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Background This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein ( SREHP ) gene as study model. Results A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica . This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites. Conclusions The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

This study addressed limitations in calcein-AM-based endothelial viability assays, specifically focusing on pre-stripped DMEK grafts. Key challenges included the suboptimal calcein staining and the incompatibility of the viability assay with subsequent immunofluorescence (IF). Using human corneal grafts, we employed two strategies to optimize calcein staining. Firstly, we improved calcein staining in corneal endothelium by adjusting calcein-AM concentration and diluent, resulting in a threefold increase in fluorescence intensity with 4 µM calcein in Opti-MEM compared to the conventional 2 µM calcein in PBS. Secondly, introducing the trypan blue (TB) post-viability assay greatly reduced non-specific fluorescence, enhancing the contrast of calcein staining. This improvement significantly and importantly decreased both inter-operator’s variability and the time required for viability counting. For the subsequent double IF, an extensive wash is recommended on the fixed and permeabilized graft after the viability assay, which was carried out using Hoechst-Calcein (HC) labeling. The simple technical tips outlined in this study are not only effective for pre-stripped DMEK grafts but may also prove beneficial for other types of corneal grafts, such as PK and DSAEK.

Curcumin, an important constituent of turmeric, is known for various biological activities, primarily due to its antioxidant mechanism. The present study focused on the antibacterial activity of curcumin I, a significant component of commercial curcumin, against four genera of bacteria, including those that are Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa). These represent prominent human pathogens, particularly in hospital settings. Our study shows the strong antibacterial potential of curcumin I against all the tested bacteria from Gram-positive as well as Gram-negative groups. The integrity of the bacterial membrane was checked using two differential permeabilization indicating fluorescent probes, namely, propidium iodide and calcein. Both the membrane permeabilization assays confirmed membrane leakage in Gram-negative and Gram-positive bacteria on exposure to curcumin I. In addition, scanning electron microscopy and fluorescence microscopy were employed to confirm the membrane damages in bacterial cells on exposure to curcumin I. The present study confirms the broad-spectrum antibacterial nature of curcumin I, and its membrane damaging property. Findings from this study could provide impetus for further research on curcumin I regarding its antibiotic potential against rapidly emerging bacterial pathogens.

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell‐derived extracellular vesicles (EVs). Here we demonstrate that nano‐sized EVs from therapy‐grade human placental‐expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell‐secreted soluble factors via tangential flow‐filtration (TFF) and subtractive tandem mass‐tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein‐based flow cytometry, super‐resolution and electron microscopy verified EV identity. PLX‐EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose‐dependently inhibited T cell proliferation in vitro. Corona removal by size‐exclusion or ultracentrifugation abrogated angiogenesis. Re‐establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super‐resolution microscopy, electron microscopy and zeta‐potential shift, and served as a proof‐of‐concept. Understanding EV corona formation will improve rational EV‐inspired nano‐therapy design.

This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn 2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.

Objectives With age, bone marrow mesenchymal stem cells (BMSC) have reduced ability of differentiating into osteoblasts but have increased ability of differentiating into adipocytes which leads to age‐related bone loss. MicroRNAs (miRNAs) play major roles in regulating BMSC differentiation. This paper explored the role of miRNAs in regulating BMSC differentiation swift fate in age‐related osteoporosis. Material and methods Mice and human BMSC were isolated from bone marrow, whose miR‐130a level was measured. The abilities of BMSC differentiate into osteoblast or fat cell under the transfected with agomiR‐130a or antagomiR‐130a were analysed by the level of ALP, osteocalcin, Runx2, osterix or peroxisome proliferator‐activated receptorγ (PPARγ), Fabp4. Related mechanism was verified via qT‐PCR, Western blotting (WB) and siRNA transfection. Animal phenotype intravenous injection with agomiR‐130a or agomiR‐NC was explored by Micro‐CT, immunochemistry and calcein double‐labelling. Results MiR‐130a was dramatically decreased in BMSC of advanced subjects. Overexpression of miR‐130a increased osteogenic differentiation of BMSC and attenuated adipogenic differentiation in BMSC, conversely, Inhibition of miR‐130a reduced osteogenic differentiation and facilitated lipid droplet formation. Consistently, overexpression of miR‐130a in elderly mice dropped off the bone loss. Furthermore, the protein levels of Smad regulatory factors 2 (Smurf2) and PPARγ were regulated by miR‐130a with an negative effect through directly combining the 3'UTR of Smurf2 and PPARγ. Conclusions The results indicated that miR‐130a promotes osteoblastic differentiation of BMSC by negatively regulating Smurf2 expression and suppresses adipogenic differentiation of BMSC by targeting the PPARγ, and supply a new target for clinical therapy of age‐related bone loss.

Accurately assessing biofilm viability is essential for evaluating both biofilm formation and the efficacy of antibacterial treatments. Traditional SYTO9 and propidium iodide (PI) live/dead staining in biofilm viability assays often ace challenges due to non-specific staining, limiting precise differentiation between live and dead cells. To address this limitation, we investigated an alternative staining method employing calcein acetoxymethyl (CAM) to detect viable cells based on esterase activity, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) to assess the remaining biofilm population. Biofilms of , , and were matured and exposed to varying concentrations of antibiotics or sterile medium. Biofilm viability was assessed using CAM/TMA-DPH or SYTO9/PIstaining, followed by analysis with confocal laser scanning microscopy (CLSM) and ImageJ-based biofilm surface coverage quantification. Viability findings were compared with colony-forming units (CFU/mL), a standard microbial viability measure. CAM/TMA-DPH staining demonstrated strong positive correlations with CFU counts across all bacterial species ( = 0.59 - 0.91), accurately reflecting biofilm vitality. In contrast, SYTO9/PI staining consistently underestimated the viability of untreated biofilms, particularly in , where a negative correlation with CFU/mL was observed ( = -0.04). Positive correlations for SYTO9/PI staining were noted in other species ( = 0.65 - 0.79). These findings underscore the limitations of membrane integrity-based staining methods and highlight the advantages of metabolic-based probes like CAM/TMA-DPH. Our findings suggest that CAM/TMA-DPH staining provides a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms, highlighting the advantages of metabolic-based probes over traditional membrane integrity assays. The consistency of CAM/TMA-DPH staining across different bacterial species underscores its potential to advance studies on biofilm and contribute to the development of more effective anti-biofilm treatments, which is essential for clinical management of biofilm-associated infections.

Coronavirus with intact infectivity attached to PPE surfaces pose significant threat to the spread of COVID-19. We tested the hypothesis that an electroceutical fabric, generating weak potential difference of 0.5 V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Porcine respiratory coronavirus AR310 particles (10 5 ) were placed in direct contact with the fabric for 1 or 5 min. Following one minute of contact, zeta potential of the porcine coronavirus was significantly lowered indicating destabilization of its electrokinetic properties. Size-distribution plot showed appearance of aggregation of the virus. Testing of the cytopathic effects of the virus showed eradication of infectivity as quantitatively assessed by PI-calcein and MTT cell viability tests. This work provides the rationale to consider the studied electroceutical fabric, or other materials with comparable property, as material of choice for the development of PPE in the fight against COVID-19.

Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry. Flow cytometry (A60-Micro, Apogee) was used to evaluate the ability of the generic EV markers calcein acetoxymethyl ester, calcein acetoxymethyl ester violet, carboxyfluorescein succinimidyl ester (CFSE), 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium (di-8-ANEPPS), and lactadherin to stain EVs from MCF7 human breast adenocarcinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM )] or platelet EVs from human plasma [integrin β3 positive (CD61 )]. Side scatter triggering was applied as a reference, and the influence of non-EV components (proteins and lipoproteins) was evaluated. Di-8-ANEPPS, lactadherin, and side scatter detected 100% of EpCAM MCF7 EVs. Lactadherin and side scatter detected 33% and 61% of CD61 EVs, respectively. Di-8-ANEPPS detected platelet EVs only if soluble protein was first removed. Because all generic markers stained proteins, at best 33% of platelet EVs in plasma were detected. The calcein markers and CFSE were either insensitive to EVs in both samples or associated with swarm detection. None of the generic markers detected all and only EVs in plasma. Side scatter triggering detected the highest concentration of plasma EVs on our A60-Micro, followed by lactadherin. The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.

Microneedle arrays (MA) have been extensively investigated in recent decades for transdermal drug delivery due to their pain-free delivery, minimal skin trauma, and reduced risk of infection. However, porous MA received relatively less attention due to their complex fabrication process and ease of fracturing. Here, we present a titanium porous microneedle array (TPMA) fabricated by modified metal injection molding (MIM) technology. The sintering process is simple and suitable for mass production. TPMA was sintered at a sintering temperature of 1250°C for 2 h. The porosity of TPMA was approximately 30.1% and its average pore diameter was about 1.3 μm. The elements distributed on the surface of TPMA were only Ti and O, which may guarantee the biocompatibility of TPMA. TPMA could easily penetrate the skin of a human forearm without fracture. TPMA could diffuse dry Rhodamine B stored in micropores into rabbit skin. The cumulative permeated flux of calcein across TPMA with punctured skin was 27 times greater than that across intact skin. Thus, TPMA can continually and efficiently deliver a liquid drug through open micropores in skin.