Confocal Scanning Microscopy Images Research Articles

Light-Modulated Circadian Synaptic Plasticity in the Somatosensory Cortex: Link to Locomotor Activity.

The circadian clock controls various physiological processes, including synaptic function and neuronal activity, affecting the functioning of the entire organism. Light is an important external factor regulating the day-night cycle. This study examined the effects of the circadian clock and light on synaptic plasticity, and explored how locomotor activity contributes to these processes. We analyzed synaptic protein expression and excitatory synapse density in the somatosensory cortex of mice from four groups exposed to different lighting conditions (LD 12:12, DD, LD 16:8, and LL). Locomotor activity was assessed through individual wheel-running monitoring. To explore daily and circadian changes in synaptic proteins, we performed double-immunofluorescence labeling and laser scanning confocal microscopy imaging, targeting three pairs of presynaptic and postsynaptic proteins (Synaptophysin 1/PSD95, Piccolo/Homer 1, Neurexins/PICK1). Excitatory synapse density was evaluated by co-labeling presynaptic and postsynaptic markers. Our results demonstrated that all the analyzed synaptic proteins exhibited circadian regulation modulated by light. Under constant light conditions, only Piccolo and Homer 1 showed rhythmicity. Locomotor activity was also associated with the circadian clock's effects on synaptic proteins, showing a stronger connection to changes in postsynaptic protein levels. Excitatory synapse density peaked during the day/subjective day and exhibited an inverse relationship with locomotor activity. Continued light exposure disrupted cyclic changes in synapse density but kept it consistently elevated. These findings underscore the crucial roles of light and locomotor activity in regulating synaptic plasticity.

From two segments and beyond: Investigating the onset of regeneration in Syllis malaquini.

Annelids feature a diverse range of regenerative abilities, but complete whole-body regeneration is less common, particularly in the context of the head and anterior body regeneration. This study provides a detailed morphological description of Syllis malaquini regenerative abilities. By replicating previous experiments and performing diverse surgical procedures, we explored the capacity of this species for whole-body regeneration. We detailed the precise timing of regeneration of particular structures such as the eyes, proventricle, pharyngeal tooth, nuchal organs, and body pigmentation after amputation. Our high-resolution scanning electron microscopy and confocal laser-scanning microscopy images provide details of the blastema region, revealing that while anal opening remains in connection to the exterior environment, oral opening is formed "de novo" during blastema differentiation. Additionally, we performed amputations to isolate fragments consisting of one, two, and three segments from the intestinal trunk region. We found that S. malaquini requires at least two to three segments to successfully regenerate the whole body. In addition, we verified a variable capacity to regenerate depending upon the gut region, with structures of the foregut greatly impairing some steps of the regenerative process. Our work notably addresses the gap in knowledge concerning gut formation and its impact on regenerative capabilities. Ongoing research is crucial to unravel the role of gut tissue specificity and plasticity during regeneration in annelids, and particularly in syllids.

Influence of Initial Root Canal Treatment on Intratubular Penetrability and Bond Strength of Nonsurgical Retreatment: An InVitro Study

IntroductionTo assess the effect of combinations of two different endodontic sealers used in initial and endodontic retreatment on the bond strength of the secondary obturation and the penetrability of the sealers. MethodsForty-eight mandibular premolars were used, receiving standardized endodontic access and biomechanical preparation. Twenty-four teeth received AH Plus sealer (AHP) in primary obturation and the others received Bio-C Sealer (BCS). Retreatment protocol was performed with an R50 instrument. The samples were further subdivided into four groups (n = 12) based on the combination of primary/secondary obturation sealers: AHP/AHP; AHP/BCS; BCS/AHP; and BCS/BCS. Four samples from each subgroup received the addition of fluorophores to the sealer for penetrability analysis using laser scanning confocal fluorescence microscopy. The root portion on the 8 push-out samples was sectioned into 6 slices of 1.0 mm. Bond strength (BS) was assessed using a universal testing machine until displacement of the filling mass. Failure pattern was evaluated under a stereomicroscope (20× magnification). BS data were analyzed using two-way analysis of variance followed by Tukey's test (P < .05), and the association between the failure pattern and BS value was assessed using the chi-square test (P < .05). Penetrability was qualitatively evaluated. ResultsThe highest BS values were observed in the AHP/AHP (4.54 ± 1.5 MPa) and BCS/AHP (5.00 ± 1.0 MPa) groups (P < .05), with a higher percentage of adhesive failures to the filling material for all groups. Laser scanning confocal fluorescence microscopy images indicated greater penetrability of AHP compared to BCS, both in initial treatment and retreatment. ConclusionAHP sealer exhibited higher BS and greater penetrability compared to BCS sealer.

Seasonal to interannual variations of daily growth rate of a Tridacna shell from Palau Island, western Pacific, and paleoclimatic implications

Seasonal-interannual climate change is an important component of Earth's climate system and has a significant impact on ecosystems and social systems. However, the short-run of modern observational data limits our understanding of the seasonal-interannual climates on long-term timescales. The natural archives can provide information on climate change before the industrial period, but most of their temporal resolution is too low to capture the seasonal-interannual climate signals. Tridacna spp. is the largest marine bivalve, and its shell has the potential to trace climatic/environmental changes on a daily to interannual scale. In this study, we investigated the daily growth pattern of a modern Tridacna gigas shell (PL-1) from Palau based on laser scanning confocal microscopy images. The results showed that the growth pattern of PL-1 was affected by effective solar radiation primarily, followed by nutrients on the seasonal timescale. On the interannual timescale, the growth rate of the shell was modulated by ENSO, which affected the effective solar radiation and nutrients accepted by Tridacna. The growth rate of Tridacna PL-1 was accelerated during the ENSO positive phase, and vice versa. However, due to a lack of situ observations, the effects of nutrients on Tridacna and the symbiotic zooxanthella in detail need to be further investigated. This study indicates that the daily growth rate of Tridacna shells in equatorial regions may have the potential to reconstruct seasonal-interannual climate/environment variations.

Neuronal Panx1 drives peripheral sensitization in experimental plantar inflammatory pain

BackgroundThe channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood.MethodsWild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund’s adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and β-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. β-galactosidase (β-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice.ResultsGlobal or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/β-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca2+ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/β-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of β-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG.ConclusionsThe present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.

Automated extraction of Tridacna shell growth patterns via machine learning for enhanced paleoclimate/paleoweather research

Tridacna spp. are valuable archives for paleoclimate and paleoweather research due to their distinct daily growth patterns and the sensitivity of the daily growth patterns to environment changes. However, manually identifying daily growth lines and measuring the daily growth increment width (DGIW) of Tridacna shells from Laser Scanning Confocal Microscopy (LSCM) images is a tedious task that has become a significant barrier to Tridacna studies. This paper addresses this challenge by integrating machine learning into Tridacna research for the first time to automate the calculation of the number of daily growth lines and DGIW of Tridacna shells. Specifically, we propose an unsupervised generative adversarial attention network called TriGAN to automatically recognize distinct daily growth lines of Tridacna shells from LSCM images. Utilizing modern Tridacna specimens collected from the South China Sea, our experimental results demonstrate that TriGAN can effectively reconstruct the ambiguous and blurred regions in LSCM images and produce higher quality images of daily growth patterns compared to existing image generation networks. Furthermore, the daily growth line number and DGIW of Tridacna shells can be counted automatically from the images recognized by TriGAN, which are in good agreement with the statistical results obtained manually from the original LSCM images (R = 0.7, p < 0.01 for the DGIW profile of T. gigas specimen MD1 and R = 0.6, p < 0.01 for T. derasa specimen XB10). This automated method provides an efficient solution for researching the laminar chronology of Tridacna.

Preparation, structure and luminescence properties of Ce3+: Y3(Al,Ga)5O12 translucent glass ceramics for AC-LEDs

Translucent glass ceramics containing Ce3+:Y3(Al,Ga)5O12 microcrystals were prepared by spontaneous crystallization in the SiO2–B2O3–Al2O3–Ga2O3–Y2O3–PbO–CeF3 glass system. The sample exhibits around 45 % transmittance in the range of 500–800 nm. X-ray diffraction patterns confirm the garnet phases. Scanning electron microscope and laser scanning confocal microscopy images suggest that about 5 μm grains are dispersed uniformly in the glass matrix. With increasing Ga/Al ratio in the components, both excitation and emission peaks blue shift. The emission intensity reaches to the maximum at Ga/Al ratio of 8/22, and the optimized Ce3+ content is 0.4 mol%. The sample exhibits yellowish-green persistent luminescence with lifetime of 47.3 ms. The glass ceramic-based device exhibits near standard white light emission with luminous efficiency of 64.7 lm/W, correlated color temperature of 6979 K, color rendering index of 65.4 and flicker index of 76.9 %. It indicates that the Ce3+:Y3(Al,Ga)5O12 glass ceramics are promising in the alternating current directly driven white light-emitting diodes (AC-LEDs).

Rheological properties and microstructure of wheat flour dough systems with enzyme-hydrolyzed mashed potatoes.

The interest in incorporating potatoes into wheat dough is increasing. However, potatoes exhibit significant viscosity during thermal processing, affecting product processing and quality. This study aims to find an effective method to reduce the viscosity of mashed potatoes. We aimed to compare the effects of different enzymes (α-amylase, β-amylase, and flavourzyme) and concentrations (0.01%, 0.05%, and 0.1%) on the micromorphology and rheological properties of mashed potatoes and potato-wheat dough. The impact of flavourzyme was the most significant (p<0.05). When enzyme concentration increased, viscosity decreased, and the degree of structural damage, indicated by increased porosity. Notably, the addition of flavourzyme can increase the content of sweet and umami free amino acids, improving the flavor of mashed potatoes. The scanning electron microscopy and confocal laser scanning microscopy images of potato-wheat dough revealed that enzyme-hydrolyzed mashed potatoes had improved homogeneity, reestablished the dough continuity, and strengthened the three-dimensional structure comprising proteins and starch. Notably, flavourzyme demonstrated the most significant effect on enhancing the protein-starch network structure. This was attributed to the exposure of functional groups resulting from protein hydrolysis, facilitating interaction with starch molecules. Our findings indicate that the addition of 0.1% flavourzyme (500 LAPU/g, pH5.5, 55±2°C, 30min treated) was the most effective in reducing viscosity and reconstructing the gluten network. Enzymatic hydrolysis plays a vital role in the production of high-quality potato products, with particular importance in the baking industry, where flavourzyme exhibits significant potential. PRACTICAL APPLICATION: Enzymatic hydrolysis plays a vital role in the production of high-quality potato products, with particular importance in the baking industry, where flavourzyme exhibits significant potential.

Superior enhancement in thermal conductivity of epoxy/graphene nanocomposites through use of dimethylformamide (DMF) relative to acetone as solvent

This method article describes the fabrication of graphene-epoxy nanocomposites using two different solvents, dimethylformamide (DMF) and acetone, and validates the resulting thermal conductivity improvements. The study compared the two solvents at a filler composition of 7 wt% and found that DMF resulted in more uniform dispersion of graphene nanoparticles in the epoxy matrix, leading to a 44% improvement in thermal conductivity compared to acetone. Laser scanning confocal microscopy (LSCM) imaging showed that DMF-based composites had more evenly dispersed graphene nanoplatelets than acetone-based composites, which exhibited larger graphene agglomerations. Effective medium theory calculations showed that DMF led to almost 35% lower interface thermal resistance between graphene and epoxy compared to acetone. The validated fabrication method and findings provide new possibilities for developing high thermal conductivity graphene-epoxy nanocomposites for various thermal management applications.•This article describes methods for fabricating graphene-epoxy composites using acetone and DMF as solvents, and validates that DMF is better for achieving higher thermal conductivity in the composite.

Single-cell transcriptome profiling of sepsis identifies HLA-DRlowS100Ahigh monocytes with immunosuppressive function

BackgroundSustained yet intractable immunosuppression is commonly observed in septic patients, resulting in aggravated clinical outcomes. However, due to the substantial heterogeneity within septic patients, precise indicators in deciphering clinical trajectories and immunological alterations for septic patients remain largely lacking.MethodsWe adopted cross-species, single-cell RNA sequencing (scRNA-seq) analysis based on two published datasets containing circulating immune cell profile of septic patients as well as immune cell atlas of murine model of sepsis. Flow cytometry, laser scanning confocal microscopy (LSCM) imaging and Western blotting were applied to identify the presence of S100A9+ monocytes at protein level. To interrogate the immunosuppressive function of this subset, splenic monocytes isolated from septic wild-type or S100a9−/− mice were co-cultured with naïve CD4+ T cells, followed by proliferative assay. Pharmacological inhibition of S100A9 was implemented using Paquinimod via oral gavage.ResultsScRNA-seq analysis of human sepsis revealed substantial heterogeneity in monocyte compartments following the onset of sepsis, for which distinct monocyte subsets were enriched in disparate subclusters of septic patients. We identified a unique monocyte subset characterized by high expression of S100A family genes and low expression of human leukocyte antigen DR (HLA-DR), which were prominently enriched in septic patients and might exert immunosuppressive function. By combining single-cell transcriptomics of murine model of sepsis with in vivo experiments, we uncovered a similar subtype of monocyte significantly associated with late sepsis and immunocompromised status of septic mice, corresponding to HLA-DRlowS100Ahigh monocytes in human sepsis. Moreover, we found that S100A9+ monocytes exhibited profound immunosuppressive function on CD4+ T cell immune response and blockade of S100A9 using Paquinimod could partially reverse sepsis-induced immunosuppression.ConclusionsThis study identifies HLA-DRlowS100Ahigh monocytes correlated with immunosuppressive state upon septic challenge, inhibition of which can markedly mitigate sepsis-induced immune depression, thereby providing a novel therapeutic strategy for the management of sepsis.

Effect of micro- and nano-sized emulsion fillers combined with trans-cinnamaldehyde on the physicochemical and rheological characteristics of myofibrillar protein gels

In this study, we investigated the influence of trans-cinnamaldehyde (CIN) and emulsion size on the physicochemical properties and functionality of emulsion gels. Myofibrillar protein or lecithin emulsions were fabricated with different ratios of canola oil and CIN and different emulsion droplet sizes. The emulsion gels showed a significant dependence on CIN concentration in terms of gel strength and rheological properties because of the improved interaction between proteins and aldehydes through the Schiff base reaction. In addition, the physical properties of the micro-sized emulsion gels were better than those of the nano-sized gels. There was no significant difference in the water-holding capacity. According to the scanning electron and confocal microscopy images, the CIN emulsion gels formed a denser microstructure and had a higher homogeneity with higher CIN concentrations. This suggested that CIN promoted protein cross-linking because the monophenol molecule had fewer structural obstacles. The structural properties were analyzed using Fourier-transform infrared spectroscopy. It was also confirmed that lipid oxidation was inhibited by CIN. The information obtained in this study could be used to develop protein-based products with novel functional properties for use in meat products and in other applications.

Analysis of grain size distribution evolution of steel during recrystallization and grain growth

Controlling the hot rolling process requires a deep understanding of the underlying metallurgical phenomena. Quantitative methods are of paramount importance for achieving the capability of controlling microstructural evolution. Since the final mechanical properties of steel result from microstructural evolution in the whole process, analysis of the microstructure provides an important input for numerical simulations that can be used for tailoring the mechanical properties of steel. The evolution of grain size distribution of a low-carbon CrNiMnB ultrahigh-steel in austenitic state is studied in hot forming and annealing using experimental data obtained with the Gleeble 3800 thermo-mechanical simulator. A general method is described that can be utilized to systematically compare the grain size distributions obtained from the experimental studies. The experimental data has been obtained from laser scanning confocal microscopy images using the mean linear intercept method. A custom-made semi-automatic software has been utilized to process the data rapidly and reliably.

Grafting of Tetraphenylethylene on Silica Surface, Characterizations, and Their Chromatographic Performance as Reversed-Phase Stationary Phases.

Surface modification is an effective way to functionalize the materials so as to get some special properties. Tetraphenylethylene (TPE) has been widely investigated as a well-known reagent which has the nature of aggregation-induced emission (AIE), but has never been reported in the liquid chromatography stationary phase. In this work, TPE-grafted silica (Sil-TPE) was obtained successfully using the derivative of 1-(4-hydroxyphenyl)-1,2,2-triphenylethylene as a ligand, and then characterized by elemental analysis, Fourier transform infrared spectra, thermogravimetric analysis, and so forth. Laser scanning confocal microscopy images reflected the AIE phenomenon of grafted TPE because the internal vibration and rotation of TPE molecules were restrained in the confined silica space. The contact angle test showed superhydrophobic properties of Sil-TPE. In order to understand thoroughly the mechanism of chromatographic performance and retention behavior for Sil-TPE, Tanaka test mixture, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), and phenols were separated. This reveals that Sil-TPE has strong aromaticity and certain shape selectivity, especially, has excellent separation performance for PAHs and phenols. The thermodynamic properties and repeatability of Sil-TPE were further studied, which showed the stability of Sil-TPE. This work shows that TPE can be successfully grafted on silica surface and it has the potential to be a new kind of promising stationary phases in the future.

Deep Association between Transglutaminase 1 and Tissue Eosinophil Infiltration Leading to Nasal Polyp Formation and/or Maintenance with Fibrin Polymerization in Chronic Rhinosinusitis with Nasal Polyps.

Transglutaminase (TGM) isoform catalyze the cross-linking reaction of identical or different substrate proteins. Eosinophil has been recognized in chronic rhinosinusitis with nasal polyps (CRSwNP) forming tissue eosinophil in nasal polyp (NP), and TGM isoforms are suggested to be associated with a critical role in asthma and other allergic conditions. The aim of this study was to reveal the association of specific TGM isoform with both the tissue eosinophil infiltration deeply concerning with the intractable severity of CRSwNP and the fibrin polymerization ability of TGM isoform associated with the tissue eosinophil infiltration, which lead to NP formation and/or maintenance in CRSwNP. NP tissues (CRSwNP group) and uncinate process (UP) (control group) were collected from patients with CRSwNP and control subjects. We examined: (1) the expression level of TGM isoforms by using a real-time polymerase chain reaction (PCR) and the comparison to the issue eosinophil count in the CRSwNP group, (2) the location of specific TGM isoform in the mucosal tissue using immunohistochemistry, (3) the inflammatory cell showing the colocalization of specific TGM isoform in Laser Scanning Confocal Microscopy (LSCM) imaging, and (4) the fibrin polymerase activity of specific TGM isoform using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A certain level of TGM 1, 2, 3, 5 expression was present in both the CRSwNP group and the control group. Only TGM 1 expression showed a positive significant correlation with the tissue eosinophil count in the CRSwNP group. The localization of TGM 1 in NP (CRSwNP) laid mainly in a submucosal layer as inflammatory cells and was at the cytoplasm in the tissue eosinophil. Fibrin polymerase activity of TGM 1 showed the same polymerase ability of factor XIIIA. TGM 1 might influence the NP formation and/or maintenance in CRSwNP related to the tissue eosinophil infiltration, which formed fibrin mesh composing NP stroma.

Metalenses with Polarization‐Insensitive Adaptive Nano‐Antennas (Laser Photonics Rev. 16(9)/2022)

Metalens Design In article number 2200268, Juntao Li, Haowen Liang, Xiao Fu, and colleagues experimentally realize a polarization-insensitive and high focus efficiency metalens at 532 nm operation for an NA = 1.48 by using dimer nano-antennas. This single layer metalens can replace a conventional objective lens and demonstrate the confocal scanning microscopic imaging with 300 nm resolution.

Bio-responsive composite liposomes against Campylobacter jejuni in vitro and its application in chicken preservation

The infection of Campylobacter jejuni (C. jejuni) in food can cause huge economic and health costs, and liposomes based on biological switch is a promising antibacterial strategy. In this study, a bio-responsive composite liposome was designed with silk fibroin and L-fucose (FLPs) based on the chemotaxis and protease of C. jejuni, and Litsea cubeba essential oil as antibacterial agent. The oil encapsulation and protein mosaic rates of the composite liposome were 70.6 ± 0.5% and 47.9 ± 1.0%, respectively. The in vitro release experiment verified that the release rates of essential oil from the liposome exceeded 95% at 5 days. Furthermore, FLPs possessed excellent antibacterial activity, the inhibition zone was 23.5 mm and 20% (v/v) of the composite liposomes could inactivate 99% C. jejuni. Moreover, laser scanning confocal microscopy and scanning electron microscopy images confirmed the antimicrobial activity of FLPs was better than that of other liposomes, and it could attach to bacteria well. Sprayed liposomes revealed sustained ability of retarding bacterial reproduction within 5 days with the refrigerated chicken meat inoculated with C. jejuni, and had no impact on the color and texture of the food. Hence, the bio-responsive composite liposome has a promising application in inhibiting C. jejuni growth in food. Industrial relevanceIn this study, the composite liposome with high encapsulation efficiency shows significant and sustained antibacterial activity, and has no negative effects on the color and texture of chicken meat. The findings of this study will facilitate the development and application of liposomes as active antimicrobial packaging in food industry.

Glucose sensing by regulator of G protein signaling 1 (RGS1) plays a crucial role in coordinating defense in response to environmental variation in tomato.

Low light intensities affect the outbreak of plant diseases. However, the underlying molecular mechanisms remain poorly understood. High-performance liquid chromatography analysis of tomato (Solanum lycopersicum) revealed that apoplastic glucose (Glc) levels decreased in response to low light. Conversely, low-light-induced susceptibility to Pseudomonas syringae pv tomato (Pst) DC3000 was significantly alleviated by exogenous Glc treatment. Using cell-based biolayer interferometry assays, we found that Glc specifically binds to the tomato regulator of G protein signaling 1 (RGS1). Laser scanning confocal microscopy imaging revealed that Glc triggers RGS1 endocytosis, which influences the uncoupling of the RGS1-Gα (GPA1) and GPA1-Gβ (SlGB1) proteins, in a dose- and duration-dependent manner. Analysis of G protein single and double mutants revealed that RGS1 negatively regulates disease resistance under low light and is required for Glc-enhanced defense. Downstream of RGS1-Glc binding, GPA1 negatively mediates the light-intensity-regulated defense, whereas SlGB1 positively regulates this process. These results reveal a novel light-intensity-responsive defense system that is mediated by a Glc-RGS1-G protein signaling pathway. This information will be critical for future investigations of how plant cells sense extracellular sugars and adjust defense under different environments, as well as for genetic engineering approaches to improve stress resilience.

Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold

PurposeTo utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct.MethodsMelt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR.ResultsCjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable.ConclusionThe 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.

Metalenses with Polarization‐Insensitive Adaptive Nano‐Antennas

AbstractMetalens research has made major advances in recent years. These advances rely on the simple design principle of arranging meta‐atoms in regular arrays to create an arbitrary phase and polarization profile. Unfortunately, the concept of equally spaced meta‐atoms reaches its limit for high deflection angles where the deflection efficiency decreases. The efficiency can be increased using nano‐antennas with multiple elements, but their polarization sensitivity hinders their application in metalenses. Here, it is shown that by designing polarization‐insensitive dimer nano‐antennas and abandoning the principle of equally spaced unit cells, polarization‐insensitive ultrahigh numerical aperture (NA = 1.48) oil‐immersion operation with an efficiency of 42% can be demonstrated. This represents a significant improvement on other polarization‐insensitive designs at visible wavelength. This single layer metalens is used to replace a conventional objective lens and demonstrates the confocal scanning microscopic imaging of a grating with a period of 300 nm at 532 nm operating wavelength. Overall, the results experimentally demonstrate a novel design concept that further improves metalens performance.

Formation of pea protein amyloid fibrils to stabilize high internal phase emulsions for encapsulation of lutein

Thermal treatment of purified pea protein in acidic environment was found to induce the hydrolysis and re-assembly of the protein to form amyloid fibrils. Pea protein amyloid fibrils showed high polymorphism with the coexistence of mature and worm-like fibrils. Pea protein amyloid fibrils can stabilize the high internal phase emulsions (HIPEs) with the internal phase volume fraction as high as 90%. The gel strength of HIPEs was enhanced with the increase of pea protein amyloid fibril contents and the dispersion speed. The typical microstructure of highly packed polyhedral geometric oil droplets appeared in both the laser scanning confocal microscopy (CLSM) and Cryo-scan electron microscopy (Cryo-SEM) images of the HIPEs samples. High loading content of lutein upto 0.2 wt% in the prepared HIPEs was realized and the stability of lutein against ultraviolet irradiation, heat and iron was promoted significantly.