Interaction of the chemotherapeutic agent oxaliplatin and the tyrosine kinase inhibitor dasatinib with the organic cation transporter 2.

Oxaliplatin (OHP) is effective in colorectal cancer treatment but induces peripheral neurotoxicity (OHP-induced peripheral neurotoxicity, OIPN), diminishing survivor quality of life. Organic cation transporter 2 (OCT2) is a key OHP uptake pathway in dorsal root ganglia. Competing for OCT2-mediated OHP uptake, such as with the tyrosine kinase inhibitor dasatinib, may mitigate OHP side effects. We investigated OHP and dasatinib interaction with OCT2 in human embryonic kidney 293 (HEK293) cells expressing OCT2 within a 10-3 to 10-7 M concentration range. Uptake competition experiments using fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+, 1 µM) and mass spectrometry (MS) to determine cellular platinum content indicated that OHP (100 µM) is an OCT2 substrate, mediating OHP cellular toxicity. ASP+ and MS analysis revealed dasatinib as a non-transported inhibitor of hOCT2 (IC50 = 5.9 µM) and as a regulator of OCT2 activity. Dasatinib reduced transporter Vmax, potentially via Y544 phosphorylation suppression. MS analysis showed cellular dasatinib accumulation independent of hOCT2. Although 3 µM dasatinib reduced 100 µM OHP accumulation in hOCT2-HEK293 cells, co-incubation with dasatinib and OHP did not prevent OHP toxicity, possibly due to dasatinib-induced cell viability reduction. In summary, this study demonstrates OHP as an OCT2 substrate and dasatinib as a non-transported inhibitor and regulator of OCT2, offering potential for OIPN mitigation.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme.

BACKGROUND AND AIMS: Blood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. METHODS: We herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. RESULTS: FVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. CONCLUSION: Our study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Quantification of Polystyrene Uptake by Different Cell Lines Using Fluorescence Microscopy and Label-Free Visualization of Intracellular Polystyrene Particles by Raman Microspectroscopic Imaging.

Environmental pollution caused by plastic is a present problem. Polystyrene is a widely used packaging material (e.g., Styrofoam) that can be broken down into microplastics through abrasion. Once the plastic is released into the environment, it is dispersed by wind and atmospheric dust. In this study, we investigated the uptake of polystyrene particles into human cells using A549 cells as a model of the alveolar epithelial barrier, CaCo-2 cells as a model of the intestinal epithelial barrier, and THP-1 cells as a model of immune cells to simulate a possible uptake of microplastics by inhalation, oral uptake, and interaction with the cellular immune system, respectively. The uptake of fluorescence-labeled beads by the different cell types was investigated by confocal laser scanning microscopy in a semi-quantitative, concentration-dependent manner. Additionally, we used Raman spectroscopy as a complementary method for label-free qualitative detection and the visualization of polystyrene within cells. The uptake of polystyrene beads by all investigated cell types was detected, while the uptake behavior of professional phagocytes (THP-1) differed from that of adherent epithelial cells.

Lysosome targeted visible light-induced photo-CORM for simultaneous CO-release and singlet oxygen generation.

We report a specific lysosome targeted light-responsive CO-releasing molecule (Lyso-CORM). Lyso-CORM is very stable under dark conditions. CO and singlet oxygen (1O2) generation was effectively triggered under one photon and two photon excitation (800 nm) conditions. The cytotoxicity results demonstrated that Lyso-CORM showed good phototoxicity due to the synergistic effect of CO and 1O2 release, and its good biocompatibility, negligible dark toxicity and specific lysosome targeting make Lyso-CORM a potent candidate for phototherapeutic applications.

Findaureus: An open-source application for locating Staphylococcus aureus in fluorescence-labelled infected bone tissue slices.

Staphylococcus aureus (S. aureus) is a facultative pathogenic bacterium that can cause infections in various tissue types in humans. Fluorescence imaging techniques have been employed to visualize the bacteria in ex-vivo samples mostly in research, aiding in the understanding of the etiology of the pathogen. However, the multispectral images generated from fluorescence microscopes are complex, making it difficult to locate bacteria across image files, especially in consecutive planes with different imaging depths. To address this issue, we present Findaureus, an open-source application specifically designed to locate and extract critical information about bacteria, especially S. aureus. Due to the limited availability of datasets, we tested the application on a dataset comprising fluorescence-labelled infected mouse bone tissue images, achieving an accuracy of 95%. We compared Findaureus with other state-of-the-art image analysis tools and found that it performed better, given its specificity toward bacteria localization. The proposed approach has the potential to aid in medical research of bone infections and can be extended to other tissue and bacteria types in the future.

Regulation of Transporters for Organic Cations by High Glucose.

Endogenous positively charged organic substances, including neurotransmitters and cationic uremic toxins, as well as exogenous organic cations such as the anti-diabetic medication metformin, serve as substrates for organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs). These proteins facilitate their transport across cell membranes. Vectorial transport through the OCT/MATE axis mediates the hepatic and renal excretion of organic cations, regulating their systemic and local concentrations. Organic cation transporters are part of the remote sensing and signaling system, whose activity can be regulated to cope with changes in the composition of extra- and intracellular fluids. Glucose, as a source of energy, can also function as a crucial signaling molecule, regulating gene expression in various organs and tissues. Its concentration in the blood may fluctuate in specific physiological and pathophysiological conditions. In this work, the regulation of the activity of organic cation transporters was measured by incubating human embryonic kidney cells stably expressing human OCT1 (hOCT1), hOCT2, or hMATE1 with high glucose concentrations (16.7 mM). Incubation with this high glucose concentration for 48 h significantly stimulated the activity of hOCT1, hOCT2, and hMATE1 by increasing their maximal velocity (Vmax), but without significantly changing their affinity for the substrates. These effects were independent of changes in osmolarity, as the addition of equimolar concentrations of mannitol did not alter transporter activity. The stimulation of transporter activity was associated with a significant increase in transporter mRNA expression. Inhibition of the mechanistic target of rapamycin (mTOR) kinase with Torin-1 suppressed the transporter stimulation induced by incubation with 16.7 mM glucose. Focusing on hOCT2, it was shown that incubation with 16.7 mM glucose increased hOCT2 protein expression in the plasma membrane. Interestingly, an apparent trend towards higher hOCT2 mRNA expression was observed in kidneys from diabetic patients, a pathology characterized by high serum glucose levels. Due to the small number of samples from diabetic patients (three), this observation must be interpreted with caution. In conclusion, incubation for 48 h with a high glucose concentration of 16.7 mM stimulated the activity and expression of organic cation transporters compared to those measured in the presence of 5.6 mM glucose. This stimulation by a diabetic environment could increase cellular uptake of the anti-diabetic drug metformin and increase renal tubular secretion of organic cations in an early stage of diabetes.

Raman Spectroscopy Profiling of Splenic T-Cells in Sepsis and Endotoxemia in Mice.

Sepsis is a life-threatening condition that results from an overwhelming and disproportionate host response to an infection. Currently, the quality and extent of the immune response are evaluated based on clinical symptoms and the concentration of inflammatory biomarkers released or expressed by the immune cells. However, the host response toward sepsis is heterogeneous, and the roles of the individual immune cell types have not been fully conceptualized. During sepsis, the spleen plays a vital role in pathogen clearance, such as bacteria by an antibody response, macrophage bactericidal capacity, and bacterial endotoxin detoxification. This study uses Raman spectroscopy to understand the splenic T-lymphocyte compartment profile changes during bona fide bacterial sepsis versus hyperinflammatory endotoxemia. The Raman spectral analysis showed marked changes in splenocytes of mice subjected to septic peritonitis principally in the DNA region, with minor changes in the amino acids and lipoprotein areas, indicating significant transcriptomic activity during sepsis. Furthermore, splenocytes from mice exposed to endotoxic shock by injection of a high dose of lipopolysaccharide showed significant changes in the protein and lipid profiles, albeit with interindividual variations in inflammation severity. In summary, this study provided experimental evidence for the applicability and informative value of Raman spectroscopy for profiling the immune response in a complex, systemic infection scenario. Importantly, changes within the acute phase of inflammation onset (24 h) were reliably detected, lending support to the concept of early treatment and severity control by extracorporeal Raman profiling of immunocyte signatures.

Identification of bacteria in mixed infection from urinary tract of patient's samples using Raman analysis of dried droplets.

Urinary tract infections (UTI) are among the most frequent nosocomial infections. A fast identification of the pathogen and assignment of Gram type could help to prescribe most suitable treatments. Raman spectroscopy holds high potential for fast and reliable bacterial pathogens identification. While most studies so far have focused on individual pathogens or artificial mixtures, this contribution aims to translate the analysis to primary urine samples from patients with suspected UTIs. For this, we have included 59 primary urine samples out of which 29 were diagnosed as mixed infections. For Raman analysis, we first trained two classification models based on principal component analysis - linear discriminant analysis (PCA-LDA) with more than 3500 Raman spectra of 85 clinical isolates from 23 species in order to (1) identify the Gram type of the bacteria and (2) assign family membership to one of the six most abundant bacterial families in urinary tract infections (Enterobacteriaceae, Morganellaceae, Pseudomonadaceae, Enterococcaceae, Staphylococcaceae and Streptococcaceae). The classification models were applied to artificial mixtures of Gram positive and Gram negative bacteria to correctly predict mixed infections with an accuracy of 75%. Raman scans of dried droplets did not yet yield optimal classification results on family level. When translating the method to primary urine samples, we observed a strong bias towards Gram negative bacteria, on family level towards Morganellaceae, which reduced prediction accuracy. Spectral differences were observed between isolates grown on standard growth medium and bacteria of the same strain when characterized directly from the patient. Thus, improvement of the classification accuracy is expected with a larger data base containing also bacteria measured directly from the urine sample.

Insights into S. aureus-Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging.

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation.

High-throughput Raman spectroscopy allows ex vivo characterization of murine small intestinal intra-epithelial lymphocytes (IEL).

T cells are considered to be critical drivers of intestinal inflammation in mice and people. The so called intra-epithelial lymphocyte (IEL) compartment largely consist of T cells. Interestingly, the specific regulation and contribution of IELs in the context of inflammatory bowel disease remains poorly understood, in part due to the lack of appropriate analysis tools. Powerful, label-free methods could ultimately provide access to this cell population and hence give valuable insight into IEL biology and even more to their disease-related functionalities. Raman spectroscopy has demonstrated over the last few years its potential for reliable cell characterization and differentiation, but its utility in regard to IEL exploration remains unknown. To address this question experimentally, we utilized a murine, T cell-driven experimental model system which is accepted to model human gut inflammation. Here, we repopulated the small intestinal IEL compartment (SI IELs) of Rag1-deficient mice endogenously lacking T cells by transferring naïve CD4+ T helper cells intraperitoneally. Using multivariate statistical analysis, high-throughput Raman spectroscopy managed to define a cell subpopulation ex vivo within the SI IEL pool of mice previously receiving T cells in vivo that displayed characteristic spectral features of lymphocytes. Raman data sets matched flow cytometry analyses with the latter identifying T cell receptor (TCR)αß+ CD4+ T cell population in SI IELs from T cell-transferred mice, but not from control mice, in an abundance comparable to the one detected by Raman spectroscopy. Hence, in this study, we provide experimental evidence for high-throughput Raman spectroscopy to be a novel, future tool to reliably identify and potentially further characterize the T cell pool of small intestinal IELs ex vivo.

Importance of ACE2 for SARS-CoV-2 Infection of Kidney Cells.

In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global concern. COVID-19 proceeds with moderate symptoms in most patients, whereas others experience serious respiratory illness that requires intensive care treatment and may end in death. The severity of COVID-19 is linked to several risk factors including male sex, comorbidities, and advanced age. Apart from respiratory complications, further impairments by COVID-19 affecting other tissues of the human body are observed. In this respect, the human kidney is one of the most frequently affected extrapulmonary organs and acute kidney injury (AKI) is known as a direct or indirect complication of SARS-CoV-2 infection. The aim of this work was to investigate the importance of the protein angiotensin-converting enzyme 2 (ACE2) for a possible cell entry of SARS-CoV-2 into human kidney cells. First, the expression of the cellular receptor ACE2 was demonstrated to be decisive for viral SARS-CoV-2 cell entry in human AB8 podocytes, whereas the presence of the transmembrane protease serine 2 (TMPRSS2) was dispensable. Moreover, the ACE2 protein amount was well detectable by mass spectrometry analysis in human kidneys, while TMPRSS2 could be detected only in a few samples. Additionally, a negative correlation of the ACE2 protein abundance to male sex and elderly aged females in human kidney tissues was demonstrated in this work. Last, the possibility of a direct infection of kidney tubular renal structures by SARS-CoV-2 was demonstrated.

Label-Free Characterization of Macrophage Polarization Using Raman Spectroscopy.

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.

Interaction of Masitinib with Organic Cation Transporters.

Tyrosine kinase inhibitors (TKI) such as Masitinib were reported to be useful as therapeutic options in malignant disorders and nonmalignant diseases, like coronavirus disease 2019 (COVID-19). Most kinases must be translocated into targeted cells by the action of specific transport proteins, as they are hydrophilic and not able to cross cell membranes freely. Accordingly, the efficacy of TKI in target cells is closely dependent on the expression of their transporters. Specifically, Masitinib is an organic cation and is expected to interact with organic cation transporters (OCT and Multidrug and Toxin Extrusion proteins-MATE-). The aim of this work was to characterize the interaction of Masitinib with different OCTs. Human embryonic kidney 293 cells stably transfected with murine or human OCT were used for the experiments. The interaction of Masitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of this drug was quantified using high performance liquid chromatography. Our results identified interactions of Masitinib with almost all investigated mouse (m) and human (h) OCTs and hMATE1 and indicated OCT1 and hOCT2 to be especially potent Masitinib translocators across cell membranes. Interestingly, some important differences were observed for the interaction with murine and human OCTs. In the future, investigations concerning further in vitro and in vivo properties of Masitinib and its efficacy related to transporter-related uptake mechanisms under pathophysiological conditions should be performed. Clinical trials in humans and other animals with Masitinib have already shown promising results. However, further research is necessary to understand the disease specific transport mechanisms of Masitinib to contribute to a successful and responsible therapy employment.

Raman Spectroscopic Differentiation of Streptococcus pneumoniae From Other Streptococci Using Laboratory Strains and Clinical Isolates.

Streptococcus pneumoniae, commonly referred to as pneumococci, can cause severe and invasive infections, which are major causes of communicable disease morbidity and mortality in Europe and globally. The differentiation of S. pneumoniae from other Streptococcus species, especially from other oral streptococci, has proved to be particularly difficult and tedious. In this work, we evaluate if Raman spectroscopy holds potential for a reliable differentiation of S. pneumoniae from other streptococci. Raman spectra of eight different S. pneumoniae strains and four other Streptococcus species (S. sanguinis, S. thermophilus, S. dysgalactiae, S. pyogenes) were recorded and their spectral features analyzed. Together with Raman spectra of 59 Streptococcus patient isolates, they were used to train and optimize binary classification models (PLS-DA). The effect of normalization on the model accuracy was compared, as one example for optimization potential for future modelling. Optimized models were used to identify S. pneumoniae from other streptococci in an independent, previously unknown data set of 28 patient isolates. For this small data set balanced accuracy of around 70% could be achieved. Improvement of the classification rate is expected with optimized model parameters and algorithms as well as with a larger spectral data base for training.

Looking Inside Non-Destructively: Label-Free, Raman-Based Visualization of Intracellular Coxiella burnetii.

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 Coxiella burnetii infection model. C. burnetii is a zoonotic obligate intracellular pathogen that causes infections in ruminant livestock and humans with an acute disease known as Q fever. Using statistical data analysis, no isolation is necessary to gain detailed information on the intracellular pathogen's metabolic state. High-quality false color image stacks with diffraction-limited spatial resolution enable a 3D spatially resolved single host cell analysis that shows excellent agreement with results from transmission electron microscopy. Quantitative analysis at different time points post infection allows to follow the infection cycle with the transition from the large cell variant (LCV) to the small cell variant (SCV) at around day 6 and a gradual change in the lipid composition during vacuole maturation. Spectral characteristics of intracellular LCV and SCV reveal a higher lipid content of the metabolically active LCV.

Ethoxy acetalated dextran-based nanocarriers accomplish efficient inhibition of leukotriene formation by a novel FLAP antagonist in human leukocytes and blood.

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.

Biochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy.

Biochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte's specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.

Expression and Function of Organic Cation Transporter 2 in Pancreas.

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 ß cells of Langerhans islets, together with GLUT2. However, a co-localization was only evident in a cell-line model of rat pancreatic ß 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.

Leukocyte Activation Profile Assessed by Raman Spectroscopy Helps Diagnosing Infection and Sepsis.

OBJECTIVES: Leukocytes are first responders to infection. Their activation state can reveal information about specific host immune response and identify dysregulation in sepsis. This study aims to use the Raman spectroscopic fingerprints of blood-derived leukocytes to differentiate inflammation, infection, and sepsis in hospitalized patients. Diagnostic sensitivity and specificity shall demonstrate the added value of the direct characterization of leukocyte's phenotype. DESIGN: Prospective nonrandomized, single-center, observational phase-II study (DRKS00006265). SETTING: Jena University Hospital, Germany. PATIENTS: Sixty-one hospitalized patients (19 with sterile inflammation, 23 with infection without organ dysfunction, 18 with sepsis according to Sepsis-3 definition). INTERVENTIONS: None (blood withdrawal). MEASUREMENTS AND MAIN RESULTS: Individual peripheral blood leukocytes were characterized by Raman spectroscopy. Reference diagnostics included established clinical scores, blood count, and biomarkers (C-reactive protein, procalcitonin and interleukin-6). Binary classification models using Raman data were able to distinguish patients with infection from patients without infection, as well as sepsis patients from patients without sepsis, with accuracies achieved with established biomarkers. Compared with biomarker information alone, an increase of 10% (to 93%) accuracy for the detection of infection and an increase of 18% (to 92%) for detection of sepsis were reached by adding the Raman information. Leukocytes from sepsis patients showed different Raman spectral features in comparison to the patients with infection that point to the special immune phenotype of sepsis patients. CONCLUSIONS: Raman spectroscopy can extract information on leukocyte's activation state in a nondestructive, label-free manner to differentiate sterile inflammation, infection, and sepsis.

Pals1 prevents Rac1-dependent colorectal cancer cell metastasis by inhibiting Arf6.

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.