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Increasing studies have highlighted the significance of milk-derived extracellular vesicles (MEV) in mother-newborn integration, as well as their application as novel drug delivery systems and diagnostic biomarkers. However, conventional ultracentrifugation (UC) often results in the co-precipitation of casein micelles in MEV pellets. In this study, we compared methods with different principles to screen the optimal pretreatment in caseins removal, and found that isoelectric precipitation by hydrochloric acid (HA) could most effectively remove caseins in porcine milk. We further characterized MEV populations isolated by UC and HA/UC from diverse aspects, including particle methodology via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), RNA and protein contents, and purity analysis. Importantly, the proliferative and anti-inflammatory effects of MEV were evaluated in vitro, showing the superiority of MEV via HA/UC in functionality compared with UC. Our results suggest that HA pretreatment before ultracentrifugation could effectively remove caseins and other protein complexes, making MEV with higher purity and more significant effects in vitro. This study provides valuable insights for the advancement of MEV isolation techniques across different species and accurate function analysis of MEV. The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. [Display omitted] [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Methods Results Conclusions The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer‐based precipitation and UC (PBP + UC).Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT‐PCR. Statistical analyses comparing groups were performed using ANOVA.All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR‐16, miR‐27a, and miR‐99a was successfully detected using all methods.The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time‐sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses. [ABSTRACT FROM AUTHOR]

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has underscored the significance of monitoring genetic variations in pathogenic viruses to manage its transmission and identify emerging variants. Nonetheless, sequencing every positive patient case is impractical for large-scale testing of the population. Here, we introduce a cost-effective tool for identifying SARS-CoV-2 mutations associated with variants in wastewater. This approach relies on a wastewater surveillance system and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), utilizing mutation-specific probes. Wastewater samples were analyzed for SARS-CoV-2 mutations corresponding to variants under monitoring (VUM) (P.2, alpha, beta, gamma, delta, and omicron) as part of a wastewater-based SARS-CoV-2 monitoring program. Our findings validate the efficacy of this method in detecting SARS-CoV-2 mutations linked to variants in wastewater, facilitating early detection of emerging variants and informing decision-making on control measures. [ABSTRACT FROM AUTHOR]

Lymphatic filariasis and onchocerciasis are neglected tropical diseases and cause significant public health problems in endemic countries, especially in sub-Saharan Africa. Since the human parasites are not viable in immune-competent mice, animal models have been developed, among them Litomosoides sigmodontis which permits a complete life cycle in BALB/c mice, including the development of patent infections with circulating microfilariae (Mf, the worm’s offspring). To investigate the immunomodulatory properties of helminths in vitro, antigenic extracts can be prepared from different life cycle stages of the L. sigmodontis model, including adult worms, but the methods to prepare these antigens differ between research groups. This study analyzed whether different centrifugation methods during the preparation of an antigenic extract, the gender of used worms, or the different fractions (soluble or pellet) altered filarial-specific CD4+ T cell responses. These cells were isolated from pre-patent or patent/chronic infected mice, hence those without and those with Mf, respectively. Ex vivo immune responses were compared at these two different time points of the infection as well as the parasitic parameters. Worm burden and cell infiltration were elevated in the thoracic cavity (TC) and draining mediastinal lymph nodes at the pre-patent stage. Within the TC, eosinophils were significantly up-regulated at the earlier time point of infection which was further reflected by the eosinophil-related eotaxin-1 levels. Regarding the production of cytokines by re-stimulated CD4+ T cells in the presence of different antigen preparations, cytokine levels were comparable for all used extracts. Our data show that immune responses differ between pre-patent and patent filarial infection, but not in response to the different antigenic extracts themselves. [ABSTRACT FROM AUTHOR]

Nanoporous membranes have a variety of applications, one of which is the size‐selective separation of nanoparticles. In drug delivery, nanoporous membranes are becoming increasingly important for the isolation of exosomes, which are bio‐nanoparticles. However, the low pore density and thickness of commercial membranes limit their efficiency. There have been many attempts to fabricate sub‐micrometer thin membranes, but the limited surface area has restricted their practicality. In this study, large‐area silicon nitride nanosieves for enhanced diffusion‐based isolation of exosomes are presented. Notably, these nanosieves are scaled to sizes of up to 4‐inch‐wafers, a significant achievement in overcoming the fabrication challenges associated with such expansive areas. The method employs a 200 nm porous sieve (38.2% porosity) for exosome separation and a 50 nm sieve (10.7% porosity) for soluble protein removal. These 300 nm thick nanosieves outperform conventional polycarbonate membranes by being 50 times thinner, thereby increasing nanoparticle permeability. The method enables a 90% recovery rate of intact exosomes from human serum and a purity ratio of 3 × 107 particles/µg protein, 4.6 times higher than ultracentrifugation methods. The throughput of the method is up to 15 mL by increasing the size of the nanosieve, making it an ideal solution for large‐scale exosome production for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Bringing effective cancer therapy in the form of chimeric antigen receptor technology to untapped markets faces numerous challenges, including a global shortage of therapeutic lentiviral or retroviral vectors on which all current clinical therapies using genetically modified T cells are based. Production of these lentiviral vectors in academic settings in principle opens the way to local production of therapeutic cells, which is the only economically viable approach to make this therapy available to patients in developing countries. The conditions for obtaining and concentrating lentiviral vectors have been optimized and described. The calcium phosphate precipitation method was found to be suitable for transfecting high cell‐density cultures, a prerequisite for high titers. We describe protocols for gradually increasing production from 6‐well plates to P100 plates, T‐175 flasks, and 5‐layer stacks while maintaining high titers, >108 transducing units. Concentration experiments using ultracentrifugation revealed the advantage of lower centrifugation speeds compared to competing protocols. The resulting batches of lentiviral vectors had a titer of 1010 infectious particles and were used to transduce primary human T lymphocytes generating chimeric antigen receptor T cells, the quality of which was checked and found potential applicability for treatment. [ABSTRACT FROM AUTHOR]

Hypertensive disorders during pregnancy pose significant risks to both maternal and fetal health, necessitating safe and effective therapeutic interventions.This study aimed to investigate the potential of an extract derived from Falcaria vulgaris (FV), loaded with exosomes to form the Exo/FV complex, as a novel therapeutic agent for the management of hypertension in pregnant mice: antioxidants, antimicrobials, and phenolic compounds present in FV lower blood pressure.The isolation of exosomes was done by ultracentrifugation methods and the FV was loaded into the exosomes by electroporation method.The Exo/FV was found to be spherical with diameter ranges from 20 to 30 nm and they were tested for biocompatibility in NHI 3T3 cell lines and found to be effective. This research investigated in vivo hypertension in mice induced by L-NAME and treated with FV and Exo/FV and found that AChE and MAO determine mice’s redox state tends to reduce blood pressure. Increased non-protein thiol (NP-SH) and decreased lipid peroxidation were also found, and PDE-5, ACE, Arginase, and MDA activity has also been tested.This analysis showed that Exo/FV effectively treated hypertension during pregnancy. [ABSTRACT FROM AUTHOR]

Lymph node metastasis (LNM) is a typical marker in oral squamous cell carcinoma (OSCC) indicating poor prognosis. Pathological examination by artificial image acquisition and analysis, as the main diagnostic method for LNM, often takes a week or longer which may cause great anxiety of the patient and also retard timely treatment. However, there are few efficient fast LNM diagnosis methods in clinical applications currently. Our previous study profiled the proteomics of extracellular vesicles (EVs) derived from postoperative drainage fluid (PDF) and showed the potential of detecting specific EVs that expressed aspartate β-hydroxylase (ASPH) for LNM diagnosis in OSCC patients. Considering that the analysis of ASPH+ PDF-EVs is challenging due to their low abundance (counting less than 10% of total EVs in PDF) and the complex EV isolation process of ultra-centrifugation, we developed a facile platform containing two microfluidic chips filled with antibody-modified microbeads to isolate ASPH+ PDF-EVs, with both the capture and retrieval rate reaching around 90%. Clinical sample analysis based on our method revealed that a mean of 6 × 106 /mL ASPH+ PDF-EVs could be isolated from LNM+ OSCC patients compared to 2.5 × 106 /mL in LNM- OSCC ones. When combined with enzyme-linked immunosorbent assay (ELISA) technique that was commonly used in clinical laboratories in hospitals, this microfluidic platform could precisely distinguish postoperative OSCC patients with LNM or not in several hours, which were validated by a double-blind test containing 6 OSCC patients. We believe this strategy has promise for early diagnosis of LNM in postoperative OSCC patients and finally helps guiding timely and reasonable treatment in clinic. [ABSTRACT FROM AUTHOR]

Human complement factor H (CFH) plays a central role in regulating activated C3b to protect host cells. CFH contain 20 short complement regulator (SCR) domains and eight N-glycosylation sites. The N-terminal SCR domains mediate C3b degradation while the C-terminal CFH domains bind to host cell surfaces to protect these. Our earlier study of Pichia-generated CFH fragments indicated a self-association site at SCR-17/18 that comprises a dimerization site for human factor H. Two N-linked glycans are located on SCR-17 and SCR-18. Here, when we expressed SCR-17/18 without glycans in an Escherichia coli system, analytical ultracentrifugation showed that no dimers were now formed. To investigate this novel finding, full-length CFH and its C-terminal fragments were purified from human plasma and Pichia pastoris respectively, and their glycans were enzymatically removed using PNGase F. Using size-exclusion chromatography, mass spectrometry, and analytical ultracentrifugation, SCR-17/18 from Pichia showed notably less dimer formation without its glycans, confirming that the glycans are necessary for the formation of SCR-17/18 dimers. By surface plasmon resonance, affinity analyses interaction showed decreased binding of deglycosylated full-length CFH to immobilized C3b, showing that CFH glycosylation enhances the key CFH regulation of C3b. We conclude that our study revealed a significant new aspect of CFH regulation based on its glycosylation and its resulting dimerization. [ABSTRACT FROM AUTHOR]

The assembly of two monomeric constructs spanning segments 1-199 (MPro1-199) and 10-306 (MPro10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro1-199 and MPro10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 μM MPro1-199. Catalytic activity arises primarily from MPro1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro1-199 and E290 of MPro10-306. Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity. [ABSTRACT FROM AUTHOR]

This study provides a preliminary background for the development of a viral vector vaccine for the dengue virus using genetic material encoded by dengue envelope ferritin nanoparticles. Adenoviruses were generated for the recombinant envelope of dengue virus 2 (DENV2) and the envelope human ferritin heavy chain using a two-vector adenovirus system. The primary immunostimulatory activity of the two viruses was analyzed in mice to determine the effect of envelope ferritin nanoparticles. Transfection of a shuttle vector delivered the target gene and packaging vector carrying the packaging signal, and recombinant adenoviruses (rAds) were generated and purified using an ultracentrifugation method. Transduction efficiencies of the generated adenoviruses were confirmed in A549 cells. Purified adenoviruses (8 × 106 PFU/mL) were immunized intramuscularly into 6 weeks old BALB/c mice. Subsequently, the DENV2-specific IgG titer was evaluated 1 and 4 weeks after immunization. Envelope ferritin-immunized mice showed a significant IgG response compared to envelope-only immunized mice at 1 and 4 weeks after immunization, revealing the persistence of the dengue virus-specific IgG response. This method demonstrated the capability of the viral vector vaccine to be used as a carrier for ferritin nanoparticles, instead of direct immunization with ferritin nanoparticles. [ABSTRACT FROM AUTHOR]

Due to the success of adeno associated viruses (AAVs) in treating single-gene diseases, improved manufacturing technology is now needed to meet their demand. The largest challenge is creating a process to separate empty and full capsids. Patients received larger capsid doses than necessary due to the presence of empty capsids. By enabling the better separation of empty and full capsids, patients would receive the greatest therapeutic benefit with the least amount of virus capsids, thus limiting potential side effects from empty capsids. The two most common empty/full separation methods used in downstream processing are ultracentrifugation and anion exchange chromatography. Both processes have limitations, leading to a need for the identification of other structural differences that can be exploited to separate empty and full capsids. Here, we describe four possible theories of the structural changes that occur when AAV capsids envelop a genome. These theories include conformational changes occurring due to either the expansion or contraction of the capsid in the presence of nucleic acids, the constraining of the N-terminus into the five-fold pore when the genome is present, and the increased number of VP3 proteins in full capsids. These theories may reveal structural differences that can be exploited to separate full and empty capsids during manufacturing. [ABSTRACT FROM AUTHOR]

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40–140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20–30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1. [ABSTRACT FROM AUTHOR]

Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation.

Abstract Objective Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles. Results To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.

Abstract Background Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization. Methods EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting. Results EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells. Conclusion Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.

Abstract Background The lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes. Results EVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates. Conclusions Our findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples.

Plant-derived exosome-like nanoparticles (ELNs) have demonstrated crosskingdom capabilities in regulating intercellular communication, facilitating drug delivery, and providing therapeutic interventions in humans. However, the functional attributes of konjac-derived ELNs (K-ELNs) remain largely unexplored. This study investigates the isolation, characterization, and functional analysis of K-ELNs, along with the profiling and differential expression analysis of associated miRNAs in both K-ELNs and Konjac tissues. K-ELNs were successfully isolated and characterized from two konjac species using ultracentrifugation, followed by Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Small RNA sequencing identified a total of 3,259 miRNAs across all samples. Differential expression analysis revealed significant differences in miRNA profiles between K-ELNs and tissue samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of target genes provided insights into their roles in modulating pathways associated with diseases such as cancer and neurodegenerative disorders. Additionally, six miRNAs were selected for validation of sequencing results via RT-qPCR. The 5'RLM-RACE method was employed to validate the cleavage sites between differentially expressed miRNAs (DEMs) and their predicted target genes, further substantiating the regulatory roles of miRNAs in konjac. The findings of this study enhance our understanding of the molecular mechanisms underlying the biological functions and applications of K-ELNs, laying the groundwork for future research into their potential therapeutic roles in human health. [ABSTRACT FROM AUTHOR]

The biggest challenge in current isolation methods for lipid bilayer‐encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large‐scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near‐native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size‐based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost‐effectiveness and rapid enrichment of the vesicles align with demands for large‐scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. [ABSTRACT FROM AUTHOR]

Dr. Helmut Cölfen, an exceptional interdisciplinary scientist, mentor, colleague, and dear friend, passed away in November 2023 at the age of 58. His untimely departure is a profound loss for the fields of analytical ultracentrifugation, colloid, crystallization, and polymer research. This obituary pays tribute to Helmut, honoring his remarkable academic career and contributions to the study of nanochemistry, biophysics, and life sciences. Helmut was renowned for his pioneering research contributions in several key research areas: (1) Development of advanced analytical techniques: Helmut made major contributions to techniques such as analytical ultracentrifugation and field flow fractionation, which are widely utilized to characterize the structure of biomolecules and the growth of nanostructured crystalline materials; (2) Study of nucleation and crystallization processes: Helmut explored the early stages of crystallization which led to the discovery of pre-nucleation clusters and mesocrystal intermediates, in the presence of additives and templates; and (3) Investigation of structure and morphogenesis of mesocrystals, examining their molecular properties. [ABSTRACT FROM AUTHOR]

In this study, the protein and salts distribution (Ca, P, Na and Mg) in processed cheese (PC) samples prepared with 180 or 360 mEq/kg of the calcium sequestering salts (CSS) disodium phosphate (DSP), disodium pyrophosphate (DSPP), sodium hexametaphosphate (SHMP) and trisodium citrate (TSC) was studied. For this purpose, a water-soluble extract (WSE) of PC samples was prepared. All PC samples contained 45–46% moisture, 26–27% fat and 20–21% protein and had a pH of 5.2 or 5.7. Ultracentrifugation slightly reduced the protein content of the WSE of PC, indicating that most protein in the WSE was non-sedimentable. At equal concentration of CSS, the protein content of the WSE was higher for PC at pH 5.7 compared to PC at pH 5.2. Approximately 55–85% of the Ca and P in the WSE of samples was 10 kDa-permeable for PC prepared with DSPP and SHMP. This suggests that the formation of non-permeable Ca–polyphosphate–casein complexes. For PC prepared with TSC, >90% of Ca in the WSE was 10 kDa-permeable, indicating that micellar disruption arises from sequestration of micellar Ca. These results indicate that the WSE method is an appropriate method to understand how salts present in PC are distributed. However, the WSE and ultracentrifugal supernatant of the WSE can include both soluble and protein-associated salts. Therefore, determining levels of salts in 10 kDa permeate of ultracentrifugal supernatant of the WSE is most appropriate. [ABSTRACT FROM AUTHOR]

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal. [ABSTRACT FROM AUTHOR]

Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflect physiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work, we present the advantages of an immunoaffinity-based method for uEVs' isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples. [ABSTRACT FROM AUTHOR]

Recombinant adeno‐associated virus, serotype 9 (rAAV9) has shown promise as a gene therapy vector for muscle and central nervous diseases. High‐dose requirements of these therapies present critical safety considerations and biomanufacturing challenges. Notably, the reduction of empty capsids (ECs), which lack therapeutic transgene, from rAAV9 products is critical to maximize efficacy. Removal of rAAV ECs from full capsids is a major downstream challenge because of their highly similar biophysical characteristics. Ultracentrifugation (UC) reduces ECs but is laborious and difficult to scale. In this paper, to replace a poorly scalable UC process, we developed an anion exchange (AEX) chromatography for rAAV9 EC reduction from full capsids. AEX load preparation by dilution incurred major product loss. The addition of histidine and surfactants to dilution buffers increased yield and reduced aggregation. Elution salts were screened and sodium acetate was found to maximize yield and EC reduction. The most promising load dilution buffer and elution salt were used in combination to form an optimized AEX method. The process reduced ECs three‐fold, demonstrated robustness to a broad range of EC load challenges, and was scaled for large‐scale manufacture. Compared to UC, the AEX method simplified scale‐up, reduced ECs to comparable levels (20%), afforded similar purity and product quality, and increased yield by 14%. [ABSTRACT FROM AUTHOR]

Adeno‐associated virus (AAV) manufacturing has traditionally focused upon lab‐scale techniques to culture and purify vector products, leading to limitations in production capacity. The tool presented in this paper assesses the feasibility of using non‐scalable technologies at high AAV demands and identifies optimal flowsheets at large‐scale that meet both cost and purity targets. The decisional tool comprises (a) a detailed process economics model with the relevant mass balance, sizing, and costing equations for AAV upstream and downstream technologies, (b) a built‐in Monte Carlo simulation to assess uncertainties, and (c) a brute‐force optimization algorithm for rapid investigation into the optimal purification combinations. The results overall highlighted that switching to more scalable upstream and downstream processing alternatives is economically advantageous. The base case analysis showed the cost and robustness advantages of utilizing suspension cell culture over adherent, as well as a fully chromatographic purification platform over batch ultracentrifugation. Expanding the set of purification options available gave insights into the optimal combination to satisfy both cost and purity targets. As the purity target increased, the optimal polishing solution moved from the non‐capsid purifying multimodal chromatography to anion‐exchange chromatography or continuous ultracentrifugation. [ABSTRACT FROM AUTHOR]

Two soy protein 11S fractions with different surface sulfhydryl contents were prepared. Utilizing analytical ultracentrifugation, the effects of storage time and hydrogen peroxide at different concentrations (0.5—100 mmol/L) on the two 11S fractions were investigated. Results show that after removing 2-mercaptoethanol (2-ME) by size exclusion chromatography, the 11S fraction with high surface sulfhydryl content (2.0 mol sulfhydryl/mol 11S) progressively formed 15S and 21S in dilute solutions during storage at 4 °C for 82 days. While, the 11S fraction with low surface sulfhydryl content (0.2 mol sulfhydryl/mol 11S) was stable under the same condition. Moreover, after treating the 11S with high surface sulfhydryl content with 1 mmol/L H2O2, the weight percentage of 15S reached the maximum value of 20%. The 15S induced by air and H2O2 could be totally converted to 11S with the addition of 10 mmol/L 2-ME, which could be attributed to that the disulfide bond linking two 11S molecules is on the surface of the 15S and easily accessible to the reducing agent 2-ME. This study helps us to deeply understand the formation mechanism of 15S and the stability of 11S. [ABSTRACT FROM AUTHOR]

Background: This study aimed to compare the lipemia removal efficiency of highspeed centrifugation, lipid scavengers, and dilution for biochemical analytes. Methods: We collected 30 cases of lipemic plasma in an emergency laboratory and divided them into 4 aliquots. Lipemia was removed by highspeed centrifugation, lipid scavenger, dilution, and ultracentrifugation, then analytes were measured by an AU5800 analyzer. Taking ultracentrifugation as reference, the efficiencies of the other three methods were evaluated based on the deviation. Results: When highspeed centrifugation was used for lipemia removal, DBIL (18.62%), and Magnesium (6.09%) could not satisfy the criterion. When lipid scavengers were applied to remove lipemia, CRP (-86.70%), TP (-8.29%), CKMB (-44.85%), DBIL (37.96%), Glu (4.20%) and phosphate (14.32%) were not suggested as lipid scavengers. For dilution, nearly half of the analytes could satisfy the criterion, including AMY (2.41%), CRP (5.54%), ALT (2.85%), GGTL (-1.73%), ALP (-0.04%), Glu (-0.84%), LDH (0.06%), CK (0.68%), BUN (3.80%), CREA (-1.54%), UA (5.42%), and magnesium (0.43%). Conclusions: Neither of the methods for lipid removal could satisfy all emergency department tests for lipid removal. This finding suggests that removing lipemia in the clinical laboratory should be based on the characteristics and the method of testing. [ABSTRACT FROM AUTHOR]

Objective: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles. Results: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies. [ABSTRACT FROM AUTHOR]

Microsomal binding (MB) is recognized as an important factor in accurately predicting drug-drug interaction. This work investigates differences among various methods employed in MB studies by: • Comparing and contrasts the protocols used and analyzing the data generated. • Identifying potential reasons for the variance in values generated across different methods. • Discussing the clinical and practical implications of selecting the most suitable method for accurate determination of MB. Accurate measurement of non-specific binding of a drug candidate to human liver microsomes (HLM) can be critical for the accurate determination of key enzyme kinetic parameters such as Michaelis-Menton (K m), reversible inhibition (K i), or inactivation (K I) constants. Several methods have been developed to determine non-specific binding of small molecules to HLM, such as rapid equilibrium dialysis (RED), ultrafiltration (UF), HLM bound to magnetizable beads (HLM-beads), ultracentrifugation (UC), the linear extrapolation stability assay (LESA), and the Transil™ system. Despite various differences in methodology between these methods, it is generally presumed that similar free fraction values (f u,mic) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high f u,mic value) and significant (low f u,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The f u,mic values were determined using a single compound concentration (1.0 µM) and three HLM concentrations (0.025, 0.50, and 1.0 mg/mL). When the HLM non-specific binding event is not extensive resulting in high f u,mic values, all methods generated similar f u,mic values. However, f u,mic values varied markedly across assay formats when high binding to HLM occurred, where f u,mic values differed by up to 33-fold depending on the method used. Potential causes for such discrepancies across the various methods employed, practical implications related to conduct the different assays, and implications to clinical drug-drug interaction (DDI) predictions are discussed. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV‐associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field‐flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high‐purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV‐associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell‐line‐derived EV markers are incompatible with the identification of plasma‐EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma‐EV markers. Benefiting from the high‐purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma‐ and serum‐derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications. [ABSTRACT FROM AUTHOR]

Background: Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization. Methods: EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting. Results: EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells. Conclusion: Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype. [ABSTRACT FROM AUTHOR]

Background: The lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes. Results: EVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates. Conclusions: Our findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples. [ABSTRACT FROM AUTHOR]

Low-density lipoprotein cholesterol (LDL-C), which makes up about 70% of the cholesterol in the blood, is critical in the formation of arteriosclerotic plaques, increasing the risk of heart disease. LDL-C levels are estimated using Friedewald, Martin and Sampson equations, though they have limitations with high triglycerides. Our aim is to compare the effectiveness of these equations versus the ultracentrifugation technique in individuals with and without dyslipidemia and identify precision. There were 113 participants, 59 healthy controls and 54 dyslipidemic patients. Samples were collected after fasting. LDL-C was estimated using the Friedewald, Martin and Sampson equations. The purified LDL-C, ultracentrifugated and dialysized control group without dyslipidemia vs. patients with coronary artery disease (CAD) showed differences in age, HDL-C, triglycerides and glucose non-HDL-C (p = 0.001 in all). There were correlations in CGWD between ultracentrifugation and Sampson R-squared (R2) = 0.791. In the dyslipidemia control group, ultracentrifugation and Friedewald R2 = 0.911. In patients with CAD, correlation between ultracentrifugation and Sampson R2 = 0.892; Bland–Altman confirmed agreement in controls without dyslipidemia. The Martin and Sampson equations are interchangeable with ultracentrifugation. Conclusion: The role of LDL analysis using precise techniques is necessary to obtain better control of disease outcomes after the use of precise therapies and suggests verifying its importance through clinical trials. [ABSTRACT FROM AUTHOR]

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed. Adaptations for distinct particle species, pitfalls, and 'forgotten' or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods. [ABSTRACT FROM AUTHOR]

A modified method for culturing, concentrating, and purifying phage ϕ24B preparations was developed. In particular, a new lysogenic phage-producing strain lacking flagella was used, induction conditions were optimized, and purification in a sucrose gradient followed by concentration by deposition on a Freon 113 cushion were used. Using this method, a preparation of the Stx-converting bacteriophage ϕ24B was obtained, suitable for CryoEM direct analysis. Based on CryoEM data for this phage, the first primary three-dimensional reconstruction of its virions was performed. The structure of the phage ϕ24B tail is described. It was shown that the adsorption apparatus of this virus is represented by six thin lateral fibrils, and an axial fibril located at the end of the tail. This arrangement of the tail structure is consistent with the previously proposed hypothesis based on analysis of the receptor binding proteins (RBPs) of this bacteriophage. [ABSTRACT FROM AUTHOR]

The approval of safe and effective LNP-mRNA vaccines during the SARS-CoV-2 pandemic is catalyzing the development of the next generation of mRNA therapeutics. Proper characterization methods are crucial for assessing the quality and efficacy of these complex formulations. Here, we show that analytical ultracentrifugation (AUC) can measure, simultaneously and without any sample preparation step, the sedimentation coefficients of both the LNP-mRNA formulation and the mRNA molecules. This allows measuring several quality attributes, such as particle size distribution, encapsulation efficiency and density of the formulation. The technique can also be applied to study the stability of the formulation under stress conditions and different buffers. [ABSTRACT FROM AUTHOR]

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide. [ABSTRACT FROM AUTHOR]

Ultracentrifugation is an attractive method for separating full and empty capsids, exploiting their density difference. Changes of the serotype/capsid, density of loading material, or the genetic information contained in the adeno-associated viruses (AAVs) require the adaptation of the harvesting parameters and the density gradient loaded onto the centrifuge. To streamline these adaptations, a mathematical model could support the design and testing of operating conditions. Here, hybrid models, which combine empirical functions with artificial neural networks, are proposed to describe the separation of full and empty capsids as a function of material and operational parameters, i.e., the harvest model. In addition, critical quality attributes are estimated by a quality model which is operating on top of the harvest model. The performance of these models was evaluated using test data and two additional blind runs. Also, a "what-if" analysis was conducted to investigate whether the models' predictions align with expectations. It is concluded that the models are sufficiently accurate to support the design of operating conditions, though the accuracy and applicability of the models can further be increased by training them on more specific data with higher variability. [ABSTRACT FROM AUTHOR]

Abstract Background Hypertriglyceridemia (HTG) is prevalent in Miniature Schnauzers, predisposing them to life‐threatening diseases. Varied responses to management strategies suggest the possibility of multiple subtypes. Hypothesis/Objective To identify and characterize HTG subtypes in Miniature Schnauzers through cluster analysis of lipoprotein profiles. We hypothesize that multiple phenotypes of primary HTG exist in this breed. Animals Twenty Miniature Schnauzers with normal serum triglyceride concentration (NTG), 25 with primary HTG, and 5 with secondary HTG. Methods Cross‐sectional study using archived samples. Lipoprotein profiles, generated using continuous lipoprotein density profiling, were clustered with hierarchical cluster analysis. Clinical data (age, sex, body condition score, and dietary fat content) was compared between clusters. Results Six clusters were identified. Dogs with primary HTG were dispersed among 4 clusters. One cluster showed the highest intensities for triglyceride‐rich lipoprotein (TRL) and low‐density lipoprotein (LDL) fractions and also included 4 dogs with secondary HTG. Two clusters had moderately high TRL fraction intensities and low‐to‐intermediate LDL intensities. The fourth cluster had high LDL but variable TRL fraction intensities with equal numbers of NTG and mild HTG dogs. The final 2 clusters comprised only NTG dogs with low TRL intensities and low‐to‐intermediate LDL intensities. The clusters did not appear to be driven by differences in the clinical data. Conclusions and Clinical Importance The results of this study support a spectrum of lipoprotein phenotypes within Miniature Schnauzers that cannot be predicted by triglyceride concentration alone. Lipoprotein profiling might be useful to determine if subtypes have different origins, clinical consequences, and response to treatment.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection primarily affecting pigs. It is caused by the porcine epidemic diarrhea virus (PEDV). PEDV targets the villus tissue cells in the small intestine and mesenteric lymph nodes, resulting in shortened intestinal villi and, in extreme cases, causing necrosis of the intestinal lining. Moreover, PEDV infection can disrupt the balance of the intestinal microflora, leading to an overgrowth of harmful bacteria like Escherichia coli. Exosomes, tiny membrane vesicles ranging from 30 to 150 nm in size, contain a complex mixture of RNA and proteins. MicroRNA (miRNA) regulates various cell signaling, development, and disease progression processes. This study extracted exosomes from both groups and performed high-throughput miRNA sequencing and bioinformatics techniques to investigate differences in miRNA expression within exosomes isolated from PEDV-infected porcine small intestine tissue compared to healthy controls. Notably, two miRNA types displayed upregulation in infected exosomes, while 12 exhibited downregulation. These findings unveil abnormal miRNA regulation patterns in PEDV-infected intestinal exosomes, shedding light on the intricate interplay between PEDV and its host. This will enable further exploration of the relationship between thesemiRNA changes and signaling pathways, enlightening PEDV pathogenesis and potential therapeutic targets. [ABSTRACT FROM AUTHOR]

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ13C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ13C values while adding only 5 atom% 13C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ13C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP. Key points: • Appropriate 13C-DNA amount was needed for DNA-SIP. • Detecting the 13C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions. • Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled. [ABSTRACT FROM AUTHOR]

Introduction: Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. Material and Methods: Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Results: Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II–III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. Conclusions: The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106 cells/mL, a plasmid concentration of 2 µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3 days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2 mM of sodium butyrate added 18 h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32 nm with 91.4% of protein similarity to exosomes. [ABSTRACT FROM AUTHOR]

Objective(s): Pediatric acute lymphoblastic leukemia (pALL) is the most prevalent neoplasm in children. pALL diagnostic process is invasive, and children need to be anesthetized for bone marrow aspiration. Exosomes are nanoparticles that reflect the status of parental cells. Given the elevated levels of exosomes in the peripheral blood of pALL patients, they can be readily extracted from blood plasma. The aim of this study was to compare the reliability of two different techniques of exosome purification in order to select the best method for clinical use in pALL. Methods: Exosomes were isolated from plasma samples using two methods: ultracentrifugation and the ExoQuick exosome isolation (EQ) kit. The performances of these methods were compared based on the nanoparticle tracking analysis (NTA), field emission electron microscopy (FESEM), and immunoblotting assays. Results: NTA results showed that exosome fractions extracted by the EQ kit were more concentrated and homogeneous compared with the ultracentrifugation method. Electron microscopy depicted spherical morphology for the isolated exosomes in both methods; however, the appearance of exosomes enriched by the commercial kit was more intact. Following the immunoblotting assays investigating the exosomal biomarkers, densitometry analysis showed that the exosome populations related to the EQ kit had higher concentrations and extreme purity. Conclusions: The data demonstrated that the commercial kit exhibited superior efficacy in isolating concentrated, intact, and pure exosomes from small quantities of patients' plasma samples when compared to the standard ultracentrifugation method. According to those findings, the ExoQuick exosome segregation kit was preferred to be used in pALL diagnostic investigations. [ABSTRACT FROM AUTHOR]

The enterovirus A71 (EV71) inactivated vaccine is an effective intervention to control the spread of the virus and prevent EV71-associated hand, foot, and mouth disease (HFMD). It is widely administered to infants and children in China. The empty particles (EPs) and full particles (FPs) generated during production have different antigenic and immunogenic properties. However, the antigen detection methods currently used were established without considering the differences in antigenicity between EPs and FPs. There is also a lack of other effective analytical methods for detecting the different particle forms, which hinders the consistency between batches of products. In this study, we analyzed the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) in characterizing the EPs and FPs of EV71. Our results showed that the proportions of the two forms could be quantified simultaneously by SV-AUC. We also determined the repeatability and accuracy of this method and found that both parameters were satisfactory. We assessed SV-AUC for bulk vaccine quality control, and our findings indicated that SV-AUC can be used effectively to analyze the percentage of EPs and FPs and monitor the consistency of the process to ensure the quality of the vaccine. [ABSTRACT FROM AUTHOR]

In the clinical laboratory, the evaluation of low-density lipoprotein cholesterol is a critical parameter in the diagnosis of cardiovascular disease. The present literature review work has a documentary design that compiled published information from the last five years on LDL cholesterol. The objective was to compare direct methods with formulas in the quantification of LDL cholesterol, in order to identify the most accurate method in clinical practice. The relevant point of this study is that laboratories frequently use formulas due to the simplicity of the mathematical calculations, however, it has been observed that in elevated triglyceride levels the formulas are inaccurate. The conclusion is that homogeneous assays are more accurate than LDL cholesterol estimation formulas. [ABSTRACT FROM AUTHOR]

Herein, TiO2 single-tube (TiO2 ST-NT) powders with and without magnetite Fe3O4 nanoparticles (TiO2 ST-NT@Fe3O4NPs) are presented for the first time as excellent photocatalysts for the degradation of one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs), naproxen (NPX). The TiO2 ST-NT powders were synthesized by anodization followed by etching of the double wall, bending, sonication, ultra-centrifugation, and finally annealing at 600°C. A part of the obtained TiO2 ST-NT powders was decorated with Fe3O4 nanoparticles using a simple one-step decoration process. The best photocatalytic performance of TiO2 ST-NT and TiO2 ST-NT@Fe3O4NPs powders was obtained under the white light (6.2 x 10-4 s-1) and the blue light (2.7 x 10-4 s-1), respectively. During NPX photodegradation using TiO2 ST-NT powders, three main NPX transformation products (P1, P2, and P3) were detected. Upon excitation with the blue light illumination, TiO2 ST-NT@ Fe3O4NPs powders exhibited higher performance (~80%) than TiO2 ST-NT powders (~23%) within 1 h, resulting in an approximately three times increased photocatalytic rate constant. Moreover, under simulated sunlight conditions, TiO2 ST-NT powders demonstrated remarkable activity, achieving a 94% NPX degradation within 1 h. TiO2 ST-NT and TiO2 ST-NT@Fe3O4NPs powders represent excellent photocatalysts for NPX degradation. [ABSTRACT FROM AUTHOR]