Determination of L-selectin in blood plasma using DNA aptamer-based surface-enhanced Raman spectroscopy assay.

In the human body, tumor cell occurrence can be indirectly monitored using the L-selectin concentration in the blood, since selectin ligands are present on the surface of tumor cells, and with tumor progression, a decrease in L-selectin levels can be expected and observed. In this study, we present a selective DNA-based surface-enhanced Raman spectroscopy (SERS) assay for the detection and determination of L-selectin in biological samples. Two calibration curves (linear in the 40-190 ng mL-1 region and exponential in the 40-500 ng mL-1 region) are fitted to the obtained SERS experimental data, i.e., the ratio of I732/I1334 band intensities (LOQ = 46 ng mL-1). Calculated determination coefficients are found to be R2 = 0.997 for the linear region of the calibration curve and R2 = 0.977 for the exponential region. Moreover, we demonstrate very good selectivity of the assay even in the presence of P- and E-selectin in a sample containing L-selectin. With our SERS assay, the L-selectin concentration in biological samples can be estimated directly from the calibration curves.

miRNA Signature of Urine Extracellular Vesicles Shows the Involvement of Inflammatory and Apoptotic Processes in Diabetic Chronic Kidney Disease.

BACKGROUND: The aim of this study was to investigate the role of urine-derived extracellular vesicles (uEVs) in diabetic kidney disease (DKD) in patients diagnosed with type 2 diabetes mellitus (T2DM). METHODS: UEVs were characterized by size distribution and microRNA content by next-generation small RNA sequencing and quantitative reverse transcription PCR. RESULTS: A subset of sixteen miRNAs enriched in T2DM patients with DKD, including hsa-miR-514a-5p, hsa-miR­451a, hsa-miR-126-3p, hsa-miR-214, or hsa-miR­503 was identified. Eight miRNAs as hsa-miR-21-3p, hsa-miR-4792, hsa-miR­375, hsa-miR-1268a, hsa-miR-501-5p, or hsa-miR-582 were downregulated. Prediction of potential target genes and pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed possible functions related to cellular processes such as apoptosis, inflammation, and tissue remodeling, that promote diabetic complications, such as DKD. Among them, hsa-miR-375, hsa-miR-503, and hsa-miR-451a make important contribution. Additionally, downregulated hsa-miR-582-5p has not been reported so far in any diabetes-related pathways. CONCLUSIONS: This study revealed the most significant miRNAs in uEVs of patients with T2DM. However, as this is a bioinformatic prediction that we performed based on the putative targets of the identified miRNAs. Thus, further in vitro functional studies are needed to confirm our findings. Knowing the fact that EVs are crucial in transferring miRNAs, there is a great need toto discover their involvement in the pathomechanism of T2DM-related kidney disease.

A National Registry-Based Epidemiological Study to Evaluate 395 646 Patients Hospitalized Due to Eye Diseases in Poland in 2019.

BACKGROUND The Nationwide General Hospital Morbidity Study Registry collects demographic, health, and medical data on patients hospitalized throughout Poland, and acts as a registry for epidemiological, public health, and hospital administrative studies. This epidemiological national registry-based study aimed to evaluate the characteristics of 395 646 patients hospitalized due to eye diseases in Poland in 2019. MATERIAL AND METHODS This study is a retrospective nationwide registry-based analysis. Data on all patients hospitalized due to eye diseases in 2019 were obtained from the Nationwide General Hospital Morbidity Study Registry. Demographic characteristics of patients hospitalized due to eye diseases as well as the hospitalization course were analyzed using descriptive epidemiology tools. RESULTS Disorders of the lens accounted for 68.6% of all hospital admissions, and disorders of the choroid and retina were the second most common (13.9%) cause of hospital admission due to eye diseases in 2019. Over three-quarters of hospital admissions (77.4%) lasted less than 24 h, and 86.8% were scheduled admissions. Most of the patients hospitalized due to eye diseases were females (59.9%) and over 70% lived in urban areas. There were regional differences in the hospital admissions rate per 100 000 inhabitants. According to the data presented in the registry, less than one-third of patients had comorbidities. CONCLUSIONS Hospital ophthalmological care in Poland is based mainly on planned hospitalizations lasting less than 24 h and related to ophthalmological surgery. Data on the comorbidity of ophthalmologic patients suggest that ophthalmologists do not pay enough attention to identifying and recording the patient's health status.

An Online Questionnaire-Based Survey of 1076 Individuals in Poland to Identify the Prevalence of Ophthalmic Symptoms in Autumn 2022.

BACKGROUND Vision health affects functioning in society, and the ability to learn and work. Ophthalmic symptoms may be caused by eye diseases, but also by environmental or lifestyle factors. This online questionnaire-based survey aimed to identify the prevalence of ophthalmic symptoms in 1076 individuals in Poland, as well as to identify factors associated with the presence of ophthalmic symptoms. MATERIAL AND METHODS An online questionnaire-based survey was carried out in December 2022 on a representative sample of 1076 adult Poles. Non-probability quota sampling was used. Respondents were asked about the presence of 16 different eye symptoms and vision problems in the last 30 days. The presence of ophthalmic symptoms was self-declared. Data were analyzed with IBM SPSS package version 28. RESULTS More than half of the respondents (57.8%) had at least 1 ophthalmic symptom in the last 30 days. Burning and stinging eyes (21.6%) and dry eyes (18.9%) were the most common ophthalmic symptoms declared by the respondents. Moreover, 21.3% of respondents reported vision deterioration in the last 30 days. Out of 10 different factors analyzed in this study, female gender, living in rural areas or small cities (below 100 000 inhabitants), living with at least 1 other person, having low economic status, having chronic diseases, and wearing spectacles/contact lenses were significantly associated (P.

SERS Signature of SARS-CoV-2 in Saliva and Nasopharyngeal Swabs: Towards Perspective COVID-19 Point-of-Care Diagnostics.

In this study, the intrinsic surface-enhanced Raman spectroscopy (SERS)-based approach coupled with chemometric analysis was adopted to establish the biochemical fingerprint of SARS-CoV-2 infected human fluids: saliva and nasopharyngeal swabs. The numerical methods, partial least squares discriminant analysis (PLS-DA) and support vector machine classification (SVMC), facilitated the spectroscopic identification of the viral-specific molecules, molecular changes, and distinct physiological signatures of pathetically altered fluids. Next, we developed the reliable classification model for fast identification and differentiation of negative CoV(-) and positive CoV(+) groups. The PLS-DA calibration model was described by a great statistical value-RMSEC and RMSECV below 0.3 and R2cal at the level of ~0.7 for both type of body fluids. The calculated diagnostic parameters for SVMC and PLS-DA at the stage of preparation of calibration model and classification of external samples simulating real diagnostic conditions evinced high accuracy, sensitivity, and specificity for saliva specimens. Here, we outlined the significant role of neopterin as the biomarker in the prediction of COVID-19 infection from nasopharyngeal swab. We also observed the increased content of nucleic acids of DNA/RNA and proteins such as ferritin as well as specific immunoglobulins. The developed SERS for SARS-CoV-2 approach allows: (i) fast, simple and non-invasive collection of analyzed specimens; (ii) fast response with the time of analysis below 15 min, and (iii) sensitive and reliable SERS-based screening of COVID-19 disease.

Methodological Aspects of Green Extraction of Usnic Acid Using Natural Deep Eutectic Solvents.

Usnic acid (UA) is a compound with multiple biological activities that make it useful in various industries, e.g., pharmaceutical, cosmetic, dentistry, and agricultural sectors. Lichens are the primary source of UA, which is primarily extracted using acetone. This study aimed to investigate the solubility of UA in numerous natural deep eutectic solvents (NADESs) and use a mixture of thymol and camphor as a NADES in the optimization of the UA extraction process with the design of experiments method. For numerical optimization, the following parameters were employed in the experiment to confirm the model: a camphor-to-thymol ratio of 0.3, a liquid-to-solid ratio of 60, and a time of 30 min. The obtained experimental results aligned well with the predicted values, with the mean experimental value falling within the confidence interval, exhibiting deviations between 11.93 and 14.96. By employing this model, we were able to optimize the extraction procedure, facilitating the isolation of approximately 91% of the total UA content through a single extraction, whereas a single acetone extraction yielded only 78.4% of UA.

Raman spectroscopy of urinary extracellular vesicles to stratify patients with chronic kidney disease in type 2 diabetes.

In this study, we verified the hypothesis that Raman signature of urinary extracellular vesicles (UEVs) can be used to stratify patients with diabetes at various stages of chronic kidney disease (CKD). Patients with type 2 diabetes diagnosed with different stages of CKD and healthy subjects were enrolled in the study. UEVs were isolated using low-vacuum filtration followed by ultracentrifugation. Correlation analysis, multiple linear regression and principal component analysis were used to find differences between spectral fingerprints of UEVs derived from both groups of patients. Electron microscopy and nanoparticle tracking analysis were applied to characterize the size and morphology of UEVs. We observed significant correlations between selected Raman bands measured for UEVs and clinical parameters. We found significant differences in the area under the specific bands originating mainly from proteins and lipids between the study groups. Based on the tryptophan and amide III bands, we were able to predict the estimated glomerular filtration rate (eGFR). Principal component analysis, partial least squares regression (PLSR) and correlation analysis of the UEV Raman spectra supported the results obtained from the direct analysis of Raman spectra. Our analysis revealed that PLSR and a regression model including tryptophan and amide III bands allows to estimate the value of eGFR.

Protein-Mediated Transformations of Superparamagnetic Nanoparticles Evidenced by Single-Particle Inductively Coupled Plasma Tandem Mass Spectrometry: A Disaggregation Phenomenon.

Progress toward translating superparamagnetic iron oxide nanoparticles (SPIONs) with specific diagnostic and therapeutic properties for clinical applications depends on developing and implementing appropriate methodologies that would allow in-depth characterizations of their behavior in a real biological environment. Herein, we report a versatile approach for studying interactions between SPIONs and proteins using single-particle inductively coupled plasma tandem mass spectrometry. By monitoring the changes in the size distribution upon exposure to human serum, the formation of stable protein corona is revealed, accompanied by particle disaggregation.

Comparative Evaluation of Different Surface Coatings of Fe3O4-Based Magnetic Nano Sorbent for Applications in the Nucleic Acids Extraction.

Nucleic acid extraction and purification are crucial steps in sample preparation for multiple diagnostic procedures. Routine methodologies of DNA isolation require benchtop equipment (e.g., centrifuges) and labor-intensive steps. Magnetic nanoparticles (MNPs) as solid-phase sorbents could simplify this procedure. A wide range of surface coatings employs various molecular interactions between dsDNA and magnetic nano-sorbents. However, a reliable, comparative evaluation of their performance is complex. In this work, selected Fe3O4 modifications, i.e., polyethyleneimine, gold, silica, and graphene derivatives, were comprehensively evaluated for applications in dsDNA extraction. A family of single batch nanoparticles was compared in terms of morphology (STEM), composition (ICP-MS/MS and elemental analysis), surface coating (UV-Vis, TGA, FTIR), and MNP charge (ζ-potential). ICP-MS/MS was also used to unify MNPs concentration allowing a reliable assessment of individual coatings on DNA extraction. Moreover, studies on adsorption medium (monovalent vs. divalent ions) and extraction buffer composition were carried out. As a result, essential relationships between nanoparticle coatings and DNA adsorption efficiencies have been noticed. Fe3O4@PEI MNPs turned out to be the most efficient nano sorbents. The optimized composition of the extraction buffer (medium containing 0.1 mM EDTA) helped avoid problems with Fe3+ stripping, which improved the validity of the spectroscopic determination of DNA recovery.

SERS-PLSR Analysis of Vaginal Microflora: Towards the Spectral Library of Microorganisms.

The accurate identification of microorganisms belonging to vaginal microflora is crucial for establishing which microorganisms are responsible for microbial shifting from beneficial symbiotic to pathogenic bacteria and understanding pathogenesis leading to vaginosis and vaginal infections. In this study, we involved the surface-enhanced Raman spectroscopy (SERS) technique to compile the spectral signatures of the most significant microorganisms being part of the natural vaginal microbiota and some vaginal pathogens. Obtained data will supply our still developing spectral SERS database of microorganisms. The SERS results were assisted by Partial Least Squares Regression (PLSR), which visually discloses some dependencies between spectral images and hence their biochemical compositions of the outer structure. In our work, we focused on the most common and typical of the reproductive system microorganisms (Lactobacillus spp. and Bifidobacterium spp.) and vaginal pathogens: bacteria (e.g., Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae), fungi (e.g., Candida albicans, Candida glabrata), and protozoa (Trichomonas vaginalis). The obtained results proved that each microorganism has its unique spectral fingerprint that differentiates it from the rest. Moreover, the discrimination was obtained at a high level of explained information by subsequent factors, e.g., in the inter-species distinction of Candida spp. the first three factors explain 98% of the variance in block Y with 95% of data within the X matrix, while in differentiation between Lactobacillus spp. and Bifidobacterium spp. (natural flora) and pathogen (e.g., Candida glabrata) the information is explained at the level of 45% of the Y matrix with 94% of original data. PLSR gave us insight into discriminating variables based on which the marker bands representing specific compounds in the outer structure of microorganisms were found: for Lactobacillus spp. 1400 cm-1, for fungi 905 and 1209 cm-1, and for protozoa 805, 890, 1062, 1185, 1300, 1555, and 1610 cm-1. Then, they can be used as significant marker bands in the analysis of clinical subjects, e.g., vaginal swabs.

Combined negative dielectrophoresis with a flexible SERS platform as a novel strategy for rapid detection and identification of bacteria.

Surface-enhanced Raman spectroscopy (SERS) is a vibrational method successfully applied in analytical chemistry, molecular biology and medical diagnostics. In this article, we demonstrate the combination of the negative dielectrophoretic (nDEP) phenomenon and a flexible surface-enhanced Raman platform for quick isolation (3 min), concentration and label-free identification of bacteria. The platform ensures a strong enhancement factor, high stability and reproducibility for the SERS response of analyzed samples. By introducing radial dielectrophoretic forces directed at the SERS platform, we can efficiently execute bacterial cell separation, concentration and deposition onto the SERS-active surface, which simultaneously works as a counter electrode and thus enables such hybrid DEP-SERS device vibration-based detection. Additionally, we show the ability of our DEP-SERS system to perform rapid, cultivation-free, direct detection of bacteria in urine and apple juice samples. The device provides new opportunities for the detection of pathogens.

Tryptophan Pathway Abnormalities in a Murine Model of Hereditary Glaucoma.

BACKGROUND: It has been shown that a possible pathogenetic mechanism of neurodegeneration in the mouse model of glaucoma (DBA/2J) may be an alteration of kynurenic acid (KYNA) in the retina. This study aimed to verify the hypothesis that alterations of tryptophan (TRP) metabolism in DBA/2J mice is not limited to the retina. METHODS: Samples of the retinal tissue and serum were collected from DBA/2J mice (6 and 10 months old) and control C57Bl/6 mice of the same age. The concentration of TRP, KYNA, kynurenine (KYN), and 3-hydroxykynurenine (3OH-K) was measured by HPLC. The activity of indoleamine 2,3-dioxygenase (IDO) was also determined as a KYN/TRP ratio. RESULTS: TRP, KYNA, L-KYN, and 3OH-K concentration were significantly lower in the retinas of DBA/2J mice than in C57Bl/6 mice. 3OH-K concentration was higher in older mice in both strains. Serum TRP, L-KYN, and KYNA concentrations were lower in DBA/2J than in age-matched controls. However, serum IDO activity did not differ significantly between compared groups and strains. CONCLUSIONS: Alterations of the TRP pathway seem not to be limited to the retina in the murine model of hereditary glaucoma.

Influence of Trace Elements on Neurodegenerative Diseases of The Eye-The Glaucoma Model.

Glaucoma is a heterogeneous group of chronic neurodegenerative disorders characterized by a relatively selective, progressive damage to the retinal ganglion cells (RGCs) and their axons, which leads to axon loss and visual field alterations. To date, many studies have shown the role of various elements, mainly metals, in maintaining the balance of prooxidative and antioxidative processes, regulation of fluid and ion flow through cell membranes of the ocular tissues. Based on the earlier and current research results, their relationship with the development and progression of glaucoma seems obvious and is increasingly appreciated. In this review, we aimed to summarize the current evidence on the role of trace elements in the pathogenesis and prevention of glaucomatous diseases. Special attention is also paid to the genetic background associated with glaucoma-related abnormalities of physiological processes that regulate or involve the ions of elements considered as trace elements necessary for the functioning of the cells.

Design and Optimization of a Biosensor Surface Functionalization to Effectively Capture Urinary Extracellular Vesicles.

For this study, we tested and optimized silicon surface functionalization procedures for capturing urinary extracellular vesicles (uEVs). The influence of the silane type (APTES or GOPS) and protein concentration on the efficiency of uEVs binding was investigated. Human lactadherin protein (LACT) was used to capture uEVs. We applied surface characterization techniques, including ellipsometry, atomic force microscopy, and time-of-flight secondary ion mass spectrometry, to observe changes in the biosensor surface after each functionalization step. uEVs were purified by a low-vacuum filtration method and concentrated by ultracentrifugation. The physical parameters of uEVs after the isolation procedure, such as morphology and size distribution, were determined using transmission electron microscopy and tunable resistive pulse sensing methods. We observed a gradual growth of the molecular layer after subsequent stages of modification of the silicon surface. The ToF-SIMS results showed no changes in the mean intensities for the characteristic peaks of amino acids and lipids in positive and negative polarization, in terms of the surface-modifying silane (APTES or GOPS) used. The most optimal concentration of LACT for the tested system was 25 µg/mL.

Analytical Problems with Preparation of Paraspinal Tissues from Patients with Spinal Fusion for Analysis of Titanium.

Preparation of paraspinal tissue of patients with implants for elemental analysis is a challenge because it contains titanium in the ionic form, as well as metallic debris. Most literature reports focus on dissolving the tissue, but the impact of digestion conditions on metallic debris of Ti has not been investigated. In our work, various digestion conditions, including systems, compositions of oxidising mixture, and time, were tested aiming (i) to digest the tissue without digestion of metallic titanium to quantify soluble Ti and (ii) to digest metallic titanium debris to asses total Ti content in tissue. The experiments were performed in a closed mode using a microwave-assisted system and a carbon heating block. Our study revealed that total digestion of titanium was impossible in the tested conditions and the maximal level of digested titanium was below 70%. The mineralisation with the use of concentrated nitric acid was optimal to prepare paraspinal samples to analyse the soluble titanium form because metallic titanium passivated and did not migrate to the solution. The elaborated conditions were applied to determine titanium ion in the periimplant tissue of patients with three different titanium-based surgical systems, including traditional growing rod (TGR), guided growth systems (GGS), and vertical expandable prosthesis titanium rib (VEPTR).

Using a lactadherin-immobilized silicon surface for capturing and monitoring plasma microvesicles as a foundation for diagnostic device development.

Microvesicles (MVs) are found in several types of body fluids and are promising disease biomarkers and therapeutic targets. This study aimed to develop a novel biofunctionalized surface for binding plasma microvesicles (PMVs) based on a lab-on-a-chip (LOC) approach. A new lactadherin (LACT)-functionalized surface was prepared and examined for monitoring PMVs. Moreover, two different strategies of LACT immobilization on a silicon surface were applied to compare different LACT orientations. A higher PMV to LACT binding efficiency was observed for LACT bonded to an αvß3 integrin-functionalized surface compared with that for LACT directly bonded to a glutaraldehyde-modified surface. Effective binding of PMVs and its components for both LACT immobilization strategies was confirmed using spectral ellipsometry and time-of-flight secondary ion mass spectrometry methods. The proposed PMV capturing system can be used as a foundation to design novel point-of-care (POC) diagnostic devices to detect and characterize PMVs in clinical samples. Graphical Abstract.

Phenolic Constituents of Lamium album L. subsp. album Flowers: Anatomical, Histochemical, and Phytochemical Study.

Flos Lamii albi has a high biological activity and is widely used in herbal medicine. The aim of the study was to characterize the secretory structures present in Lamium album subsp. album corolla and the location of phenolic compounds. Additionally, we carried out qualitative phytochemical analyses of flavonoids and phenolic acids. Light, fluorescence, and scanning electron microscopy were used to analyze the structure of the floral organs. The main classes of phenolic compounds and their localization were determined histochemically. Phytochemical analyses were performed with high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). Six types of glandular trichomes were found which contained flavonoids, phenolic acids, and tannins. The phytochemical studies demonstrated the presence of caffeic, chlorogenic, ferulic, gallic, p-coumaric, protocatechuic, syringic, gentisic, and vanillic phenolic acids as well as rutoside, isoquercetin, and quercetin flavonoids. The corolla in L. album subsp. album has antioxidant properties due to the presence of various polyphenols, as shown by the histo- and phytochemical analyses. The distribution and morphology of trichomes and the content of phenolic compounds in the corolla have taxonomic, pharmacognostic, and practical importance, facilitating the identification of the raw material.

Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures.

The surface-enhanced Raman spectroscopy (SERS)-based analysis of bacteria suffers from the lack of a standard SERS detection protocol (type of substrates, excitation frequencies, and sampling methodologies) that could be employed throughout laboratories to produce repeatable and valuable spectral information. In this work, we have examined several factors influencing the spectrum and signal enhancement during SERS studies conducted on both Gram-negative and Gram-positive bacterial species: Escherichia coli and Bacillus subtilis, respectively. These factors can be grouped into those which are related to the structure and types of plasmonic systems used during SERS measurements and those that are associated with the culturing conditions, types of culture media, and method of biological sample preparation.

Correction to: Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures.

The authors would like to call the reader's attention to the fact that unfortunately following information was missing in the original article: "Evelin Witkowska is supported by the Foundation of Polish Science (FNP)."

Raman spectral signatures of urinary extracellular vesicles from diabetic patients and hyperglycemic endothelial cells as potential biomarkers in diabetes.

Raman spectroscopy was applied to the measurement of urinary and in vitro endothelium-derived extracellular vesicles (EVs) isolated by hydrostatic filtration dialysis (HFD) method. Raman spectra obtained for urinary EVs (UEVs) showed distinct differences in the fingerprint region. In contrast, average Raman spectra of endothelium-derived EVs samples were almost identical. Cluster Analysis of UEVs significantly discriminated diabetic samples from control, moreover endothelium-derived EVs revealed stronger similarity between long hyperglycemia and normoglycemia samples compared to short hyperglycemia. Results obtained from Partial Least Squares analysis corresponded well with integral intensities of selected bands. Our proof-of-concept approach demonstrates the potential for Raman spectroscopy to be used both for identification of EVs molecular signatures in urine samples from patients with type 2 diabetes mellitus and good glycemic control and unsatisfactory glycemic control as well as for in vitro hyperglycemic model. This noninvasive technique may be useful in identifying new biomarkers of diabetes and renal complications.