Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.

Pushing Boundaries: What's Next in Metal-Free C-H Functionalization for Sulfenylation?

The synthesis of thioether derivatives has been explored widely due to the potential application of these derivatives in medicinal chemistry, pharmaceutical industry and material chemistry. Within this context, there has been an increasing demand for the environmentally benign construction of C-S bonds via C-H functionalization under metal-free conditions. In the present article, we highlight recent developments in metal-free sulfenylation that have occurred in the past three years. The synthesis of organosulfur compounds via a metal-free approach using a variety of sulfur sources, including thiophenols, disulfides, sulfonyl hydrazides, sulfonyl chlorides, elemental sulfur and sulfinates, is discussed. Non-conventional strategies, which refer to the development of thioether derivatives under visible light and electrochemically mediated conditions, are also discussed. The key advantages of the reviewed methodologies include broad substrate scope and high reaction yields under environmentally benign conditions. This comprehensive review will provide chemists with a synthetic tool that will facilitate further development in this field.

Low concentration of peroxymonosulfate coupled with visible light triggers oxygen reactive species generation over constructed Bi25FeO40/BiOCl Z-scheme heterojunction for various tetracycline antibiotics removal.

Photocatalytic peroxymonosulfate (PMS) oxidation systems demonstrate significant potential and promising prospects through the interconnection of photocatalytic and PMS oxidation for simultaneously achieving efficient pollutant removal and reduction of PMS dosage, which prevents resource wastage and secondary pollution. In this study, a Z-scheme Bi25FeO40/BiOCl (BOFC) heterojunction was constructed to carry out the photocatalytic PMS oxidation process for tetracyclines (TCs) pollutants at low PMS concentrations (0.08 mM). The photocatalytic PMS oxidation rate of Bi25FeO40/BiOCl composites for tetracycline hydrochloride (TCH), chlortetracycline (CTC), oxytetracycline (OTC) and doxycycline (DXC) reaches 86.6%, 83.6%, 86.7%, and 88.0% within 120 min. Simultaneously, the BOFC/PMS system under visible light (Vis) equally displayed the practical application prospects for the solo and mixed simulated TCs antibiotics wastewater. Based on the electron spin resonance (ESR) and X-ray photoelectron spectroscopy (XPS) valence band spectrum, a Z-scheme electron migration pathway was proposed to elucidate the mechanism underlying the performance enhancement of BOFC composites. Bi25FeO40 in BOFC composites can serve as active site for activating PMS by the formation of Fe3+/Fe2+ cycle. Toxicity estimation software tool (T.E.S.T.) and mung beans planting experiment demonstrates that BOFC/PMS/Vis system can reduce toxicity of TCs wastewater. Therefore, BOFC/PMS/Vis system achieves efficient examination in different water environments and efficient utilization of PMS, which displays a scientific reference for achieving environmentally-friendly and resource-saving handling processes.

Highly visible-light-active sulfur and carbon co-doped TiO2 (SC-TiO2) heterogeneous photocatalysts prepared by underwater discharge plasma.

To promptly and simply create highly crystalline S/C co-doped TiO2 (SC-TiO2) photocatalysts at room temperature and atmospheric pressure, we suggest a novel plasma-assisted sol-gel synthesis method. This method is a simultaneous synthetic process, in which an underwater plasma undergoes continuous reactions to generate high-energy atomic and molecular species that enable TiO2 to achieve crystallinity, a large surface area, and a heterogeneous structure within a few minutes. In particular, it was demonstrated that the heterogeneously structured TiO2 was formed by doping that sulfur and carbon replace O or Ti atoms in the TiO2 lattice depending on the composition of the synthesis solution during underwater plasma treatment. The resultant SC-TiO2 photocatalysts had narrowed bandgap energies and extended optical absorption scope into the visible range by inducing the intermediate states within bandgap due to generation of oxygen vacancies on the surface of TiO2 through synthesis, crystallization, and doping. Correspondingly, SC-TiO2 showed a significant degradation efficiency ([k] = 6.91 h-1) of tetracycline (TC, antibiotics) under solar light irradiation, up to approximately 4 times higher compared to commercial TiO2 ([k] = 1.68 h-1), resulting in great water purification. Therefore, we anticipate that this underwater discharge plasma system will prove to be an advantageous technique for producing heterostructural TiO2 photocatalysts with superior photocatalytic efficiency for environmental applications.

Non-toxic orange carbon dots stimulate photosynthesis and CO2 assimilation in hydroponically cultivated green beans (Phaseolus vulgaris).

Continuous increasing leaf photosynthesis may enhance plant yield. As an evolutionary property, plants use less photosynthetic capacity than is theoretically possible. Plant nanobionics is a bioengineering field that improves plant functions using nanoparticles. We applied orange carbon dots (o-CDs) onto the foliage of green beans (Phaseolus vulgaris ) grown in hydroponics to improve their photosynthetic performance and CO2 assimilation. Photosynthesis parameters, photosynthetic pigments content, total phenolic content (TPC) and antioxidative activity (TAA) were measured. Results show that photosynthetic pigments remained unchanged, while photosynthesis was improved. Both o-CDs concentrations decreased TPC and TAA. The light response curve showed higher CO2 assimilation at both o-CDs concentrations, particularly at lower light intensity. Correlation analysis confirmed increased CO2 binding and assimilation at 1mg L-1 . This study demonstrated the potential of using o-CDs as a safe biostimulator through photosynthesis increase and CO2 assimilation without toxic effects on plants. This may stimulate yield increase that paves the way for their agricultural application.

Modulatory effects of low-intensity retinal ultrasound stimulation on rapid and non-rapid eye movement sleep.

Prior investigations have established that the manipulation of neural activity has the potential to influence both rapid eye movement and non-rapid eye movement sleep. Low-intensity retinal ultrasound stimulation has shown effectiveness in the modulation of neural activity. Nevertheless, the specific effects of retinal ultrasound stimulation on rapid eye movement and non-rapid eye movement sleep, as well as its potential to enhance overall sleep quality, remain to be elucidated. Here, we found that: In healthy mice, retinal ultrasound stimulation: (i) reduced total sleep time and non-rapid eye movement sleep ratio; (ii) changed relative power and sample entropy of the delta (0.5-4 Hz) in non-rapid eye movement sleep; and (iii) enhanced relative power of the theta (4-8 Hz) and reduced theta-gamma coupling strength in rapid eye movement sleep. In Alzheimer's disease mice with sleep disturbances, retinal ultrasound stimulation: (i) reduced the total sleep time; (ii) altered the relative power of the gamma band during rapid eye movement sleep; and (iii) enhanced the coupling strength of delta-gamma in non-rapid eye movement sleep and weakened the coupling strength of theta-fast gamma. The results indicate that retinal ultrasound stimulation can modulate rapid eye movement and non-rapid eye movement-related neural activity; however, it is not beneficial to the sleep quality of healthy and Alzheimer's disease mice.

Improving strawberry plant resilience to salinity and alkalinity through the use of diverse spectra of supplemental lighting.

BACKGROUND: This study explores the impact of various light spectra on the photosynthetic performance of strawberry plants subjected to salinity, alkalinity, and combined salinity/alkalinity stress. We employed supplemental lighting through Light-emitting Diodes (LEDs) with specific wavelengths: monochromatic blue (460 nm), monochromatic red (660 nm), dichromatic blue/red (1:3 ratio), and white/yellow (400-700 nm), all at an intensity of 200 µmol m-2 S-1. Additionally, a control group (ambient light) without LED treatment was included in the study. The tested experimental variants were: optimal growth conditions (control), alkalinity (40 mM NaHCO3), salinity (80 mM NaCl), and a combination of salinity/alkalinity. RESULTS: The results revealed a notable decrease in photosynthetic efficiency under both salinity and alkalinity stresses, especially when these stresses were combined, in comparison to the no-stress condition. However, the application of supplemental lighting, particularly with the red and blue/red spectra, mitigated the adverse effects of stress. The imposed stress conditions had a detrimental impact on both gas exchange parameters and photosynthetic efficiency of the plants. In contrast, treatments involving blue, red, and blue/red light exhibited a beneficial effect on photosynthetic efficiency compared to other lighting conditions. Further analysis of JIP-test parameters confirmed that these specific light treatments significantly ameliorated the stress impacts. CONCLUSIONS: In summary, the utilization of blue, red, and blue/red light spectra has the potential to enhance plant resilience in the face of salinity and alkalinity stresses. This discovery presents a promising strategy for cultivating plants in anticipation of future challenging environmental conditions.

LAGSwin: Local attention guided Swin-transformer for thermal infrared sports object detection.

Compared with visible light images, thermal infrared images have poor resolution, low contrast, signal-to-noise ratio, blurred visual effects, and less information. Thermal infrared sports target detection methods relying on traditional convolutional networks capture the rich semantics in high-level features but blur the spatial details. The differences in physical information content and spatial distribution of high and low features are ignored, resulting in a mismatch between the region of interest and the target. To address these issues, we propose a local attention-guided Swin-transformer thermal infrared sports object detection method (LAGSwin) to encode sports objects' spatial transformation and orientation information. On the one hand, Swin-transformer guided by local attention is adopted to enrich the semantic knowledge of low-level features by embedding local focus from high-level features and generating high-quality anchors while increasing the embedding of contextual information. On the other hand, an active rotation filter is employed to encode orientation information, resulting in orientation-sensitive and invariant features to reduce the inconsistency between classification and localization regression. A bidirectional criss-cross fusion strategy is adopted in the feature fusion stage to enable better interaction and embedding features of different resolutions. At last, the evaluation and verification of multiple open-source sports target datasets prove that the proposed LAGSwin detection framework has good robustness and generalization ability.

Dissection of Daytime and Nighttime Thermoresponsive Hypocotyl Elongation in Arabidopsis.

Hypocotyl elongation in Arabidopsis is widely utilized as a readout for phytochrome B (phyB) signaling and thermomorphogenesis. Hypocotyl elongation is gated by the circadian clock and, therefore, it occurs at distinct times depending on day length or seasonal cues. In short-day conditions, hypocotyl elongation occurs mainly at the end of nighttime when phyB reverts to the inactive form. In contrast, in long-day conditions, hypocotyl elongation occurs during the daytime when phyB is in the photoactivated form. Warm temperatures can induce hypocotyl growth in both long-day and short-day conditions. However, the corresponding daytime and nighttime temperature responses reflect distinct underpinning mechanisms. Here, we describe assays for dissecting the mechanisms between daytime and nighttime thermoresponsive hypocotyl elongation.

Measurement of Phytochrome B Thermal Reversion Rates In Vivo.

Thermal reversion of phytochromes is the light-independent but strongly temperature-dependent relaxation of the light-activated Pfr form of phytochromes back into the inactive Pr ground state. The thermal reversion rates of different phytochromes vary considerably. For phytochrome B (phyB), thermal reversion represents a critical parameter affecting phyB activity as it reduces the active phyB Pfr pool, accelerated by increasing temperatures. Phytochromes are dimers existing in three different states: Pfr-Pfr homodimer, Pfr-Pr heterodimer, and Pr-Pr homodimer. Consequently, thermal reversion occurs in two steps, with Pfr-Pfr to Pfr-Pr reversion being much slower than reversion from Pfr-Pr to Pr-Pr. To measure thermal reversion in vivo, the relative proportion of Pfr in relation to the total amount of phytochrome (Ptot) must be determined in living samples. This is accomplished by in vivo spectroscopy utilizing dual wavelength ratiospectrophotometers, optimized for assaying phytochromes in highly scattering plant material. The method is depending on the photoreversibility of phytochromes displaying light-induced absorbance changes in response to actinic irradiation. In this chapter, we describe the experimental design and explain step-by-step the calculations necessary to determine the thermal reversion rates of phyB in vivo, taking into account phytochrome dimerization.

Characterizing the Synergistic Response Induced by Warm Temperatures and Shade.

Plants exhibit an impressive capability to detect and respond to neighboring plants by closely monitoring changes in the light spectrum. They possess the ability to perceive adjustments in the ratio of red (R) to far-red (FR) light (R/FR) triggered by the presence of nearby plants, even before experiencing complete shading. When the R/FR ratio falls below 1, plants activate a shade avoidance response that manifests as hypocotyl elongation. Furthermore, elevated ambient temperatures can also stimulate hypocotyl elongation. As hypocotyl elongation is a visible characteristic, it is a valuable indicator for monitoring shade avoidance response, warm ambient temperature response, and the interplay between the two.

Phase Separation of Phytochrome B in HEK293T Cells.

In this method, we employed HEK293T cells to express the plant photoreceptor phytochrome B (phyB). Through the application of various treatments such as phycocyanobilin (PCB) supplementation, red light exposure, and temperature adjustments, the phyB proteins exhibited liquid-liquid phase separation, leading to the formation of biomolecular condensates. Here, we present a comprehensive description of the protein expression, cell treatment, and imaging capture procedures. This detailed guide provides step-by-step instructions on how to induce phase separation of phyB proteins in HEK293T cells. By utilizing this approach, researchers can investigate the physicochemical characteristics and dynamic formation process of phyB photobodies with precision.

Characterization of Thermoresponsive Photobody Dynamics.

Photobodies (PBs) are subnuclear membraneless organelles that self-assemble via the condensation of the plant photoreceptor and thermosensor phytochrome B (phyB). Changes in the light and temperature environment directly modulate PB formation and maintenance by altering the number and size of PBs. In thermomorphogenesis, increases in the ambient temperature incrementally reduce the number of PBs, suggesting that individual PBs possess distinct thermostabilities. Here, we describe a detailed protocol for characterizing cell type-specific PB dynamics induced by warm temperatures in Arabidopsis.

Analysis of Phase Separation of EARLY FLOWERING 3.

Phase separation is an important mechanism for regulating various cellular functions. The EARLY FLOWERING 3 (ELF3) protein, an essential element of the EVENING COMPLEX (EC) involved in circadian clock regulation, has been shown to undergo phase separation. ELF3 is known to significantly influence elongation growth and flowering time regulation, and this is postulated to be due to whether the protein is in the dilute or phase-separated state. Here, we present a brief overview of methods for analyzing ELF3 phase separation in vitro, including the generation of phase diagrams as a function of pH and salt versus protein concentrations, optical microscopy, fluorescence recovery after photobleaching (FRAP), and turbidity assays.

Isolation of Phytochrome B Photobodies.

Phytochrome B (phyB), a plant photoreceptor, forms a membraneless organelle known as a photobody. Here, we present a protocol for the isolation of phyB photobodies through fluorescence-activated particle sorting from mature transgenic Arabidopsis leaves expressing phyB-GFP. This protocol involves the isolation of nuclei from frozen ground leaves using sucrose gradient centrifugation, the disruption of nuclear envelopes by sonication, and the subsequent isolation of phyB photobodies through fluorescence-activated particle sorting. We include experimental tips and notes for each step.

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

Optical penetration models for practical prediction of femtosecond laser ablation of dental hard tissue.

OBJECTIVES: To develop and practically test high-precision femtosecond laser ablation models for dental hard tissue that are useful for detailed planning of automated laser dental restorative treatment. METHODS: Analytical models are proposed, derived, and demonstrated for practical calculation of ablation rates, ablation efficiency and ablated morphology of human dental enamel and dentin using femtosecond lasers. The models assume an effective optical attenuation coefficient for the irradiated material. To achieve ablation, it is necessary for the local energy density of the attenuated pulse in the hard tissue to surpass a predefined threshold that signifies the minimum energy density required for material ionization. A 1029 nm, 40 W carbide 275 fs laser was used to ablate sliced adult human teeth and generate the data necessary for testing the models. The volume of material removed, and the shape of the ablated channel were measured using optical profilometry. RESULTS: The models fit with the measured ablation efficiency curve against laser fluence for both enamel and dentin, correctly capturing the fluence for optimum ablation and the volume of ablated material per pulse. The detailed shapes of a 400-micrometer wide channel and a single-pulse width channel are accurately predicted using the superposition of the analytical result for a single pulse. CONCLUSIONS: The findings have value for planning automated dental restorative treatment using femtosecond lasers. The measurements and analysis give estimates of the optical properties of enamel and dentin irradiated with an infrared femtosecond laser at above-threshold fluence and the proposed models give insight into the physics of femtosecond laser processing of dental hard tissue.

Bumblebee nest departures under low light conditions at sunrise and sunset.

Only a few diurnal animals, such as bumblebees, extend their activity into the time around sunrise and sunset when illumination levels are low. Low light impairs viewing conditions and increases sensory costs, but whether diurnal insects use low light as a cue to make behavioural decisions is uncertain. To investigate how they decide to initiate foraging at these times of day, we observed bumblebee nest-departure behaviours inside a flight net, under naturally changing light conditions. In brighter light bees did not attempt to return to the nest and departed with minimal delay, as expected. In low light the probability of non-departures increased, as a small number of bees attempted to return after spending time on the departure platform. Additionally, in lower illumination bees spent more time on the platform before flying away, up to 68 s. Our results suggest that bees may assess light conditions once outside the colony to inform the decision to depart. These findings give novel insights into how behavioural decisions are made at the start and the end of a foraging day in diurnal animals when the limits of their vision impose additional costs on foraging efficiency.

Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Comparing resistance of bacterial spores and fungal conidia to pulsed light and UVC radiation at a wavelength of 254 nm.

Pulsed light (PL) inactivates microorganisms by UV-rich, high-irradiance and short time pulses (250 µs) of white light with wavelengths from 200 nm to 1100 nm. PL is applied for disinfection of food packaging material and food-contact equipment. Spores of seven Bacillus ssp. strains and one Geobacillus stearothermophilus strain and conidia of filamentous fungi (One strain of Aspergillus brasiliensis, A. carbonarius and Penicillium rubens) were submitted to PL (fluence from 0.23 J/cm2 to 4.0 J/cm2) and UVC (at λ = 254 nm; fluence from 0.01 J/cm2 to 3.0 J/cm2). One PL flash at 3 J/cm2 allowed at least 3 log-reduction of all tested microorganisms. The emetic B. cereus strain F4810/72 was the most resistant of the tested spore-forming bacteria. The PL fluence to 3 log-reduction (F3 PL) of its spores suspended in water was 2.9 J/cm2 and F3 UVC was 0.21 J/cm2, higher than F3 PL and F3 UVC of spores of B. pumilus SAFR-032 2.0 J/cm2 and 0.15 J/cm2, respectively), yet reported as a highly UV-resistant spore-forming bacterium. PL and UVC sensitivity of bacterial spores was correlated. Aspergillus spp. conidia suspended in water were poorly sensitive to PL. In contrast, PL inactivated Aspergillus spp. conidia spread on a dry surface more efficiently than UVC. The F2 PL of A. brasiliensis DSM1988 was 0.39 J/cm2 and F2 UVC was 0.83 J/cm2. The resistance of spore-forming bacteria to PL could be reasonably predicted from the knowledge of their UVC resistance. In contrast, the sensitivity of fungal conidia to PL must be specifically explored.