Increase In Fluorescence Signal Research Articles

Temporal Progression of Excitotoxic Calcium Following Distal Middle Cerebral Artery Occlusion in Freely Moving Mice.

Ischemic stroke is recognized as one of the leading causes of adult disability, morbidity, and death worldwide. Following stroke, acute neuronal excitotoxicity can lead to many deleterious consequences, one of which is the dysregulation of intracellular calcium ultimately culminating in cell death. However, to develop neuroprotective treatments that target neuronal excitotoxicity, it is essential to know the therapeutic time window for intervention following an ischemic event. To address this question, the current study aimed to characterize the magnitude and temporal progression of neuronal intracellular calcium observed following distal middle cerebral artery occlusion (dMCAO) in mice. Using the calcium fluorescence indicator, GCaMP, we tracked neuronal population response in freely moving animals immediately following dMCAO in both the core infarct and peri-infarct regions. Our results demonstrate that calcium excitotoxicity following artery occlusion can be generally characterized by two phases: a transient increase in activity that lasts tens of minutes, followed by a long, slow sustained increase in fluorescence signal. The first phase is primarily thought to represent neuronal hyperexcitability, defining our therapeutic window, while the second may represent gradual cell death. Importantly, we show that the level of intracellular calcium following artery occlusion correlated with the infarct size at 24 h demonstrating a direct connection between excitotoxicity and cell death in our stroke model. In addition, we show that administration of the NMDA antagonist MK-801 resulted in both a decrease in calcium signal and a subsequent reduction in the infarct size. Altogether, this study represents the first demonstration in freely moving animals characterizing the temporal progression of toxic calcium signaling following artery occlusion. In addition, these results define a critical time window for neuroprotective therapeutic intervention in mice.

Modular DNA-Incorporated Aggregation-Induced Emission Probe for Sensitive Detection and Imaging of DNA Methyltransferase.

DNA adenine methylation (Dam) MTase serves a very important epigenetic process that transfers a methyl group on an adenine residue including N6-methyladenosine (m6A). A variety of evidence have demonstrated that m6A methylation plays a significant role in modulating genes in human disease and development. Hence, a modular DNA-incorporated AIEgen probe (TPE-Py-DNA) was specifically developed for detection and imaging of Dam MTase. TPE-Py-DNA consisted of two modules: a "turn-on" fluorescent AIEgen (TPE-Py) and a DNA sequence (Alk-DNA) involved in specific recognition of the targeted strand. The TPE-Py-DNA probe was dispersed and almost nonfluorescent in an aqueous environment. On the contrary, the TPE-Py-DNA molecule was digested based on the target-recycling strategy in assistance with exonuclease III (Exo III) when Dam MTase was presented, finally releasing aggregated AIEgens to produce a remarkably increased fluorescence signal. Therefore, the detection limit toward Dam MTase was as low as 3.1 × 10-5 U mL-1, and the fluorescent signal could be used to detect Dam MTase activities in real samples and screen its inhibitors. More importantly, the Dam MTase expression was visualized in E. coli cells via CLMS imaging and confirmed in E. coli cell-bearing tissues. In this vein, our results demonstrated that the TPE-Py-DNA probe is a potent tool for the Dam MTase detection and imaging as well as offers an efficient biosensing platform for further investigation of disease pathway and carcinogenesis.

An Evolutionary Strategy for Identification of Higher Order, Green Fluorescent Host-Guest Pairs Compatible with Living Systems.

Engineered miniprotein host-small-molecule guest pairs could be utilized to design new processes within cells as well as investigate fundamental aspects of cell signaling mechanisms. However, the development of host-guest pairs capable of functioning in living systems has proven challenging. Moreover, few examples of host-guest pairs with stoichiometries other than 2:1 exist, significantly hindering the ability to study the influence of oligomerization state on signaling fidelity. Herein, we present an approach to identify host-guest systems for relatively small green fluorescent guests by incorporation into cyclic peptides. The optimal host-guest pair produced a 10-fold increase in green fluorescence signal upon binding. Biophysical characterization clearly demonstrated higher order supramolecular assembly, which could be visualized on the surface of living yeast cells using a turn-on fluorescence readout. This work further defines evolutionary design principles to afford host-guest pairs with stoichiometries other than 2:1 and enables the identification of spectrally orthogonal host-guest pairs.

Whole brain irradiation in mice causes long-term impairment in astrocytic calcium signaling but preserves astrocyte-astrocyte coupling.

Whole brain irradiation (WBI) therapy is an important treatment for brain metastases and potential microscopic malignancies. WBI promotes progressive cognitive dysfunction in over half of surviving patients, yet, the underlying mechanisms remain obscure. Astrocytes play critical roles in the regulation of neuronal activity, brain metabolism, and cerebral blood flow, and while neurons are considered radioresistant, astrocytes are sensitive to γ-irradiation. Hallmarks of astrocyte function are the ability to generate stimulus-induced intercellular Ca2+ signals and to move metabolic substrates through the connected astrocyte network. We tested the hypothesis that WBI-induced cognitive impairment associates with persistent impairment of astrocytic Ca2+ signaling and/or gap junctional coupling. Mice were subjected to a clinically relevant protocol of fractionated WBI, and 12 to 15months after irradiation, we confirmed persistent cognitive impairment compared to controls. To test the integrity of astrocyte-to-astrocyte gap junctional coupling postWBI, astrocytes were loaded with Alexa-488-hydrazide by patch-based dye infusion, and the increase of fluorescence signal in neighboring astrocyte cell bodies was assessed with 2-photon microscopy in acute slices of the sensory-motor cortex. We found that WBI did not affect astrocyte-to-astrocyte gap junctional coupling. Astrocytic Ca2+ responses induced by bath administration of phenylephrine (detected with Rhod-2/AM) were also unaltered by WBI. However, an electrical stimulation protocol used in long-term potentiation (theta burst), revealed attenuated astrocyte Ca2+ responses in the astrocyte arbor and soma in WBI. Our data show that WBI causes a long-lasting decrement in synaptic-evoked astrocyte Ca2+ signals 12-15months postirradiation, which may be an important contributor to cognitive decline seen after WBI.

Photodynamic inactivation of circulating tumor cells: An innovative approach against metastatic cancer

The spread of a primary malignant tumor is the major reason for most of the cancer-associated deaths. To this day, treatment regimen and available drugs are still insufficient to manage these conditions. In this work, a new therapeutic concept based on photodynamic therapy (PDT) of metastasis-initiating cells is introduced. To address this issue, an experimental model was developed to simulate the movement and photodynamic inactivation of circulating tumor cells (CTCs) in vitro. Using curcumin loaded poly(lactic-co-glycolic acid) nanoparticles, a significant reduction in the cell viability of human breast cancer cells (MDA-MB-231) could be achieved after 30 min laser irradiation (λ = 447 nm, P = 100mW) under flow conditions (5 cm s−1). Confocal laser scanning microscopy images confirmed the immediate accumulation of curcumin on the cell membrane and an increased fluorescence signal after irradiation. PDT caused time-dependent morphological cell alterations (i.e. membrane evaginations and disruption) indicating apoptosis and early necrosis. During the photoactivation of curcumin, a blue shift in the absorption spectra and a decrease in the curcumin content could be determined. This study confirms that the presented experimental model is suitable for in vitro investigations of CTCs under in vivo-like conditions, at the same time encouraging the clinical implementation of PDT as an innovative strategy against metastasis.

Fluorescence-Based Detection of Cholesterol Using Inclusion Complex of Hydroxypropyl-β-Cyclodextrin and l-Tryptophan as the Fluorescence Probe

The study is inspired by the fact that intravenous administration of 2-hydroxypropyl-beta-cyclodextrins (HP-β-CD) in rats leads to a rapid decrease in plasma cholesterol levels. Further, HP-β-CD being more water soluble than native β-CD, HP-β-CD forms soluble complex with cholesterol and higher affinity of cholesterol towards HP-β-CD than native β-CD leads to increase sensitivity. Hence, present work reports potential use of HP-β-CD as a chemosensor to detect cholesterol aided with l-tryptophan (l-Trp) as a fluorescence probe. The purpose of the study is to develop fast, cost-effective, and high throughput fluorescence-based chemosensor as convenient point-of-care diagnostic method for cholesterol detection. The host-guest interaction of l-Trp with HP-β-CD was elaborately studied using characterization techniques such as FT-IR, NMR, and DSC. Time and concentration dependent interactions were studied to determine the stoichiometry of molecular inclusion complexes of HP-β-CD with l-Trp. Further, the inclusion complex of HP-β-CD with l-tryptophan is explored to detect cholesterol based on the competitive displacement phenomenon and further explored for detecting different concentrations of cholesterol. Fluorescence spectroscopic data revealed that HP-β-CD served as a quencher for l-Trp. Also, promising results of the displacement assay proved that the fluorescence of l-Trp was quenched by HP-β-CD followed by increase in fluorescence signals in quantitative relation with cholesterol as it replaces l-Trp from the HP-β-CD host. The present investigation is a proof-of-concept that proves the potential of HP-β-CD with l-Trp as a novel fluorescence–based technique for cholesterol detection which is developed using competitive host–guest interaction.

Determination of heavy metal tolerance threshold in a bamboo species (Arundinaria pygmaea) as treated with silicon dioxide nanoparticles

This research was carried out in a tissue culture study to analyze the impact of silicon dioxide nanoparticles on growth, photosynthesis and protective enzymes of a bamboo species (Arundinaria pygmaea) under three various heavy metal stresses (copper (Cu), manganese (Mn), and cadmium (Cd)). The treatments consisted of four concentrations of heavy metals (50, 100, 200, 400 μM) in combination with 100 μM silicon dioxide nanoparticles in comparison to controls. The results indicated that silicon dioxide nanoparticles under heavy metals (Cu and Mn) increased plant growth relative to the control, and they alleviated Cu and Mn toxicity, as reflected by significant increases in protective enzymes, chlorophyll content and fluorescence, as well as plant biomass and shoot length. However, the treatments under Cd concentrations resulted in a significantly decreased biomass and shoots length, which suggested that 100 μM silicon dioxide nanoparticles treatment was unable to prevent Cd accumulation in leaves through internal and external mechanisms. Overall, we concluded that 100 μM silicon dioxide nanoparticles increased the tolerance of bamboo species to heavy metals stress (Cu and Mn) and promoted plant growth indexes by evoking key mechanisms, including inhibiting metal accumulation in the leaves by the adsorption of the metal ions onto silicon dioxide nanoparticles, increasing levels of protective enzymes, and improving photosynthetic properties such as enhanced light-use efficiency and light transmission. We suggest that the combination of 100 μM silicon dioxide nanoparticles with concentrations of 100 and 200 μM heavy metals (Cu and Mn) can confer optimal growth rate, representing the threshold of the plant tolerance to heavy metals in a combination with 100 μM silicon dioxide nanoparticles.

Photoacoustic Molecular Imaging for the Identification of Lymph Node Metastasis in Head and Neck Cancer Using an Anti-EGFR Antibody-Dye Conjugate.

The presence of lymph node (LN) metastases is an essential prognostic indicator in patients with head and neck squamous cell carcinoma (HNSCC). This study assessed photoacoustic molecular imaging (PAMI) of the antiepidermal growth factor receptor antibody (panitumumab) conjugated to a near-infrared fluorescent dye, IRDye800CW (panitumumab-IRDye800CW; pan800), for the identification of occult metastatic LNs in patients with HNSCC (n = 7). Methods: After in vitro photoacoustic imaging characterization of pan800, PAMI was performed on excised neck specimens from patients infused with pan800 before surgery. Freshly obtained neck specimens were imaged with 3-dimensional, multiwavelength spectroscopic PAMI (wavelengths of 680, 686, 740, 800, 860, 924, and 958 nm). Harvested LNs were then imaged with a closed-field near-infrared fluorescence imager and histologically examined by the pathologist to determine their metastatic status. Results: In total, 53 LNs with a maximum diameter of 10 mm were analyzed with photoacoustic and fluorescence imaging, of which 4 were determined to be metastatic on the final histopathologic report. Photoacoustic signals in the LNs corresponding to accumulated pan800 were spectrally unmixed using a linear least-square-error classification algorithm. The average thresholded photoacoustic signal intensity corresponding to pan800 was 5-fold higher for metastatic LNs than for benign LNs (2.50 ± 1.09 arbitrary units [a.u.] vs. 0.53 ± 0.32 a.u., P < 0.001). Fluorescence imaging showed that metastatic LNs had a 2-fold increase in fluorescence signal compared with benign LNs ex vivo (P < 0.01, 0.068 ± 0.027 a.u. vs. 0.035 ± 0.018 a.u.). Moreover, the ratio of the average of the highest 10% of the photoacoustic signal intensity over the total average, representative of the degree of heterogeneity in the pan800 signal in LNs, showed a significant difference between metastatic LNs and benign LNs (11.6 ± 13.4 vs. 1.8 ± 0.7, P < 0.01) and an area under the receiver-operating-characteristic curve of 0.96 (95% CI, 0.91-1.00). Conclusion: The data indicate that PAMI of IRDye800-labeled tumor-specific antibody may have the potential to identify occult LN metastasis perioperatively in HNSCC patients.

Zinc Oxide Nanocomposites-Extracellular Synthesis, Physicochemical Characterization and Antibacterial Potential.

This research presents, for the first time, the potential of the Lactobacillus paracasei LC20 isolated from sweet whey as a novel, effective and accessible source for post-cultured ZnO nanocomposites synthesis. The obtained nanocomposites were subjected to comprehensive characterization by a broad spectrum of instrumental techniques. Results of spectroscopic and microscopic analysis confirmed the hexagonal crystalline structure of ZnO in the nanometer size. The dispersion stability of the obtained nanocomposites was determined based on the zeta potential (ZP) measurements—the average ZP value was found to be −29.15 ± 1.05 mV in the 7–9 pH range. The ZnO nanocomposites (NCs) demonstrated thermal stability up to 130 °C based on the results of thermogravimetric TGA/DTG) analysis. The organic deposit on the nanoparticle surface was recorded by spectroscopic analysis in the infrared range (FT-IR). Results of the spectrometric study exhibited nanostructure-assisted laser desorption/ionization effects and also pointed out the presence of organic deposits and, what is more, allowed us to identify the specific amino acids and peptides present on the ZnO NCs surfaces. In this context, mass spectrometry (MS) data confirmed the nano-ZnO formation mechanism. Moreover, fluorescence data showed an increase in fluorescence signal in the presence of nanocomposites designed for potential use as, e.g., biosensors. Despite ZnO NCs’ luminescent properties, they can also act as promising antiseptic agents against clinically relevant pathogens. Therefore, a pilot study on the antibacterial activity of biologically synthesized ZnO NCs was carried out against four strains (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa) by using MIC (minimal inhibitory concentration). Additionally, the colony forming units (CFU) assay was performed and quantified for all bacterial cells as the percentage of viable cells in comparison to a control sample (untreated culture) The nanocomposites were effective among three pathogens with MIC values in the range of 86.25–172.5 μg/mL and showed potential as a new type of, e.g., medical path or ointment formulation.

Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves Salix safsaf.

Organic fractions and extracts of willow (Salix safsaf) leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl3) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive™ HF Hybrid Quadrupole-Orbitrap™ coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS H2O2 with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.

Abstract P009: Differential Redox Signaling In Urine Derived Renal Proximal Tubule Cells Isolated From Inverse Salt Sensitive Vs Salt Resistant Clinical Study Participants

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. We demonstrated that single nucleotide polymorphisms (SNPs) in the dopamine type 2 receptor (D2R) as well as lower expression of cellular and plasma membrane D2R are associated with inverse salt sensitivity (ISS, a paradoxical increase in blood pressure while on the low sodium diet). Urine derived human renal proximal tubule cells (RPTC) were cultured from 3 separate ISS and 3 salt resistant (SR, resists blood pressure change on both low and high sodium diets) participants. D2R reduces cellular redox signaling so we hypothesized that an aberrant redox state occurs in ISS and SR RPTC exposed to different NaCl concentrations. RPTC incubated in 90 mM NaCl (low salt - LS) for 2 hours in both ISS and SR RPTCs had a significant reduction of monochlorobimane (mBCl - reduced glutathione indicator) compared to 140 mM NaCl (normal salt -NS) (SR -7.1± 2.1% and ISS -9.7 ± 1.6% vs NS, n=12, p&lt;0.05, t-test). Only ISS RPTC had a significant increase in mBCl fluorescence in 190 mM NaCl (ISS +7.0 ± 2.5% vs NS, n=12, p&lt;0.05, t-test) and mBCl fluorescence was not affected by incubation with losartan (angiotensin type 1 receptor inhibitor). 3-Aminobenzamide (3AB, 1 mM) inhibits Parp1 (poly[ADP-ribose] synthase), a protein necessary for the transcriptional repressor CTCF insulator function. 3AB reversed the lower mBCl fluorescence only in the SR RPTC, suggesting that reducing CTCF insulator function only affects D2R wild type cells. mBCl fluorescence in ISS RPTC showed a greater reduction by LOS vs SR RPTC (ISS -49.3 ± 1.5% vs SR -37.3 ± 4.8%, n=12, p&lt;0.05, t-test), which was blocked by nemonapride (NEM, 1 uM, D2R antagonist) and further reduced the mBCl fluorescence only in the SR RPTC. Whether these differences in salt sensitivities are due to differential effects of CTCF insulator function at the homozygous variant or wild type D2R warrants further studies.

Nanoporous layer fiber biosensing platform for real time culture- and separation-free detecting bacterial pathogens and measuring their susceptibility to antibiotics

Simple, rapid, and sensitive detection of bacterial pathogens in environmental and clinical samples is a key requirement because of the ensuing high morbidity and mortality. In this study, a nanoporous layer fiber biosensing platform (called NFB) was developed for the rapid culture- and separation-free detection of bacterial pathogens through integrating evanescent wave fluorescence technology, the size effect of nanoporous layer, and real-time aptasensors. The in-situ etched nanoporous layer of the fiber probe can not only effectively distinguish free Cy5.5-aptasensors and Cy5.5-aptasensors bound to pathogens but also enhance evanescent wave fluorescence. NFB can selectively and sensitively quantify Escherichia coli O157:H7 and Salmonella typhimurium species in environmental and blood samples within 18 min and 32 min, respectively. NFB detected a significant increase in fluorescence when the mixture of pathogens and Cy5.5-aptasensors was exposed to antibiotics. Our results clearly demonstrated that the portable NFB can serve as a powerful tool for real time detection of pathogens and measurement of their response to antibiotics.

Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria

We hypothesized that NO• is generated in isolated cardiac mitochondria as the source for ONOO− production during oxidative stress. We monitored generation of ONOO− from guinea pig isolated cardiac mitochondria subjected to excess Ca2+ uptake before adding succinate and determined if ONOO− production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O2•−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO− to form dityrosine (diTyr) in proportion to the ONOO− present. We found that exposing mitochondria to excess CaCl2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H2O2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa2+ and succinate, increased mitochondrial ONOO− production via NO• and O2•−. Changes in mitochondrial ONOO− production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO− production, PTIO decreased production of ONOO−, and TEMPOL decreased ONOO− levels by converting more O2•− to H2O2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO− into the buffer is dependent both on O2•− released from mitochondria and NO• derived from a mtCa2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.

Drying methods differentially alter volatile profiles of edible locusts and silkworms

In this study, the effect of freeze-drying, oven- and microwave-drying on the volatile profiles and sensory properties of edible locust and silkworm was studied. Fifty-five headspace volatiles were detected, including alkanes, alcohols, aldehydes, esters, and ketones, with variations in intensity depending on the insect species and drying method. Volatiles from microwave-dried locusts and silkworms were typical for the Maillard reaction, that is in accordance with the significant increase of fluorescence of advanced Maillard products (2.9 and 5.5 times higher than in raw silkworms and locusts, respectively). Comparison of fatty acid composition and Fourier transform infrared spectra revealed signs of oxidation process at different levels depending on the insect. Changes in aroma and its intensity caused by different drying methods were also detected by sensory panellists who rated the samples of oven-dried locusts (2.4) and microwave-dried silkworms (1.9) with the lowest scores. An addition 15% of dried insects to cookies decreased the diversity of the volatiles, changed their intensity and lessened the dissimilarities between cookies with differently dried insect powders. Sensory panellists determined the presence of additional ingredients by higher odour intensity and general changes in overall liking scores of the insect-based cookies. Thus, drying can modify odour and sensory characteristics of locusts and silkworms and, consequently, cookies with these insect species, that should be considered in the processing of insects and formulation of novel insect-based foods.

Synthesis and Initial Evaluation of a Novel Fluorophore for Selective FMDV 3C Protease Detection.

The development and evaluation of a Boc-AL(Boc)Q(Trt)-AMC fluorophore to detect 3C Protease, produced by Foot and Mouth Disease Virus (FMDV) is reported, with a view to a potential use as a rapid screen for FMDV infected livestock The peptide-linked conjugate fluorophore is evaluated in vitro for sensitivity, specificity, stability and rapidity and shows statistically significant increases in fluorescence when exposed to physiologically relevant concentrations of 3C Protease and selectivity when compared with other common proteases likely to be located, typically in the absence of FMDV. The stability of deprotected Boc-AL(Boc)Q(Trt)-AMC is reported as a limitation of this probe.

Biophysical Insights into Implications of PEG-400 on the α-Crystallin Structure: Multispectroscopic and Microscopic Approach.

Aggregation and precipitation of α-crystallin play a vital role in the cataract development. This study was targeted to delineate the effect of PEG-400 on the structural integrity of α-crystallin employing a multispectroscopic and microscopic approach. Intrinsic fluorescence and UV–vis spectroscopy suggested alterations in the tertiary structure of α-crystallin, namely global transition of native α-crystallin to a non-native form in the presence of PEG-400. Circular dichroism spectroscopy suggested secondary structural transition in a native conformation of α-crystallin in the presence of PEG-400. Loss in the native conformation of α-crystallin is implicated in cataract developments, thus highlighting the clinical significance of this work. Further, a significant increase in ANS fluorescence of PEG-400-incubated α-crystallin (7 days) suggested this non-native form to be molten globule (MG)-like state. Increased Thioflavin T fluorescence (ThT) and congo red (CR) absorbance along with transmission electron microscopy (TEM) confirmed the formation of the aggregates of α-crystallin after prolonged incubation with PEG-400. Insights into PEG-400-induced structural alterations can provide a platform to search for new therapeutic molecules that can combat α-crystallin-directed eye diseases.

New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

A DNA molecular diagnostic technology with LAMP-like sensitivity based on one pair of hairpin primers-mediated isothermal polymerization amplification

With the rapid development of isothermal amplification technology, DNA molecular diagnosis has become an important reference for clinical treatment. In this work, we have designed a DNA molecular diagnostic technology with LAMP-like sensitivity for nucleic acid analysis and detection based on only one pair of hairpin primers. This DNA molecular diagnostic technology consists of Bst DNA polymerase and one pair of hairpin primers, which are designed easily by adding a stem-loop structure to a target binding domain. When the target is present, the polymerization reaction between the hairpin primers and the target generates a specific dumbbell DNA similar to LAMP, which triggers cyclic amplification reactions to extend a series of long dsDNA products with repeated sequences by inserting fluorescent dye Eva Green observed the increase in fluorescence signal. In our method, using the hairpin primers-mediated isothermal polymerization amplification, we can specifically monitor 3–5 copies of the target nucleic acid in the system without labeling and temperature cycling in the reaction. In addition, serum samples from 13 patients with suspected schistosomiasis were targeted; we further demonstrated the ability of the technology to detect complex clinic samples, and its potentially inestimable applicability in clinic early molecular diagnostic research.

Visualization of GLUT1 Trafficking in Live Cancer Cells by the Use of a Dual-Fluorescence Reporter.

Glucose metabolism is an essential process for energy production and cell survival for both normal and abnormal cellular metabolism. Several glucose transporter/solute carrier 2A (GLUT/SLC2A) superfamily members, including glucose transporter 1 (GLUT1), have been shown to mediate the cellular uptake of glucose in diverse cell types. GLUT1-mediated glucose uptake is a transient and rapid process; thus, the real-time monitoring of GLUT1 trafficking is pivotal for a better understanding of GLUT1 expression and GLUT1-dependent glucose uptake. In the present study, we established a rapid and effective method to visualize the trafficking of GLUT1 between the plasma membrane (PM) and endolysosomal system in live cells using an mCherry-EGFP-GLUT1 tandem fluorescence tracing system. We found that GLUT1 localized at the PM exhibited both red (mCherry) and green (EGFP) fluorescence (yellow when overlapping). However, a significant increase in red punctate fluorescence (mCherry is resistant to acidic pH), but not green fluorescence (EGFP is quenched by acidic pH), was observed upon glucose deprivation, indicating that the mCherry-EGFP-GLUT1 functional protein was trafficked to the acidic endolysosomal system. Besides, we were able to calculate the relative ratio of mCherry to EGFP by quantification of the translocation coefficient, which can be used as a readout for GLUT1 internalization and subsequent lysosomal degradation. Two mutants, mCherry-EGFP-GLUT1-S226D and mCherry-EGFP-GLUT1-ΔC4, were also constructed, which indirectly confirmed the specificity of mCherry-EGFP-GLUT1 for monitoring GLUT1 trafficking. By using a series of endosomal (Rab5, Rab7, and Rab11) and lysosomal markers, we were able to define a model of GLUT1 trafficking in live cells in which upon glucose deprivation, GLUT1 dissociates from the PM and experiences a pH gradient from 6.8–6.1 in the early endosomes to 6.0–4.8 in the late endosomes and finally pH 4.5 in lysosomes, which is appropriate for degradation. In addition, our proof-of-concept study indicated that the pmCherry-EGFP-GLUT1 tracing system can accurately reflect endogenous changes in GLUT1 in response to treatment with the small molecule, andrographolide. Since targeting GLUT1 expression and GLUT1-dependent glucose metabolism is a promising therapeutic strategy for diverse types of cancers and certain other glucose addiction diseases, our study herein indicates that pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening.

Abstract B15: Metabolic imaging of patient-derived tumor organoids provides a fast and dynamic readout of drug response

Abstract Patient-derived tumor organoids are a more physiologically relevant 3D cell culture model system amenable to drug screening. However, several challenges exist with this model system, including the ability to maintain the heterocellular complexity of the original tumor microenvironment (TME) as well as the ability to interrogate this complexity in a quantitative and dynamic manner. To overcome these challenges, we employ fluorescence lifetime imaging microscopy (FLIM) to measure the metabolic activity of unlabeled patient-derived colorectal cancer (CRC) organoids in response to drug perturbations. FLIM relies on the exponential decay rates of fluorescent molecules. By measuring autofluorescent signals from coenzyme NADH, we determined the metabolic state of individual organoids by representing the ratio of free and protein-bound NADH on a 2D phasor plot. Using a confocal microscope, we acquired z-stack images of patient-derived organoids at multiple time points in the presence and absence of clinically relevant chemotherapy and targeted agents (i.e., 5-fluorouracil, SN-38 [active metabolite of irinotecan], and cetuximab). Unlike traditional viability assays that measure the metabolic activity of a population of organoids and provide a single readout at a fixed time point, we can use FLIM to dynamically measure the metabolic activity of individual organoids and capture intra- and interpatient heterogeneity. We were able to detect drug responses in a dose-dependent manner by measuring a shift towards oxidative phosphorylation prior to an increase in fluorescent signal from a vital dye detecting cell death. Thus, FLIM can capture early drug-mediated changes in organoids on the order of hours compared to vital dyes, which are on the order of days. Additionally, cancer cells are in physical and biochemical contact with many different stromal cell types native to the host environment. Cancer-associated fibroblasts (CAFs) are the dominant stromal cell type within the TME and have been linked with increased tumor cell survival and protection against drug-induced apoptosis. When we cocultured organoids with CAFs, we were able to distinguish the metabolic signatures of each cell type without the need for fluorescent labels. Moreover, we quantified the drug-induced metabolic shifts at the IC50 value when CAFs were present. In conclusion, our imaging-based approach has advantages over traditional drug screening methods (e.g., ATP measurements, phototoxic dyes) by capturing the dynamics and heterogeneity of patient-specific drug responses. We are implementing this workflow to better understand the interactions between cancer cells and their microenvironment in the context of drug response. Citation Format: Emma J. Fong, Seungil Kim, Shannon M. Mumenthaler. Metabolic imaging of patient-derived tumor organoids provides a fast and dynamic readout of drug response [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B15.