Efficient Isolation Research Articles

Efficient and Rapid Enrichment of Extracellular Vesicles Using DNA Nanotechnology-Enabled Synthetic Nano-Glue.

Swift and efficient enrichment and isolation of extracellular vesicles (EVs) are crucial for enhancing precise disease diagnostics and therapeutic strategies, as well as elucidating the complex biological roles of EVs. Conventional methods of isolating EVs are often marred by lengthy and laborious processes. In this study, we introduce an innovative approach to enrich and isolate EVs by leveraging the capabilities of DNA nanotechnology. We have developed a novel multivalent cholesterol-modified paranemic crossover DNA (PX-DNA-chol) construct, which is a four-stranded DNA structure containing adjacent double helices intertwined with their local helix axes parallel and serves as an effective synthetic nano-glue. This construct promotes the rapid coalescence of nanoscale EVs into clusters of micrometer scale, thereby streamlining their enrichment. Utilizing a conventional low-speed centrifuge, this intriguing methodology achieves a rapid concentration of EVs within minutes, bypassing the laborious and high-speed centrifugation steps typically required. The quality of EVs isolated by our technique is comparable to that obtained through ultracentrifugation methods. Given these advancements, our PX-DNA-chol-facilitated EVs enrichment protocol is poised to advance the field of EVs research, providing a robust and accessible tool for in-depth studies of EVs.

Demystifying EV heterogeneity: emerging microfluidic technologies for isolation and multiplexed profiling of extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous lipid containers carrying complex molecular cargoes, including proteins, nucleic acids, glycans, etc. These vesicles are closely associated with specific physiological characteristics, which makes them invaluable in the detection and monitoring of various diseases. However, traditional isolation methods are often labour-intensive, inefficient, and time-consuming. In addition, single biomarker analyses are no longer accurate enough to meet diagnostic needs. Routine isolation and molecular analysis of high-purity EVs in clinical applications is even more challenging. In this review, we discuss a promising solution, microfluidic-based techniques, that combine efficient isolation and multiplex detection of EVs, to further demystify EV heterogeneity. These microfluidic-based EV multiplexing platforms will hopefully facilitate development of liquid biopsies and offer promising opportunities for personalised therapy.

Optimized separation of astaxanthin stereoisomers from microbial sources using chiral HPLC.

Astaxanthin (AST) is a high-value antioxidant, and its efficient isolation and utilization are challenging owing to the presence of different stereoisomers from various sources. In the present study, a semi-preparative HPLC method for the efficient separation of AST stereoisomers using a Chiralpak IC chiral column with good loading capacity and chiral recognition ability was successfully developed. The mobile phase was methanol-methyl tert-butyl ether (90 : 10, v/v), with a flow rate of 3.06 mL min-1 and a maximum injection volume of 0.32 mg. The results indicated that the purity of all-trans AST was 97.9% for Haematococcus pluvialis and 97.5% for Phaffia rhodozyma. Additionally, molecular weights and fragmentation patterns analyzed using mass spectrometry were consistent with those of all-trans AST. Linearity validation and reproducibility experiments revealed that all calibration curves had coefficients of determination (R2) greater than 0.999 and a relative standard deviation (RSD) of <3.8%. This is because all-trans AST stereoisomers could undergo specific rotations or spins due to π-π interactions, hydrogen bonding, and inclusion interactions. This process allowed the successful separation of the three all-trans AST optical isomers and provides a theoretical basis for large-scale preparation of all-trans AST stereoisomers from different sources.

Using Callus as an Ex Vivo System for Chromatin Analysis.

Next-generation sequencing has revolutionized epigenetics research, enabling a comprehensive analysis of DNA methylation and histone modification profiles to explore complex biological systems at unprecedented depth. Deciphering the intricate epigenetic mechanisms that regulate gene activity presents significant challenges, including the issue of analyzing heterogeneous cell populations in bulk. Bulk analysis introduces bias and can obscure crucial information by averaging readouts from distinct cells. Various approaches have been developed to address this issue, such as cell-type-specific enrichment or single-cell sequencing techniques. However, the need for transgenic lines with fluorescent markers, along with technical challenges such as efficient protoplast isolation and low yield, limits their widespread adoption and use in multi-omic studies. This review discusses the pros and cons of these approaches, providing a valuable basis for selecting the most suitable strategy to minimize heterogeneity. We will also highlight the use of cotyledon-derived callus as an ex vivo system as a simple, accessible, and robust platform for enabling high-throughput multi-omic analyses.

Screening and assessment of PGP and biocontrol properties of Azotobacter species isolated from agriculture soils of North Karnataka

Plant growth-promoting rhizobacteria (PGPR) are recognized for their ability to produce phytohormones, root-stimulating compounds, anti-fungal compounds and other secondary metabolites, making them potential biocontrol agents in agriculture. In the present study, 85 Azotobacter isolates were isolated from the agricultural soils of Raichur and Chikkabalapura locations. The isolates were further accessed for morphological, biochemical and plant growth-promotion (PGP) properties. All the isolated strains showed brown to black colour colonies on the Waksmann 77 media plate. Similar biochemical results were obtained for all the Azotobacter isolates. The isolates such as Azt-85 recorded the highest N2 fixation (33.36 µgN/mL/Day), Azt-69 produced IAA (24.67 µg/mL), and Azt-51 produced GA (23.7 µg/25mL). The anti-fungal efficacy studies were conducted using the dual culture technique using the efficient PGPR Azotobacter isolates against fungal species (Fusarium oxysporum f. sp. lini, Fusarium oxysporum f. sp. ciceris and Aspergillus flavus). After the incubation period, the Azt-41 isolate showed the highest zone of inhibition (18 mm) against Fusarium oxysporum f. sp. lini. Similarly, Azotobacter isolates viz., Azt-25, 38 and 41 showed maximum growth inhibition (9 mm) against Fusarium oxysporum f. sp. ciceris. Similarly, the Azt-31 isolate recorded a moderate (13 mm) zone of inhibition against Aspergillus flavus. Integrating sustainable biocontrol strategies by injecting beneficial microbes like Azotobacter can enhance resilient food production systems and reduce reliance on chemical inputs through PGPR properties.

Biodegradation of Organophosphorus Insecticides by Bacillus Species Isolated From Soil.

This study investigates the biodegradation of methyl parathion, an organophosphate pesticide used in paddy fields. Microbial degradation transforms toxic pesticides into less harmful compounds, influenced by the microbial community in the soil. To isolate different microbial colonies, soil samples from an organophosphorus-treated groundnut field were plated on nutrient agar and MSM with 1% glucose and 0.25 mM methyl parathion. Biodegradation efficiency was determined by estimating the OP hydrolase enzyme activity spectrophotometrically. HPLC was used to quantify residual methyl parathion concentrations in the culture medium. The identified isolate effectively degraded methyl parathion in MSM with 0.25 mM methyl parathion which showed peak hydrolase activity (2.02 µmol/min/mg) after 96 h of incubation and the residual methyl parathion level was determined as 6.2 µmol by HPLC quantification. The efficient isolate was identified as Bacillus cereus by using a 16S rRNA molecular marker and the sequence was subjected to MEGA11 phylogenetic tree construction. The results show that the SM6 clade shared with B. cereus 16S rRNA sequence. B. cereus (SM6) showed substantial enzyme activity and the specific reported opdA gene-coded protein is involved in ATP hydrolysis. This OP hydrolase makes it a strong candidate for bioremediation of methyl parathion. Molecular analysis suggested that the opdA gene, likely chromosomally located, plays a key role in degradation, with potential involvement of the "Cell division protein FtsK" gene responsible for hydrolase activity. Organophosphorus compounds, widely used in agriculture, pose environmental concerns due to their persistence. This study focuses on isolating pesticide-degrading bacteria to expedite bioremediation, aiming for efficient degradation. This study highlights the cross-adaptation phenomenon, where B. cereus strains degrade similar compounds, improving bioremediation strategies.

Development of a Collection Kit for Efficient Isolation of SARS-CoV-2 RNA from Urine and Stool Samples

Background Information: Consider adding a brief sentence about the significance of detecting SARS-CoV-2 in urine and stool samples. For example, mention how these samples can serve as alternative sources for diagnosis, potentially leading to better patient outcomes. Objective Statement: This study aimed to develop a collection kit for the effective extraction of SARS-CoV-2 RNA from urine and stool samples. The study presents a significant advancement in the field of viral diagnostics by introducing a novel collection kit specifically designed for the extraction of SARS-CoV-2 RNA from urine and stool samples. The demonstrated efficacy of the kit in isolating the virus with high success rates from alternative biological fluids, as well as the superior RNA. Methodology: The kit contains a swab of medical polyester and three tubes, two of them used to inactivate the virus, one contains 3% phenol and the other 1% phenol, and the third tube contains 2 ml of (PBS) Phosphate Buffer Saline added to it 4% of skim milk. Urine or stool samples from Covid-19 patients who reported positive by nasopharyngeal swab during RT-PCR was adde consequently in these three tubes then assessed through RT-PCR testing. Results: The kit succeeded in isolating coronavirus from the urine and stool of patients who reported positive by nasopharyngeal samples. From 20 positive nasopharyngeal samples, there was 18 (90%) of them were positive by using stool sample using this kit, and 15 (75%) of them were give positive result when urine samples were used by this kit. The concentration of RNA of coronavirus which was isolated from urine and feces was higher than in the nasopharyngeal swab during RT-PCR. Mean Ct value of the 20 samples was showed that the mean Ct of nasopharyngeal was 31.5, while urine and stool samples Ct means less concentration of virus. were 25.75 and 24.32 respectively. High The kit was able to serve RNA that was isolated from urine and feces for about 5,10, and 30 days in a percentage of 100%, 94.1%, and 94.1% respectively. Conclusion and Implications: The kit is produce to confirm that the patient is infected with by COVID-19 and to preserve the virus in stool or urine samples of the infected person for a long period until laboratory tests through RT-PCR are conducted on the samples. The kit showed great ability to test SARS-CoV-2, even if it was at low concentrations of virus compared to the examination of nasopharyngeal swabs.

A large-scale proteomics resource of circulating extracellular vesicles for biomarker discovery in pancreatic cancer

Pancreatic cancer has the worst prognosis of all common tumors. Earlier cancer diagnosis could increase survival rates and better assessment of metastatic disease could improve patient care. As such, there is an urgent need to develop biomarkers to diagnose this deadly malignancy. Analyzing circulating extracellular vesicles (cEVs) using ‘liquid biopsies’ offers an attractive approach to diagnose and monitor disease status. However, it is important to differentiate EV-associated proteins enriched in patients with pancreatic ductal adenocarcinoma (PDAC) from those with benign pancreatic diseases such as chronic pancreatitis and intraductal papillary mucinous neoplasm (IPMN). To meet this need, we combined the novel EVtrap method for highly efficient isolation of EVs from plasma and conducted proteomics analysis of samples from 124 individuals, including patients with PDAC, benign pancreatic diseases and controls. On average, 912 EV proteins were identified per 100 µL of plasma. EVs containing high levels of PDCD6IP, SERPINA12, and RUVBL2 were associated with PDAC compared to the benign diseases in both discovery and validation cohorts. EVs with PSMB4, RUVBL2, and ANKAR were associated with metastasis, and those with CRP, RALB, and CD55 correlated with poor clinical prognosis. Finally, we validated a seven EV protein PDAC signature against a background of benign pancreatic diseases that yielded an 89% prediction accuracy for the diagnosis of PDAC. To our knowledge, our study represents the largest proteomics profiling of circulating EVs ever conducted in pancreatic cancer and provides a valuable open-source atlas to the scientific community with a comprehensive catalogue of novel cEVs that may assist in the development of biomarkers and improve the outcomes of patients with PDAC.

A large-scale proteomics resource of circulating extracellular vesicles for biomarker discovery in pancreatic cancer.

Pancreatic cancer has the worst prognosis of all common tumors. Earlier cancer diagnosis could increase survival rates and better assessment of metastatic disease could improve patient care. As such, there is an urgent need to develop biomarkers to diagnose this deadly malignancy. Analyzing circulating extracellular vesicles (cEVs) using 'liquid biopsies' offers an attractive approach to diagnose and monitor disease status. However, it is important to differentiate EV-associated proteins enriched in patients with pancreatic ductal adenocarcinoma (PDAC) from those with benign pancreatic diseases such as chronic pancreatitis and intraductal papillary mucinous neoplasm (IPMN). To meet this need, we combined the novel EVtrap method for highly efficient isolation of EVs from plasma and conducted proteomics analysis of samples from 124 individuals, including patients with PDAC, benign pancreatic diseases and controls. On average, 912 EV proteins were identified per 100 µL of plasma. EVs containing high levels of PDCD6IP, SERPINA12, and RUVBL2 were associated with PDAC compared to the benign diseases in both discovery and validation cohorts. EVs with PSMB4, RUVBL2, and ANKAR were associated with metastasis, and those with CRP, RALB, and CD55 correlated with poor clinical prognosis. Finally, we validated a seven EV protein PDAC signature against a background of benign pancreatic diseases that yielded an 89% prediction accuracy for the diagnosis of PDAC. To our knowledge, our study represents the largest proteomics profiling of circulating EVs ever conducted in pancreatic cancer and provides a valuable open-source atlas to the scientific community with a comprehensive catalogue of novel cEVs that may assist in the development of biomarkers and improve the outcomes of patients with PDAC.

Freeze-Thaw-Induced Patterning of Extracellular Vesicles with Artificial Intelligence for Breast Cancers Identifications.

Extracellular vesicles (EVs) play a crucial role in the occurrence and progression of cancer. The efficient isolation and analysis of EVs for early cancer diagnosis and prognosis have gained significant attention. In this study, for the first time, a rapid and visually detectable method termed freeze-thaw-induced floating patterns of gold nanoparticles (FTFPA) is proposed, which surpasses current state-of-the-art technologies by achieving a 100 fold improvement in the limit of detection of EVs. Notably, it allows for multi-dimensional visualizations of EVs through site-specific oligonucleotide incorporation. This capability empowers FTFPA to accurately identify EVs derived from subtypes of breast cancers with artificial intelligence algorithms. Intriguingly, learning the freezing-thawing-microstructures of EVs with a random forest algorithm is not only able to distinguish their original cell lines (with an accuracy of 95.56%), but also succeed in processing clinical samples (n = 156) to identify EVs by their healthy donors, breast lump and breast cancer subtypes (Luminal A, Triple-negative breast cancer, and Luminal B) with an accuracy of 83.33%. Therefore, this AI-empowered micro-visualization method establishes a rapid and precise point-of-care platform that is applicable to both fundamental research and clinical settings.

Screening of agroresidues for economical and sustainable Phytase Production by Bacteria

Phytase, a phytate hydrolysing enzyme has substantial application in food, feed and agriculture sector for improvising the availability of phosphorus and reduces the environmental pollution. The present study emphasizes on cost effective production of bacterial phytase. Total 25 agroresidues were screened for economic production of phytase using bacteria. A total of 30 bacterial isolates were obtained from poultry and goat shed soil and litter sample showing highest phytase activity. The efficient isolates SP-46 and SP-52 were screened for selection of efficient combination bacterial isolate and agroresidues for cost-effective production of phytase. The results showed that utilization of agroresidues not only serve as efficient substrate but also contribute to sustainable agro-waste valorisation and reduces need of expensive synthetic media for phytase production.

Native and Non-Native Soil and Endophytic Trichoderma spp. from Semi-Arid Sisal Fields of Brazil Are Potential Biocontrol Agents for Sisal Bole Rot Disease.

Sisal (Agave sisalana) bole rot caused by Aspergillus welwitschiae is the main phytosanitary problem affecting sisal in the Brazilian semi-arid region. The aim of this study was to evaluate Trichoderma spp. as biocontrol agents for sisal bole rot. Native and non-native species, both soil inhabitants and endophytes, and isolated from different plant hosts were tested. Anatomical studies of the interaction among A. sisalana, Trichoderma spp., and A. welwitschiae were performed. T. cf. asperellum (isolate F12), an endophyte of sisal leaves; T. cf. asperellum (TCS83) from banana plant soil; T. lentiforme (TCS15) and T. harzianum (species complex) (TCS35 and TCS76) from sisal root soil; T. spirale (R62) and T. saturnisporum (R75), endophytes of sisal roots, were the most efficient isolates, with inhibition of A. welwitschiae mycelial growth by up to 70%, and inhibition of sporulation and spore germination by 99%. A reduction in disease incidence of 70 to 93% and in disease severity of 97% was achieved. T. lentiforme (TCS1), T. harzianum (species complex) (TCS35 and R72), and T. koningiopsis (R78) showed mycoparasitism. An increase in cell wall thickness of bole tissue colonized by these Trichoderma species indicated that induced plant defense responses occurred, preventing pathogen colonization, which should be further investigated. Native and non-native Trichoderma species can control sisal bole rot disease.

Single-Cell Isolation Chip Integrated with Multicolor Barcode Array for High-Throughput Single-Cell Exosome Profiling in Tissue Samples.

Exosomes, functional biomarkers involved in cancer progression, have gained widespread attention for promoting tumor formation, growth, and metastasis. Current bulk exosome detections in bodily fluids enable cancer functional analysis, but average secretion levels from cell populations, losing parent cell information and ignoring exosome heterogeneity from diverse cell subgroups, necessitating an effective platform for analyzing single-cell exosome functional heterogeneity. Here, a high-throughput platform is presented, capable of efficient single-cell isolation and multi-color exosome phenotype analysis, as well as quantifying trace exosomes secreted by single cells. Photothermal-driven single-cell chips achieve significant single-cell isolation efficiency (≈97%) within 5 min, facilitating the ultra-high throughput single-cell exosome analysis. By conducting mass spectrometry and protein interaction of breast cancer exosome phenotypic proteins, key exosome phenotypes are identified. Tens of thousands of single cells from breast cancer cell lines, and clinical tissues are analyzed, revealing various subgroup differences. The study finds more CD44 and EGFR co-expressing exosome subgroups in breast cancer cell lines, while immune-evasion PD-L1 high-phenotype exosome subgroups are primarily presented in complex tumor microenvironments, especially in HER2-positive tissues. This platform offers powerful single-cell isolation, exosome quantification, and phenotypic analysis capabilities, making it a powerful tool for advancing single-cell exosome analysis in cancer research.

Purification and Characterization of Kyasanur Forest Disease Virus EDIII domain of major Envelope Glycoprotein

Current research efforts are underway to create novel approaches for the efficient diagnosis, monitoring, and mitigation of Kyasanur Forest Disease Virus (KFDV) infections. Flavivirus subunit-based vaccines based on envelope glycoprotein EDIII are now in preclinical and clinical research stages. Efficient purification and isolation methods for surface immunogenic viral antigens, including the recombinant envelope immunoglobulin-like domain III (rEDIII) protein, are crucial for the production and manufacturing of promising vaccine candidates that have been extensively assessed in previous literature. Here, we describe a method for high-yield expression, purification, and refolding of a KFDV rEDIII protein from a bacterial expression system. The KFDV rEDIII protein is extracted from the inclusion bodies in urea denaturing buffer followed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The purified, denatured KFDV rEDIII protein was subsequently refolded using a step-wise gradient urea dilution via the dialysis method. The circular dichroism and Fourier transform infrared spectroscopy analysis confirms that the refolded KFDV rEDIII maintains the native secondary conformation majorly containing β-strands. Our study provides valuable insights into the design and expression strategies of rEDIII as a novel subunit vaccine candidate against KFDV.

Biodegradation of Di-2-Ethylhexyl Phthalate by Mangrove Sediment Microbiome Impacted by Chronic Plastic Waste.

Plastic pollution through the leaching of di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, has led to the emergence of mangrove pollution. This study aimed to assess the DEHP removal efficiency of indigenous mangrove sediment microbiomes and identify key DEHP degraders using microcosm construction and metagenomic analysis. During the 35-day incubation period, the indigenous mangrove sediment microbiome, affected by chronic plastic pollution, demonstrated a 99% degradation efficiency of 200mg/kg DEHP. Spearman's correlation analysis suggested that Myxococcales, Methyloligellaceae, Mycobacterium, and Micromonospora were potentially responsible for DEHP degradation. Based on PICRUSt2, the DEHP-degrading pathway in the sediment was predicted to be an anaerobic process involving catechol metabolism through catC, pcaD, pcaI, pcaF, and fadA. Efficient bacterial isolates from the mangrove sediment, identified as Gordonia sp. and Gordonia polyisoprenivorans, were able to degrade DEHP (65-97%) within 7days and showed the ability to degrade other phthalate esters (PAEs).

Enhanced Electrical Performance of InAs Nanowire Field-Effect Transistors Based on the Y2O3 Isolation Layer.

Nanowire (NW) field-effect transistors (FETs) have great potential in next-generation integrated circuits. InAs NWs are suitable for N-type transistors because of their excellent electrical properties. However, unlike the Si/SiO2 system, the loose and defective native oxide of InAs is unable to passivate the channel surface and serve as an efficient isolation layer (IL) in the gate stack. Here, we, for the first time, demonstrate that using a stable dielectric Y2O3 to replace the native oxide IL can effectively passivate the NW surface, isolate the channel surface from high-k HfO2, and improve the electrical properties of InAs NW FETs significantly. First, top-gate devices with Y2O3 IL outperform the devices with native oxide or chemically generated oxide of InAs as the IL, exhibiting a small subthreshold swing (SS) of 77 mV/dec, a large on-off ratio, a high field-effect mobility (μFE) of 1845 cm2/V·s, and an extremely low interface trap density (Dit) of 9.1 × 1011 eV-1 cm-2. Double-gate FETs with Y2O3 IL also show excellent gate control (drain-induced barrier lowering (DIBL) down to 35 mV/V) and a high μFE of 2711 cm2/V·s (the highest in the multigate structure). In addition, InAs NW FETs with Y2O3 IL show greater stability after long-term air exposure than those with native oxide IL, demonstrating retained on-off ratio, DIBL, transconductance, and even smaller SS (from 89 to 77 mV/dec). Finally, the results of low-temperature measurement suggest that Y2O3 IL can effectively improve interface qualities. Our results indicate that Y2O3/HfO2 InAs NW FETs have great potential in digital units of integrated circuits for future nodes. Our strategy for improving the interface between InAs NW and the dielectric layer is also valuable for other III-V NW devices.

Prevalence of Cronobacter spp. in tropical seafood from Mumbai, India: Comparative study of isolation media and PCR detection.

Cronobacter species are opportunistic emerging pathogens associated with diverse foods of plant and animal origin. Considering the diversity of the Cronobacter group of bacteria and their co-existence with closely related Enterobacterales in the aquatic environment, their isolation from fish and shellfish is a challenge. This study aimed to investigate the incidence of Cronobacter in finfish, shellfish, and dried fish, and to compare nine combinations of enrichment broth-selective isolation media for efficient isolation of Cronobacter spp. Seventy-five seafood samples collected from five different retail markets were subjected to multiple selective-enrichment methods to isolate Cronobacter, which were presumptively identified by biochemical tests followed by confirmation with genus- and species-specific PCRs. Of 75 seafood samples analyzed, 24 (32%) were positive for Cronobacter spp. The highest incidence was in dried fish (21 samples, 47.72%), followed by 19 (43.18%) fresh finfish and four (9.09%) shellfish samples. Forty-four isolates from these samples were identified as Cronobacter spp. by PCR. Species-specific PCR further categorized these as C. sakazakii (25), C. malonaticus (16) and C. turicensis (1), while two isolates remained unidentified at species level. Enrichment in Cronobacter screening broth or Rappaport Vassiliadis medium, followed by isolation on the chromogenic Cronobacter sakazakii agar was found to be the most effective combination for the isolation of Cronobacter spp. from seafood. Dried fish is an important reservoir of C. sakazakii owing to its desiccation tolerance and absence of competing microbiota in dried fish. Although C. sakazakii is the only known pathogen among Cronobacter spp., improved and specific methods to identify diverse members of this genus are needed.

Bifunctional Nanomaterial Enabled High-specific Isolation of Urinary Exosomes for Cervical Cancer Metabolomics Analysis and Biomarker Discovery

Cervical cancer (CC) remains a critical public health issue, highlighting the importance of early detection. However, current methods such as cytological and HPV testing face challenges of invasiveness and low patient compliance. Exosomes, emerging as crucial in cancer diagnosis, offer promise due to their noninvasive, highly specificity, and abundant biomarkers. However, isolating exosomes efficiently remains challenging. In this study, we designed and synthesized a bifunctional affinity nanomaterial Fe3O4 @CD63-CLIKKPF, based on the synergistic interaction between its modified aptamer CD63 and peptide CLIKKPF, and CD63 protein and PS of exosomes which can achieve high specificity and high yield separation of urinary exosomes. Notably, the co-modified aptamer CD63 and peptide CLIKKPF not only enable efficient exosome isolation by leveraging dual-affinity mechanisms through a synergistic "AND" logic analysis, but also could be achieved on the Fe3O4 in one-step reaction at room temperature via Fe-S bonding. Combined with LC-MS/MS, we conducted exosome metabolomics analysis in healthy individuals and CC patients across various stages, and machine learning models demonstrated accurate classification (accuracy > 0.822) and prediction capabilities for CC. Furthermore, six key metabolites indicative of CC progression were identified and validated in additional patient samples, highlighting their potential as biomarkers. Overall, this study establishes a novel method for exosome metabolomics in CC, offering insights for non-invasive early diagnosis and progression prediction on a large scale.

Interkingdom Communication via Extracellular Vesicles: Unraveling Plant and Pathogen Interactions and Its Potential for Next-Generation Crop Protection.

Recent advancements in the field of plant-pathogen interactions have spotlighted the role of extracellular vesicles (EVs) as pivotal mediators of cross-kingdom communication, offering new vistas for enhancing crop protection strategies. EVs are instrumental in the transport of small regulatory RNAs (sRNAs) and other bioactive molecules across species boundaries, thus playing a critical role in the molecular warfare between plants and pathogens. This review elucidates the sophisticated mechanisms by which plants utilize EVs to dispatch sRNAs that silence pathogenic genes, fortifying defenses against microbial threats. Highlighting both eukaryotic and prokaryotic systems, this review delves into the biogenesis, isolation, and functional roles of EVs, illustrating their importance not only in fundamental biological processes but also in potential therapeutic applications. Recent studies have illuminated the significant role of EVs in facilitating communication between plants and pathogens, highlighting their potential in host-defense mechanisms. However, despite these advancements, challenges remain in the efficient isolation and characterization of plant-derived EVs. Overcoming these challenges is critical for fully harnessing their potential in developing next-generation crop protection strategies. This review proposes innovative strategies for utilizing RNA-based interventions delivered via EVs to bolster plant resilience against diseases. By integrating the latest scientific findings with practical applications in agriculture, this review aims to enhance the connection between fundamental plant biology and the development of innovative crop management technologies.

Scratch-Based Isolation of Primary Cells (SCIP): A Novel Method to Obtain a Large Number of Human Dental Pulp Cells Through One-Step Cultivation.

Background: Dental pulp (DP) is a connective tissue composed of various cell types, including fibroblasts, neurons, adipocytes, endothelial cells, and odontoblasts. It contains a rich supply of pluripotent stem cells, making it an important resource for cell-based regenerative medicine. However, current stem cell collection methods rely heavily on the enzymatic digestion of dissected DP tissue to isolate and propagate primary cells, which often results in low recovery rates and reduced cell survival, particularly from deciduous teeth. Methods: We developed a novel and efficient method to obtain a sufficient number of cells through a one-step cultivation process of isolated DP. After the brief digestion of DP with proteolytic enzymes, it was scratched onto a culture dish and cultured in a suitable medium. By day 2, the cells began to spread radially from DP, and by day 10, they reached a semi-confluent state. Cells harvested through trypsinization consistently yielded over 1 million cells, and after re-cultivation, the cells could be propagated for more than ten passages. Results: The proliferative and differentiation capacities of the cells after the 10th passage were comparable to those from the first passage. The cells expressed alkaline phosphatase as an undifferentiation marker. Similarly, they also maintained the constitutive expression of stem cell-specific markers and differentiation-related markers, even after the 10th passage. Conclusions: This method, termed "scratch-based isolation of primary cells from human dental pulps (SCIP)", enables the efficient isolation of a large number of DP cells with minimal equipment and operator variability, while preserving cell integrity. Its simplicity, high success rate, and adaptability for patients with genetic diseases make it a valuable tool for regenerative medicine research and clinical applications.