The effects of different pH incubation values and K+ on yeast plasma membrane potential (PMP) were studied both by the fluorescence changes and the accumulation of thioflavin T (ThT), a method that has been shown most adequate for both procedures. By the changes in fluorescence of ThT, the qualitative observation of PMP at the 3 evaluated pHs indicated that cells at pH 4.0 maintain a PMP lower, but close to the observed at pH 6.0 and 7.0. By measuring the accumulation of ThT and applying the Nernst equation on the different concentrations in and out, the values of PMP could also be estimated at the different pHs, resulting in values in mV, in agreement with our observations by following the fluorescence. Yeast cells at their native niches, or during fermentations must cope with low pHs, so the importance to maintain a robust PMP to survive. The contribution of bicarbonate, derived from the fermentation to the establishment of the PMP is also described. The experiments showed once more the efficacy of the methods used with this dye.

Amyloids, with their β-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17-20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vitro. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

Leveraging the fluorescence enhancement effect of the G-triplex (G3)/thioflavin T (ThT) catalyzed by the adjacent double-stranded DNA positioned at the 5\' terminus of the G3, the G3-specific oligonucleotide (G3MB6) was utilized to facilitate the rapid detection of mercury (Hg(II)) through thymine-Hg(II)-thymine (T-Hg(II)-T) interactions. G3MB6 adopted a hairpin structure in which partially complementary strands could be disrupted with the presence of Hg(II). It prompted the formation of double-stranded DNA by T-Hg(II)-T, inducing the unbound single strand of G3MB6 to spontaneously form a parallel G3 structure, producing a solid fluorescence signal by ThT. Conversely, fluorescence was absent without Hg(II), since no double strand and formation of G3 occurred. The fluorescence intensity of G3MB6 exhibited a positive correlation with Hg(II) concentrations from 17.72 to 300 nM (R2 = 0.9954), boasting a notably low quality of limitation (LOQ) of 17.72 nM. Additionally, it demonstrated remarkable selectivity for detecting Hg(II). Upon application to detect Hg(II) in milk samples, the recovery rates went from 100.3% to 103.2%.

This study investigates the interaction between daphnetin and ovalbumin (OVA) as well as its potential to inhibit OVA fibrillation using both spectroscopic and computational analysis. A moderate binding affinity of 1 × 104 M-1 was observed between OVA and daphnetin, with a static quenched mechanism identified during the fluorescence quenching processes. Metal ions\' (Cu2+ and Zn2+) presence led to an increase in the binding affinities of daphnetin toward OVA, mirroring a similar trend observed with the pH variation. Synchronous and 3D fluorescence studies indicated an increase in the polarity of the microenvironment surrounding the Trp residues during binding. Interestingly, circular dichroism and Fourier transform infrared studies showed a significant change in the secondary structure of OVA upon binding with daphnetin. The efficacy of daphnetin in inhibiting protein fibrillation was confirmed through thioflavin T and Congo Red binding assays along with fluorescence microscopic imaging analysis. The thermodynamic assessment showed positive ΔH° [+(29.34 ± 1.526) kJ mol-1] and ΔS° [+(181.726 ± 5.465) J mol-1] values, indicating the presence of the hydrophobic forces, while negative ΔG° signifies spontaneous binding interactions. These experimental findings were further correlated with computational analysis, revealing daphnetin dynamics within the binding site of OVA.

Nucleolar stress induced by stressors like hypoxia, UV irradiation, and heat shock downregulates ribosomal RNA transcription, thereby impairing protein synthesis capacity and potentially contributing to cell senescence and various human diseases such as neurodegenerative disorders and cancer. Live-cell imaging of the nucleolus may be a feasible strategy for investigating nucleolar stress, but currently available nucleolar stains are limited for this application. In this study using mouse hippocampal HT22 cells, we demonstrate that thioflavin T (ThT), a benzothiazole dye that binds RNA with high affinity, is useful for nucleolar imaging in cells where RNAs predominate over protein aggregates. Nucleoli were stained with high intensity simply by adding ThT to the cell culture medium, making it suitable for use even in damaged cells. Further, ThT staining overlapped with specific nucleolar stains in both live and fixed cells, but did not overlap with markers for mitochondria, lysosomes, endoplasmic reticulum, and double-stranded DNA. Ferroptosis, an iron-dependent nonapoptotic cell death pathway characterized by lipid peroxide accumulation, reduced the number of ThT-positive puncta while endoplasmic reticulum stress did not. These findings suggest that ferroptosis is associated with oxidative damage to nucleolar RNA molecules and ensuing loss of nucleolar function.

Deposition of extracellular Amyloid Beta (Aβ) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer\'s Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aβ and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aβ42 oligomers (AβOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.

Hypochlorous acid (HClO), as an essential reactive oxygen species (ROS) in biological systems, plays a pivotal role in processes of physiology and pathology. Abnormal fluctuations in HClO concentration can lead to various diseases, such as inflammation, cardiovascular diseases, and neurodegeneration. Therefore, developing an approach to rapidly and sensitively quantify ClO- content is vital to biomedicine development and bioassays. Herein, we fabricated a novel \"turn-on\" label-free fluorescence DNA probe to specifically detect hypochlorite ion (ClO-) based on G-quadruplex formation. To this end, we designed a G-rich signal DNA sequence (S-DNA) and a block DNA sequence (B-DNA), followed by the introduction of ClO--responsive phosphorothioate (PS) into B-DNA. In the absence of ClO-, B-DNA hybridized with S-DNA, preventing G-quadruplex formation from S-DNA; this resulted in the relatively low fluorescence intensity of ThT. Once ClO- was added, the hydrolysis between PS and ClO- split the B-DNA into two fragments, resulting in B-DNA breaking away from S-DNA, allowing G-quadruplex formation from S-DNA and increasing the fluorescence intensity of ThT. Using this method, we can detect ClO- without the interference of additional reactive oxygen species. The detection limit of ClO- was as low as 10 nM. Furthermore, this method facilitates the detection of ClO- within the tissues of rats with stress-induced hypertension.

Three thioflavin T (ThT) derivatives, namely ThT/ethylenediaminetetraacetic acid conjugates (E1T, E2T, and E1T1P), were designed and synthesized as sensing components for divalent metal ion detection. Furthermore, these ThT derivatives were used to design lantern-type G-quadruplex (G4) fluorescent sensors. The fluorescence intensities of the ThT derivatives decreased by 1.2- to 5.6-folds in the presence of Ni2+ and Cu2+, respectively, regardless of the topology of the utilized G4. Conversely, when Mn2+ and Zn2+ coexisted in antiparallel G4, the fluorescence intensities of E2T increased to approximately 3.3- and 2.3-folds, respectively, depending on the concentration of the divalent metal ion, allowing for quantitative analyses. The Job plot analysis revealed that the binding ratio of G4 and E2T changed from 2:1 to 1:2 with the increasing concentration of the divalent metal ions. These results indicated that the basic principle of such a lantern-type G4 sensor can be applied to the detection of divalent metal ions and other types of targets, such as proteins, and small molecules via ThT derivatization.

Legume proteins can be induced to form amyloid-like fibrils upon heating at low pH, with the exact conditions greatly impacting the fibril characteristics. The protein extraction method may also impact the resulting fibrils, although this effect has not been carefully examined. Here, the fibrillization of lentil protein prepared using various extraction methods and the corresponding fibril morphology were characterized. It was found that an acidic, rather than alkaline, protein extraction method was better suited for producing homogeneous, long, and straight fibrils from lentil proteins. During alkaline extraction, co-extracted phenolic compounds bound proteins through covalent and non-covalent interactions, contributing to the formation of heterogeneous, curly, and tangled fibrils. Recombination of isolated phenolics and proteins (from acidic extracts) at alkaline pH resulted in a distinct morphology, implicating a role for polyphenol oxidase also in modifying proteins during alkaline extraction. These results help disentangle the complex factors affecting legume protein fibrillization.

Amyloid deposits can be the cause of many chronic diseases. Primary localized cutaneous amyloidosis (PLCA) is a chronic dermatologic condition with amyloid deposits in the papillary dermis. The most common types of the keratinocyte-derived form of PLCA include macular (MA), lichen (LA), and biphasic (BA) amyloidosis. The estimated prevalence of PLCA in the Asian population is 0.98/10,000, which is higher than in the European population; thus, epidemiologic data on PLCA in the Caucasian population are limited. We performed a retrospective single-center study analyzing epidemiologic characteristics of a Central European PLCA population. Epidemiologic data regarding age, sex, skin phototype (Fitzpatrick scale I-VI), disease duration, comorbidities, history of atopy, and family history of PLCA were collected. Clinical characteristics, localization of PLCA lesions, applied therapies and treatment outcomes were also analyzed. Dermoscopic characteristics were also evaluated. A total of 41 patients diagnosed with PLCA were included, with 22 presenting with macular, 18 with lichen, and 1 with biphasic amyloidosis. The male/female ratio was 16/25, and mean age at diagnosis was 54.6 ± 15.2 years (range 27-87 years). The mean age at the onset of PLCA was 53 ± 16.1 years (range 19-79 years) in MA, 46.7 ± 18.2 years (range 14-73 years) in LA, and 26 years in BA. The interscapular region in MA and the extensor surface of the lower extremities in LA proved to be localization-related areas. In our center, a wide range of therapeutic options was applied, with the most prescribed being topical corticosteroids in all types of PLCA. We presented a retrospective, monocentric study on the epidemiology of PLCA in the Central European region. By examining the medical data of a significant number of PLCA patients, we compared our epidemiologic data with that of the Asian PLCA population. Due to the rarity of the condition, further randomized controlled trials and guidelines are needed to improve therapeutic outcomes.