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Journal of Extracellular Biology Dec 2023Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising biomarkers for early cancer diagnosis. However, existing EV-miRNA extraction technologies have a...
Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising biomarkers for early cancer diagnosis. However, existing EV-miRNA extraction technologies have a complex two-step process that results in low extraction efficiency and inconsistent results. This study aimed to develop and evaluate a new single-step extraction method, called miRQuick, for efficient and high-recovery extraction of EV-miRNAs from samples. The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. A membrane lysate is then added to extract only miRNA. The entire process can be completed within an hour using standard laboratory equipment. We validated the miRQuick method using various analytical techniques and compared its performance to other methods for plasma, urine and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected nine biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick. Our results demonstrate that miRQuick is a rapid and efficient method for EV-miRNA extraction with excellent repeatability, making it suitable for various applications including cancer diagnosis.
PubMed: 38938899
DOI: 10.1002/jex2.126 -
Journal of Extracellular Biology Apr 2024Isolation of extracellular vesicles (EV) has been developing rapidly in parallel with the interest in EVs. However, commonly utilized protocols may not suit more...
Isolation of extracellular vesicles (EV) has been developing rapidly in parallel with the interest in EVs. However, commonly utilized protocols may not suit more challenging sample matrixes and could potentially yield suboptimal results. Knowing and assessing the pitfalls of isolation procedure to be used, should be involved to some extent for EV analytics. EVs in cow milk are of great interest due to their abundancy and large-scale availability as well as their cross-species bioavailability and possible use as drug carriers. However, the characteristics of milk EVs overlap with those of other milk components. This makes it difficult to isolate and study EVs individually. There exists also a lack of consensus for isolation methods. In this study, we demonstrated the differences between various differential centrifugation-based approaches for isolation of large quantities of EVs from cow milk. Samples were further purified with gradient centrifugation and size exclusion chromatography (SEC) and differences were analyzed. Quality measurements were conducted on multiple independent platforms. Particle analysis, electron microscopy and RNA analysis were used, to comprehensively characterize the isolated samples and to identify the limitations and possible sources of contamination in the EV isolation protocols. Vesicle concentration to protein ratio and RNA to protein ratios were observed to increase as samples were purified, suggesting co-isolation with major milk proteins in direct differential centrifugation protocols. We demonstrated a novel size assessment of vesicles using a particle mobility analyzer that matched the sizing using electron microscopy in contrast to commonly utilized nanoparticle tracking analysis. Based on the standards of the International Society for Extracellular Vesicles and the quick checklist of EV-Track.org for EV isolation, we emphasize the need for complete characterization and validation of the isolation protocol with all EV-related work to ensure the accuracy of results and allow further analytics and experiments.
PubMed: 38938848
DOI: 10.1002/jex2.149 -
JACS Au Jun 2024The characterization of intrinsically disordered regions (IDRs) in membrane-associated proteins is of crucial importance to elucidate key biochemical processes,...
The characterization of intrinsically disordered regions (IDRs) in membrane-associated proteins is of crucial importance to elucidate key biochemical processes, including cellular signaling, drug targeting, or the role of post-translational modifications. These protein regions pose significant challenges to powerful analytical techniques of molecular structural investigations. We here applied magic angle spinning solid-state nuclear magnetic resonance to quantitatively probe the structural dynamics of IDRs of membrane-bound α-synuclein (αS), a disordered protein whose aggregation is associated with Parkinson's disease (PD). We focused on the mitochondrial binding of αS, an interaction that has functional and pathological relevance in neuronal cells and that is considered crucial for the underlying mechanisms of PD. Transverse and longitudinal N relaxation revealed that the dynamical properties of IDRs of αS bound to the outer mitochondrial membrane (OMM) are different from those of the cytosolic state, thus indicating that regions generally considered not to interact with the membrane are in fact affected by the spatial proximity with the lipid bilayer. Moreover, changes in the composition of OMM that are associated with lipid dyshomeostasis in PD were found to significantly perturb the topology and dynamics of IDRs in the membrane-bound state of αS. Taken together, our data underline the importance of characterizing IDRs in membrane proteins to achieve an accurate understanding of the role that these elusive protein regions play in numerous biochemical processes occurring on cellular surfaces.
PubMed: 38938811
DOI: 10.1021/jacsau.4c00323 -
JACS Au Jun 2024MeXpose is an end-to-end image analysis pipeline designed for mechanistic studies of metal exposure, providing spatial single-cell metallomics using laser...
MeXpose is an end-to-end image analysis pipeline designed for mechanistic studies of metal exposure, providing spatial single-cell metallomics using laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS). It leverages the high-resolution capabilities of low-dispersion laser ablation setups, a standardized approach to quantitative bioimaging, and the toolbox of immunohistochemistry using metal-labeled antibodies for cellular phenotyping. MeXpose uniquely unravels quantitative metal bioaccumulation (sub-fg range per cell) in phenotypically characterized tissue. Furthermore, the full scope of single-cell metallomics is offered through an extended mass range accessible by ICP-TOFMS instrumentation (covering isotopes from / 14-256). As a showcase, an human skin model exposed to cobalt chloride (CoCl) was investigated. For the first time, metal permeation was studied at single-cell resolution, showing high cobalt (Co) accumulation in the epidermis, particularly in mitotic basal cells, which correlated with DNA damage. Significant Co deposits were also observed in vascular cells, with notably lower levels in dermal fibers. MeXpose provides unprecedented insights into metal bioaccumulation with the ability to explore relationships between metal exposure and cellular responses on a single-cell level, paving the way for advanced toxicological and therapeutic studies.
PubMed: 38938797
DOI: 10.1021/jacsau.4c00154 -
BioImpacts : BI 2024The endothelial cells derived from the human vein cord (HUVECs) are used as in-vitro models for studying cellular and molecular pathophysiology, drug and hormones...
INTRODUCTION
The endothelial cells derived from the human vein cord (HUVECs) are used as in-vitro models for studying cellular and molecular pathophysiology, drug and hormones transport mechanisms, or pathways. In these studies, the proliferation and quantity of cells are important features that should be monitored and assessed regularly. So rapid, easy, noninvasive, and inexpensive methods are favorable for this purpose.
METHODS
In this work, a novel method based on fast Fourier transform square-wave voltammetry (FFTSWV) combined with a 3D printed electrochemical cell including two inserted platinum electrodes was developed for non-invasive and probeless rapid in-vitro monitoring and quantification of human umbilical vein endothelial cells (HUVECs). The electrochemical cell configuration, along with inverted microscope images, provided the capability of easy use, online in-vitro monitoring, and quantification of the cells during proliferation.
RESULTS
HUVECs were cultured and proliferated at defined experimental conditions, and standard cell counts in the initial range of 12 500 to 175 000 were prepared and calibrated by using a hemocytometer (Neubauer chamber) counting for electrochemical measurements. The optimum condition, for FFTSWV at a frequency of 100 Hz and 5 mV amplitude, were found to be a safe electrochemical measurement in the cell culture medium. In each run, the impedance or admittance measurement was measured in a 5 seconds time window. The total measurements were fulfilled at 5, 24, and 48 hours after the seeding of the cells, respectively. The recorded microscopic images before every electrochemical assay showed the conformity of morphology and objective counts of cells in every plate well. The proposed electrochemical method showed dynamic linearity in the range of 12 500-265 000 HUVECs 48 hours after the seeding of cells.
CONCLUSION
The proposed electrochemical method can be used as a simple, fast, and noninvasive technique for tracing and monitoring of HUVECs population in in-vitro studies. This method is highly cheap in comparison with other traditional tools. The introduced configuration has the versatility to develop electrodes for the study of various cells and the application of other electrochemical designations.
PubMed: 38938755
DOI: 10.34172/bi.2023.28854 -
BioImpacts : BI 2024A new analytical method based on the coupling of microextraction and microfluidics was developed and investigated for the pre-concentration, separation, and...
INTRODUCTION
A new analytical method based on the coupling of microextraction and microfluidics was developed and investigated for the pre-concentration, separation, and electrochemical detection of fenitrothion (FT) and parathion (PA) at the sub-ppm concentrations.
METHODS
In the first step, the microchip capillary electrophoresis technique was used to serve as a separation and detection system. Analytes were injected in the 40 mm long microchannel with 10 mm sidearms. Then, they were separated by applying a direct electrical field (+1800 V) between the buffer and detection reservoirs. 2-(n-morpholino)ethanesulfonic acid (MES) buffer (20 mM, pH 5) was used as a running buffer. The electrochemical detection was performed using three Pt microelectrodes with the width of working, counter, and reference electrodes (50, 250, and 250 µm, respectively) in the out-channel approach.
RESULTS
The system was devised to have the optimum detection potential equal to -1.2 V vs. pseudo-reference electrode. The dimensions of the SU-8 channel have 20 µm depth and 50 µm width. In the second step, an air-assisted liquid-liquid microextraction technique was used to extract and preconcentration of analytes from human blood plasma. Then, 1, 2 di-bromoethan was used as extractant solvent, the analytes were preconcentrated, and the sedimented solvent (50 µL) was evaporated in a 60 ˚C water bath followed by substitution of running buffer containing 10% ethanol. The optimal extraction cycles were found to be 8 with adding 1% NaCl to the aqueous phase. Analyzing time of the mentioned analytes was less than 100s, the precision range was 3.3 - 8.2 with a linear range of 0.8-100 ppm and 1.2-100 ppm for FT and PA, respectively. The extraction recoveries were about 91% and 87% for FT and PA, respectively. The detection limits for FT and PA were 240 and 360 ppb, respectively. Finally, the reliability of the method was investigated by GC-FID.
CONCLUSION
The proposed method and device were validated and can be used as in situ and portable detection systems for detecting fenitrothion and parathion insecticides.
PubMed: 38938753
DOI: 10.34172/bi.2023.25288 -
Biomedical Reports Aug 2024Type 2 diabetes mellitus (T2DM) is a major global health problem. Response to first-line therapy is variable. This is partially due to interindividual variability across...
Type 2 diabetes mellitus (T2DM) is a major global health problem. Response to first-line therapy is variable. This is partially due to interindividual variability across those genes codifying transport, metabolising, and drug activation proteins involved in first-line pharmacological treatment. Single nucleotide polymorphisms (SNPs) of genes and affect metformin therapeutic response in patients with T2DM patients. The present study investigated allelic and genotypic frequencies of organic cation (OCT)1, OCT2, and OCT3 polymorphisms among metformin-treated patients with type 2 diabetes mellitus (T2DM). It also reports the association between clinical and genetic variables with glycated haemoglobin (HbA1c) control in 59 patients with T2DM. Patients were genotyped through real-time PCR (TaqMan assays). Metformin plasmatic levels were determined by mass spectrometry. Neither the analysis of HbA1c control by SNPs in , and , nor the dominant genotypic model analysis yielded statistical significance between genotypes in polymorphisms rs72552763 (P=0.467), rs622342 (P=0.221), rs316019 (P=0.220) and rs2076828 (P=0.215). HbA1c levels were different in rs72552763 [GAT/GAT, 6.0 (5.7-6.6), GAT/del=6.5 (6.2-9.0), del/del=6.5 (6.4-6.8); P=0.022] and rs622342 [A/A=6.0 (5.8-6.5), A/C=6.4 (6.1-7.7), C/C=6.8 (6.4-9.3); P=0.009] genotypes. The dominant genotypic model found the lowest HbA1c levels in GAT/GAT (P=0.005) and A/A (P=0.010), in rs72552763 (GAT/GAT vs. GAT/del + del/del) and rs622342 (A/A vs. A/C + CC), respectively. There was a significant correlation between HbA1c levels and metformin dosage amongst del allele carriers in rs72552763 (β=0.14, P<0.001, r=0.387), as opposed to GAT/GAT in rs72552763. There were no differences between HbA1c values in the test set and those predicted by machine learning models employing a simple linear regression based on metformin dosage. Therefore, rs72552763 and rs622342 polymorphisms in may affect metformin response determined by HbA1c levels in patients with T2DM. The del allele of SNP rs72552763 may serve as a metformin response biomarker.
PubMed: 38938740
DOI: 10.3892/br.2024.1806 -
Kidney Medicine Jul 2024In the wake of the coronavirus disease 2019 (COVID-19) pandemic, the United States federal government expanded originating telemedicine sites to include outpatient...
RATIONALE & OBJECTIVE
In the wake of the coronavirus disease 2019 (COVID-19) pandemic, the United States federal government expanded originating telemedicine sites to include outpatient dialysis units. For the first time, nephrology practitioners across the United States could replace face-to-face visits with telemedicine for patients receiving in-center hemodialysis. This study describes patients' perspectives on the use of telemedicine during in-center hemodialysis.
STUDY DESIGN
A qualitative study.
SETTING & PARTICIPANTS
Thirty-two patients from underserved populations (older, less educated, unemployed, persons of color) receiving in-center hemodialysis who used telemedicine with their nephrologist during the COVID-19 pandemic.
ANALYTICAL APPROACH
Telephone semistructured interviews were conducted in English or Spanish. Transcripts were thematically analyzed.
RESULTS
We identified 6 themes with subthemes: adapting to telemedicine (gaining familiarity and confidence, overcoming and resolving technical difficulties, and relying on staff for communication); ensuring availability of the physician (enabling an immediate response to urgent medical needs, providing peace of mind, addressing patient needs adequately, and enhanced attention and contact from physicians); safeguarding against infection (limiting COVID-19 exposures and decreasing use); straining communication and physical interactions (loss of personalized touch, limited physical examination, and unable to reapproach physicians about forgotten issues); maintaining privacy (enhancing privacy and projecting voice enables others to hear); and supporting confidence in telemedicine (requiring established rapport with physicians, clinical stabilty of health, and ability to have in-person visits when necessary).
LIMITATIONS
Interviews were conducted later in the pandemic when some nephrology care providers were using telemedicine infrequently.
CONCLUSIONS
Patients receiving in-center hemodialysis adapted to telemedicine visits by their nephrologists in the context of the COVID-19 pandemic and observed its benefits. However, further considerations regarding communication, privacy, and physical assessments are necessary. Integrating telemedicine into future in-center hemodialysis care using a hybrid approach could potentially build trust, optimize communication, and augment care.
PubMed: 38938646
DOI: 10.1016/j.xkme.2024.100848 -
Frontiers in Plant Science 2024Global warming has led to increased environmental stresses on plants, notably drought. This affects plant distribution and species adaptability, with some medicinal...
INTRODUCTION
Global warming has led to increased environmental stresses on plants, notably drought. This affects plant distribution and species adaptability, with some medicinal plants showing enhanced drought tolerance and increased medicinal components. In this pioneering study, we delve into the intricate tapestry of , a medicinal plant renowned for its resilience in arid environments. By fusing a rich historical narrative with cutting-edge analytical methodologies, this research endeavors to demystify the plant's intricate response to drought stress, illuminating its profound implications for medicinal valorization.
METHODS
The methodology includes a comprehensive textual research and resource investigation of , regionalization studies, field sample distribution analysis, transcriptome and metabolome profiling, rhizosphere soil microbiome analysis, and drought stress experiments. Advanced computational tools like ArcGIS, MaxEnt, and various bioinformatics software were utilized for data analysis and modeling.
RESULTS
The study identified significant genetic variations among samples from different regions, correlating with environmental factors, particularly precipitation during the warmest quarter (BIO18). Metabolomic analysis revealed marked differences in metabolite profiles, including shikonin content, which is crucial for the plant's medicinal properties. Soil microbial community analysis showed variations that could impact plant metabolism and stress response. Drought stress experiments demonstrated 's resilience and its ability to modulate metabolic pathways to enhance drought tolerance.
DISCUSSION
The findings underscore the complex interplay between genetic makeup, environmental factors, and microbial communities in shaping 's adaptability and medicinal value. The study provides insights into how drought stress influences the synthesis of active compounds and suggests that moderate stress could enhance the plant's medicinal properties. Predictive modeling indicates future suitable growth areas for , aiding in resource management and conservation efforts. The research contributes to the sustainable development of medicinal resources and offers strategies for improving the cultivation of .
PubMed: 38938629
DOI: 10.3389/fpls.2024.1395046 -
Health Care Science Dec 2023This study aims to develop and validate a Structured Training Effectiveness Evaluation (STEE) tool based on the Kirkpatrick model for newly graduated registered nurses...
BACKGROUND
This study aims to develop and validate a Structured Training Effectiveness Evaluation (STEE) tool based on the Kirkpatrick model for newly graduated registered nurses in the operating room in China.
METHODS
The first phase will involve focus group and individual interviews with nursing educators and newly graduated registered nurses selected using purposive sampling. The data will be analyzed thematically to identify key components necessary to develop the STEE tool. The second phase will develop and validate the STEE tool through a panel of experts using the Delphi method. The item weights will be determined with the analytic hierarchy process technique. The third phase will involve implementation and evaluation of the STEE tool with an exploratory, nonexperimental, and comparative analysis. Descriptive and inferential statistical analyses will be performed with SPSS version 23.
RESULTS
The STEE tool for newly graduated registered nurses in the operating room will be useful for evaluating training effectiveness during standardized training. The results obtained with this tool will clarify the effectiveness of training, thereby helping transform nursing students into competent nurses.
CONCLUSION
In this way, this study will provide practical guidance for improving standardized training programs and help newly graduated nurses manage their transition to the clinical work environment and remain in their posts.
PubMed: 38938624
DOI: 10.1002/hcs2.75