early KPS, VAS improvements, and safety for pancreatic cancer; however, long-term outcomes favor surgery. For III-stage disease, HIFU was noninferior to surgery in overall survival.

MPGAN: Multi Pareto Generative Adversarial Network for the denoising and quantitative analysis of low-dose PET images of human brain.

Positron emission tomography (PET) imaging is widely used in medical imaging for analyzing neurological disorders and related brain diseases. Usually, full-dose imaging for PET ensures image quality but raises concerns about potential health risks of radiation exposure. The contradiction between reducing radiation exposure and maintaining diagnostic performance can be effectively addressed by reconstructing low-dose PET (L-PET) images to the same high-quality as full-dose (F-PET). This paper introduces the Multi Pareto Generative Adversarial Network (MPGAN) to achieve 3D end-to-end denoising for the L-PET images of human brain. MPGAN consists of two key modules: the diffused multi-round cascade generator (GDmc) and the dynamic Pareto-efficient discriminator (DPed), both of which play a zero-sum game for n(n∈1,2,3) rounds to ensure the quality of synthesized F-PET images. The Pareto-efficient dynamic discrimination process is introduced in DPed to adaptively adjust the weights of sub-discriminators for improved discrimination output. We validated the performance of MPGAN using three datasets, including two independent datasets and one mixed dataset, and compared it with 12 recent competing models. Experimental results indicate that the proposed MPGAN provides an effective solution for 3D end-to-end denoising of L-PET images of the human brain, which meets clinical standards and achieves state-of-the-art performance on commonly used metrics.

Novel Dissymmetric Formal Quinoidal Molecules End-Capped by Dicyanomethylene and Triphenylphosphonium.

A number of quinoidal molecules with symmetric end-capping groups, particularly dicyanomethylene units, have been synthesized for organic optoelectronic materials. In comparison, dissymmetric quinoidal molecules, characterized by end-capping with different groups, are less explored. In this paper, we present the unexpected formation of new formal quinoidal molecules, which are end-capped with both dicyanomethylene and triphenylphosphonium moieties. The structures of these dissymmetric quinoidal molecules were firmly verified by single crystal structural analyses. On the basis of the control experiments and DFT calculations, we proposed the reaction mechanism for the formation of these dissymmetric quinoidal molecules. The respective zwitterionic forms should make contributions to the ground state structures of these quinoidal molecules based on the analysis of their bond lengths and aromaticity and Mayer Bond Orbital (MBO) calculation. This agrees well with the fact that negative solvatochromism was observed for these quinoidal molecules. Although these new quinoidal molecules are non-emissive both in solutions and crystalline states, they become emissive with quantum yields up to 51.4 % after elevating the solvent viscosity or dispersing them in a PMMA matrix. Interestingly, their emissions can also be switched on upon binding with certain proteins, in particular with human serum albumin.

A Perovskite Material Screening and Performance Study Based on Asymmetric Convolutional Blocks.

This study introduces an innovative method for identifying high-efficiency perovskite materials using an asymmetric convolution block (ACB). Our approach involves preprocessing extensive data on perovskite oxide materials and developing a precise predictive model. This system is designed to accurately predict key properties such as band gap and stability, thereby eliminating the reliance on traditional feature importance filtering. It exhibited outstanding performance, achieving an accuracy of 96.8% and a recall of 0.998 in classification tasks, and a coefficient of determination (R2) value of 0.993 with a mean squared error (MSE) of 0.004 in regression tasks. Notably, DyCoO3 and YVO3 were identified as promising candidates for photovoltaic applications due to their optimal band gaps. This efficient and precise method significantly advances the development of advanced materials for solar cells, providing a robust framework for rapid material screening.

Silver Nanocomposites with Enhanced Shelf-Life for Fruit and Vegetable Preservation: Mechanisms, Advances, and Prospects.

Reducing fruit and vegetable waste and maintaining quality has become challenging for everyone. Nanotechnology is a new and intriguing technology that is currently being implemented in fruit and vegetable preservation. Silver nanomaterials provide superior antibacterial qualities, biodegradability, and biocompatibility, which expands their potential applications in fruit and vegetable preservation. Silver nanomaterials include silver nanocomposites and Ag-MOF, of which silver nanocomposites are mainly composed of silver nanoparticles. Notably, not all kinds of silver nanoparticles utilized in the preservation of fruits and vegetables are thoroughly described. Therefore, the synthesis, mechanism of action, and advancements in research on silver nanocomposites for fruit and vegetable preservation were discussed in this study.

Effect of Multimodal Intervention in Individuals with Mild Cognitive Impairment: A Randomized Clinical Trial in Shanghai.

Background: Previous trials have indicated that multimodal training could improve cognitive functions and moods in individuals with mild cognitive impairment (MCI). However, evidence was mainly obtained from studies in high-income countries. Objective: This trial aims to investigate the efficacy, safety, and potential mechanism of a multimodal intervention on cognitive function in individuals with MCI living in a community. Methods: In this single-blind, randomized controlled trial, 120 participants with MCI were randomly assigned to either the intervention group or the control group. The intervention group received the multimodal intervention, while the control group received regular health education. Neuropsychological tests and magnetic resonance imaging (MRI) were conducted at baseline and after the 12-week intervention. Results: Fifty-nine and fifty-seven participants respectively in the intervention and control groups completed the trial. The intervention group shown improvements in primary outcome, Mini-Mental State Exam (MMSE) total score (mean difference -0.96, 95% CI [-1.58, -0.34], p = 0.003), and secondary outcomes: MMSE recall (-0.39, 95% CI [-0.71, -0.07], p = 0.019), MMSE language (-0.26, 95% CI [-0.44, -0.07], p = 0.007), Auditory Verbal Learning Test instantaneous memory (-3.30, 95% CI [-5.70, -0.89], p = 0.008), Digit Symbol Substitution Test total score (-2.91, 95% CI [-5.67, -0.15], p = 0.039), digit span forwards (-1.25, 95% CI [-1.93, -0.56], p < 0.001), and Digit Span Test (-1.33, 95% CI [-2.33, -0.34], p = 0.009) compared to the control group. Improvements were observed in structural and functional connectivity related to language, concentration, executive function, memory, and recall functioning via MRI in the intervention group. Conclusions: The multimodal intervention improved cognitive function in individuals with MCI in cognitive performance and neuroimaging.

Anisotropic Chitosan-nanocellulose/Zeolite imidazolate frameworks-8 aerogel for sustainable dye removal.

Assembling microscopic metal-organic frameworks into macroscopic polymeric scaffolds to develop highly renewable materials has been a promising yet challenging area of research. Herein, chitosan (CS) blended with nano-cellulose (NC) was unidirectionally transformed into an aerogel with oriented macropores and then biomineralized with zeolite imidazolate frameworks-8 (ZIF-8) to form a hierarchical structured chitosan-nanocellulose/zeolite imidazolate frameworks-8 (CS-NC-ZIF-8) hybrid aerogel. Incorporating ZIF-8 significantly increases the versatility and mechanical strength with a Young's modulus of 14.18 MPa of the CS-NC aerogel. The incorporation of ZIF-8 into the aerogel not only enhances its adsorption capacity for methylene blue, rhodamine B, acid fuchsin, and methyl orange, but also facilitates the generation of electrons from water that can be transferred to degrade > 90 % of malachite green within 90 min in each catalytic cycle, and this capability was maintained for at least 10 consecutive cycles. Remarkably, the hybrid aerogel was highly renewable after the adsorption of cationic dyes and catalytic removal of malachite green. With its facile production process, high removal efficiency, affordable and green nature, and excellent regeneration feasibility, the CS-NC-ZIF-8 aerogel stands as a promising solution for addressing challenges associated with dye-contaminated water treatment.

Sex-dependent nonlinear Granger connectivity patterns of brain aging in healthy population.

BACKGROUND: Brain aging is a complex process that involves functional alterations in multiple subnetworks and brain regions. However, most previous studies investigating aging-related functional connectivity (FC) changes using resting-state functional magnetic resonance images (rs-fMRIs) have primarily focused on the linear correlation between brain subnetworks, ignoring the nonlinear casual properties of fMRI signals. METHODS: We introduced the neural Granger causality technique to investigate the sex-dependent nonlinear Granger connectivity (NGC) during aging on a publicly available dataset of 227 healthy participants acquired cross-sectionally in Leipzig, Germany. RESULTS: Our findings indicate that brain aging may cause widespread declines in NGC at both regional and subnetwork scales. These findings exhibit high reproducibility across different network sparsities, demonstrating the efficacy of static and dynamic analysis strategies. Females exhibit greater heterogeneity and reduced stability in NGC compared to males during aging, especially the NGC between the visual network and other subnetworks. Besides, NGC strengths can well reflect the individual cognitive function, which may therefore work as a sensitive metric in cognition-related experiments for individual-scale or group-scale mechanism understanding. CONCLUSION: These findings indicate that NGC analysis is a potent tool for identifying sex-dependent brain aging patterns. Our results offer valuable perspectives that could substantially enhance the understanding of sex differences in neurological diseases in the future, especially in degenerative disorders.

The antagonistic role of an E3 ligase pair in regulating plant NLR-mediated autoimmunity and fungal pathogen resistance.

Plant immune homeostasis is achieved through a balanced immune activation and suppression, enabling effective defense while averting autoimmunity. In Arabidopsis, disrupting a mitogen-activated protein (MAP) kinase cascade triggers nucleotide-binding leucine-rich-repeat (NLR) SUPPRESSOR OF mkk1/2 2 (SUMM2)-mediated autoimmunity. Through an RNAi screen, we identify PUB5, a putative plant U-box E3 ligase, as a critical regulator of SUMM2-mediated autoimmunity. In contrast to typical E3 ligases, PUB5 stabilizes CRCK3, a calmodulin-binding receptor-like cytoplasmic kinase involved in SUMM2 activation. A closely related E3 ligase, PUB44, functions oppositely with PUB5 to degrade CRCK3 through monoubiquitylation and internalization. Furthermore, CRCK3, highly expressed in roots and conserved across plant species, confers resistance to Fusarium oxysporum, a devastating soil-borne fungal pathogen, in both Arabidopsis and cotton. These findings demonstrate the antagonistic role of an E3 ligase pair in fine-tuning kinase proteostasis for the regulation of NLR-mediated autoimmunity and highlight the function of autoimmune activators in governing plant root immunity against fungal pathogens.

Ginkgo biloba leaf polysaccharide-stabled selenium nanozyme as an efficient glutathione peroxidase mimic for the preservation of bananas and cherry tomatoes.

To develop functional, sustainable and eco-friendly active packaging materials as alternatives to plastic films, we successfully prepared Ginkgo biloba leaf polysaccharide-stabilized selenium nanomaterials (Se-GBLP). Se-GBLP with glutathione peroxidase-like activity could efficiently remove harmful reactive oxygen species. As a functional additive, Se-GBLP was incorporated into degradable chitosan (CS) to fabricate CS/Se-GBLP films. The addition of Se-GBLP improved the mechanical properties, UV-visible light barrier performance, water vapor permeability, and antioxidant activity of the films. Preservation experiments demonstrated CS/Se-GBLP film could maintain quality and prolong the storage time of bananas and cherry tomatoes. It was the first time to use selenium-based nanozyme for fruit preservation. This work offered a cost-effective solution to reduce post-harvest losses, increasing sustainability and profitability. Future research should focus on more factors affecting freshness such as variety, maturity, harvest and storage conditions to improve preservation, as well as on the material's safety concern and environmental impact.

Recent advance for animal-derived polysaccharides in nanomaterials.

Inspired by the structure characteristics of natural products, the size and morphology of particles are carefully controlled using a bottom-up approach to construct nanomaterials with specific spatial unit distribution. Animal polysaccharide nanomaterials, such as chitosan and chondroitin sulfate nanomaterials, exhibit excellent biocompatibility, degradability, customizable surface properties, and novel physical and chemical properties. These nanomaterials hold great potential for development in achieving a sustainable bio-economy. This paper provides a summary of the latest research results on the preparation of nanomaterials from animal polysaccharides. The mechanism for preparing nanomaterials through the bottom-up method from different sources of animal polysaccharides is introduced. Furthermore, this paper discusses the potential hazards posed by industrial applications to the environment and human health, as well as the challenges and future prospects associated with using animal polysaccharides in nanomaterials.

Electronic symptom monitoring after lung cancer surgery: establishing a core set of patient-reported outcomes for surgical oncology care in a longitudinal cohort study.

BACKGROUND: Electronic symptom monitoring via patient-reported outcome in surgical oncology is limited owing to lengthy instruments and non-specific items in common patient-reported outcome instruments. To establish electronic symptom monitoring through a clinically relevant and fit-for-purpose core set of patient-reported outcome in patients undergoing lung cancer surgery. MATERIALS AND METHODS: One qualitative (Cohort 1) and two prospective studies (Cohorts 2 and 3) were conducted between 2018 and 2023. Patients undergoing lung cancer surgery were recruited. Items of symptoms and daily functioning were generated through extensive interviews in Cohort 1 and incorporated into a smartphone-based platform to establish the electronic Perioperative Symptom Assessment for Lung surgery (ePSA-Lung). This tool was finalized and validated in Cohort 2. Patients in Cohort 3 were longitudinally monitored for the first year post-surgery using the validated ePSA-Lung. RESULTS: In total, 1,037 patients scheduled for lung cancer surgery were recruited. The 11-item draft PSA-Lung was generated based on qualitative interview with 39 patients and input from a Delphi study involving 42 experts. A 9-item ePSA-Lung was finalized by assessing 223 patients in the validation cohort; the results supported the instrument's understandability, reliability, sensitivity, and surgical specificity. In Cohort 3 (n=775), compliance ranged from 63.21% to 84.76% during the one-year follow-up after discharge. Coughing, shortness of breath, and disturbed sleep were the most severe symptoms after discharge. Longitudinally, patients who underwent single-port video-assisted thoracic surgery had a lower symptom burden than those who underwent multi-port video-assisted thoracic surgery or thoracotomy (all symptoms, P<0.001). CONCLUSION: The ePSA-Lung is valid, concise, and clinically applicable as it supports electronic symptom monitoring in surgical oncology care. The need for long-term extensive care was identified for patients after discharge, even in early-stage cancer with potential curative treatment.

[Preparation of peptide-functionalized affinity materials for the highly specific capture and analysis of mitochondria].

Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders. The specific isolation and purification of intact, high-purity, and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks. In this study, a mitochondrial penetrating peptide (MPP) with the sequence FrFKFrFK(Ac) was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material. The multiple aromatic phenylalanine (F) residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane, whereas the basic residues (D-arginine and lysine) render the MPP surface positively charged, thereby promoting the binding of negatively charged mitochondria. After the derivatization of the N terminal of MPP with an oligoglycine spacer, the peptide ligands were conjugated to matrix beads (MB) with surface aldehyde functional groups. Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads. The generated Schiff bases were reduced, affording stable covalent bonds. The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography (HPLC), zeta potential assay, and scanning electron microscopy (SEM). The immobilization efficiency of the peptide ligands was 1.47 µmol/g, and the surface potential of MB@MPP was 11 mV. MB@MPP was used for the direct isolation of mitochondria after cell homogenization. As observed by SEM, mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface. Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes, the specific binding and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions. Western blot assays were employed to characterize the protein markers of the mitochondria (citrate synthase (CS) and voltage-dependent anion channel protein (VDAC)) and cytoplasmic protein (vinculin), and examine the integrity and purity of the captured mitochondria. The results showed that the lysates released from MB@MPP had high CS and VDAC contents. By contrast, vinculin, which is highly abundant in whole-cell lysates, was barely detected in the lysates from MB@MPP. These results suggest that MB@MPP isolates mitochondria with high affinity, specificity, and antifouling ability by using the targeting peptide as the capture handle. A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity. Given the superior separation performance of MB@MPP, the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to detect tryptophan (Trp) and riboflavin in the mitochondria. Quantification was performed in multiple-reaction monitoring (MRM) mode. Owing to the high purity of the mitochondria, the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg, respectively. The metabolic response of mitochondria to external stimuli was further examined using acadesine, an adenosine 5'-monophosphate (AMP)-activated protein kinase activator with a wide range of metabolic effects, to treat cells. After cell homogenization, MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment, followed by LC-MS/MS analysis. The quantification results demonstrated that acadesine induced a 14% upregulation of Trp content in the mitochondria. By contrast, the riboflavin content decreased to 0.48 nmol/mg, which is 72% of that in untreated mitochondria. The changes in Trp and riboflavin contents could influence their metabolic pathways and, thus, the levels of their metabolites, such as nicotinamide adenine dinucleotide, flavin mononucleotide, and flavin adenine dinucleotide, which are essential coenzymes in mitochondria. Peptide-functionalized affinity microbeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria, and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.

A broad neutralizing nanobody against SARS-CoV-2 engineered from an approved drug.

SARS-CoV-2 infection is initiated by Spike glycoprotein binding to the human angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain. Blocking this interaction has been proven to be an effective approach to inhibit virus infection. Here we report the discovery of a neutralizing nanobody named VHH60, which was directly produced from an engineering nanobody library based on a commercialized nanobody within a very short period. VHH60 competes with human ACE2 to bind the receptor binding domain of the Spike protein at S351, S470-471and S493-494 as determined by structural analysis, with an affinity of 2.56 nM. It inhibits infections of both ancestral SARS-CoV-2 strain and pseudotyped viruses harboring SARS-CoV-2 wildtype, key mutations or variants at the nanomolar level. Furthermore, VHH60 suppressed SARS-CoV-2 infection and propagation 50-fold better and protected mice from death for twice as long as the control group after SARS-CoV-2 nasal infections in vivo. Therefore, VHH60 is not only a powerful nanobody with a promising profile for disease control but also provides evidence for a highly effective and rapid approach to generating therapeutic nanobodies.