Using an ER-specific optogenetic mechanostimulator to understand the mechanosensitivity of the endoplasmic reticulum.

The ability of cells to perceive and respond to mechanical cues is essential for numerous biological activities. Emerging evidence indicates important contributions of organelles to cellular mechanosensitivity and mechanotransduction. However, whether and how the endoplasmic reticulum (ER) senses and reacts to mechanical forces remains elusive. To fill the knowledge gap, after developing a light-inducible ER-specific mechanostimulator (LIMER), we identify that mechanostimulation of ER elicits a transient, rapid efflux of Ca2+ from ER in monkey kidney COS-7 cells, which is dependent on the cation channels transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and polycystin-2 (PKD2) in an additive manner. This ER Ca2+ release can be repeatedly stimulated and tuned by varying the intensity and duration of force application. Moreover, ER-specific mechanostimulation inhibits ER-to-Golgi trafficking. Sustained mechanostimuli increase the levels of binding-immunoglobulin protein (BiP) expression and phosphorylated eIF2α, two markers for ER stress. Our results provide direct evidence for ER mechanosensitivity and tight mechanoregulation of ER functions, placing ER as an important player on the intricate map of cellular mechanotransduction.

Genetic-algorithm-based waveguide display system with a multiplexed volume holographic grating.

Most of the current holographic waveguide display systems are designed based on the center beam. When the incident beam consists of rays with different angles, the field of view and optical efficiency would greatly reduce. The heavy angular dependence of the volume holographic grating (VHG) and the back-coupling loss are two main reasons. This paper proposes a design method of the waveguide display system with multiplexed VHG, which is based on a genetic algorithm to optimize and calculate the parameters both of the VHG and the waveguide. The simulation results show that the diagonal field of view of the holographic waveguide system is increased to 28°, and its optical efficiency is improved by 30%. The design method of the waveguide system with the multiplexed grating proposed in this paper can effectively expand the field of view and improve the optical efficiency.

Reproductive toxic effects of chronic exposure to bisphenol A and its analogues in marine medaka (Oryzias melastigma).

As awareness of BPA's health risks has increased, many countries and regions have implemented strict controls on its use. Consequently, bisphenol analogues like BPF and BPAF are being increasingly used as substitutes. However, these compounds are also becoming increasingly prevalent in the environment due to production, use and disposal processes. The oceans act as a repository for various pollutants, and recent studies have revealed the extensive presence of bisphenols (BPs, including BPA, BPF, BPAF, etc.) in the marine environment, posing numerous health hazards to marine wildlife. Nevertheless, the reproductive toxicity of these chemicals on marine fish is not comprehensively comprehended yet. Thus, the histological features of the gonads and the gene expression profiles of HPG (Hypothalamic-Pituitary-Gonadal) axis-related genes in marine medaka (Oryzias melastigma) were studied after exposure to single and combined BPs for 70 days. The effects of each exposure group on spawning, embryo fertilization, and hatching in marine medaka were also assessed. Furthermore, the impacts of each exposure group on the genes related to methylation in the F2 and F3 generations were consistently investigated. BPs exposure was found to cause follicular atresia, irregular oocytes, and empty follicles in the ovary; but no significant lesions in the testis were observed. The expression of several HPG axis genes, including cyp19b, 17ßhsd, 3ßhsd, and fshr, resulted in significant changes compared to the control group. The quantity of eggs laid and fertilization rate decreased in all groups treated with BPs, with the BPAF-treated group showing a notable reduction in the number of eggs laid. Additionally, the hatching rate showed a more significant decline in the BPF-treated group. The analysis of methylated genes in the offspring of bisphenol-treated groups revealed significant changes in the expression of genes including amh, dnmt1, dnmt3ab, mbd2, and mecp2, indicating a potential transgenerational impact of bisphenols on phenotype through epigenetic modifications. Overall, the potential detrimental impact of bisphenol on the reproduction of marine medaka emphasizes the need for caution in considering the use of BPAF and BPF as substitutes.

A high-quality chromosome-level genome assembly of the Chinese medaka Oryzias sinensis.

Oryzias sinensis, also known as Chinese medaka or Chinese ricefish, is a commonly used animal model for aquatic environmental assessment in the wild as well as gene function validation or toxicology research in the lab. Here, a high-quality chromosome-level genome assembly of O. sinensis was generated using single-tube long fragment read (stLFR) reads, Nanopore long-reads, and Hi-C sequencing data. The genome is 796.58 Mb, and a total of 712.17 Mb of the assembled sequences were anchored to 23 pseudo-chromosomes. A final set of 22,461 genes were annotated, with 98.67% being functionally annotated. The Benchmarking Universal Single-Copy Orthologs (BUSCO) benchmark of genome assembly and gene annotation reached 95.1% (93.3% single-copy) and 94.6% (91.7% single-copy), respectively. Furthermore, we also use ATAC-seq to uncover chromosome transposase-accessibility as well as related genome area function enrichment for Oryzias sinensis. This study offers a new improved foundation for future genomics research in Chinese medaka.

Toxic effects of environmental concentration Bisphenol AF exposure on the survival, growth and reproduction of adult male Oryzias curvinotus.

Bisphenol AF (BPAF) is a novel environmental endocrine disruptor, and is widely detected in the aquatic environment, which is a potential threat to the health of fish. In this study, male Oryzias curvinotus were exposed to environmental concentrations (0.93 and 9.33 µg/L) of BPAF for 21 days. The effects of BPAF on survival, growth, reproduction, liver and testis histology, and gene transcriptional profiles of O. curvinotus were investigated. The results showed that the survival rate of male O. curvinotus slight decrease with increasing BPAF concentration, and there was no significant effect on body length, body weight, and K-factor. BPAF (9.33 µg/L) caused significant changes in testicular structure and reduced spermatid count in O. curvinotus. Changes in transcript levels of some antioxidant-related genes in gills and liver following BPAF exposure, imply an effect of BPAF on the immune system. After BPAF exposure, chgs and vtgs were up-regulated, validating the estrogenic effect of BPAF. In the hypothalamic - pituitary - gonadal axis (HPG) results, erα, erγ and cyp19a1b were all up-regulated in the brain, and the 0.93 µg/L BPAF group was more up-regulated than the 9.33 µg/L BPAF group. In testis, BPAF significantly up-regulated the mRNA expression level of cyp17a1 and cyp11b, while significantly down-regulated mRNA expression level of cyp11a, and cyp19a1 was significantly down-regulated only in the 0.93 µg/L BPAF group. In conclusion, environmental levels of BPAF have adverse effects on the survival and reproduction of O. curvinotus, and the potential toxic effects of environmental levels of BPAF cannot be ignored.

High-Performance Gel-Free and Label-Free Size Fractionation of Extracellular Vesicles with Two-Dimensional Electrophoresis in a Microfluidic Artificial Sieve.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Toxic Effects of Bisphenol AF Exposure on the Reproduction and Liver of Female Marine Medaka (Oryzias melastigma).

In recent years, bisphenol AF (BPAF) in aquatic environments has drawn attention to its ecological risks. This study aims to investigate the toxic effects of BPAF (188.33 µg/L) exposure for 30 days on female marine medaka (Oryzias melastigma). On the 10th and 30th day of exposure, the toxicity was evaluated using histological analysis of the liver and ovaries and the transcription levels of genes related to the antioxidant system, immune system, and hypothalamic-pituitary-gonadal (HPG) axis. Findings revealed that (1) BPAF exposure caused vacuolation, karyopyknosis and karyolysis in the liver of marine medaka, and the toxic impact augmented with duration; (2) exposure to BPAF for 10 days facilitated the growth and maturation of primary ova, and this exposure had a comparatively inhibitory effect after 30 days; (3) exposure to BPAF resulted in a biphasic regulation of the transcriptional abundance of genes involved in antioxidant and inflammatory response (e.g., il-8, cat), with an initial up-regulation followed by down-regulation. Additionally, it disrupted the transcriptional pattern of HPG axis-related genes (e.g., 3ßhsd, arα). In conclusion, 188.33 µg/L BPAF can alter the expression levels of functionally related genes, impair the structural integrity of marine organisms, and pose a threat to their overall health.

Coordination of RAB-8 and RAB-11 during unconventional protein secretion.

Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C. elegans-polarized intestine epithelia, we recently have revealed that the small GTPase RAB-8/Rab8 serves as an important player in the process. Nonetheless, its function and the relevant UcPS itinerary remain poorly understood. Here, we show that deregulated RAB-8 activity resulted in impaired apical UcPS, which increased sensitivity to infection and environmental stress. We also identified the SNARE VTI-1/Vti1a/b as a new RAB-8-interacting factor involved in the apical UcPS. Besides, RAB-11/Rab11 was capable of recruiting RABI-8/Rabin8 to reduce the guanine nucleotide exchange activity of SMGL-1/GEF toward RAB-8, indicating the necessity of a finely tuned RAB-8/RAB-11 network. Populations of RAB-8- and RAB-11-positive endosomal structures containing the apical UcPS cargo moved toward the apical side. In the absence of RAB-11 or its effectors, the cargo was retained in RAB-8- and RAB-11-positive endosomes, respectively, suggesting that these endosomes are utilized as intermediate carriers for the UcPS.

TMED10 mediates the trafficking of insulin-like growth factor 2 along the secretory pathway for myoblast differentiation.

The insulin-like growth factor 2 (IGF2) plays critical roles in cell proliferation, migration, differentiation, and survival. Despite its importance, the molecular mechanisms mediating the trafficking of IGF2 along the secretory pathway remain unclear. Here, we utilized a Retention Using Selective Hook system to analyze molecular mechanisms that regulate the secretion of IGF2. We found that a type I transmembrane protein, TMED10, is essential for the secretion of IGF2 and for differentiation of mouse myoblast C2C12 cells. Further analyses indicate that the residues 112-140 in IGF2 are important for the secretion of IGF2 and these residues directly interact with the GOLD domain of TMED10. We then reconstituted the release of IGF2 into COPII vesicles. This assay suggests that TMED10 mediates the packaging of IGF2 into COPII vesicles to be efficiently delivered to the Golgi. Moreover, TMED10 also mediates ER export of TGN-localized cargo receptor, sortilin, which subsequently mediates TGN export of IGF2. These analyses indicate that TMED10 is critical for IGF2 secretion by directly regulating ER export and indirectly regulating TGN export of IGF2, providing insights into trafficking of IGF2 for myoblast differentiation.

Zoledronic Acid Accelerates Bone Healing in Carpal Navicular Fracture via Silencing Long Non-coding RNA Growth Arrest Specificity 5 to Modulate MicroRNA-29a-3p Expression.

Carpal navicular fractures are the most common carpal fractures. This study intends to explore the specific mechanism of Zoledronic Acid (ZA) in carpal navicular fracture healing via long non-coding RNA (lncRNA) growth arrest specificity 5 (GAS5) to mediate microRNA (miR)-29a-3p. A fractured rat model was constructed. Two weeks later, a subcutaneous injection of systemic ZA was implemented, and an injection of plasmid vectors interfered with GAS5 or miR-29a-3p expression was performed on the fracture site. Osteocalcin (OCN) and bone morphogenetic protein-2 (BMP-2) were determined, as well as serum levels of alkaline phosphatase (ALP), osteopontin (OPN) and osteoprotegerin (OPG) and bone mineral density. MC3T3-E1 cells were transfected with plasmid vectors interfering with GAS5 or miR-29a-3p, and cell proliferation and apoptosis were analyzed. GAS5 and miR-29a-3p expression in fractured rats was tested, together with their binding relationship. ZA promoted OCN and BMP-2 expression, increased bone mineral density and serum levels of ALP, OPN and OPG in fractured rats. GAS5 was upregulated and miR-29a-3p was down-regulated in fractured rats. Downregulation of GAS5 or upregulation of miR-29a-3p further promoted bone healing in fractured rats. GAS5 targets miR-29a-3p, and down-regulation of miR-29a-3p can reverse the effect of down-regulation of GAS5 on bone healing in fractured rats. ZA promoted the proliferation of MC3T3-E1 cells and inhibited apoptosis by regulating the GAS5/miR-29a-3p axis. ZA regulates miR-29a-3p expression by down-regulating GAS5 to promote carpal navicular fracture healing, promote MC3T3-E1 cell proliferation, and inhibit cell apoptosis.

The first high-quality chromosome-level genome of Eretmochelys imbricata using HiFi and Hi-C data.

Eretmochelys imbricata, a critically endangered sea turtle inhabiting tropical oceans and protected across the world, had an unknown genome sequence until now. In this study, we used HiFi reads and Hi-C technology to assemble a high-quality, chromosome-level genome of E. imbricata. The genome size was 2,138.26 Mb, with contig N50 length of 123.49 Mb and scaffold N50 of 137.21 Mb. Approximately 97.52% of the genome sequence was anchored to 28 chromosomes. A total of 20,206 protein-coding genes were predicted. We also analyzed the evolutionary relationships, gene family expansions, and positive selection of E. imbricata. Our results revealed that E. imbricata diverged from Chelonia mydas 38 million years ago and had enriched olfactory receptors and aging-related genes. Our genome will be useful for studying E. imbricata and its conservation.

The First High-Quality Chromosome-Level Genome of the Lutjanus erythropterus (Bloch, 1790) Using Single-Tube Long Fragment Reads and Hi-C Technologies.

Lutjanus erythropterus (Bloch, 1790), a Perciformes from the Lutjanidae family, is a commercially important species because of its taste and abundance. Despite the increase in genome resources in recent years, few genome assemblies are available within this fish family for comparative and functional studies. In this study, we determined the chromosomal genome of Crimson snapper using high-throughput Single-Tube Long Fragment Reads sequencing technology and Hi-C data. The final assembly size was 973.04 Mb with contig and scaffold N50 values of 1.51 and 40.65 Mb, respectively. We successfully scaffolded 95.84% of the genome sequence onto 24 chromosomes ranging in length from 19.37 to 49.48 Mb. A total of 22,663 genes and 13,877 gene families were identified in the genome, with 29 gene families being L. erythropterus-specific. A phylogenetic analysis using single-copy gene families showed that L. erythropterus and Larimichthys crocea had the closest genetic relationship with a divergence time of ∼47.7 Ma. This new genomic resource will facilitate comparative genomic studies as well as genetic breeding programs for L. erythropterus.

Intestinal Villi-Inspired Mathematically Base-Layer Engineered Microneedles (IMBEMs) for Effective Molecular Exchange during Biomarker Enrichment and Drug Deposition in Diversified Mucosa.

The mucosa-interfacing systems based on bioinspired engineering design for sampling/drug delivery have manifested crucial potential for the monitoring of infectious diseases and the treatment of mucosa-related diseases. However, their efficiency and validity are severely restricted by limited contact area for molecular transfer and dissatisfactory capture/detachment capability. Herein, inspired by the multilayer villus structure of the small intestine that enables high nutrient absorption, a trigonometric function-based periodic pattern was fabricated and integrated on the base layer of the microneedle patch, exhibiting a desirable synergistic effect with needle tips for deep sample enrichment and promising molecular transfer, significantly improving the device-mucosa bidirectional interaction. Moreover, mathematical modeling and finite element analysis were adopted to visualize and quantify the microcosmic molecular transmission process, guiding parameter optimization in actual situation. Encouragingly, these intestinal villi-inspired mathematically base-layer engineered microneedles (IMBEMs) have demonstrated distinguished applicability among mucosa tissue with varying surface curvatures, tissue toughness, and local environments, and simultaneously, have gained favorable support from healthy volunteers receiving preliminary test of IMBEMs patches. Overall, validated by numerous in vitro and in vivo tests, the IMBEMs were confirmed to act as a promising candidate to facilitate mucosa-based sampling and topical drug delivery, indicating highly clinical translation potential.

Effects of bisphenols on lipid metabolism and neuro-cardiovascular toxicity in marine medaka larvae.

Bisphenols are environmental endocrine disruptors that have detrimental effects on aquatic organisms. Using marine medaka larvae, this study explored the effects of bisphenol compounds [bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF)] on the early growth and development of aquatic organisms. Marine medaka larvae were exposed to bisphenol compounds at concentrations of 0.05, 0.5, and 5 µM for 72 h, and changes in heartbeat rate, behavior, hormone levels, and gene expression were determined. Bisphenols were shown to have a toxic effect on the cardiovascular system of larvae and can cause neurotoxicity and endocrine disruption, such as changes to thyroid-related hormones. Functional enrichment showed that bisphenols mainly affect lipid metabolism and cardiac muscle contraction of larvae, which implied that the main toxic effects of bisphenols on marine medaka larvae targeted the liver and heart. This study provides a theoretical foundation for evaluating the toxicological effects of bisphenols on the early development of aquatic organisms.

Helicobacter pylori plus N-Methyl-N'-nitro-N-nitrosoguanidine: DNA damage and repair: Malignant transformation of human esophageal epithelial cells.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), found in pickled foods and in chlorinated water, has been used to induce malignant transformation and gastrointestinal cancer in rats. Helicobacter pylori (HP) is implicated in human gastric cancer and possibly also in esophageal cancer. These two agents - one chemical and the other biological - might act together to induce esophageal cancer. In this study, human esophageal epithelial cells (HEECs) were divided into four groups: HP, MNNG, HP + MNNG, and control. The HP-to-HEEC ratio was 100:1. Cells were exposed for 6 h and then passaged until malignant transformation. HEEC at early, intermediate, and late stages of malignant transformation were used for proliferation, cell-cycle, and invasion assays. The alkaline comet assay was performed and expression of proteins, including γ-H2AX and PAXX, was studied by western blotting, to explore DNA damage and repair processes. Measurements of cell morphology, soft-agar clone formation, and invasiveness, and a nude mouse xenograft model, were used to examine malignancy. The effect of HP was stronger than that of MNNG. The combination HP + MNNG exerted a stronger malignant transformation effect than either HP or MNNG alone. Mechanisms of this combined carcinogenesis may include promotion of cell proliferation, perturbation of the cell cycle, promotion of invasiveness, DNA double-strand break induction, or PAXX inhibition.

Single-frame deep-learning super-resolution microscopy for intracellular dynamics imaging.

Single-molecule localization microscopy (SMLM) can be used to resolve subcellular structures and achieve a tenfold improvement in spatial resolution compared to that obtained by conventional fluorescence microscopy. However, the separation of single-molecule fluorescence events that requires thousands of frames dramatically increases the image acquisition time and phototoxicity, impeding the observation of instantaneous intracellular dynamics. Here we develop a deep-learning based single-frame super-resolution microscopy (SFSRM) method which utilizes a subpixel edge map and a multicomponent optimization strategy to guide the neural network to reconstruct a super-resolution image from a single frame of a diffraction-limited image. Under a tolerable signal density and an affordable signal-to-noise ratio, SFSRM enables high-fidelity live-cell imaging with spatiotemporal resolutions of 30 nm and 10 ms, allowing for prolonged monitoring of subcellular dynamics such as interplays between mitochondria and endoplasmic reticulum, the vesicle transport along microtubules, and the endosome fusion and fission. Moreover, its adaptability to different microscopes and spectra makes it a useful tool for various imaging systems.

[Serological characteristics of ABO blood group and molecular genetic analysis of a Chinese pedigree with cisAB09 subtype].

OBJECTIVE: To explore the serological characteristics of ABO blood group and molecular genetic mechanism for a Chinese pedigree with cisAB09 subtype. METHODS: A pedigree undergoing ABO blood group examination at the Department of Transfusion, Zhongshan Hospital Affiliated to Xiamen University on February 2, 2022 was selected as the study subjects. Serological assay was carried out to determine the ABO blood group of the proband and his family members. Activities of A and B glycosyltransferases in the plasma of the proband and his mother were measured with an enzymatic assay. Expression of A and B antigens on the red blood cells of the proband was analyzed by flow cytometry. Peripheral blood samples of the proband and his family members were collected. Following extraction of genomic DNA, exons 1 to 7 of the ABO gene and their flanking introns were sequenced, and Sanger sequencing of exon 7 was carried out for the proband, his elder daughter and mother. RESULTS: The results of serological assay suggested that the proband and his elder daughter and mother had an A2B phenotype, whilst his wife and younger daughter had an O phenotype. Measurement of plasma A and B glycosyltransferase activity suggested that the titers of B-glycosyltransferase activity were 32 and 256 for the proband and his mother, which were respectively below and above that of A1B phenotype-positive controls (128). Flow cytometry analysis showed that the expression of A antigen on the red blood cell surface of the proband has decreased, whilst the expression of B antigen was normal. Genetic sequencing confirmed that, in addition to an ABO*B.01 allele, the proband, his elder daughter and mother have harbored a c.796A>G variant in exon 7, which has resulted in substitution of the methionine at 266th position of the B-glycosyltransferase by valine and conformed to the characteristics of ABO*cisAB.09 allele. The genotypes of the proband and his elder daughter were determined as ABO*cisAB.09/ABO*O.01.01, his mother was ABO*cisAB.09/ABO*B.01, and his wife and younger daughter were ABO*O.01.01/ABO*O.01.01. CONCLUSION: The c.796A>G variant of the ABO*B.01 allele has resulted in an amino acid substitution p.Met266Val, which probably underlay the cisAB09 subtype. The ABO*cisA B.09 allele encodes a special glycosyltransferase which can synthesize normal level of B antigen and low level of A antigen on the red blood cells.

Molecular Basis of KAT2A Selecting Acyl-CoA Cofactors for Histone Modifications.

Emerging discoveries about undocumented acyltransferase activities of known histone acetyltransferases (HATs) advance our understandings in the regulation of histone modifications. However, the molecular basis of HATs selecting acyl coenzyme A (acyl-CoA) substrates for histone modification is less known. We here report that lysine acetyltransferase 2A (KAT2A) as an illustrative instance of HATs can selectively utilize acetyl-CoA, propionyl-CoA, butyryl-CoA, and succinyl-CoA to directly deposit 18 histone acylation hallmarks in nucleosome. By analyzing the co-crystal structures of the catalytic domain of KAT2A in complex with acetyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-CoA, succinyl-CoA, and glutaryl-CoA, we conclude that the alternative substrate-binding pocket of KAT2A and the length and electrostatic features of the acyl chain cooperatively determine the selection of the acyl-CoA substrates by KAT2A. This study reveals the molecular basis underlying the pluripotency of HATs that selectively install acylation hallmarks in nucleosomes, which might serve as instrumental mechanism to precisely regulate histone acylation profiles in cells.

Continuous-flow label-free size fractionation of extracellular vesicles through electrothermal fluid rolls and dielectrophoresis synergistically integrated in a microfluidic device.

Extracellular vesicles (EVs) are cell-derived bioparticles that play significant roles in various biological processes including cell-to-cell communication and intercellular delivery. Additionally, they hold great potential as liquid biopsy biomarkers for pre-diagnostic applications. However, the isolation of EV subpopulations, especially exosomes from a biological fluid remains a challenge due to their submicron range. Here, we demonstrate continuous-flow label-free size fractionation of EVs for the first time through a synergistic combination of electrothermal fluid rolls and dielectrophoresis in a microfluidic device. The device features three dimensional microelectrodes with unique sidewall contours that give rise to effective electrothermal fluid rolls in cooperation with dielectrophoretic forces for the electrokinetic manipulation and size separation of submicron particles. We first validate the device functionality by separating submicron polystyrene particles from binary mixtures with a cut-off size of ∼200 nm and then isolate intact exosomes from cell culture medium or blood serum with a high recovery rate and purity (∼80%). The device operation in a high-conductivity medium renders the method ideal for the purification of target bioparticles directly from physiological fluids, and may offer a robust and versatile platform for EV related diagnostic applications.