Differential Fluorescence Research Articles

Enhanced thermal stability photophysical properties of photoselective PMMA/ITO nanohybrid films for greenhouse cooling in hot climates

The current research proposed innovative photoselective greenhouse cooling films made from PMMA/ITO nanohybrids, incorporating varying concentrations of Indium tin oxide nanocrystals (ITO NCs) and the fluorescent organic dye Lumogen F Red300, utilizing the solvent casting method. The morphology and structure were examined using transmission electron microscopy (TEM) and X-ray diffraction (XRD), demonstrating good homogeneity and amorphous nature. The impact of different ITO NC concentrations on physical properties was examined through differential scanning calorimetry (DSC), optical absorption, transmission, reflection, fluorescence, and Fourier transform infrared (FT-IR) spectroscopy. Integrating ITO NCs into the PMMA matrix showed enhanced thermal insulation capabilities of PMMA films while maintaining their transparency to photosynthetically active radiation (PAR). Maintaining this balance is crucial because it enables the films to selectively filter and reflect infrared radiation leading to lower greenhouse temperatures while still allowing the essential light needed for plant growth to pass through. This research is particularly significant for Sustainable Development Goals (SDGs) 2 and 13, especially in hot, water-scarce regions, as it protects plants from thermal stress, promotes growth, and supports food security in developing countries.

Fruit waste-derived carbon dots with rhodamine B for the ratiometric detection of Fe3+ and Cu2.

A green and eco-friendly solvothermal approach is proposed for the synthesis of carbon quantum dots (CQDs) from watermelon rind. The as-prepared CQDs exhibited superior teal fluorescence in aqueous solutions, with a quantum yield of 13.9%. The CQDs and rhodamine B (RhB) were demonstrated to selectively react with Fe3+ and Cu2+, leading to a fluorescence (FL) quenching effect, which was successfully used for constructing "double-response-off" type ratiometric FL probes. A comparative study was conducted to assess the sensitivity and accuracy of ratiometric fluorescent probes, specifically those based on CQDs alone and in combination with RhB, for the selective detection of Fe3+ and Cu2+. By plotting the ratio of the differential fluorescence (ΔF) signals of CQDs to that of RhB against the practical application analyte concentration, the detection limits for Fe3+ (1.75 μM) and Cu2+ (0.43 μM) were markedly improved. The quenching mechanism was further explored, and the detection of Fe3+ and Cu2+ in surface water was demonstrated, showcasing the potential of efficient and effective nanosensors based on a static quenching effect. Futhermore, the addition of ascorbic acid can restore the fluorescence quenched by Fe3+. Therefore, in the presence of copper and iron, the ratiometric probe can demonstrate the ability to identify two different metals.

1,3,4-oxadiazoles as inhibitors of the atypical member of the BET family bromodomain factor 3 from Trypanosoma cruzi (TcBDF3).

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions globally, with increasing urban cases outside of Latin America. Treatment is based on two compounds, namely, benznidazole (BZ) and nifurtimox, but chronic cases pose several challenges. Targeting lysine acetylation, particularly bromodomain-containing proteins, shows promise as a novel antiparasitic target. Our research focuses on TcBDF3, a cytoplasmic protein, which is crucial for parasite differentiation that recognizes acetylated alpha-tubulin. In our previous study, A1B4 was identified as a high-affinity binder of TcBDF3, showing significant trypanocidal activity with low host toxicity in vitro. In this report, the binding of TcBDF3 to A1B4 was validated using differential scanning fluorescence, fluorescence polarization, and molecular modeling, confirming its specific interaction. Additionally, two new 1,3,4-oxadiazoles derived from A1B4 were identified, which exhibited improved trypanocide activity and cytotoxicity profiles. Furthermore, TcBDF3 was classified for the first time as an atypical divergent member of the bromodomain extraterminal family found in protists and plants. These results make TcBDF3 a unique target due to its localization and known functions not shared with higher eukaryotes, which holds promise for Chagas disease treatment.

Mixtures of Intrinsically Disordered Neuronal Protein Tau and Anionic Liposomes Reveal Distinct Anionic Liposome-Tau Complexes Coexisting with Tau Liquid-Liquid Phase-Separated Coacervates.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein that binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full-length human protein tau and other negatively charged binding substrates, as revealed by differential interference contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase-separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes (i.e. tau-lipoplexes) exhibited distinct types of morphologies. This included large ∼20-30 μm tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles and tau-mediated finite-size assemblies of small liposomes. As the salt concentration was increased to near and above 150 mM for 1:1 electrolytes, AL-tau complexes remained stable, while tau self-coacervate droplets were found to dissolve, indicative of the breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen-facing monolayer of the axon's plasma membrane, suggesting the possibility of transient yet robust interactions near relevant ionic strengths found in neurons.

(1-Deoxy)ceramides in bilayers containing sphingomyelin and cholesterol

The discovery of a novel sphingolipid subclass, the (1-deoxy)sphingolipids, which lack the 1-hydroxy group, attracted considerable attention in the last decade, mainly due to their involvement in disease. They differed in their physico-chemical properties from the canonical (or 1-hydroxy) sphingolipids and they were more toxic when accumulated in cells, inducing neurodegeneration and other dysfunctions. (1-Deoxy)ceramides, (1-deoxy)dihydroceramides, and (1- deoxymethyl)dihydroceramides, the latter two containing a saturated sphingoid chain, have been studied in this work using differential scanning calorimetry, confocal fluorescence and atomic force microscopy, to evaluate their behavior in bilayers composed of mixtures of three or four lipids. When compared to canonical ceramides (Cer), a C16:0 (1-deoxy)Cer shows a lower miscibility in mixtures of the kind C16:0 sphingomyelin/cholesterol/XCer, where XCer is any (1-deoxy)ceramide, giving rise to the coexistence of a liquid-ordered phase and a gel phase. The latter resembles, in terms of thermotropic behavior and nanomechanical resistance, the gel phase of the C16:0 sphingomyelin/cholesterol/C16:0 Cer mixture [Busto et al., Biophys. J. 2014, 106, 621–630]. Differences are seen between the various C16:0 XCer under study in terms of nanomechanical resistance, bilayer thickness and bilayer topography. When examined in a more fluid environment (bilayers based on C24:1 SM), segregated gel phases are still present. Probably related to such lateral separation, XCer preserve the capacity for membrane permeation, but their effects are significantly lower than those of canonical ceramides. Moreover, C24:1 XCer show significantly lower membrane permeation capacity than their C16:0 counterparts. The above data may be relevant in the pathogenesis of certain sphingolipid-related diseases, including certain neuropathies, diabetes, and glycogen storage diseases.

Cryo-Fluorescence Tomography as a Tool for Visualizing Whole-Body Inflammation Using Perfluorocarbon Nanoemulsion Tracers.

We explore the use of intravenously delivered fluorescent perfluorocarbon (PFC) nanoemulsion tracers and multi-spectral cryo-fluorescence tomography (CFT) for whole-body tracer imaging in murine inflammation models. CFT is an emerging technique that provides high-resolution, three-dimensional mapping of probe localization in intact animals and tissue samples, enabling unbiased validation of probe biodistribution and minimizes reliance on laborious histological methods employing discrete tissue panels, where disseminated populations of PFC-labeled cells may be overlooked. This methodology can be used to streamline the development of new generations of non-invasive, cellular-molecular imaging probes for in vivo imaging. Mixtures of nanoemulsions with different fluorescent emission wavelengths were administered intravenously to naïve mice and models of acute inflammation, colitis, and solid tumor. Mice were euthanized 24 h post-injection, frozen en bloc, and imaged at high resolution (~ 50 µm voxels) using CFT at multiple wavelengths. PFC nanoemulsions were visualized using CFT within tissues of the reticuloendothelial system and inflammatory lesions, consistent with immune cell (macrophage) labeling, as previously reported in in vivo magnetic resonance and nuclear imaging studies. The CFT signals show pronounced differences among fluorescence wavelengths and tissues, presumably due to autofluorescence, differential fluorescence quenching, and scattering of incident and emitted light. CFT is an effective and complementary methodology to in vivo imaging for validating PFC nanoemulsion biodistribution at high spatial localization, bridging the resolution gap between in vivo imaging and histology.

Long-term escape behavior and release mechanism of organic emissions from asphalt materials

In the context of carbon neutrality and carbon peaking, investigating the long-term escape behavior and release mechanism of organic emissions from asphalt materials can contribute to the development of environmentally sustainable asphalt pavements. In this study, the long-term emission behavior and release mechanism of organic emissions from asphalt materials were unraveled by combining the headspace gas chromatography–mass spectrometry (HS-GC–MS), differential scanning calorimetry (DSC), fluorescence microscopy (FM), and Fourier transform infrared spectroscopy (FTIR) tests. The results demonstrate that the pressure aging vessel (PAV) test and the ultraviolet (UV) aging test can result in a notable reduction in the concentration of organic emissions from asphalt materials, respectively. This indicates that asphalt pavements can potentially release a substantial quantity of organic emissions during their long-term service life. Besides, the aging mechanism of asphalt materials is established to unravel the release mechanism of organic emissions from asphalt materials. Aging increases the probability of organic emissions being released and volatilized from asphalt materials, which leads to the organic emissions from asphalt materials being more likely to be released and volatilized. Consequently, the aging process facilitates a greater release and volatilization of organic emissions from asphalt materials, resulting in a decrease in the detected concentration of these emissions after aging.

Characterization and utilization capabilities of industrial wastes for green bricks production

BackgroundThe goal of this study is to develop a feasible and sustainable solution to manage the use of industrial wastes of ground granulated blast-furnace steel slag (GGBS) activated by cement kiln dust (CKD) and quicklime (QL). Using activated GGBS in the manufacture of stabilized green bricks is still uncommon in Egypt in such applications. Five clay-based mixtures, each with varying replacement ratios (5–10, wt.%) of CKD and QL, were studied. Laboratory tests were conducted on cylindrical specimens made from these mixtures, which were left to cure for periods of up to 60 days. The raw materials and lab-made specimens were analyzed using particle size analysis, differential thermal analysis, X-ray fluorescence, and X-ray diffraction techniques. The physical and mechanical properties of the cured specimens were also determined and evaluated according to standard specifications. Furthermore, the durability of the cured specimens was evaluated against collapsibility in water.ResuItsIt has been observed that adding QL and CKD to the stabilized green specimens of different mixes can enhance their engineering properties with curing age increasing. This is due to the pozzolanic reaction, which fills the pore structure with calcium silicate hydrates and calcium aluminate hydrates gel. The ratio of QL and CKD used significantly affected the engineering properties of the specimens. The study found that using 20% GGBS and 5% QL led to an increase in compressive strength (266 kg/cm2) at the density of (2.15 g/cm3), while also water absorption was reduced (8%) to give superior results. When GGBS and CKD were combined, a higher content of CKD (10 wt.%) gave better results compared to (5 wt.%) CKD. Furthermore, the physical and mechanical properties of the tested specimens (MD 1, MD II, MD III and MD IV) met the acceptable limits of dry compressive strength (30–70 kg/cm2), water absorption (8–15%), and density (1.7–2 gm/cm3), as specified by the Egyptian standard specifications for buildings used compressed earth blocks.ConclusionThe CKD and QL act as alkali activators for GGBS and can be utilized in masonry construction.

Effect of treatment conditions on the morphology of date empty fruit bunch lignocellulosic fiber for biocomposite applications

The fiber of the empty date fruit is one of the waste products left after the date fruit is harvested from the date palm. Optimization of the treatment parameters as well as the economics of the treatment process are critical to the successful commercialization of the treatment of natural fibers for the production of biocomposites. Of the natural fiber treatment methods described in previous work, hot water and alkali treatments are the most cost-effective, which justified the choice of these treatment methods in this work. Here, date empty fruit bunch fibers were pulverized to a particle size of 0–500 µm and then treated with boiling water at 100 °C for 3 h or with sodium hydroxide at different concentrations (2, 5, 10%) for 3 h at room temperature with constant stirring at 800 rpm. To confirm the effect of temperature on the fiber treatment, another fiber sample was treated at a 5% concentration of sodium hydroxide solution at an elevated temperature of 80 °C for the same period and stirring condition. The untreated and treated DEFB fibers were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), X-ray fluorescence (XRF), and scanning electron microscopy (SEM). Chemical analysis of the fibers showed that DEFB fiber treated with 5% sodium hydroxide solution at room temperature had the highest cellulose content (52.0%), which was also confirmed by the XRD analysis. The DEFB fiber treated with boiling water exhibited higher thermal stability while the sodium hydroxide-treated fiber exhibited higher crystallinity index and cellulose content. Apparently, DEFB fiber treated with sodium hydroxide solution at 5% concentration for 3 h at room temperature for continuous stirring at 800 rpm offered the best properties. The treated fibers are expected to be viable reinforcement in the production of biocomposites with the goal of achieving energy efficiency. The developed materials could be used in industrial applications such as insulating building systems, automotive parts, and home furniture.

Differential fluorescence features and recovery speeds of different scorpion exoskeleton parts during the molting process

Scorpion fluorescence under ultraviolet light is a well-known phenomenon, but its features under excitation in the UVA, UVB and UVC bands have not been characterized. Systematic fluorescence characterization revealed indistinguishable fluorescence spectra with a peak wavelength of 475 nm for whole exuviae from second-, third- and fifth-instar scorpions under different ultraviolet light ranges. In-depth investigations of the chelae, mesosoma, metasoma and telson of adult scorpions further indicated heterogeneity in the typical fluorescence spectrum within the visible light range and in the newly reported fluorescence spectrum with a peak wavelength of 320 nm within the ultraviolet light range, which both showed excitation wavelength-independent features. Dynamic fluorescence changes during the molting process of third-instar scorpions revealed the fluorescence heterogeneity-dependent recovery speed of scorpion exoskeletons. The typical fluorescence spectra of the molted chelae and telson rapidly recovered approximately 6 h after ecdysis under UVA light and approximately 36 h after ecdysis under UVB and UVC light. However, it took approximately 12 h and 24 h to obtain the typical fluorescence spectra of the molted metasoma and mesosoma, respectively, under UVA irradiation and 72 h to obtain the typical fluorescence spectra under UVB and UVC irradiation. The fluorescence heterogeneity-dependent fluorescence recovery of the scorpion exoskeleton was further confirmed by tissue section analysis of different segments from molting third-instar scorpions. These findings reveal novel scorpion fluorescence features and provide potential clues on the biological function of scorpion fluorescence.

The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell–cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.

High fluorescent lymphocytes in dengue infection; A navigator for hematopathologists.

Objective: The aim of this research is the early detection of dengue infection on the basis of presence of high fluorescent lymphocyte (HFL) which enables the clinicians to tailor further investigations and early commencement of treatment to prevent complications and thus can reduce mortality and morbidity in dengue patients. Study Design: Prospective Research. Setting: Fauji Foundation Hospital in Rawalpindi. Period: February 2022 to July 2022. Material & Methods: Samples of Full blood counts (FBC) received in Pathology laboratory were processed on automated Hematology analyzer Sysmex XN-series (XN-1000). All the FBCs that were of known dengue patients were excluded from the study. The FBCs with High Fluorescent Lymphocytes on White blood cell differential fluorescence (WDF) scattergram were selected and evaluated. Detection of dengue infection was done by using Dengue Virus ELISA (IgM and IgG) test. Results: Among one hundred samples with High Fluorescence Lymphocyte there were 77% were Dengue Positive. Binomial test did give a significant association of Dengue with High Fluorescence Lymphocyte (p<0.01). Conclusion: We draw the conclusion that presence of high fluorescent lymphocytes helps in the early detection of dengue positive cases.

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence.

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Differential Fluorescent Chemosensing of Antibiotics Using a Luminescent Zn(II) Coordination Polymer Based on a 4-Amino-1,8-naphthalimide Tröger’s Base Fluorophore

The design and development of useful chemosensors for the ultra-trace detection of environmental pollutants and contaminants is a topical area of research. Herein, we report a new nanoscale emissive Zn(II) coordination polymer (TB-Zn-CP) for differential fluorescence sensing of various antibiotics in water. TB-Zn-CP was synthesized using a unique V-shaped green emitting 4-amino-1,8-naphthalimide Tröger’s base (TBNap) fluorophore. The structural and morphological features of TB-Zn-CP were characterized by various standard spectroscopic and microscopy techniques. The fluorescence titration studies in water demonstrated a remarkable sensitivity and differential fluorescence sensing properties of TB-Zn-CP for the fast detection of different antibiotics. Among different antibiotics, chloramphenicol (CRP), 1,2-dimethyl-5-nitroimidazole (DMZ), and sulfamethazine (SMZ) displayed the highest fluorescence-quenching efficiency and superior sensitivity in their detection. The differential sensing capability of TB-Zn-CP was also indicated by visualizable color changes. The Stern–Volmer quenching constant KSV was determined to be in the order of 103–104 M−1, and the sensitivity was shown to be at a nanomolar (10−9 M) level. All these results confirm that TB-Zn-CP can be a potential and practically useful polymeric sensor for differential fluorescence and visual detection of different antibiotics in water.

A study of bread wheat lines from crosses with the synthetic form Avrodes in regard to their yellow rust resistance

The genome-substituted synthetic form Avrodes (AABBSS) was used for transferring resistance to yellow rust (Puccinia striiformis f. sp. tritici Eriks.) from Aegilops speltoides Tausch, (2n = 14) to bread wheat. The study involved 24 introgressive lines of bread wheat obtained using the Avrodes form. Yellow rust resistant lines P07-L.02, P07-L.1, P07-L.17, P07-L.43, P07-L.19, AS12-88, AS12-06, AS12-07, AS12- 51, Asp81-21, Asp63-21, Asp053-21, Asp04-21, Asp022-19, Asp023-19 and Asp029-20 were selected and can be used as new donors of disease resistance. The use of differential chromosome staining (C-banding) and fluorescence in situ hybridization (FISH) identified the genetic material of Ae. speltoides transmitted in the form of 5S(5D) chromosome substitution and translocations of T5BS.5BL-5SL, T2DL.2DS-2SS, T5D, as well as translocation of T1BL.1RS from Secale cereale L. The work revealed that the lines with single translocations of T1BL.1RS and T5BS.5BL-5SL were susceptible to yellow rust, while the lines in which the T2DL.2DS-2SS translocation and 5S(5D) substitutions were identified, as well as the lines with translocations of T1BL.1RS, T2DL.2DS-2SS and T5D showed resistance to the disease. Presumably, the selected introgression lines, obtained by means of crosses with Avrodes, may carry new genes or loci for yellow rust resistance.

Dendrimer Induced Bilayer Disintegration of Hybrid Vesicles.

Physicochemical investigations on the inclusion of anionic polyamidoaminesuccinamic acid dendrimer, generation 5 (PAMAM-SA, G5) with positively charged hybrid vesicles (HCV), prepared using soylecithin, ion pair amphiphile (IPA), cholesterol and dihexadecyldimethylammonium bromide, were investigated by dynamic light scattering, transmission electron/atomic force microscopy (TEM/AFM), differential scanning calorimetry, fluorescence spectroscopy and surface pressure-time isotherm studies. Adsorption of dendrimer onto vesicle surface and subsequent bilayer disruption strongly depends on the bilayer composition and dendrimer concentration. Change in the zeta potential value with increasing dendrimer concentration suggests the dendrimer-vesicle interaction to be electrostatic in nature. AFM studies also confirm the adsorption of dendrimer as well as hole formation in the bilayer. Impact of the inclusion of dendrimer into the bilayer were further investigated through differential scanning calorimetry by monitoring the chain melting temperature and enthalpy of the chain melting processes. Dendrimer at low concentration does not alter bilayer integrity, while hole formations are noted at higher dendrimer concentration. Fluorescence anisotropy studies confirm the adsorption and subsequent bilayer disruption due to dendrimer inclusion. Dendrimer induced vesicle disintegration kinetics conclusively illustrate the transformation of cationic bilayer to monolayer and thereby exposing the role of IPA. In vitro cytotoxicity studies on PAMAM-SA, G5 and HCVs mixtures against human breast cancer cell line suggest that dendrimer-liposome aggregates (dendriosomes) exhibit substantial anticancer activities with insignificant side effects. It is expected that the dendriosomes may have application to host and deliver anticancer drug in the field of targeted drug delivery.

The impact of palmitoyl glycyl-histidyl-lysine on phospholipid model membranes

BackgroundPalmitoyl glycyl-histidyl-lysine (Pal-GHK), a peptide amphiphile derived from the GHK tripeptide, has gained attention for its diverse bioactive properties and applications in skincare formulations. This study delves into the interactions and effects of Pal-GHK on phospholipid model membranes. Using a thin film hydration-sonication method, Pal-GHK and hydrogenated soy phosphatidylcholine (HSPC) were combined to create lipid vesicles. MethodsVarious analytical techniques, including dynamic light scattering (DLS), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), fluorescence polarization (FP), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), were employed to comprehensively assess the impact of Pal-GHK on the lipid bilayer domain. Significant FindingsThe results reveal insights into the size, zeta potential, storage stability, morphology, thermotropic phase behavior, molecular arrangement, and lipid ordering within the HSPC membranes in the presence of Pal-GHK. The introduction of Pal-GHK increased vesicle size, surface charge density, and storage stability in HSPC-rich vesicles. The addition of Pal-GHK dominated the endothermic phase behavior of the lipid vesicles and increased the proportion of gauche conformation in the acyl chains of HSPC, inducing molecular fluidity in the hydrophobic region of the lipid membrane in its gel state. These findings contribute to our understanding of the interactions between Pal-GHK and lipid model membranes, holding significance for various scientific disciplines and potential applications.

Conventional properties, rheological characteristics, and thermal performance of waste polyurethane modified asphalt with butyl rubber

To enhance the low-temperature performance of waste polyurethane (WPU) modified asphalt, a composite modifier incorporating butyl rubber (BR) was investigated. In this study, recycled waste polyurethane for industrial filling is selected, which is belonging to the Polyether polyols. Various concentrations of BR were introduced to WPU modified binder, with and without stabilizer. The blends underwent conventional asphalt performance tests, dynamic shear rheometer (DSR), and bending beam rheometer (BBR) tests. Thermal performance and morphology were analyzed using differential scanning calorimeter (DSC) and fluorescence microscopy (FM). Fourier transform infrared spectroscopy (FTIR) experiments revealed the mechanism of WPU/BR modified asphalt. Results indicated that adding 3% BR to 15% WPU increased ductility at 5 ℃ by over 5 times, elevating the performance grade from PG-22 to PG-28. BR addition lowered the glass transition temperature, enhancing stability at higher temperatures. Besides improving low-temperature performance, BR also enhanced high-temperature performance and deformation resistance. Functional group analysis suggested a chemical modification process. Considering overall performance and modification effects, the optimal composite modifier was found to be 15% WPU and 3% BR.

Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae, and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum, and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.

Detection of CAR-T Cell Persistence with Digital Droplet PCR: Correlation of In Vivo Expansion with Clinical Outcomes in a Cohort of B-Cell Lymphoma Patients

Introduction: In vivo CAR-T cell expansion is of central importance to clinical response and toxicity in patients with hematologic malignancies. In clinical trials, the area under the curve (AUC) within the first 28 days of infusion was considered a valid tool to describe the concentration-time profile of CAR-T cells in peripheral blood (PB). However, several longitudinal assessments performed by either flow cytometry or quantitative real-time polymerase chain reaction (qPCR) were required. This study evaluates the clinical utility of assessing CAR-T concentration by digital droplet PCR (ddPCR) in patients with non-Hodgkin lymphoma (B-NHL). Methods: Patients with B-NHL ddPCR test collected on day 28 ± 7 post-commercial CD19 CAR T infusion, and clinical outcome data was extracted retrospectively. In this ddPCR assay, the PCR primers target the mammalian expression vector present in the CAR constructs as well as the ATP2B4 reference gene. The CAR-T and reference gene signals are segregated by differential fluorescence signals of tagged probes. The number of CAR constructs and reference gene signals per microliter (uL) are calculated from sample droplets and data fitting to a Poisson distribution. The assay sensitivity is ~0.003% of CAR construct per reference gene. Results: Between October 2020 and February 2022, 66 patients (62% male) with diffuse large B cell lymphoma (DLBCL, 73%), Burkitt lymphoma (1%), mantle cell lymphoma (MCL, 24%) and follicular lymphoma (FL, 12%) received axicabtagene ciloleucel (83%), brexucabtagene autoleucel (12%), or tisagenlecleucel (5%), with fludarabine/cyclophosphamide (49%) or bendamustine (51%) lymphodepletion and had testing within the timeframe. Bridging therapy was administered in 82%, and elevated serum LDH prior to CAR T was present in 31%. With a median follow-up of 4.5 months (IQR 3.1-6.2 mo), the day 28 overall response rate was 80%, complete remission 57%, and progressive disease 18%. Median CAR copies/microL was 2.3 (range 0-474); of those with a detectable value, the median was 3.57 copies/microL. Excluding the 7 patients who had progression at the same time the sample was drawn, day 28 landmarked 3- and 6-month progression-free survival (PFS) for patients with non-detectable (22%) vs. detectable (78%) ddPCR construct was 31% vs 81% and 31% vs 73%, respectively (p=0.012, Figure 1B). Three- and 6-month PFS for non-detectable (22%), detectable <= 3.57 (41%), and detectable >3.57 (37%) was 31%, 77%, 86% and 31%, 62%, and 86%, respectively (p=0.039, Figure 1A). Univariable analysis of factors predicting detection of CAR T construct by ddPCR at Day 28, including age, sex, gender, elevated LDH, lines of therapy, bridging, CAR T construct, grade 2+ cytokine release syndrome (CRS), and grade 2+ ICANS was significant only for grade 2+ CRS. Patients with grade 0-1 CRS (44%) vs grade 2+ CRS (56%) had a median of 0 (range 0 - 474) vs 4 (0 - 307) CAR copies/microL (p<0.001). Conclusion: Assessment of PB CAR-T concentration performed by ddPCR at a single timepoint reliably predicted CD19 CAR-T cell efficacy in patients with B-NHL, with detectable CAR construct at day 28 predicting better 6-month PFS. Further research is needed to define the best timepoint for testing and the impact of using strategies to mitigate CRS and/or ICANS.