Fluorescence Microscopy Research Articles

Multivariate Analysis of Individual Bacterial Outer Membrane Vesicles Using Fluorescence Microscopy.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. OMVs have emerged as promising therapeutic agents for various biological applications such as vaccines and targeted drug delivery. However, the full potential of OMVs is currently constrained by inherent heterogeneities, such as size and cargo differences, and traditional ensemble assays are limited in their ability to reveal OMV heterogeneity. To overcome this issue, we devised an innovative approach enabling the identification of various characteristics of individual OMVs. This method, employing fluorescence microscopy, facilitates the detection of variations in size and surface markers. To demonstrate our method, we utilize the oral bacterium Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) which produces OMVs with a bimodal size distribution. As part of its virulence, A. actinomycetemcomitans secretes leukotoxin (LtxA) in two forms: soluble and surface associated with the OMVs. We observed a correlation between the size and toxin presence where larger OMVs were much more likely to possess LtxA compared to the smaller OMVs. In addition, we noted that, among the smallest OMVs (<100 nm diameter), the fractions that are toxin positive range from 0 to 30%, while the largest OMVs (>200 nm diameter) are between 70 and 100% toxin positive.

Microfiber emission from a municipal wastewater treatment plant in Hungary.

Since the ingestion of both natural and anthropogenic microfibers produces a deleterious effect on aquatic organisms, it is crucial to explore the emission of these pollutants by WWTPs into the receiving water bodies, such as rivers. Cellulose- and petroleum-based microfibers, as well as microplastic particles, were collected from the effluent of a municipal WWTP operating with activated sludge technology in Budapest, Hungary. During two sampling campaigns organized in February and April of 2023 on different working days and at different times of the day, 123-145 L of effluent was sieved and filtered. The organic matter was removed by hydrogen-peroxide treatment. All fibers and particles larger than 10µm were counted, and using a fluorescence microscope, the fibers were geometrically characterized in terms of length and diameter. Each fiber was individually identified by transflection-FT-IR method. The fiber concentration varied in the range of 1.88-2.84 and 4.25-6.79 items/L during the 7th and the 16th week of 2023, respectively. In February and April, the proportion of microfibers in the solid particles was 78.3 and 94.7%, respectively. In the effluent the cellulose-based microfibers were dominant (53-91%), while among the petroleum-based microfibers, polyester occurred most often. The median length of cellulose-based fibers was considerably higher in April than in February (650 vs. 1250µm), and simultaneously the median diameter also increased from 21 to 29µm. This behaviour was also seen, albeit to a lesser extent, in connection to microfibers derived from petroleum. The treated wastewater's daily microfiber transport to the Danube River varied between 0.44 -0.69 and 0.94-1.53 billion in February and April 2023, respectively.

Mucin-Inspired Polymeric Fibers for Herpes Simplex Virus Type 1 Inhibition.

Mucus lines the epithelial cells at the biological interface and is the first line of defense against multiple viral infections. Mucins, the gel-forming components of mucus, are high molecular weight glycoproteins and crucial for preventing infections by binding pathogens. Consequently, mimicking mucins is a promising strategy for new synthetic virus inhibitors. In this work, synthetic mucin-inspired polymers (MIPs) as potential inhibitors of herpes simplex virus 1 (HSV-1) are investigated. By using a telechelic reversible addition-fragmentation chain-transfer (RAFT) polymerization technique, a new dendronized polysulfatep(G1AAm-OSO3)PDSwith an amide-backbone similar to the native mucin glycoproteins is synthesized.p(G1AAm-OSO3)PDSshows mucin-like elongated fiber structure, as revealed in cryo-electron microscopy (cryo-EM) imaging, and its HSV-1 inhibition activity together with its previously reported methacrylate analoguep(G1MA-OSO3)PDS is tested. Both of the sulfated MIPs show strong HSV-1 inhibition in plaque reduction assays with IC50 values in lower nanomolar range (<3× 10-9 m) and demonstrate a high cell compatibility (CC50>1.0mgmL-1) with lower anticoagulant activity than heparin. In addition, the prophylactic and therapeutic activity of both MIPs is assessed in pre- and post-infection inhibition assays and clearly visualize their high potential for application using fluorescent microscopy imaging of infected cells.

Pixel-wise programmability enables dynamic high-SNR cameras for high-speed microscopy.

High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera (~2-3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions.

Widefield Super-Resolution Infrared Spectroscopy and Imaging of Autofluorescent Biological Materials and Photosynthetic Microorganisms Using Fluorescence Detected Photothermal Infrared (FL-PTIR).

We have demonstrated high-speed, super-resolution infrared (IR) spectroscopy and chemical imaging of autofluorescent biomaterials and organisms using camera-based widefield photothermal detection that takes advantage of temperature-dependent modulations of autofluorescent emission. A variety of biological materials and photosynthetic organisms exhibit strong autofluorescence emission under ultraviolet excitation and the autofluorescent emission has a very strong temperature dependence, of order 1%/K. Illuminating a sample with pulses of IR light from a wavelength-tunable laser source causes periodic localized sample temperature increases that result in a corresponding transient decrease in autofluorescent emission. A low-cost light-emitting diode-based fluorescence excitation source was used in combination with a conventional fluorescence microscopy camera to detect localized variations in autofluorescent emission over a wide area as an indicator of localized IR absorption. IR absorption image stacks were acquired over a range of IR wavelengths, including the fingerprint spectral range, enabling extraction of localized IR absorption spectra. We have applied widefield fluorescence detected photothermal IR (FL-PTIR) to an analysis of autofluorescent biological materials including collagen, leaf tissue, and photosynthetic organisms including diatoms and green microalgae cells. We have also demonstrated the FL-PTIR on live microalgae in water, demonstrating the potential for label-free dynamic chemical imaging of autofluorescent cells.

Effect of treatment conditions on the morphology of date empty fruit bunch lignocellulosic fiber for biocomposite applications

The fiber of the empty date fruit is one of the waste products left after the date fruit is harvested from the date palm. Optimization of the treatment parameters as well as the economics of the treatment process are critical to the successful commercialization of the treatment of natural fibers for the production of biocomposites. Of the natural fiber treatment methods described in previous work, hot water and alkali treatments are the most cost-effective, which justified the choice of these treatment methods in this work. Here, date empty fruit bunch fibers were pulverized to a particle size of 0–500 µm and then treated with boiling water at 100 °C for 3 h or with sodium hydroxide at different concentrations (2, 5, 10%) for 3 h at room temperature with constant stirring at 800 rpm. To confirm the effect of temperature on the fiber treatment, another fiber sample was treated at a 5% concentration of sodium hydroxide solution at an elevated temperature of 80 °C for the same period and stirring condition. The untreated and treated DEFB fibers were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), X-ray fluorescence (XRF), and scanning electron microscopy (SEM). Chemical analysis of the fibers showed that DEFB fiber treated with 5% sodium hydroxide solution at room temperature had the highest cellulose content (52.0%), which was also confirmed by the XRD analysis. The DEFB fiber treated with boiling water exhibited higher thermal stability while the sodium hydroxide-treated fiber exhibited higher crystallinity index and cellulose content. Apparently, DEFB fiber treated with sodium hydroxide solution at 5% concentration for 3 h at room temperature for continuous stirring at 800 rpm offered the best properties. The treated fibers are expected to be viable reinforcement in the production of biocomposites with the goal of achieving energy efficiency. The developed materials could be used in industrial applications such as insulating building systems, automotive parts, and home furniture.

The chemotrophic behaviour of Aspergillus niger: Mapping hyphal filaments during chemo-sensing; the first step towards directed materials formation

In the development of fungal based materials for applications in construction through to biomedical materials and fashion, understanding how to regulate and direct growth is key for gaining control over the form of material generated. Here, we show how simple ‘chemical food’ cues can be used to manipulate the growth of fungal networks by taking Aspergillus niger as an exemplar species. Chemotrophic responses towards a range of nitrogen and carbon containing biomolecules including amino acids, sugars and sugar alcohols were quantified in terms of chemotrophic index (CI) under a range of basal media compositions (low and high concentrations of N and C sources). Growth of filamentous networks was followed using fluorescence microscopy at single time points and during growth by an AI analytical approach to explore chemo sensing behaviour of the fungus when exposed to pairs (C-C, C-N, N-N) of biomolecules simultaneously. Data suggests that the directive growth of A. niger can be controlled towards simple biomolecules with CI values giving a good approximation for expected growth under a range of growth conditions. This is a first step towards identifying conditions for researcher-led directed growth of hyphae to make mycelial mats with tuneable morphological, physicochemical, and mechanical characteristics.

Collagen hydrogel protects intestinal epithelial cells from indomethacin-induced damage: results of an in vitro experiment

INTRODUCTION. Indomethacin is a derivative of indoleacetic acid and has anti-inflammatory, analgesic and antipyretic effects. However, the results of numerous studies show that indomethacin, like many other nonsteroidal anti-inflammatory drugs (NSAIDs), have an inhibitory effect on the viability and functional activity of enterocytes. In this regard, the search for new ways to reduce the severity of side effects from the use of NSAIDs remains relevant. One of these approaches may be to enrich patients’ diets with non-drug biologically active compounds, including proteins. However, the effect of dietary proteins and biologically active peptides on NSAID-induced damage to the wall of the small intestine and stomach has not been sufficiently studied. AIM. To evaluate the ability of a collagen-containing dietary supplement to protect human duodenal epithelial cells (HuTu-80 line) from indomethacin-induced damage. MATERIALS AND METHODS. The composite collagen-containing hydrogel was provided by «FIRST ALIVE COLLAGEN» LLC (Russia) and is a registered dietary supplement. The work used a commercial culture of human skin fibroblast cells and human duodenal epithelial cells (line HuTu-80). The viability of intestinal cells and fibroblasts was assessed using light and fluorescence microscopy and flow cytometry methods. RESULTS AND DISCUSSION. It has been established that indomethacin inhibits cell growth, causes apoptosis and death of enterocytes, and also leads to the accumulation of cells in the S-phase, which indicates a disruption in the regulation of the cell cycle. It was revealed that collagen hydrogel prevents cell death caused by indomethacin and reduces the number of apoptotic cells in the population. The protective effect of collagen hydrogel is characterized by normalization of the cell cycle of enterocytes and restoration of their growth and proliferative activity. CONCLUSION. Thus, collagen hydrogel, in vitro, is able to reduce the pathogenic effect of indomethacin on human intestinal epithelial cells. The protective effect of collagen hydrogel is characterized by maintaining viability, inhibiting apoptotic processes, and maintaining cell cycle stability. The results obtained indicate the prospects of using a dietary supplement based on a composite collagen hydrogel as a prophylactic agent to reduce the risk of NSAID-associated gastrointestinal diseases. However, to confirm the therapeutic effectiveness of the dietary supplement, further research is necessary, both using experimental animal modeling of NSAID-associated diseases of the human gastrointestinal tract, and clinical studies.

Fluorophore multimerization on a PEG backbone as a concept for signal amplification and lifetime modulation

Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels’ fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.

Environment modulates protein heterogeneity through transcriptional and translational stop codon readthrough

Stop codon readthrough events give rise to longer proteins, which may alter the protein’s function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

Research on Low-Cost High-Viscosity Asphalt and Its Performance for Porous Asphalt Pavement.

To develop a cost-effective, high-viscosity asphalt for porous asphalt pavement, we utilized SBS, tackifier, and solubilizer as the main raw materials, identified the optimal composition through an orthogonal experiment of three factors and three levels, and prepared a low-cost high-viscosity asphalt. We compared its conventional and rheological properties against those of rubber asphalt, SBS modified asphalt, and matrix asphalt, employing fluorescence microscopy and Fourier transform infrared spectroscopy for microstructural analysis. The results indicate that the optimal formula composition for high-viscosity asphalt was 4-5% styrene-butadiene-styrene (SBS) + 1-2% tackifier +0-3% solubilizer +0.15% stabilizer. The components evenly dispersed and the performances were enhanced with chemical and physical modification. Compared with SBS modified asphalt, rubber asphalt, and matrix asphalt, the softening point, 5 °C ductility, and 60 °C dynamic viscosity of high-viscosity asphalt were significantly improved, while the 175 °C Brookfield viscosity was equivalent to SBS modified asphalt. In particular, the 60 °C dynamic viscosity reaches 383,180 Pa·s. Rheological tests indicate that the high- and low-temperature grade of high-viscosity asphalt reaches 88-18 °C, and that high-viscosity asphalt has the best high-temperature resistance to permanent deformation and low-temperature resistance to cracking. It can save about 30% cost compared to commercially available high-viscosity asphalt, which is conducive to the promotion and application of porous asphalt pavement.

Correlating semiconductor nanoparticle architecture and applicability for the controlled encoding of luminescent polymer microparticles

Luminophore stained micro- and nanobeads made from organic polymers like polystyrene (PS) are broadly used in the life and material sciences as luminescent reporters, for bead-based assays, sensor arrays, printable barcodes, security inks, and the calibration of fluorescence microscopes and flow cytometers. Initially mostly prepared with organic dyes, meanwhile luminescent core/shell nanoparticles (NPs) like spherical semiconductor quantum dots (QDs) are increasingly employed for bead encoding. This is related to their narrower emission spectra, tuneability of emission color, broad wavelength excitability, and better photostability. However, correlations between particle architecture, morphology, and photoluminescence (PL) of the luminescent nanocrystals used for encoding and the optical properties of the NP-stained beads have been rarely explored. This encouraged us to perform a screening study on the incorporation of different types of luminescent core/shell semiconductor nanocrystals into polymer microparticles (PMPs) by a radical-induced polymerization reaction. Nanocrystals explored include CdSe/CdS QDs of varying CdS shell thickness, a CdSe/ZnS core/shell QD, CdSe/CdS quantum rods (QRs), and CdSe/CdS nanoplatelets (NPLs). Thereby, we focused on the applicability of these NPs for the polymerization synthesis approach used and quantified the preservation of the initial NP luminescence. The spectroscopic characterization of the resulting PMPs revealed the successful staining of the PMPs with luminescent CdSe/CdS QDs and CdSe/CdS NPLs. In contrast, usage of CdSe/CdS QRs and CdSe QDs with a ZnS shell did not yield luminescent PMPs. The results of this study provide new insights into structure–property relationships between NP stained PMPs and the initial luminescent NPs applied for staining and underline the importance of such studies for the performance optimization of NP-stained beads.

#944 Soluble uric acid inhibits neutrophil functions during infections in patients with kidney disease

Abstract Background and Aims Chronic kidney disease (CKD) is one of the most common diseases affecting 8-16% of the world population. Beside cardiovascular complications, infections including sepsis are the second most common cause of death in patients with acute kidney injury (AKI) and CKD. However, the underlying pathomechanisms that contribute to the secondary immunodeficiency related to kidney disease [1] are not well understood. Recent evidence suggests that CKD-related hyperuricemia characterised by elevated serum uric acid (UA) levels suppresses immune cell functions during sterile inflammation [2]. Whether this is also the case during host defense is subject of our current investigation. In this study, we hypothesized that soluble UA inhibits neutrophil effector functions and therefore contributes to the increased infection risk in patients with kidney disease. Method Blood neutrophils were isolated from healthy individuals as well as from patients with kidney diseases, and incubated ex vivo in the presence or absence of 10 mg/dl soluble UA prior to stimulation with bacterial peptides including LPS. Immune cell functions including cytoskeletal changes, immune cell activation, maturation, endo- and phagocytosis (pHrodo Dextran particles, IgG-FITC beads, pHrodo E. coli bioparticles), ROS production, and neutrophil extracellular trap (NET) formation were analysed and quantified using flow cytometry, fluorescence and colorimetric assays, and fluorescence microscopy. In addition, to verify our results we stimulated neutrophils from healthy individuals with sera from hyperuricemic CKD patients. Results Our results show for the first time that soluble UA significantly inhibits the ability of neutrophils to endocytose small particles and phagocytose beads in neutrophils from healthy individuals comparable to the inhibitory effect of Cytochalasin D, an inhibitor of endo- and phagocytosis. Interestingly, unlike neutrophils from AKI patients, neutrophils from CKD patients were significantly less able to phagocytose beads compared to healthy controls with and without stimulation. To mimic the uptake of pathogens more physiologically, pHrodo E. coli bioparticles were used. We found that soluble UA inhibited the phagocytosis of such bioparticles in neutrophils. In addition, our investigation into the formation of ROS showed that soluble UA inhibited specifically nitric oxide, peroxynitrite, hydroxyl radicals and hydrogen peroxide, but to a lower extent the formation of superoxides in neutrophils from healthy individuals, similar to the effectiveness of the radical scavenger N-acetylcysteine. These data were comparable with our cohort study of neutrophils from CKD patients. To examine the mechanism of reduced ROS production, we inhibited NADPH oxidase (DPI) and MPO (4-ABAH) in neutrophils and found that soluble UA has similar inhibitory effects on ROS production like DPI whereas 4-ABAH was more potent. This indicated that soluble UA modulates NADPH oxidase and ROS production, which subsequently resulted in reduced NET formation in neutrophils. Conclusion Our data identify soluble UA as potential immune regulator of the secondary immunodeficiency in patients with kidney disease by inhibiting the endo- and phagocytosis of particles, NADPH-mediated ROS production and NET formation in neutrophils, processes that are important to kill pathogens and fight an infection. Thus, specifically targeting UA with urate-lowering therapy might overcome the suppressed host defence against infection in patients with kidney disease.

Prediction of Nili-Ravi buffalo bull fertility through Fourier harmonic analysis of sperm

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0–5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0–5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (−0.0002 ± 0.07 vs. −0.266 ± 0.09), kurharm0 (−0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0–5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. −0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.

Time-Separated Pulse Release-Activation of an Enzyme from Alginate-Polyethylenimine Hydrogels Using Electrochemically Generated Local pH Changes.

β-Glucosidase (EC 3.2.1.21) from sweet almond was encapsulated into pH-responsive alginate-polyethylenimine (alginate-PEI) hydrogel. Then, electrochemically controlled cyclic local pH changes resulting from ascorbate oxidation (acidification) and oxygen reduction (basification) were used for the pulsatile release of the enzyme from the composite hydrogel. Activation of the enzyme was controlled by the very same pH changes used for β-glucosidase release, separating these two processes in time. Importantly, the activity of the enzyme, which had not been released yet, was inhibited due to the buffering effect of PEI present in the gel. Thus, only a portion of the released enzyme was activated. Both enzymatic activity and release were monitored by confocal fluorescence microscopy and regular fluorescent spectroscopy. Namely, commercially available very little or nonfluorescent substrate 4-methylumbelliferyl-β-d-glucopyranoside was hydrolyzed by β-glucosidase to produce a highly fluorescent product 4-methylumbelliferone during the activation phase. At the same time, labeling of the enzyme with rhodamine B isothiocyanate was used for release observation. The proposed work represents an interesting smart release-activation system with potential applications in biomedical field.

#328 Non-invasive assessment of urinary exfoliated proximal tubule cells using multispectral autofluorescence imaging features for early detection of CKD

Abstract Background and Aims Early detection of CKD is important to allow for timely intervention which may reduce adverse clinical outcomes and cost of renal replacement therapies. Histopathological diagnosis of CKD remains the gold standard, but routine kidney biopsy is costly and associated with risks such as pain and bleeding. There is a need to develop accurate non-invasive methods for early diagnosis and prognostication of CKD. Kidney cell exfoliation into urine is an active process, which may generate significant information regarding an individual's kidney status. In particular, proximal tubule cells (PTCs) make up majority of the kidney mass and represents the hallmark of CKD in reflecting the degree of tubulointerstitial fibrosis and atrophy. Multispectral assessment of native cell autofluorescence has been specifically shown to be sensitive to metabolic changes and oxidative stress, which are key factors involved in kidney function decline. Hence, we evaluated whether multispectral autofluorescence signals in urinary exfoliated PTCs reflect kidney function as determined by eGFR. Method Individuals aged 18 or above were included and informed consent was obtained from all participants involved in this study. Spot urine samples were collected and stored at -80°C. On assessment, exfoliated PTCs were extracted from thawed urine using a validated specific immunomagnetic separation method based on anti-CD13 and anti-SGLT2 antibodies. Multispectral autofluorescence imaging was performed using a custom-made autofluorescence microscopy system (standard Olympus iX83™ fluorescence microscope and multispectral excitation lamp from Quantitative™, AU). Images were captured by a Nüvü™ EMCCD camera HNü 1024 cooled to -65°C in our laboratory to reduce sensor-induced noise. For the 34 image channels taken per sample, image acquisition times of up to 5 seconds per channel, with multiple averaging (3 times) was applied to optimize image quality. Image analysis was conducted through a workflow encompassing data preprocessing, data normalization and outlier handling, followed by basic model training and evaluation. An entropy-based image feature selection methodology was initially completed by hand, with results subsequently compared to that generated from the AutoGluon machine-learning framework [1]. The models developed from this process were assessed using ROC (Receiver Operating Characteristic) curves and AUC (Area Under the Curve) values. Two discriminative studies were performed – an overall study between exfoliated PTCs of individuals eGFR≥60 and eGFR &amp;lt; 60 ml/min/1.73 m2 and a subgroup study between that of individuals eGFR≥90 and eGFR 60-90 ml/min/1.73 m2. Results 40 individuals were included in our study. 20 individuals had eGFR≥60 and 20 had eGFR &amp;lt; 60 ml/min/1.73 m2. Multispectral assessment of cell autofluorescence features differentiated between exfoliated PTCs of these 2 groups with AUC value of 0.97 (95% CI 0.94-1.00) (Fig. 1), model accuracy being 82%. Subgroup analysis included 20 individuals of which 10 had eGFR≥90 and 10 had eGFR 60-90 ml/min/1.73 m2. Multispectral assessment of cell autofluorescence features differentiated between exfoliated PTCs of these 2 groups with AUC value of 0.99 (95% CI 0.99-1.00) (Fig. 2), model accuracy being 85%. Conclusion An excellent degree of differentiation between urinary PTCs derived from those with normal or high eGFR and mild degrees of CKD is now reliably demonstrated, complementing conclusions from our group's original small pilot study which evaluated between urinary PTCs from CKD and non-CKD cohorts [2]. Further studies are required to determine whether our novel method can be used to non-invasively predict individuals at risk of progressive kidney disease and to monitor whether intervention is effective.

Multiscale enhancement mechanism of low-temperature performance for degraded recycled waste rubber asphalt binders: MD simulation and microscopic investigation

Desulfurization and degradation are important processes to facilitate the storage of waste rubber asphalt and improve its utilization. However, this process is extremely complex and the mechanisms underlying the resulting performance enhancement remain unclear. This study aims to investigate the multiscale mechanism of rubber degradation and its impact on the low-temperature performance of degraded rubber modified asphalt (DRMA) binder through differential scanning calorimetry (DSC), bending beam rheometer (BBR), fluorescence microscopy (FM) and Fourier transform infrared spectroscopy (FTIR) tests and molecular dynamics (MD) simulations. The results showed that as the degradation of rubber powder deepens, lightweight components decomposed from the rubber are more easily dissolved into the virgin asphalt, thus improving its dispersion. In addition, the small molecular structure after rubber degradation penetrates and fills the aggregation space between asphalt molecules, effectively increasing the molecular activity of asphalt, thus enhancing the low-temperature performance of asphalt.

Low density granulocytes and neutrophil extracellular trap formation are increased in incomplete systemic lupus erythematosus.

To investigate the proportion of low-density granulocytes (LDGs), circulating plasma neutrophil extracellular traps (NETs), and serum-induced NET formation in patients with incomplete systemic lupus erythematosus (iSLE) and systemic lupus erythematosus (SLE). LDGs were measured cross-sectionally in 18 iSLE patients, 11 SLE patients and 14 healthy controls (HCs), whereas circulating NETs and serum-induced NET formation were assessed in 35 iSLE patients, 41 SLE patients and 16 HCs. LDGs (CD14lowCD15+) were measured in PBMCs using flow cytometry and circulating plasma NETs were measured using anti-myeloperoxidase-DNA, anti-citrullinated histone H3 and anti-elastase-DNA complex ELISAs. Serum-induced NET formation was assessed by incubating healthy neutrophils with serum from iSLE patients, SLE patients or HCs and visualizing NETs with fluorescence microscopy. Proportions of LDGs and circulating plasma NETs were similarly elevated in iSLE and SLE patients compared with those in HCs. Furthermore, patients under hydroxychloroquine (HCQ) treatment had lower proportions of LDGs than those without. Serum from iSLE and SLE patients similarly induced NET formation in healthy neutrophils. In iSLE patients, myeloperoxidase-DNA complexes were correlated with proportions of age-associated B-cells, memory B-cells and negatively with naïve B-cells, while we did not find associations between measures of NETs or serum-induced NET formation and interferon score or clinical parameters. These results show that neutrophil dysfunction, including higher proportions of LDGs, and increased NET formation, already occur in iSLE, similar to SLE, despite differences in disease manifestations. Thereby, neutrophil dysfunction may contribute to sustained exposure to autoantigens and autoreactivity in early stages of SLE.

Tracking Drugs and Lipids: Quantitative Mass Spectrometry Imaging of Liposomal Doxorubicin Delivery and Bilayer Fate in Three-Dimensional Tumor Models.

Targeted therapy to the tumor would greatly advance precision medicine. Many drug delivery vehicles have emerged, but liposomes are cited as the most successful to date. Recent efforts to develop liposomal drug delivery systems focus on drug distribution in tissues and ignore liposomal fate. In this study, we developed a novel method to elucidate both drug and liposomal bilayer distribution in a three-dimensional cell culture model using quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI qMSI) alongside fluorescence microscopy. Imaging liposomal distribution in a cell culture model is challenging, as lipids forming the bilayer are endogenous to the model system. To resolve this issue, we functionalized the bilayer by chemically cross-linking a fluorescent tag to the alkyne-containing lipid hexynoyl phosphoethanolamine (HPE). We synthesized liposomes incorporating the tagged HPE lipid and encapsulated within them doxorubicin, yielding a theranostic liposome capable of both drug delivery and monitoring liposomal uptake. We employed an "in-tissue" MALDI qMSI approach to generate a calibration curve with R2 = 0.9687, allowing for quantification of doxorubicin within spheroid sections at multiple time points. After 72 h of treatment with the theranostic liposomes, full doxorubicin penetration was observed. The metabolites doxorubicinone and 7-deoxydoxorubicinone were also detected after 48 h. Modification of the bilayer allowed for fluorescence microscopy tracking of liposomes, while MALDI MSI simultaneously permitted the imaging of drugs and metabolites. While we demonstrated the utility of our method with doxorubicin, this system could be applied to examine the uptake, release, and metabolism of many other liposome-encapsulated drugs.

Revealing operando surface defect–dependent electrocatalytic performance of Pt at the subparticle level

Understanding the operando defect-tuning performance of catalysts is critical to establish an accurate structure-activity relationship of a catalyst. Here, with the tool of single-molecule super-resolution fluorescence microscopy, by imaging intermediate CO formation/oxidation during the methanol oxidation reaction process on individual defective Pt nanotubes, we reveal that the fresh Pt ends with more defects are more active and anti-CO poisoning than fresh center areas with less defects, while such difference could be reversed after catalysis-induced step-by-step creation of more defects on the Pt surface. Further experimental results reveal an operando volcano relationship between the catalytic performance (activity and anti-CO ability) and the fine-tuned defect density. Systematic DFT calculations indicate that such an operando volcano relationship could be attributed to the defect-dependent transition state free energy and the accelerated surface reconstructing of defects or Pt-atom moving driven by the adsorption of the CO intermediate. These insights deepen our understanding to the operando defect-driven catalysis at single-molecule and subparticle level, which is able to help the design of highly efficient defect-based catalysts.