Fluorescence-based Method Research Articles

Naltrexone blocks alcohol-induced effects on kappa-opioid receptors in the plasma membrane

Naltrexone (NTX), a homolog of the opiate antidote naloxone, is an orally active long-acting general opioid receptor antagonist used in the treatment of opiate dependence. NTX is also found to relieve craving for alcohol and is one of few FDA-approved medications for treatment of alcohol use disorder (AUD). While it was early on established that NTX acts by blocking the binding of endogenous opioid peptide ligands released by alcohol, experimental evidence emerged that could not be fully accounted for by this explanation alone, suggesting that NTX may have additional modes of action. Mu- and kappa-opioid receptors (MOP and KOP, respectively) are structurally related G-protein-coupled receptors (GPCRs), but they are anatomically differently distributed and functionally distinct, often mediating opposite responses, with MOP typically promoting euphoria and reward, while KOP is associated with dysphoria and aversive states. While the actions of NTX on MOP are extensively characterized, the interactions with KOP are not. Here, we used sensitive fluorescence-based methods with single-molecule sensitivity to study in live cells the influence of alcohol (ethanol, EtOH) on KOP and the interaction between KOP and NTX. Our data show that alcohol, at relevant concentrations (10–40 mM), alters KOP interactions with the lipid environment in the plasma membrane. The counteracting effects of NTX are exerted by both its canonical action on KOP and its hitherto unrevealed effects on the lateral dynamics and organization of lipids in the plasma membrane. The KOP-specific antagonist LY2444296, in clinical trial for major depressive disorder (MDD), blocks KOP but does not show the full action profile of NTX. The therapeutic effect of NTX treatment in AUD may in part be due to direct actions on KOP and in part due to its effect on the surrounding lipid environment.

Sclareolide as Antifungal Strategy Against Cryptococcus neoformans: Unveiling Its Mechanisms of Action.

Cryptococcal infection commonly begins as an opportunistic infection in humans, however, this can escalate to a systemic or life-threatening form in immunocompromised individuals. Here, we aim to identify novel antifungal molecules from plants resources. Sclareolide, a phytochemical classified as a sesquiterpene lactone, was assessed against Cryptococcus neoformans H99. Sclareolide exhibited promising antifungal properties with a minimum inhibitory concentration (MIC) of 16 µg/mL. Additionally, the C. neoformans growth rate was significantly affected by sclareolide treatment in a concentration-dependent manner, as observed through a time killing assay, with a significant reduction at MIC × 8 compared to the control by 48 h. To elucidate the underlying mechanisms of sclareolide antifungal activity, fluorescence-based methods were employed. Propidium iodide (PI) accumulation assay indicated a reduction in C. neoformans membrane integrity, with values as low as 6.62 ± 0.18% after treatment. Moreover, sclareolide at MIC × 4 and MIC × 8 significantly increased the production of reactive oxygen species (ROS) and reduced the mitochondrial membrane potential (MMP), suggesting oxidative stress and mitochondrial dysfunction in C. neoformans. Sclareolide did not induce caspase-dependent apoptosis, suggesting a non-apoptotic mechanism. Further, a checkerboard experiment was performed to assess potential synergistic interaction with Amphotericin B, however, no synergism was observed. Moving on, sclareolide at 128 µg/mL did not exhibit toxicity in Galleria mellonella, further supporting its potential as a safe antifungal agent. These findings suggest that the antifungal activity of sclareolide against C. neoformans is mediated by oxidative stress. Further in vivo and pharmacokinetic studies are recommended to explore the potential of sclareolide as a prototype for the development of novel anti-cryptococcal therapies.

Establishing a dominant early larval sex-selection strain in the Asian malaria vector Anopheles stephensi

BackgroundGenetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions.MethodsTo address the challenge of sex-sorting in the Asian malaria vector Anopheles stephensi, we engineer Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter (SEPARATOR). This dominant fluorescent-based method, previously proven effective in Aedes aegypti, exploits sex-specific alternative splicing of a reporter to ensure exclusive male-specific expression early in development. The sex-specific alternative RNA splicing of the doublesex gene was selected as a target for engineering SEPARATOR due to its evolutionary conservation in insects. To expand SEPARATOR’s applicability for genetic sexing, we assessed the cross-species sex-specific RNA splicing activity of the An. gambiae doublesex (AngDsx) splicing module in An. stephensi. Male-specific enhanced green fluorescent protein (EGFP) expression was verified throughout the mosquito life cycle using a fluorescent stereomicroscope.ResultsOur results confirm that SEPARATOR regulates male-specific EGFP expression in An. stephensi and enables reliable positive male selection from the first instar larval stages. Molecular analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. Additionally, the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species.ConclusionsSEPARATOR’s independence from sex-chromosome linkage provides resistance to breakage that could be mediated by meiotic recombination and chromosomal rearrangements, making it highly suitable for mass male releases. By enabling precise male selection from the first instar larval stages, SEPARATOR represents a significant advancement that will aid in the genetic biocontrol for Anopheles mosquitoes.Graphical

Eco-Friendly Analytical Approach for Sensitive Spectrofluorimetric Determination of the Flavonoid Chrysin in Capsules and Human Plasma.

Chrysin is a plant flavonoid that has different therapeutic effects as anti-inflammatory, anti-cancer, anti-oxidant, and immune booster. Spectrofluorimetry has received a lot of interest lately because of itsecological greenness and analytical performance. This approach employed the native fluorescence of chrysin at 339nm following excitation at 231nm in distilled water. Modern advances in analytical chemistry have been used to lessen occupational and environmental concerns by employing distilled water as a dilution solvent throughmethod development and application. The approach was found to be excellent green supported byeco-scale score of 97and 0.94AGREErating, in addition to an overall whiteness score of 88.80. The design aimed to analyze chrysin in raw materials, Chrysin® capsules and human plasma. The method was linear over0.5-7.0ngmL⁻1 chrysin, with LOD of 0.06ngmL⁻1 and LOQ of 0.20ngmL⁻1. The offered methodwas effectively applied for determination of chrysin inthe commercial capsules Chrysin® and spiked human plasma samples with average recoveries of 99.76%and 99.98%, respectively for capsules andspiked human plasma. Up to date, no spectrofluorimetric method has been described for chrysin analysis, then, this presented an opportunity to develop a sensitive, quick, reliable, environmentally friendly, and valid fluorescence-based method.

Cellular Phosphate Sensing and Anion Binding by an Azacrown-Calixpyrrole Hybrid.

A hybrid receptor-sensor for anions originating from the merging of positively charged ammonium moieties for electrostatic attraction/stronger binding of azacrowns with directionality of calixpyrrole hydrogen bond donors for selectivity is investigated. As demonstrated this hybrid receptor-sensor shows a remarkable selectivity for orthophosphate even in the presence of other phosphates and anions found in cellular materials (Kassoc H2PO4 ->H2P2O7 2->AMP-≫ADP2- or ATP3- over halides, nitrate, or hydrogen sulfate; all Na+ salts in water) but also cellular polyphosphate or phospholipids. This selectivity is harnessed in a real-time monitoring of cell lysis by lysozyme, which releases orthophosphate and other phosphates and anions from the cells. This sensitive (LOD 0.4 μM) fluorescence-based microscale method compares favorably with the state-of-the-art techniques but can easily be practiced in a high-throughput screening (HTS) manner. The anion binding and selectivity in aqueous solutions were investigated by NMR and put in context with phosphate binding of the parent calix[4]pyrrole. The microscopic understanding of anion binding by the hybrid receptor was then obtained from a combination of density functional theory (DFT), classical molecular dynamics (MD) with explicit water solvation, and ab initio MD (AIMD) simulations. Correlating the NMR and fluorescence binding data with studies of solvation of the receptor, phosphate anion, and the resulting complex confirms the binding is largely driven by entropic component (TΔS) associated with receptor and anion desolvation.

Nuclease-induced stepwise photodropping (NISP) to precisely investigate single-stranded DNA degradation behaviors of exonucleases and endonucleases.

Here, we employed a fluorescence-based single molecule method called nuclease-induced stepwise photodropping (NISP) to measure in realtime the DNA degradation mediated by mitochondrial genome maintenance exonuclease 1 (MGME1), a bidirectional single-stranded DNA (ssDNA)-specific exonuclease. The method detects a stepwise decrease in fluorescence signals from Cy3 fluorophores labeled on an immobilized DNA substrate. Using NISP, we successfully determined the DNA degradation rates of 6.3±0.4and 2.0±0.1 nucleotides (nt) s-1 for MGME1 in the 5'-to-3' and 3'-to-5' directions, respectively. These results provide direct evidence of the stronger 5' directionality of MGME1, consistent with its established role in mitochondrial DNA maintenance. Importantly, when we employed NISP to investigate mung bean nuclease, an ss-specific endonuclease, we observed a markedly different NISP pattern, suggesting a distributive cleavage activity of the enzyme. Furthermore, we applied NISP to determine the ssDNA degradation behavior of the double-stranded-specific exonuclease, λexonuclease. These findings underscore the capability of NISP to accurately and reliably measure the degradation of ssDNA by both exo- and endonucleases. Here, we demonstrate NISP as a powerful tool for investigating the ssDNA degradation behavior of nucleases at the single-molecule level.

Novel Fluorescence-Based Methods to Determine Infarct and Scar Size in Murine Models of Reperfused Myocardial Infarction.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson's (r = 0.9920) and Gomori's Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

Visual Detection of Dopamine with CdS/ZnS Quantum Dots Bearing by ZIF-8 and Nanofiber Membranes.

Dopamine (DA) is a widely present, calcium cholinergic neurotransmitter in the body, playing important roles in the central nervous system and cardiovascular system. Developing fast and sensitive DA detection methods is of great significance. Fluorescence-based methods have attracted much attention due to their advantages of easy operation, a fast response speed, and high sensitivity. This study prepared hydrophilic and high-performance CdS/ZnS quantum dots (QDs) for DA detection. The waterborne CdS/ZnS QDs were synthesized in one step using the amphiphilic polymer PEI-g-C14, obtained by grafting tetradecane (C14) to polyethyleneimine (PEI), as a template. The polyacrylonitrile nanofiber membrane (PAN-NFM) was prepared by electrospinning (e-spinning), and a metal organic frame (ZIF-8) was deposited in situ on the surface of the PAN-NFM. The CdS/ZnS QDs were loaded onto this substrate (ZIF-8@PAN-NFM). The results showed that after the deposition of ZIF-8, the water contact angle of the hydrophobic PAN-NFM decreased to within 40°. The nanofiber membrane loaded with QDs also exhibited significant changes in fluorescence in the presence of DA at different concentrations, which could be applied as a fast detection method of DA with high sensitivity. Meanwhile, the fluorescence on this PAN-NFM could be visually observed as it transitioned from a blue-green color to colorless, making it suitable for the real-time detection of DA.

Imaging and proteomics toolkits for studying organelle contact sites.

Organelle contact sites are regions where two heterologous membranes are juxtaposed by molecular tethering complexes. These contact sites are important in inter-organelle communication and cellular functional integration. However, visualizing these minute foci and identifying contact site proteomes have been challenging. In recent years, fluorescence-based methods have been developed to visualize the dynamic physical interaction of organelles while proximity labeling approaches facilitate the profiling of proteomes at contact sites. In this review, we explain the design principle for these contact site reporters: a dual-organelle interaction mechanism based on how endogenous tethers and/or tethering complexes localize to contact sites. We classify the contact site reporters into three categories: (i) single-protein systems, (ii) two-component systems with activated reporter signal upon organelle proximity, and (iii) reporters for contact site proteomes. We also highlight advanced imaging analysis with high temporal-spatial resolution and the use of machine-learning algorithms for detecting contact sites.

Establishing a coumarin production platform by protein and metabolic engineering

Coumarins are a vast family of natural products with diverse biological activities. Cinnamyl-CoA ortho-hydroxylases (CCHs) catalyze the gateway and rate-limiting step in coumarin biosynthesis. However, engineering CCHs is challenging due to the large size of the substrates and the vague structure-activity relationship. Herein, directed evolution and structure-guided engineering were performed to engineer a CCH (AtF6′H from Arabidopsis thaliana) using a fluorescence-based screening method, yielding the transplantable surface mutations and the substrate-specific pocket mutations with improved activity. Structural analysis and molecular dynamics simulations elucidated the conformational changes that led to increased catalytic efficiency. Applying appropriate variants with the optimized upstream biosynthetic pathways improved the titers of three simple coumarins by 5 to 22-fold. Further introducing glycosylation modules resulted in the production of four coumarin glucosides, among which the titer of aesculin was increased by 15.7-fold and reached 3 g/L in scale-up fermentation. This work unleashed the potential of CCHs and established an Escherichia coli platform for coumarins production.

Fluorescence-based determination of tobramycin using a portable smartphone-based device

The study presents the development of a robust portable smartphone-based device for determining tobramycin in drugs and urine by a fluorescence-based method. As existing analytical methods for the determination of tobramycin often require complex equipment and time-consuming procedures, the proposed approach offers simplicity, portability and real-time monitoring capabilities. The spectrofluorimetric method is based on the reaction of tobramycin and fluorescamine in an alkaline medium. The measurement of analytical signals was enabled with a specially designed device integrating a 3D printed adapter, LEDs, a macro lens, an Arduino-based microcontroller, and asmartphone. The traditional spectrofluorimetry was used as a reference method. Preliminary and optimisation studies were conducted to select the most suitable experimental conditions (e.g. reagent concentration and reaction time) as well as instrumental parameters (e.g. selection of the appropriate RGB model component, ISO, and white balance). The performance of the method was validated, and then verified against conventional spectrofluorimetry, showing comparable accuracy and precision. The method implemented in a smartphone-based device showed a linear range of 0.10–1.00 mgL-1 with a detection limit of 0.03 mgL-1. Synthetic samples were tested to verify analytical usability, followed by analysis of pharmaceutical and urine samples. The method showed good precision (CV<3.4 %) and recovery values (92.3–114.1 %), consistent with spectrofluorimetric results (CV<5.9 %, recovery 92.3–119.8 %). Comparison with other methods highlighted the simplicity and efficiency of the developed approach. The research reveals that it is competitive with existing well-established methods. The proposed device offers reliable results with low reagent and sample consumption, making itsuitable for on-the-go analysis in non-laboratory conditions.

Measuring concrete air-entraining admixture adsorption on coal ash using three-phase equilibrium and fluorescence-based methods

This manuscript presents two novel methods of evaluating the adsorption of air-entraining admixtures (AEAs) by coal ashes used in cementitious mixtures. A developed three-phase equilibrium (TPE) method measures fly ash adsorption capacity accounting for carbon adsorption, Ca2+ interactions with AEA, and the equilibrium between the two. A fluorescence-based method (FBM) was also developed, utilizing a non-ionic NP-10 surfactant as a representative for AEA. This study verifies the applicability and accuracy of the TPE and FBM methods using seven class C and F coal ashes with a wide range of loss on ignition values, varying from 0.2 to 15.6 %, and three commercial AEAs. Verified with foam index test results, the TPE method was applicable to all tested AEAs and coal ashes. The results were consistent between the FBM and TPE methods when comparing rosin- and fatty acid-based AEAs, but less consistent when using a sulfonate-based AEA. These findings help us understand the applicability and limitations of the TPE and FBM methods and provide two methods for quantifying adsorption in fly ash samples.

Biogenic and Anthropogenic Secondary Organic Aerosols Become Fluorescent after Highly Acidic Aging.

Primary biological aerosol particles (PBAPs) and secondary organic aerosol (SOA) both contain organic compounds that share similar chemical and optical properties. Fluorescence is often used to characterize PBAPs; however, this may be hindered due to interferences from fluorophores in SOA. Despite extensive efforts to understand the aging of SOA under elevated particle acidity conditions, little is known about how these processes affect the fluorescence of SOA and thereby their interference with the measurements of PBAPs. The objective of this study is to investigate the fluorescence of SOA and understand the influence of acidity on the optical properties of organic aerosols and potential interference for the analysis of bioaerosols. The SOA was generated by O3- or OH-initiated oxidation of d-limonene or α-pinene, as well as by OH-initiated oxidation of toluene or xylene. The SOA compounds were then aged by exposure to varying concentrations of aqueous H2SO4 for 2 days. Absorption and fluorescence spectrophotometry were used to examine the changes in the optical properties before and after aging. The key observation was the appearance of strongly light-absorbing and fluorescent compounds at pH = ∼-1, suggesting that acidity is a major driver of SOA aging. The aged SOA from biogenic precursors (d-limonene and α-pinene) resulted in stronger fluorescence than the aged SOA from toluene and xylene. The absorption spectra of the aged SOA changed drastically in shape upon dilution, whereas the shapes of the fluorescence spectra remained the same, suggesting that the fluorophores and chromophores in SOA are separate sets of species. The fluorescence spectra of aged SOA overlapped with the fluorescence spectra of PBAPs, suggesting that SOA exposed to highly acidic conditions can be confused with PBAPs detected by fluorescence-based methods. These processes are likely to play a role in the atmospheric regions where high concentrations of H2SO4 persist, such as the upper troposphere and lower stratosphere.

Excreted albumin of diabetic microalbuminuria cases exhibits pseudo esterase activity: A new way to explore microalbuminuria, perhaps with more information

BackgroundMicroalbuminuria is associated with several clinical conditions of public health concern. Particularly in diabetic patients, there is routine microalbuminuria screening to understand whether the renal complication has progressed to the microalbuminuria stage or not. Therefore, microalbuminuria detection is a matter of considerable interest. For such detection, the clinical labs rely on immunochemical methods. Nevertheless, the immunochemical methods are believed to be less sensitive for the purpose. So, the need arises for continuous research in the field. We believe that pseudoesterase activity of the excreted albumin in microalbuminuria cases is a potential target. This aspect is investigated here and it is shown that the excreted albumin in diabetic microalbuminuria cases retains its pseudoesterase activity, unlike the overt albuminuria cases. MethodsThe cases of diabetic nephropathy and healthy controls were included in the study. The patients were divided into diabetic controls microalbuminuria, and overt albuminuria group considering the albumin to creatinine ratio (ACR). The urinary proteins of the cases were isolated by centrifugation. The obtained protein pellet was then checked for pseudoesterase activity by electrophoretic and fluorescence-based methods. The CD spectroscopy and LC-MS study was carried out to show the suitability of the substrate for the detection of albumin pseudoesterase activity. To further, understand the structure–function relation, molecular docking studies were carried out. ResultsFrom the CD and LC-MS study the suitability of the used substrate was confirmed. The electrophoretic and fluorescence study showed that the protein of the microalbuminuria group retained the pseudoesterase activity whereas the same is lost in the overt albuminuria group. The molecular docking studies indicated that a change in albumin structure may result in a change in its pseudoesterase activity. ConclusionThe urinary protein of diabetic microalbuminuria cases exhibits pseudoesterase activity. It distinguishes the excreted protein in the diabetic albuminuria group and the overt albuminuria group. This is the first study that showed the retention of pseudoesterase property in excreted albumin. Further, in this study a simple test is developed that distinguishes the excreted albumin in the microalbuminuria group and overt albuminuria group.

Templated 3ʹ terminal fluorescent labeling of RNA using Klenow DNA polymerase

A long-standing challenge in the study of RNA structure-function dynamics using fluorescence-based methods has been the precise attachment of fluorophores to structured RNA molecules. Despite significant advancements in the field, existing techniques have limitations, especially for 3ʹ end labeling of long, structured RNAs. In response to this challenge, we developed a chemo-enzymatic method that uses Klenow DNA polymerase to label RNAs. In this method:•Klenow DNA polymerase adds an amino-modified nucleotide to the 3ʹ end of the RNA, guided by the DNA oligonucleotide template.•An NHS-ester dye is then conjugated to the amino-modified RNA, forming a covalent amide bond.•For highly structured RNAs, DNA oligonucleotides complementary to the RNA disrupt pre-existing intramolecular RNA structures.This methodological advancement enables site-specific incorporation of a single modified nucleotide at the 3′ terminus of various RNA substrates, irrespective of their length or secondary structure. The user-friendly nature of the technique, with minimal modifications required for different RNA targets, makes it readily adaptable by a broad range of researchers. This approach has the potential to significantly improve the development of functionalized RNA for various applications.

Single 5-nm quantum dot detection via microtoroid optical resonator photothermal microscopy

Label-free detection techniques for single particles and molecules play an important role in basic science, disease diagnostics, and nanomaterial investigations. While fluorescence-based methods are tools for single molecule detection and imaging, they are limited by available molecular probes and photoblinking and photobleaching. Photothermal microscopy has emerged as a label-free imaging technique capable of detecting individual nanoabsorbers with high sensitivity. Whispering gallery mode (WGM) microresonators can confine light in a small volume for enhanced light-matter interaction and thus are a promising ultra-sensitive photothermal microscopy platform. Previously, microtoroid optical resonators were combined with photothermal microscopy to detect 250 nm long gold nanorods and 100 nm long polymers. Here, we combine microtoroids with photothermal microscopy to spatially detect single 5 nm diameter quantum dots (QDs) with a signal-to-noise ratio exceeding 104. Photothermal images were generated by point-by-point scanning of the pump laser. Single particle detection was confirmed for 18 nm QDs by high sensitivity fluorescence imaging and for 5 nm QDs via comparison with theory. Our system demonstrates the capability to detect a minimum heat dissipation of 0.75 pW. To achieve this, we integrated our microtoroid based photothermal microscopy setup with a low amplitude modulated pump laser and utilized the proportional-integral-derivative controller output as the photothermal signal source to reduce noise and enhance signal stability. The heat dissipation of these QDs is below that from single dye molecules. We anticipate that our work will have application in a wide variety of fields, including the biological sciences, nanotechnology, materials science, chemistry, and medicine.

Electrokinetics with Surface-Enhanced Raman Spectroscopy for Bacterial Identification in Wastewater

Bacterial presence in wastewater serves as a crucial bioindicator for monitoring population-level infections and offering early disease outbreak warnings [1,2]. However, identifying bacteria in wastewater presents outstanding challenges; notably, current culturing or fluorescence-based methods [3] to identify bacteria are slow and expensive, unsuitable for high-throughput screening of diverse bacterial species, and may not work well in the complex wastewater matrix. Here, we harness the potential of surface chemistry and electrokinetics to introduce an innovative electro-optical approach that has the promise to detect a broad spectrum of pathogenic bacteria in wastewater.We combine surface-enhanced Raman spectroscopy (SERS) [4] with alternating current electric fields, complemented by machine learning models [5], to enable rapid and amplification-free bacteria detection in filtered wastewater, at cell concentrations as low as 104 cells/mL. First, we synthesize gold nanorods that can electrostatically bind to bacteria surfaces, allowing for SERS from cell surfaces. We collect SERS from bacteria spiked into filtered wastewater, including Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, spanning concentrations from 109 cells/mL to 104 cells/mL. Spectral clustering analysis shows that bacteria signals become more indistinguishable from wastewater as the concentrations decrease. Further, we incorporate electrokinetic effects into SERS by employing gold microelectrodes to apply electric fields, utilizing dielectrophoresis [6] to rapidly displace and concentrate bacteria based on their dielectric responses within minutes. The enrichment of bacteria with nanorods is directly visualized through microscopy, resulting in Raman signal intensities increased by up to tenfold under electrical fields for bacterial concentrations ranging from 106 down to 104 cells/mL. Such enhancement may enable the detection sensitivity to reach environmentally-relevant concentrations. Moreover, employing data science analysis, we identify biologically-relevant “fingerprint” Raman peaks characterizing proteins, nucleic acids, and lipids from bacteria surfaces, allowing for rapid identification of bacteria species in wastewater. This novel method exhibits potential for generalized pathogen detection and molecular recognition in complex liquid samples, such as wastewater, blood, and seawater.[1] Hellmér, et al. Applied and environmental microbiology. (2014).[2] Keshaviah, et al. The Lancet Global Health. (2023).[3] Jahn, et al. Nature Microbiology. (2022).[4] Tadesse, et al., Nano Lett. (2020).[5] Ho, et al., Nat. Comm. (2019).[6] Pethig. John Wiley & Sons (2010).

Plasmid-Stabilizing Strains for Antibiotic-Free Chemical Fermentation.

Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168. The application of EcΔ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in EcΔ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of BsΔ44 was due to the deletion of the phage SPP1 surface receptor gene yueB, resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain BsΔyueB exhibited a 61.99% higher acetoin titer compared to strain Bs168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain BsΔyueB achieved an 80.63% higher titer than Bs168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for E. coli and B. subtilis, highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.

Determination of bactericidal activity against 3HC-2-Tre-labelled Mycobacterium abscessus (Mycobacteroides abscessus) by automated fluorescence microscopy

The minimum bactericidal concentration (MBC) of antibiotics is an important parameter for the potency of a drug in eradicating a bacterium as well as an important measure of the potential of a drug candidate in research and development. We have established a fluorescence-based microscopy method for the determination of MBCs against the non-tuberculous mycobacterium Mycobacterium abscessus (Mycobacteroides abscessus) to simplify and accelerate the performance of MBC determination compared to counting colony forming units on agar. Bacteria are labelled with the trehalose-coupled dye 3HC-2-Tre and analysed in a 96-well plate. The results of the new method are consistent with MBC determination by plating on agar. The method was used to evaluate the bactericidality of the antibiotics rifabutin, moxifloxacin, amikacin, clarithromycin and bedaquiline. Bactericidal effects against M. abscessus were observed, which are consistent with literature data.

A Fluorescence-based Method to Reaccess Root Canals in Endodontically Treated Teeth: A Micro–Computed Tomography Tridimensional Assessment

IntroductionThe aim of this study was to evaluate the volume of dentin removal and the volume of remnants of restorative material after the removal of an esthetic restorative coronal set and cervical barrier in endodontically treated mandibular molars with the aid of different magnification methods using 3-dimensional (3D) micro–computed tomographic (micro-CT) morphometric analysis. MethodsA sample of 30 mandibular first molars (N = 30) was used. All teeth were endodontically treated, and the specimens were initially scanned using micro-CT imaging and reconstructed. The molars were filled by a single-cone technique, and immediately the material at the initial 2-mm cervical level was removed. Cervical barriers were confected using ionomer glass cement with fluorescein 0.1%, filling the 2 mm at the cervical level of the canals and an additional 2 mm as the base. The coronal restoration set was performed using esthetic resin composites. A simulated tooth aging process was performed with 20,000 thermocycling cycles. The sample was distributed into the following 3 groups (n = 10) for the removal of the restoration set and cervical barrier with diamond burs based on the magnification aid: no magnification aid (naked eye), operative microscope aid, and REVEAL device (Design for Vision Inc, Bohemia, NY) aid. After removal, the final 3D micro-CT scanning and reconstruction were conducted with the same parameters as the initial scanning, and superposition of the final and initial scanning was performed. Morphometric analysis was conducted using CTAn software (Bruker microCT, Kontich, Belgium) to assess the volume of remnant restorative material (mm³), the volume of dentin removal (mm³), and the direction and site of dentin removal. Data were analyzed using 1-Way analysis of variance (P < .05). ResultsThe REVEAL group showed better results regarding the volume of remnant material (3.17 ± 1.65) and the percentage of dentin removal (2.56 ± 1.34). The microscope group showed no statistical difference compared with the REVEAL group regarding dentin removal (3.30 ± 1.48) and was statistically similar to the naked eye group in the volume of remnant material (9.63 ± 4.33). The naked eye group showed the worst results for the volume of remnant material (7.60 ± 2.68) and the percentage of dentin removal (6.60 ± 3.70). ConclusionsThe use of fluorescence associated with magnification was the method that presented the best results, with lower percentages of dentin removal and smaller volumes of remaining restorative material. This is an innovative technology in endodontics that shows potential to overcome the challenge of reaccessing root canals in the context of endodontic retreatment.