Fluorescence Intensity Measurements Research Articles

#2615 Altered peroxidasin expression pattern in IgA nephropathy

Abstract Background and Aims PXDN is a multifunctional heme peroxidase responsible for the crosslinking of type IV collagen protomers at basement membranes, playing a crucial role in the extracellular matrix dynamics. In renal pathophysiology, increased PXDN expression has been associated with pro-fibrotic and pro-carcinogenic mechanisms and we have shown that PXDN is expressed throughout all kidney compartments since the early stages of nephrogenesis. However, the role of PXDN in kidney health and disease remains largely unknown. In this work we performed a comparative analysis of PXDN expression between renal biopsy samples from IgA nephropathy (IgAN) patients and healthy kidney biopsy samples. IgAN was the selected CKD model due to its straightforward diagnosis by standard protocols for histopathological evaluation of kidney biopsies, and to the fact that the renal outcomes are consistently associated with several key pathologic features, including tubular atrophy and interstitial fibrosis. Correlations between PXDN expression and IgAN disease and kidney function parameters as well as with ECM proteins were also explored. Method Fluorescence immunohistochemistry for PXDN was performed in biopsy samples from IgAN patients (n = 20) and in biopsies from healthy kidney donors (n = 7). Proximal tubules were assessed by anti-CD15 fluorescent staining. Collagen III (COLIII) deposition was investigated in IgAN samples (n = 7) by immunofluorescence. Brightfield images were manually segmented into glomeruli, proximal and distal tubules, and interstitium, using QuPath software, and the fluorescence intensity measurements were obtained with ImageJ/Fiji. Results Results showed that, in comparison with healthy kidney samples, PXDN is significantly decreased in proximal and distal tubules of IgAN patients (P < 0.001 and P < 0.05, respectively) whereas it is increased in IgAN interstitium (P < 0.001). When IgAN samples were stratified by the presence or absence of chronic lesions (glomerular sclerosis and interstitial fibrosis) we observed that in the proximal tubules, PXDN is decreased even in areas without lesions while in the distal tubules the expression is compromised only in the lesioned areas. In the interstitium, PXDN shows a tendency of increase from healthy tissue to IgAN in areas without and with tubulointerstitial fibrosis, presenting a significant increase in the lesioned areas (P < 0.001). Concerning ECM deposition, results showed a significant correlation between the expression of PXDN and COLIII in sclerotic glomeruli (R = 0.8214; P = 0.0341) and in the tubulointerstitial fibrotic areas (R = 0.93; P = 0.0067). Moreover, although no differences were observed in the glomerular expression of PXDN between healthy and IgAN samples, when considering eGFR, a significant increase of PXDN was observed in both non-lesioned and sclerotic glomeruli from IgAN patients with eGFR bellow 60 ml/min/1,73 m2. In the interstitium, the expression of PXDN was also increased in tubulointerstitial fibrotic areas of IgAN patients with eGFR bellow 60 ml/min/1,73 m2, in comparison with the same areas in patients with higher eGFR values. Conclusions Overall, our study shows a differential expression of PXDN in IgAN, with loss in the tubular region that relates with the state of interstitial fibrosis, and gain in the glomerular and interstitial region where it correlates with COLIII compartment-specific deposition and with kidney function as assessed by eGFR. These data support, for the first time, the enrollment of PXDN in the topographic mechanism of IgA-mediated kidney lesion that deserves further investigations. Given that sclerosis and fibrosis are a common pathogenic mechanism of CKD, irrespective of the underlying etiology, we postulate that PXDN may serve as a novel therapeutic target for this disease.

Continuous distribution of cancer cells in the cell cycle unveiled by AI-segmented imaging of 37,000 HeLa FUCCI cells

Classification of live or fixed cells based on their unlabeled microscopic images would be a powerful tool for cell biology and pathology. For such software, the first step is the generation of a ground truth database that can be used for training and testing AI classification algorithms. The Application of cells expressing fluorescent reporter proteins allows the building of ground truth datasets in a straightforward way. In this study, we present an automated imaging pipeline utilizing the Cellpose algorithm for the precise cell segmentation and measurement of fluorescent cellular intensities across multiple channels. We analyzed the cell cycle of HeLa–FUCCI cells expressing fluorescent red and green reporter proteins at various levels depending on the cell cycle state. To build the dataset, 37,000 fixed cells were automatically scanned using a standard motorized microscope, capturing phase contrast and fluorescent red/green images. The fluorescent pixel intensity of each cell was integrated to calculate the total fluorescence of cells based on cell segmentation in the phase contrast channel. It resulted in a precise intensity value for each cell in both channels. Furthermore, we conducted a comparative analysis of Cellpose 1.0 and Cellpose 2.0 in cell segmentation performance. Cellpose 2.0 demonstrated notable improvements, achieving a significantly reduced false positive rate of 2.7 % and 1.4 % false negative. The cellular fluorescence was visualized in a 2D plot (map) based on the red and green intensities of the FUCCI construct revealing the continuous distribution of cells in the cell cycle. This 2D map enables the selection and potential isolation of single cells in a specific phase. In the corresponding heatmap, two clusters appeared representing cells in the red and green states. Our pipeline allows the high-throughput and accurate measurement of cellular fluorescence providing extensive statistical information on thousands of cells with potential applications in developmental and cancer biology. Furthermore, our method can be used to build ground truth datasets automatically for training and testing AI cell classification. Our automated pipeline can be used to analyze thousands of cells within 2 h after putting the sample onto the microscope.

In Vitro and In Vivo Behavioral Evaluation of Condensed Lipid-Coated Perfluorocarbon Nanodroplets

BackgroundPhase-shift contrast agents consist of a liquid perfluorocarbon core that can be vaporized by ultrasound to generate echogenic contrast with excellent spatiotemporal control. The purpose of the present work was to evaluate the in vitro and in vivo behavior of condensed lipid-shelled nanodroplets using different analytical procedures. MethodsIn this study, perfluorobutane nanodroplets were prepared by condensation of precursor fluorescently labeled lipid shelled microbubbles and were characterized in terms of size distribution, gas core content, in vitro stability in blood as well as for their acoustic vaporization behavior using a custom-made set-up. In particular, their in vivo behavior was thoroughly investigated after intravenous bolus injection in rats. To this end, we report, for the first time, the efficient use of three complementary detection procedures to assess the NDs in vivo persistence namely 1) Ultrasound contrast imaging of vaporized NDs, 2) Gas chromatography-mass spectrometry to determine the perfluorobutane core content and 3) Fluorescence intensity measurement in the collected blood samples. ResultsThe Coulter Counter Multisizer results confirmed the size distribution shift post-condensation. Furthermore, similar PFB concentration from MBs and NDs suspensions was obtained, thus demonstrating an exceptionally low rate of microbubbles breaking and spontaneous nanodroplets vaporization. As expected, these nanoscale droplets display longer circulation time compared to clinically approved microbubbles and only slight variations of half-life were observed between the three monitoring procedures. Finally, echogenic signal observed in focal area of the liver and spleen after vaporization was confirmed by accumulation of fluorescent nanodroplets in these organs. ConclusionThese results further contribute to deepen our understanding of both in vitro and in vivo behavior of sonoresponsive nanodroplets, which is key to enable efficient clinical translation.

Oxalohydrazide‐Based Chemosensor for Detection of Cu2+/Fe3+ Ions and Its Application in Live Cell Imaging

AbstractThe N1,N2‐diphenyloxalohydrazide OX(HA)2 chemosensor was synthesized and used as a colorimetric and fluorescent receptor to detect Cu2+ and Fe3+ ions in the presence of other metal ions. A range of physicochemical approaches were utilized to characterize the symmetrical oxalohydrazide‐based receptor, including FT‐IR, 1H and 13C NMR, TGA, and mass spectrometry. According to single crystal X‐ray diffraction investigation, OX(HA)2 crystallizes in monoclinic with space group C2/c. UV‐Vis and fluorescence spectroscopy were used to thoroughly explore OX(HA)2’s sensing capabilities towards diverse cations. Using measurements of fluorescence intensity, the binding constant for OX(HA)2 with Cu2+/Fe3+ ions was calculated to be 2.78×103 M−1 and 1.12×103 M−1 with the lower detection limit of 0.43 μM and 0.2 μM respectively. With a 1 : 1 binding mechanism validated by Job's plot, mass spectral analysis, FT‐IR spectra, and density functional theory, the dual‐sensing receptor exhibits remarkable selectivity and sensitivity towards Cu2+ and Fe3+ ions. The average fluorescence lifetimes of OX(HA)2, OX(HA)2+Cu2+, and OX(HA)2+Fe3+ were calculated to be 2.19 ns, 1.73 ns, and 1.26 ns, respectively. This fluorescence lifetime measurement results strongly support the complexation of Cu2+ and Fe3+. When Cu2+ and Fe3+ ions bind with OX(HA)2, they induce the ‘Turn‐Off’ signal. Then, fluorescence imaging of the OX(HA)2 receptor in MDA‐MB‐231 demonstrated that the receptor holds a lot of promise for usage in bioimaging and their IC50 value for cytotoxicity is 96.96 μg. Furthermore, fluorescence microscopy experiments demonstrate that OX(HA)2 can be utilized as a fluorescent probe for detecting Cu2+ and Fe3+ ions in living cells, making it potentially helpful as an anticancer medication.

Abstract 3196: Overcoming lung cancer drug resistance: Enhanced cellular delivery using lipid-extracted nanoliposomes

Abstract Background: Lung cancer causes substantial cancer deaths in the U.S. Drug resistance remains a major issue. Nanoliposomes demonstrate enhanced drug targeting and resistance-overcoming potential. Incorporating lipid extracts derived from target cells shows improved delivery over conventional nanoliposomes. This study examined the effects of lung cancer cell lipid extract nanoliposomes against target and non-target cell populations from different environments. Method: Cellular Membrane Lipid-Extracted Nanoliposomes (CLENs) were fabricated with DOPC, DOTAP, ChaGo-K-1 lipid extract, cholesterol, PEG5000, and rhodamine fluorescent label was incorporated for uptake studies. The liposome size was reduced by probe sonication. Particle size and polydispersity index (PDI) were measured by dynamic light scattering. Zeta potential was quantified by laser Doppler micro-electrophoresis. Four cell lines were utilized: ChaGo-K-1 NSCLC target, A549 pulmonary microenvironment control, 4T1 extra-pulmonary control, and normal lung fibroblasts off-target control. All cell lines were cultured in 48-well plates at a seeding density of 10,000 cells/mL and incubated for 24 hours. Freshly prepared CLENs were added to all cell lines and incubated for an additional 24 hours. Cells were washed with PBS, and fluorescence intensity was then quantified and qualified by a microplate reader to measure the cellular uptake of the CLENs. Results: The mean particle size and PDI of the ChaGo-K-1 CLENs were 117 ± 0.7 nm and 0.216 ±0.009, compared to 126 ± 0.5 nm and 0.195 ± 0.008 for the control, respectively. Both formulations were within the optimal size range for drug delivery and tumor cell penetration. The zeta potential of the ChaGo-K-1 CLENs was -1.75 ± 1.81 mV compared to -1.74 ± 6 mV for the control. Quantification and qualification of cellular uptake using fluorescence intensity measurements revealed a 3.2-fold increase in ChaGo-K-1 CLENs compared to conventional liposomes without ChaGo-K-1 lipid extracts. This demonstrates that the inclusion of the lipid extracts in the CLEN significantly enhanced selectivity and delivery to target cancer cell populations. Conclusion: Higher uptake occurred with target cells using CLENs with lipid extracts versus standard liposomes. NSCLC-derived CLENs show potential to improve selective delivery and overcome drug resistance. Further, in vivo studies evaluating selective NSCLC tumor accumulation and efficacy enhancement are warranted. Citation Format: Mohammed Alqaryan, Rashad Allahyani, Dayeoun Lee, Robert B. Campbell. Overcoming lung cancer drug resistance: Enhanced cellular delivery using lipid-extracted nanoliposomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3196.

Laccase‐mediated polymerization of tannins from a pine bark extract: Toward an eco‐friendly valorization of forest wastes

AbstractThe valorization of tannins from forest by‐products is a major opportunity for the sustainable development of the forestry industry. Herein we report the laccase‐mediated polymerization of tannins obtained from an aqueous pine bark extract. The reaction was conducted under an oxygen atmosphere at 50°C for 60 min and followed by UV–Vis, fluorescence intensity, phenol content, and viscosity measurements. An insoluble polymer was obtained after 45 min, which was attributed to coupling reactions triggered by phenoxyl radicals within the tannins mixture. The polymer showed a marked increase in thermal stability compared with the precursor pine bark extract, with a total mass loss of less than 12% at temperatures up to 500°C, suggesting potential practical applications in flame retardant materials. Additionally, the polymer showed an 85% DPPH activity and a 60% attenuation of singlet oxygen release upon light irradiation, which supports antioxidant and photoprotective properties for the prepared material. To the best of our knowledge, this is the first report to describe the formation of an insoluble polymer by direct enzymatic reaction of an aqueous tannin extract. In this case, soluble tannins are the sole reactants used to produce a promising versatile material for forest waste valorization.

Theranostic potential of a novel aptamer specifically targeting HER2 in breast cancer cells.

The overexpression of HER2 is correlated with poorer outcomes and therapeutic resistance in breast cancer patients. While HER2-targeted therapies have shown improvement, prognosis remains poor for HER2-positive breast cancer patients, and these treatments have limitations. Therefore, it is crucial to explore effective molecular strategies for early detection and treatment of HER2-positive breast cancers. In this study, we employed the cell-SELEX method to generate a selective aptamer capable of recognizing HER2 in its native conformation within breast cancer cells, for theranostic applications. Utilizing an adherent cell-SELEX approach, we developed and explored a DNA aptamer, named HMAP7, which can specifically target HER2 in the MDA-MB-453 and SK-BR-3 human breast cancer cell lines. After sequencing, the binding affinities of 10 candidate aptamers to HER2 receptors were evaluated by measuring fluorescence intensities within intact cells using near-infrared optical imaging. The dissociation constant of HMAP7 was determined to be in the nanomolar range in both cell lines. The cell-SELEX-derived aptamer sequence, HMAP7 (41-mer), exhibited the highest binding affinity and specificity for HER2. HMAP7 was rapidly internalized into breast cancer cells overexpressing HER2 but showed no uptake in the HER2 receptor-deficient breast cancer cell line MDA-MB-231. Moreover, HMAP7 demonstrated remarkable selectivity for HER2, rendering it suitable for use in complex biological systems. Our findings suggest that the novel DNA aptamer HMAP7 holds promise for both therapeutic and diagnostic applications, enabling selective delivery of therapeutic agents or imaging of HER2-positive breast tumors.

Aptamer-enhanced organic electrochemical transistors for ultra-sensitive dopamine detection

In this study, we present the development of an ultrasensitive dopamine (DA) aptasensor based on organic electrochemical transistors (OECTs). The aptasensor utilizes a highly efficient DA aptamer immobilized on the surface of a gold gate electrode in the OECT. The functionalization of the aptamer was confirmed through fluorescence intensity measurements and transfer characteristics of the OECT device. Real-time detection of DA was achieved, and the sensor exhibited a linear response in the concentration range of 10 fM to 10 nM, with a detection limit of 10 fM. The aptasensor's working mechanism was elucidated, involving mutual binding between DA and the aptamer that leads to a shift in the transfer characteristics. Our aptasensor shows great promise for sensitive and rapid detection of DA, which is crucial in diagnosing neurological disorders such as Parkinson's and Alzheimer's diseases. The combination of OECTs and aptamer-based recognition elements offers a cost-effective and efficient platform for detecting biomolecules like DA in various clinical applications.

Rapid, label-free, contactless measurement of membrane potential in excitable H9c2 cardiomyoblasts using ζ-potential

The measurement of cell membrane potential (V m) is important for understanding ion channel function. V m plays a role in several routine cellular functions and diseases, particularly in excitable cells such as muscle and nerve. However, measuring V m is difficult, relying either on labour-intensive direct measurement of single cells (intracellular electrodes, patch clamp) or indirect measurement of fluorescence intensity, using V m-sensitive labels. Here we demonstrate a direct measurement technique based on determination of the cell’s ζ-potential, the electrical potential at the hydrodynamic shear plane, approximately 1 nm beyond the cell surface. We demonstrate this principle using excitable H9c2 cardiomyoblasts, measured in both polarised and depolarised states, before and after extracellular intervention to alter cell ion concentration. Given widespread availability of ζ-potential measurement apparatus (most typically in chemistry and materials science settings), this offers a new method of measuring V m without the need for fluorescence measurements or calibration curves.

Measuring Antibody-Mediated Tau Fibril Uptake in Microglia by Flow Cytometry.

Microglia are brain-resident phagocytic cells, considered to be the innate immune cells of the central nervous system. Microglia respond to both infectious pathogens in the brain and sterile cellular debris, including the proteinaceous aggregates that accumulate in the brains of patients with Alzheimer's disease (AD). Microtubule-associated protein Tau is an intracellular protein that self-aggregates into neurofibrillary tangles in Alzheimer's disease and many other neurodegenerative diseases. Ongoing clinical trials are testing whether therapeutic antibodies specific to Tau protein aggregates can reduce pathological protein deposition and improve the course of disease. Data suggest that Tau-specific antibodies act on extracellular Tau aggregates by promoting uptake into microglia cells and thus preventing its prion-like spread to unaffected neurons. Here we describe a protocol to test the effect of Tau-specific antibodies on Tau uptake into microglia by flow cytometry. Recombinant Tau protein is fibrillized in vitro and tagged with a fluorescent label. Then, fibrillized Tau is incubated with the antibody of interest and applied to microglial cells in culture. Uptake of Tau into microglia is then assessed by measuring fluorescence intensity by flow cytometry. With slight modifications, this assay can be used to test effects of many antibodies, various Tau protein compositions, and different microglial sources in a high-throughput format.

Efficient monodisperse upconversion composite prepared using high-density local field and its dual-mode temperature sensing.

Enhanced upconversion via plasmonics has considerable potential in biosensors and solar cells; however, conventional plasmonic configurations such as core-shell assemblies or nanoarray platforms still suffer from the compromise between the enhancement factor and monodispersity, which has failed to meet the requirement of the materials for the in vivo all-solution-prepared solar cells and biosensors. We herein report a monodisperse metal-dielectric-metal (MDM) type upconverted hybrid material with high efficiency. The lanthanide-doped upconversion nanoparticles (UCNPs) were sandwiched by two gold nanodisk mirrors, and the highly localized excitation field around the UCNPs together with the efficient coupling enhanced the upconversion. The upconversion intensity can then be effectively regulated and improved by three to four orders of magnitude. As per the measurement of the temperature-dependent fluorescence intensity and spectra shift, a dual-mode nanothermometer based on our proposed hybrid materials was demonstrated. This MDM-type upconverted hybrid material demonstrated the merits of high efficiency and monodispersity, which demonstrated promise in in vivo biosensors and solar cell fabrication techniques such as spin-coating and roll-to-roll.

The influence of powdery mildew (Sawadaea bicornis (Wallr.) Miyabe) on chlorophyll fluorescence in Norway maple (Acer platanoides L.) seedlings

The article presents the results of research on impact of Sawadaea bicornis (Wallr.) Miyabe on the state of photosynthetic apparatus in Acer platanoides L. seedlings using a technique of chlorophyll fluorescence induction (ChlF) measurement, which at the present time can be implemented through the use of biosensors. The research was conducted in September 2022 in the territory of the Botanical Garden of Oles Honchar Dnipro National University. To diagnose a violation of the native chlorophyll photosynthesis in fresh leaves of A. platanoides, a portable fluorometer “Floratest” was used (selected spectral range for fluorescence intensity measurement was 670–800 nm). The research was carried out on fresh leaves of Norway maple seedlings both not unaffected and affected with powdery mildew. Analysis of the data obtained indicates a high sensitivity of the parameters of chlorophyll fluorescence induction to damage by the disease regardless of environmental conditions of local growth of A. platanoides seedlings. The high informativeness of induction changes in chlorophyll fluorescence in the structural organization of chloroplasts in Norway maple leaves determined by the parameters Fo, Fm, Fv, Fv/Fо, Fv/Fm, (Fm–Fst)/Fst, (Fp–Fо)/Fv was revealed. This study demonstrated the effectiveness of using the studied chlorophyll fluorescence parameters to detect severe stress in Norway maple seedlings caused by powdery mildew exposure when the fungus affects more than 50% of the leaf blade area. It is further necessary to conduct dynamic studies throughout the growing season to determine the effectiveness of using these parameters to detect mild stress in the early stages of infection. Therefore, our research showed that the method of induction of chlorophyll fluorescence using the “Floratest” device allows you to quickly determine the general condition of the plant by evaluating photosynthesis, which is the main life process. Analysis of chlorophyll fluorescence parameters is a powerful and effective tool for determining the effect of a parasitic fungus on a plant.

A solid-phase fluorescence sensor for measuring chemical species in water

In this study, the first of its kind, a solid-phase fluorescence sensing platform was developed to quantify contaminants in water. ZnO quantum dots (QDs) were combined with molecularly imprinted polymers (MIPs) to form fluorescence sensing materials. Solid sensing layers were formed via a straightforward spin-coating method, which demonstrated a strong attachment to the sensor substrate while maintaining the integrity of the sensing materials. The developed sensing platform comprised a portable fluorescence detector to measure fluorescence intensity, instead of traditional fluorescence spectroscopy. The solid sensing platform was first tested with 2,4-dichlorophenoxyacetic acid (2,4-D), demonstrating high sensitivity (0.0233) and a very strong correlation (0.98) between the target molecule concentration and sensor signal. Further, the sensing platform was successfully adapted to measure a substance with a different molecular mass and chemical structure, the algae toxin microcystin-LR (MCLR); this demonstrated the sensor's versatility in quantifying target molecules. Tap water samples spiked with MCLR were also used to test the sensor's practical application. Finally, the working mechanism of the sensing platform was established, and the key information for using the sensor to measure various contaminants was determined. With its high performance, broad applicability, and ease of use, the developed platform provides a suitable basis for lab-on-chip image-based sensing devices for environmental monitoring.

Single‐molecule Analysis of Protein Glycosylation Patterns for Early Detection of Alzheimer’s Disease Biomarkers

AbstractBackgroundIn recent years, it has become apparent that protein glycosylation plays a variety of roles in the development of Alzheimer’s disease (AD). For instance, characteristic glycosylation patterns of tau and APP have been found in AD patient samples, and O‐GlcNAcylation of alpha‐synuclein (aSyn) has been indicated as a therapeutic anti‐AD target. By extension, the presence of specific glycoforms in patient samples could constitute an important biomarker for early detection purposes (Zhou et al., 2023, Alzheimer’s Dement., https://doi.org/10.1002/alz.13024). However current state‐of‐the‐art methods using mass spectrometry (MS) typically detect proteins starting at millions of copies, thus they lack the sensitivity to perform detection in early disease stages. Moreover, they are prohibitively expensive for routine clinical analysis.MethodWe have developed an ultra‐sensitive protein analysis method that can be used to localize post‐translational modifications in full‐length proteins, including glycans, at single‐molecule resolution (Filius et al., 2021, Nano letters, https://doi.org/10.1021/acs.nanolett.1c00725). Using Förster resonance energy transfer (FRET), a fluorescence‐based distance measurement approach with sub‐nanometer resolution, our method can determine both presence and approximate location of glycans on the protein. Contrary to MS, it does not require complex sample preparation, such as enzymatic digestion or peptide enrichment. We validated this approach by detecting O‐GlcNAcylation on aSyn.ResultBased on measured fluorescence intensities and without using prior information on aSyn’s O‐GlcNAcylation tendencies, we deduced that molecules are doubly O‐GlcNAcylated. Using a calibration construct, we then inferred that the modified residues are in between residues 42 and 78. This is in accordance with MS measurements on bulk samples, which indicate that double glycosylation had taken place at Thr54 and Thr64 respectively. Our results demonstrate for the first time that O‐GlcNAc can be localized on full‐length proteins with single molecule sensitivity.ConclusionUsing our highly sensitive protein analysis approach, we have demonstrated the detection and location of glycans on full‐length aSyn at single‐molecule resolution. We believe that the high resolution of our method will allow for the detection of biomarkers in patient samples in early stages of disease. In future work, we will expand the proof‐of‐concept presented here for such usage by detecting AD‐associated tau and APP glycoforms.

Evaluation of topotecan and 10-hydroxycamptothecin on Toxoplasma gondii: Implications on baseline DNA damage and repair efficiency

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes toxoplasmosis in humans and animals worldwide. Despite its prevalence, there is currently no effective vaccine or treatment for chronic infection. Although there are therapies against the acute stage, prolonged use is toxic and poorly tolerated. This study aims to explore the potential of repurposing topotecan and 10-hydroxycamptothecin (HCPT) as drugs producing double strand breaks (DSBs) in T. gondii. DSBs are mainly repaired by Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ). Two T. gondii strains, RHΔHXGPRT and RHΔKU80, were used to compare the drug's effects on parasites. RHΔHXGPRT parasites may use both HRR and NHEJ pathways but RHΔKU80 lacks the KU80 protein needed for NHEJ, leaving only the HRR pathway. Here we demonstrate that topotecan and HCPT, both topoisomerase I venoms, affected parasite replication in a concentration-dependent manner. Moreover, variations in fluorescence intensity measurements for the H2A.X phosphorylation mark (γH2A.X), an indicator of DNA damage, were observed in intracellular parasites under drug treatment conditions. Interestingly, intracellular replicative parasites without drug treatment show a strong positive staining for γH2A.X, suggesting inherent DNA damage. Extracellular (non-replicating) parasites did not exhibit γH2A.X staining, indicating that the basal level of DNA damage is likely to be associated with replicative stress. A high rate of DNA replication stress possibly prompted the evolution of an efficient repair machinery in the parasite, making it an attractive target. Our findings show that topoisomerase 1 venoms are effective antiparasitics blocking T. gondii replication.

Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature.

Fluorescence microscopy is of vital importance for understanding biological function. However, most fluorescence experiments are only qualitative inasmuch as the absolute number of fluorescent particles can often not be determined. Additionally, conventional approaches to measuring fluorescence intensity cannot distinguish between two or more fluorophores that are excited and emit in the same spectral window, as only the total intensity in a spectral window can be obtained. Here we show that, by using photon number resolving experiments, we are able to determine the number of emitters and their probability of emission for a number of different species, all with the same measured spectral signature. We illustrate our ideas by showing the determination of the number of emitters per species and the probability of photon collection from that species, for one, two and three otherwise unresolvable fluorophores. The convolution binomial model is presented to represent the counted photons emitted by multiple species. Then, the expectation-maximization (EM) algorithm is used to match the measured photon counts to the expected convolution binomial distribution function. In applying the EM algorithm, to leverage the problem of being trapped in a sub-optimal solution, the moment method is introduced to yield an initial guess for the EM algorithm. Additionally, the associated Cramér-Rao lower bound is derived and compared with the simulation results.

Molecular Accessibility and Diffusion of Resorufin in Zeolite Crystals.

We have used confocal laser scanning microscopy on the small, fluorescent resorufin dye molecule to visualize molecular accessibility and diffusion in the hierarchical, anisotropic pore structure of large (~10 μm-sized) zeolite-β crystals. The resorufin dye is widely used in life and materials science, but only in its deprotonated form because the protonated molecule is barely fluorescent in aqueous solution. In this work, we show that protonated resorufin is in fact strongly fluorescent when confined within zeolite micropores, thus enabling fluorescence microimaging experiments. We find that J-aggregation guest-guest interactions lead to a decrease in the measured fluorescence intensity that can be prevented by using non-fluorescent spacer molecules. We characterized the pore space by introducing resorufin from the outside solution and following its diffusion into zeolite-β crystals. The eventual homogeneous distribution of resorufin molecules throughout the zeolite indicates a fully accessible pore network. This enables the quantification of the diffusion coefficient in the straight pores of zeolite-β without the need for complex analysis, and we found a value of 3×10-15 m2 s-1 . Furthermore, we saw that diffusion through the straight pores of zeolite-β is impeded when crossing the boundaries between zeolite subunits.

Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 biocontrol Dickeya soft rot diseases by quenching virulence factor modulating quorum sensing signal.

Virulence factor modulating (VFM) is a quorum sensing (QS) signal shared by and specific to Dickeya bacteria, regulating the production of plant cell wall degrading enzymes (PCWDEs) and virulence of Dickeya. High polarity and trace of VFM signal increase the difficulty of signal separation and structure identification, and thus limit the development of quorum quenching strategy to biocontrol bacterial soft rot diseases caused by Dickeya. In order to high-throughput screen VFM quenching bacteria, a vfmE-gfp biosensor VR2 (VFM Reporter) sensitive to VFM signal was first constructed. Subsequently, two bacterial strains with high quenching efficiency were screened out by fluorescence intensity measurement and identified as Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 using multilocus sequence analysis (MLSA). L5 and L95 supernatants reduced the expression of vfm genes, and both strains also decreased the production of PCWDEs of D. zeae MS2 and significantly reduced the virulence of D. oryzae EC1 on rice seedlings, D. zeae MS2 on banana seedlings, D. dadantii 3937 on potato and D. fangzhongdai CL3 on taro. Findings in this study provide a method to high-throughput screen VFM quenching bacteria and characterize novel functions of P. chlororaphis and E. asburiae in biocontrolling plant diseases through quenching VFM QS signal.

An emission based optical CO2 sensor fabricated on grating-like TiO2 substrates using HPTS

TiO2 is a widely used material in various applications, including photo-catalysis, sensing, and dye-sensitized solar cells. In this work, we presented a stable and sensitive CO2 sensor working on principle of emission signals of the HPTS dye immobilized on TiO2 substrates whose effectiverefractive indicesin the parallel and perpendicular grating orientations evidenced earlier. The HPTS exhibited 8.4-fold enhanced emission signal on the offered substrates with respect to the previously reported intensities recorded for the polymeric supports. The emission peaks of the HPTS exhibited CO2 induced intensity quenching when excited at 466 nm. Calibration sensitivity of the offered composites has been tested exposing the sensor materials to varying concentrations of the CO2 between 0.0 and 100.0 % pCO2 and correlating the measured fluorescence intensity with the corresponding CO2 levels. The dye revealed enhanced CO2 induced response exhibiting the I0/100 values of 11.9, 13.3, 11.2 and 6.5. We also recorded bi-exponential excited state lifetimes for the HPTS on four different test materials both in the absence and presence of the CO2. The emission based variations were followed as the analytical signal due to higher CO2-induced relative signal changes than that of the lifetime based variations. Herein we used the ion pair form of the HPTS in a protective chemical microenvironment and obtained considerable long term stability extending to16 mounts which can be attributed to the presence of the 1-butyl-3-methylimidazolium tetrafluoroborate as an internal buffering system in the sensing composition as well as the inert and robust structure of the substrate. The extremely high optical response, advantage of excitation with blue LEDs and long-term stability observed on grating-like TiO2 surfaces make the proposed system a promising design for the quantification of CO2 for further applications.

Measurement of Blood-Brain Barrier Hyperpermeability Using Evans Blue Extravasation Assay.

Blood-brain barrier (BBB) dysfunction and hyperpermeability have been implicated in a myriad of brain pathologies. TheEvans Blue assay is one of the most popular methods for studying BBB integrity and permeability in rodent models of brain disorders. Under normal physiological conditions, the BBB is impermeable to albumin, so Evans Blue when injected intravenously binds to serum albumin and remains restricted within blood vessels. In traumatic and ischemic injuries, and other brain pathologies that result in BBB hyperpermeability, neighboring endothelial cells partially lose their close contacts to each other, and the BBB becomes permeable to proteins such as albumin. This paracellular leak of Evans blue-bound albumin is considered a reliable indicator of BBB dysfunction and hyperpermeability. Here, we describe the procedures for the evaluation of BBB integrity and hyperpermeability using Evans Blue extravasation assay in a mouse model of traumatic brain injury. The method described here focuses on intravenous injection of Evans Blue followed by Evans Blue dye extraction. This is followed by the measurement of fluorescence intensity of Evans Blue to determine the dye extravasation as a direct indicator of BBB hyperpermeability.