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Intestinal inflammation-driving T cells within the epithelial layer of the small intestine can be identified and further characterized by Raman spectroscopy ex vivo.

The life cycle of intracellular pathogens is often complex and can include different morphoforms. Treatment of intracellular infections and unperturbed studying of the pathogen inside the host cell are frequently challenging. Here, we present a Raman-based, label-free, non-invasive, and non-destructive method to localize, visualize, and even quantify intracellular bacteria in 3D within intact host cells in a

Macrophages are important cells of the innate immune system that play many different roles in host defense, a fact that is reflected by their polarization into many distinct subtypes. Depending on their function and phenotype, macrophages can be grossly classified into classically activated macrophages (pro-inflammatory M1 cells), alternatively activated macrophages (anti-inflammatory M2 cells), and non-activated cells (resting M0 cells). A fast, label-free and non-destructive characterization of macrophage phenotypes could be of importance for studying the contribution of the various subtypes to numerous pathologies. In this work, single cell Raman spectroscopic imaging was applied to visualize the characteristic phenotype as well as to discriminate between different human macrophage phenotypes without any label and in a non-destructive manner. Macrophages were derived by differentiation of peripheral blood monocytes of human healthy donors and differently treated to yield M0, M1 and M2 phenotypes, as confirmed by marker analysis using flow cytometry and fluorescence imaging. Raman images of chemically fixed cells of those three macrophage phenotypes were processed using chemometric methods of unmixing (N-FINDR) and discrimination (PCA-LDA). The discrimination models were validated using leave-one donor-out cross-validation. The results show that Raman imaging is able to discriminate between pro- and anti-inflammatory macrophage phenotypes with high accuracy in a non-invasive, non-destructive and label-free manner. The spectral differences observed can be explained by the biochemical characteristics of the different phenotypes.

Loss of apical-basal polarity and downregulation of cell-cell contacts is a critical step during the pathogenesis of cancer. Both processes are regulated by the scaffolding protein Pals1, however, it is unclear whether the expression of Pals1 is affected in cancer cells and whether Pals1 is implicated in the pathogenesis of the disease. Using mRNA expression data and immunostainings of cancer specimen, we show that Pals1 is frequently downregulated in colorectal cancer, correlating with poorer survival of patients. We further found that Pals1 prevents cancer cell metastasis by controlling Rac1-dependent cell migration through inhibition of Arf6, which is independent of the canonical binding partners of Pals1. Loss of Pals1 in colorectal cancer cells results in increased Arf6 and Rac1 activity, enhanced cell migration and invasion in vitro and increased metastasis of transplanted tumor cells in mice. Thus, our data reveal a new function of Pals1 as a key inhibitor of cell migration and metastasis of colorectal cancer cells. Notably, this new function is independent of the known role of Pals1 in tight junction formation and apical-basal polarity. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-021-01354-2.

In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of K D value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.

Under hemolytic conditions, toxic heme is scavenged by hemopexin. Recently, the heme-binding properties of hemopexin have been reassessed, which revealed a KD of ~ 0.32 nM as well as a stoichiometry of one to two heme molecules binding to hemopexin. A 66mer hemopexin-derived peptide that spans over three heme-binding motifs was used to verify the earlier suggested heme-recruiting mechanism. Herein, we employed spectroscopic and computational methods to substantiate the hypothesis of more than one heme molecule binding to hemopexin and to analyze the heme-binding mode. Both, hemopexin and the 66mer peptide, were found to bind heme in mixed penta- and hexacoordinated states, which strongly indicates that heme binding follows distinct criteria and increases rigidity of the peptide-heme complex. Additional in silico molecular dynamics simulations support these experimental findings and, thus, contribute to our understanding of the molecular basis of the heme-hemopexin interaction. This analysis provides further details for consideration of hemopexin in biomedical applications.

Lysosomes, the acidic degradation compartments of eukaryotic cells, play an essential role in many physiological processes. Their dysfunction is associated with a number of diseases, which are often related to an altered localization or luminal pH. Thus, the in-depth characterization of lysosomes within the intact eukaryotic cell is of utmost interest. For microscopic evaluation of lysosomal distribution and acidity, a number of labels have been developed, but many showed poor organelle specificity or rapid clearing from lysosomes, rendering them unsuitable for long-term observations. Here, we describe the synthesis and spectroscopic properties of a novel small molecule marker for lysosomes based on naphthalene monoimide with reversible, pH-dependent spectral shifts in both the absorption and the emission spectrum and acidity-associated changes in fluorescence lifetime. The dye can be excited either with single- or two-photon excitation and appears to be very stably associated with lysosomes for several days. We used this chromophore to detect chemically-induced changes of lysosomal pH in HeLa cells by ratiometric and FLIM imaging.

Self-assembly of polyesters like PlLA and PdLA into stereocomplexes (SCs) is an interesting approach to tailor physical properties of polymeric nanoparticles without affecting their hydrophilicity....

Neutrophils are important cells of the innate immune system and the major leukocyte subpopulation in blood. They are responsible for recognizing and neutralizing invading pathogens, such as bacteria or fungi. For this, neutrophils are well equipped with pathogen recognizing receptors, cytokines, effector molecules, and granules filled with reactive oxygen species (ROS)-producing enzymes. Depending on the pathogen type, different reactions are triggered, which result in specific activation states of the neutrophils. Here, we aim to establish a label-free method to indirectly detect infections and to identify the cause of infection by spectroscopic characterization of the neutrophils. For this, isolated neutrophils from human peripheral blood were stimulated in an

Leukotrienes are pro-inflammatory lipid mediators generated by 5-lipoxygenase aided by the 5-lipoxygenase-activating protein (FLAP). BRP-201, a novel benzimidazole-based FLAP antagonist, inhibits leukotriene biosynthesis in isolated leukocytes. However, like other FLAP antagonists, BRP-201 fails to effectively suppress leukotriene formation in blood, which limits its therapeutic value. Here, we describe the encapsulation of BRP-201 into poly(lactide-co-glycolide) (PLGA) and ethoxy acetalated dextran (Ace-DEX) nanoparticles (NPs), aiming to overcome these detrimental pharmacokinetic limitations and to enhance the bioactivity of BRP-201. NPs loaded with BRP-201 were produced via nanoprecipitation and the physicochemical properties of the NPs were analyzed in-depth using dynamic light scattering (size, dispersity, degradation), electrophoretic light scattering (effective charge), NP tracking analysis (size, dispersity), scanning electron microscopy (size and morphology), UV–VIS spectroscopy (drug loading), an analytical ultracentrifuge (drug release, degradation kinetics), and Raman spectroscopy (chemical attributes). Biological assays were performed to study cytotoxicity, cellular uptake, and efficiency of BRP-201-loaded NPs versus free BRP-201 to suppress leukotriene formation in primary human leukocytes and whole blood. Both PLGA- and Ace-DEX-based NPs were significantly more efficient to inhibit leukotriene formation in neutrophils versus free drug. Whole blood experiments revealed that encapsulation of BRP-201 into Ace-DEX NPs strongly increases its potency, especially upon pro-longed (≥ 5 h) incubations and upon lipopolysaccharide-challenge of blood. Finally, intravenous injection of BRP-201-loaded NPs significantly suppressed leukotriene levels in blood of mice in vivo. These results reveal the feasibility of our pharmacological approach using a novel FLAP antagonist encapsulated into Ace-DEX-based NPs with improved efficiency in blood to suppress leukotriene biosynthesis.

Dilated cardiomyopathy (DCM) is a leading cardiomyopathy condition and is the leading reason for heart transplantation. Due to high etiologic and genetic heterogeneity of the pathologies, different therapeutic treatment strategies are available and have been successful for different treatments. Immunoadsorption (IA) therapy removes the circulating anticardiac antibodies and improves the left ventricular function in substantial proportion of DCM patients. Powerful, non-invasive analytical tools are highly desired to investigate the efficiency and success of IA therapy. In this contribution, we followed the changes of a female DCM patient undergoing IA therapy at different treatment time points in a label-free, non-invasive manner from blood samples (plasma and serum) on the basis of vibrational spectroscopy (Raman scattering and IR absorption). Chemometric methods, including dimension reduction and statistical modeling, were used to interpret spectral data. The impact of different time points of the IA treatment can be identified in both the plasma and serum, using both techniques, with high accuracy. The removal of antibodies of immunoglobulin G (IgG) group during IA therapy and their restoration was reflected in both Raman and FTIR spectra. Relative changes in the spectral bands assigned to IgG agreed well with the immunoturbidimetry measurement of total IgG. Successful clinical treatment was accompanied by spectral differences between vibrational spectra obtained at initial disease state and 11 months after the IA treatment. The long-term follow-up of the patient reveals the stabilization of the health state after therapy. It is noteworthy that the treatment time points were distinguished with a better accuracy using spectra from plasma compared to those from serum samples, which might indicate the involvement of corresponding proteins in the coagulation. Vibrational spectroscopy is a powerful tool for personalized medicine to follow-up the treatment success of IA therapy for the DCM disorder.

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.

We introduce a colorimetric as well as fluorometric dual response CO-releasing molecule as Mn(I) tricarbonyl complex with a nitrobenzoxadiazole (NBD) fluorophore. DFT and TD-DFT studies corroborate that extended conjugation in the principal ligand frame will help to release CO at low energy radiation. A very low toxicity of NBD-CORM was observed against LX-2 and HepaRG® human cell lines.

Molecular mechanisms through which Gram-positive bacteria induce the canonical inflammasome are poorly understood. Here, we studied the effects of Group B streptococci (GBS) and Staphylococcus aureus (SA) on inflammasome activation in human macrophages. Dinucleotide binding small RNA aptamers released by SA and GBS were shown to trigger increased IL-1β generation by inflammasomes. The stimulator of interferon genes-STING as a central mediator of innate immune responses has been identified as the key target of pathogenic RNA. Multi-lamellar lipid bodies (MLBs) produced by SA function as vehicles for the RNA aptamers. Notably, expression of RNA aptamers is controlled by an accessory gene regulator quorum sensing system of the bacteria. These findings have been translated to patients with Gram-positive sepsis showing hallmarks of MLB-RNA-mediated inflammasome activation. Together our findings may provide a new perspective for the pathogenicity of Gram-positive bacterial infection in man.

Organic cation transporters (OCT) play an important role in mediating cellular uptake of several pharmaceuticals, such as the antidiabetic drug metformin and the platinum-derived chemotherapeutics. Since these drugs can also affect the pancreas, here it was investigated whether these transporters are expressed in this organ. An interaction between OCT2 and the glucose transporter 2 (GLUT2), which is expressed with important functional consequences in the kidneys and in the pancreas, has already been demonstrated elsewhere. Therefore, here it was further investigated whether the two proteins have a functional relationship. It was demonstrated that OCT2 is expressed in pancreas, probably in β cells of Langerhans islets, together with GLUT2. However, a co-localization was only evident in a cell-line model of rat pancreatic β cells under incubation with high glucose concentration. High glucose stimulated OCT2 expression and activity. On the other side, studies conducted in human embryonic kidney cells stably expressing OCT2, showed that overexpression of GLUT2 decreased OCT2 activity. Unfortunately, pull-down experiments aimed to confirm a physical OCT2/GLUT2 interaction were not successful. Renal glucose excretion was reduced in mice with genetic deletion of OCT2. Nonetheless, in these mice no regulation of known kidney glucose transporters was measured. Therefore, it may be speculated that OCT2 may influence cellular trafficking of GLUT2, without changing its amount. OCT2 may play a role in drug uptake of the pancreas, and its activity may be regulated by glucose and GLUT2. Vice versa, GLUT2 activity may be regulated through an interaction with OCT2.

In recent years, the quality of fluorescence microscopic imaging produced by a laser scanning microscope (LSM) system has been increased through the use of optical imaging techniques being used for small biological or nonbiological particles like instance, bacteria or cells in tissue samples. Currently, multiple manufactures provide LSM equipment able to capture single-layer or stacked images. However, the manufactures still using distinct data formats including proprietary ones, limiting the interoperability with third systems. In the clinical environment, the scanners need to be integrated with a vendor-neutral archive, storing images in a standard format and interface, making them accessible to healthcare professionals regardless of what proprietary system created it. Having distinct software solutions to manage the data is tiresome and time-consuming. This article proposes a normalization pipeline that can convert distinct vendor formats in a standard DICOM structure including pixel data and metadata. After conversion, the images are sent to a DICOM-compliant repository being able to be consumed in the network using normalized communication and visualization processes. The proposed solution is reliable and uses efficiently the less memory, a critical issue since the resolution of pathology whole-slide images can reach several gigapixels.

Bacteriophages, or "phages" for short, are viruses that replicate in bacteria. The therapeutic and biotechnological potential of phages and their lytic enzymes is of interest for their ability to selectively destroy pathogenic bacteria, including antibiotic-resistant strains. Introduction of phage preparations into medicine, biotechnology, and food industry requires a thorough characterization of phage-host interaction on a molecular level. We employed Raman tweezers to analyze the phage-host interaction of

In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity in a 1:1 ratio. Here we show that hemopexin binds heme with lower affinity than previously assumed and that the interaction ratio tends to 2:1 (heme:hemopexin) or above. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. In addition,in silicomolecular modelling with a newly created homology model of human hemopexin allowed us to propose a recruiting mechanism by which heme consecutively binds to several histidine residues and is finally funnelled into the high-affinity binding pocket. Our findings have direct implications for the biomedical application of hemopexin and its potential administration in hemolytic disorders.

Multidirectional imaging flow cytometry (mIFC) extends conventional imaging flow cytometry (IFC) for the image-based measurement of 3D-geometrical features of particles. The innovative core is a flow rotation unit in which a vertical sample lamella is incrementally rotated by 90 degrees into a horizontal lamella. The required multidirectional views are generated by guiding all particles at a controllable shear flow position of the parabolic velocity profile of the capillary slit detection chamber. All particles pass the detection chamber in a two-dimensional sheet under controlled rotation while each particle is imaged multiple times. This generates new options for automated particle analysis. In an experimental application, we used our system for the accurate classification of 15 species of pollen based on 3D-morphological information. We demonstrate how the combination of multi directional imaging with advanced machine learning algorithms can improve the accuracy of automated bio-particle classification. As an additional benefit, we significantly decrease the number of false positives in the classification of foreign particles, i.e. those elements which do not belong to one of the trained classes by the 3D-extension of the classification algorithm.

Escherichia coli KI683 was isolated from blood of a patient who developed septicemia as a complication of a urinary tract infection. Genome sequencing resulted in three contigs with a total genome size of 5,243,173 bp encoding 5,143 genes.

The development of antibiotic resistances in common pathogens is an increasing challenge for therapy of infections and especially severe complications like sepsis. To prevent administration of broad-spectrum and potentially non-effective antibiotics, the susceptibility spectrum of the pathogens underlying the infection has to be determined. Current phenotypic standard methods for antibiotic susceptibility testing (AST) require the isolation of pathogens from the patient and the subsequent culturing in the presence of antibiotics leading to results only after 24-72 h. Since the early initialization of an effective antibiotic therapy is crucial for positive treatment result in severe infections, faster methods of AST are urgently needed. A large number of different assay systems are currently tested for their practicability for fast detection of antibiotic resistance profiles. They can be divided into genotypic ones which detect the presence of certain genes or gene products associated with resistances and phenotypic assays which determine the effect of antibiotics on the pathogens. In this mini-review, we summarize current developments in fast phenotypic tests that use photonic approaches and critically discuss their status. We further outline steps that are required to bring these assays into clinical practice.

Targeted administration of carbon monoxide with CO releasing molecules (CORMs) inside of cells proved to be very challenging. Consequently, there are only very few reports on intracellular uptake of CORMs requiring high extracellular CORM loading because an equilibrium between extra- and intracellular concentrations can be assumed. Here we present a strategy for a targeted intracellular administration of manganese(I)-based CORMs that are altered inside of cells to trap these complexes. Thereafter, carbon monoxide can be liberated by irradiation (photoCORMs). To achieve this innovative task, acetoxymethyl (AM) groups are attached at the periphery of the hydrophobic manganese(I) carbonyl complexes to not influence the CO release behavior. Inside of cells these AM substituents are cleaved by esterases yielding hydrophilic manganese(I) carbonyl compounds which are captured inside of cells. This objective is realized by using the bidentate bases 4-(acetoxymethoxycarbonyl)phenyl-bis(3,5-dimethylpyrazolyl)methane (1) and 4-(acetoxymethoxy)phenyl-bis(3,5-dimethylpyrazolyl)methane (4) at facial (OC)3 MnBr fragments yielding CORM-AM1 (2) and CORM-AM2 (5), respectively. Besides synthesis, crystal structures and spectroscopic properties we present targeted administration and intracellular accumulation of these AM-containing CORMs.

Co-registered molecular logic gates combine two different inputs and outputs, such as light and matter. We introduce a biocompatible CO-releasing molecule (CORM, A) as Mn(I) tricarbonyl complex with the ligand 5-(dimethylamino)-N, N-bis(pyridin-2-ylmethyl) naphthalene-1-sulfonamide (L). CO release is chaperoned by turn-on fluorescence and can be triggered by light (405 nm) as well as with hydrogen peroxide in aqueous phosphate buffer. Complex A behaves as a logic "OR" gate via co-registering the inputs of irradiation (light) and peroxide (matter) into the concomitant outputs fluorescence (light) and CO (matter). Cell viability assays confirm the low toxicity of A toward different human cell lines. The CORM has been used to track the inclusion of A into cancer cells.

In this study, we report a method for the suppression of Escherichia coli (E. coli) vitality by means of therapeutic ultrasound irradiation (USI) using biocompatible silicon nanoparticles as cavitation sensitizers. Silicon nanoparticles without (SiNPs) and with polysaccharide (dextran) coating (DSiNPs) were used. Both types of nanoparticles were nontoxic to Hep 2 cells up to a concentration of 2 mg/mL. The treatment of bacteria with nanoparticles and application of 1 W/cm2 USI resulted in the reduction of their viabilities up to 35 and 72% for SiNPs and DSiNPs, respectively. The higher bacterial viability reduction for DSiNPs as compared with SiNPs can be explained by the fact that the biopolymer shell of the polysaccharide provides a stronger adhesion of nanoparticles to the bacterial surface. Transmission electron microscopy (TEM) studies showed that the bacterial lipid shell was partially perforated after the combined treatment of DSiNPs and USI, which can be explained by the lysis of bacterial membran...

A Raman-based, strain-independent, semi-automated method is presented that allows the rapid (

Renal drug transporters such as the organic cation transporters (OCTs), organic anion transporters (OATs) and multidrug resistance proteins (MRPs) play an important role in the tubular secretion of many drugs influencing their efficacy and safety. However, only little is known about the distinct protein abundance of these transporters in human kidneys, and about the impact of age and gender as potential factors of inter-subject variability in their expression and function. The aim of this study was to determine the protein abundance of MDR1, MRP1-4, BCRP, OAT1-3, OCT2-3, MATE1, PEPT1/2, and ORCTL2 by liquid chromatography-tandem mass spectrometry-based targeted proteomics in a set of 36 human cortex kidney samples (20 males, 16 females, median age 53 and 55 years, respectively). OAT1 and 3, OCT2 and ORCTL2 were found to be most abundant renal SLC transporters while MDR1, MRP1 and MRP4 were the dominating ABC transporters. Only the expression levels of MDR1 and ORCTL2 were significantly higher abundant in older donors. Moreover, we found several significant correlations between different transporters, which may indicate their functional interplay in renal vectorial transport processes. Our data may contribute to a better understanding of the molecular processes determining renal excretion of drugs.

Raman spectroscopy has been widely used in clinical and molecular biological studies, providing high chemical specificity without the necessity of labels and with little-to-no sample preparation. However, currently performed Raman-based studies of eukaryotic cells are still very laborious and time-consuming, resulting in a low number of sampled cells and questionable statistical validations. Furthermore, the approach requires a trained specialist to perform and analyze the experiments, rendering the method less attractive for most laboratories. In this work, we present a new high-content analysis Raman spectroscopy (HCA-RS) platform that overcomes the current challenges of conventional Raman spectroscopy implementations. HCA-RS allows sampling of a large number of cells under different physiological conditions without any user interaction. The performance of the approach is successfully demonstrated by the development of a Raman-based cell viability assay, i.e., the effect of doxorubicin concentration on monocytic THP-1 cells. A statistical model, principal component analysis combined with support vector machine (PCA-SVM), was found to successfully predict the percentage of viable cells in a mixed population and is in good agreement to results obtained by a standard cell viability assay. This study demonstrates the potential of Raman spectroscopy as a standard high-throughput tool for clinical and biological applications.

Raman spectroscopy has been applied to investigate the suitability of the drop coating deposition technique to study plasma samples from healthy donors and a patient with underlying cardiac condition. When blood plasma is deposited on a solid substrate, a droplet with coffee-ring is formed, and the plasma proteins will distribute inhomogeneously depending on the chemical and physical properties of the proteins. Changes in the fingerprint region of the Raman spectra were observed from the outer-ring and central zone of the droplet through a systematic investigation. For complete characterization of the sample, optimum measurement scheme has been proposed. To obtain clinically relevant information of the effects of immunoadsorption (IA) treatment of dilated cardiomyopathy (DCM) patient’s, Raman spectral information from outer-ring as well as from the central zone is required.