Performance comparison of Findaures and Otsu’s thresholding method on simulated infected mouse bone tissue images (Accuracy, Precision, Sensitivity, and F1 Score) across four trials.

Performance comparison of Findaures and Otsu’s thresholding method on simulated infected mouse bone tissue images (Accuracy, Precision, Sensitivity, and F1 Score) across four trials.</p

Raman Spectroscopic Characterization of Packaged <i>L. pneumophila</i> Strains Expelled by <i>T. thermophila</i>

The intracellular lifestyle of <i>L. pneumophila</i> within protozoa is considered to be a fundamental process that supports its survival in nature. However, after ingesting the cells of <i>L. pneumophila</i>, some protozoa expel them as compressed live cells in the form of small rounded pellets. The pellets of tightly packaged viable but not culturable forms (VBNCFs) as well as highly infectious mature intracellular forms (MIFs) of <i>L. pneumophila</i> are considered as infectious particles most likely capable to cause human infection. Since <i>L. pneumophila</i> cells are hardly culturable from these pellets, detection methods for packaged live <i>L. pneumophila</i> forms remaining in water should be cultivation free. Hence, we demonstrate the potential of Raman microspectroscopy to directly sort pellets containing <i>L. pneumophila</i> cells, expelled by <i>T. thermophila</i>, and to characterize them on the basis of their Raman spectra

Simple Ciprofloxacin Resistance Test and Determination of Minimal Inhibitory Concentration within 2 h Using Raman Spectroscopy

Resistant bacteria are spreading worldwide, which makes fast antibiotic susceptibility testing and determination of the minimal inhibitory concentration (MIC) urgently necessary to select appropriate antibiotic therapy in time and, by this, improve patient’s outcome and, at the same time, avoid inappropriate treatment as well as the unnecessary use of broad spectrum antibiotics that would foster further spread of resistant bacteria. Here, a simple and fast Raman spectroscopy-based procedure is introduced to identify antimicrobial susceptibilities and determine the MIC within only 2 h total analysis, marking a huge time savings compared to established phenotypic methods nowadays used in diagnostics. Sample preparation is fast and easy as well as comparable to currently established tests. The use of a dielectrophoresis chip allows automated collection of the bacteria in a micron-sized region for high-quality Raman measurement directly from bacterial suspensions. The new Raman spectroscopic MIC test was validated with 13 clinical E. coli isolates that show a broad range of ciprofloxacin resistance levels and were collected from patients with blood-stream infection. Micro-Raman spectroscopy was able to detect ciprofloxacin-induced changes in E. coli after only 90 min interaction time. Principal component analysis as well as a simple computed ratio of the Raman marker bands at 1458 and 1485 cm^–1 show a clear concentration-dependent effect. The MIC values determined with the new Raman method are in good agreement with MICs obtained by reference methods (broth microdilution, Vitek-2, E-test) and can be used to provide a classification as sensitive, intermediate, or resistant using the clinical breakpoints provided by EUCAST

Label-Free Imaging and Spectroscopic Analysis of Intracellular Bacterial Infections

<i>Staphylococcus aureus</i> is one of the most frequent human pathogens that can also act as a facultative intracellular pathogen causing infections that are extremely difficult to treat. Only little is known about the pathogen’s intracellular adaptation strategies to escape the host’s response. Here, we present an advanced Raman-based imaging approach providing high quality false-color images to specifically identify intracellular <i>S. aureus</i> and to localize them exactly in three dimensions within endothelial cells. At the same time unprecedented insights into the metabolic characteristics of the pathogen are provided in a label-free and nondestructive manner. The spectral information reveals that the intracellular bacteria are in the exponential growth phase with a reduced replication rate and biochemically different from extracellular bacteria proving their adaptation to the host’s conditions. This powerful biophotonic analysis tool paves the way for further mechanistic studies of difficult-to-investigate infection processes

IR Spectroscopic Methods for the Investigation of the CO Release from CORMs

Carbon monoxide (CO) is a toxic gas for mammals, and despite this fact, it is naturally produced in these organisms and has been proven to be beneficial in medical treatments, too. Therefore, CO-releasing molecules (CORMs) are intensively developed to administer and dose CO for physiological applications. Nearly all of these compounds are metal carbonyl complexes, which have been synthesized and investigated. However, for most of these CORMs, the exact reaction mechanisms of CO release is not completely elucidated, although it is of utmost importance. The widely used myoglobin assay for testing the CO release has several disadvantages, and therefore, different methods have to be applied to characterize CORMs. In this work, different setups of IR absorption spectroscopy are used to analyze and quantify the CO release during the decay of various CORMs: IR spectroscopy of the gas phase is applied to follow the CO liberation, and attenuated total reflection (ATR) IR spectroscopy is used to record the decay of the metal carbonyl. IR spectroscopy supported by DFT calculations yields valuable insights in the CO release reaction mechanism. The focus is set on two different CORMs: CORM-2 (Ru₂(CO)₆Cl₄) and on the photoactive CORM-S1 (photoCORM [Fe­(CO)₂(SCH₂CH₂NH₂)₂]). Our results indicate that the CO liberation from CORM-2 strongly depends on sodium dithionite, which is required for the commonly applied myoglobin assay and that CORM-S1 loses all its bound CO molecules upon irradiation with blue light

Kidney Transplantation Down-Regulates Expression of Organic Cation Transporters, Which Translocate β‑Blockers and Fluoroquinolones

Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), β-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, β-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug–drug competition at the transporter can also alter drug excretion rate

Co-Registered Molecular Logic Gate with a CO-Releasing Molecule Triggered by Light and Peroxide

Co-registered molecular logic gates combine two different inputs and outputs, such as light and matter. We introduce a biocompatible CO-releasing molecule (CORM, <b>A</b>) as Mn­(I) tricarbonyl complex with the ligand 5-(dimethylamino)-<i>N</i>, <i>N</i>-bis­(pyridin-2-ylmethyl) naphthalene-1-sulfonamide (<b>L</b>). 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 <b>A</b> 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 <b>A</b> toward different human cell lines. The CORM has been used to track the inclusion of <b>A</b> into cancer cells

CORM-EDE1: A Highly Water-Soluble and Nontoxic Manganese-Based photoCORM with a Biogenic Ligand Sphere

[Mn­(CO)₅Br] reacts with cysteamine and 4-amino-thiophenyl with a ratio of 2:3 in refluxing tetrahydrofuran to the complexes of the type [{(OC)₃Mn}₂­(μ-SCH₂CH₂NH₃)₃]­Br₂ (<b>1</b>, CORM-EDE1) and [{(OC)₃­Mn}₂(μ-SC₆H₄-4-NH₃)₃]­Br₂ (<b>2</b>, CORM-EDE2). Compound <b>2</b> precipitates during refluxing of the tetrahydrofuran solution as a yellow solid whereas <b>1</b> forms a red oil that slowly solidifies. Recrystallization of <b>2</b> from water yields the HBr-free complex [{(OC)₃Mn}₂(μ-S-C₆H₄-4-NH₂)₂­(μ-SC₆H₄-4-NH₃)] (<b>3</b>). The <i>n</i>-propylthiolate ligand (which is isoelectronic to the bridging thiolate of <b>1</b>) leads to the formation of the di- and tetranuclear complexes [(OC)₄Mn­(μ-S-<i>n</i>Pr)₂]₂ and [(OC)₃Mn­(μ-S-<i>n</i>Pr)]₄. CORM-EDE1 possesses ideal properties to administer carbon monoxide to biological and medicinal tissues upon irradiation (photoCORM). Isolated crystalline CORM-EDE1 can be handled at ambient and aerobic conditions. This complex is nontoxic, highly soluble in water, and indefinitely stable therein in the absence of air and phosphate buffer. CORM-EDE1 is stable as frozen stock in aqueous solution without any limitations, and these stock solutions maintain their CO release properties. The reducing dithionite does not interact with CORM-EDE1, and therefore, the myoglobin assay represents a valuable tool to study the release kinetics of this photoCORM. After CO liberation, the formation of MnHPO₄ in aqueous buffer solution can be verified