Red Intensity Research Articles

P117 Transglutaminase 2 is elevated in Crohn’s disease associated strictures and exerts profibrotic activities in myofibroblasts and experimental intestinal fibrosis

Abstract Background Intestinal fibrosis is a significant clinical problem in Inflammatory bowel disease (IBD). This may lead to intestinal strictures induced by excessive accumulation of extracellular matrix (ECM) produced by human intestinal myofibroblasts (HIMF). Transglutaminase (TG)2 covalently cross-links ECM-associated proteins, increases ECM stiffness and has direct pro-fibrotic effects. We investigated the role of TG2 in intestinal fibrosis in human tissues, HIMF and transgenic mouse models. Methods TG2 was detected in human tissues from Crohn’s disease (CD) strictures (CDs; n=10), CD non-strictured (CDns; n=20), ulcerative colitis (UC; n=10) and non-IBD controls (NL; n=14) by immunofluorescence (IF), qPCR, in-situ hybridization (ISH), and biotinylated cadaverin incorporation (BCI). Relative contribution of TG2 was measured by BCI using a TG2-selective blocking antibody (TAb). The cellular source of TG2 was evaluated by IF. HIMF ECM production was measured using a novel ECM deposition assay and TG2-inhibitors. HIMF were exposed to anti-TG2 antibody or isotype control and bulk RNA-sequencing (RNAseq) analysis was performed. Tamoxifen-inducible global Cre/TG2 floxed conditional KO (cKO) mice were used in the DSS and TNBS fibrosis models. Results Total TG activity was increased in CDs and UC vs. NL in the submucosa (SM) and muscularis propria (MP; P≤ 0.04). Strikingly 90-99% of TG activity were attributable to TG2 in all groups. TG2 activity correlated with fibrosis scores (P≤0.02), but not inflammation scores. TG2 amount was elevated in CDs patients vs. NL in qPCR and ISH (P≤0.05). IF revealed HIMF as the major source of TG2 in the intestine as indicated by colocalization with alpha-SMA. HIMF, invitro, produced 82–97% of active TG2. Inhibition of TG2 by cell-permeable inhibitor or Tab reduced fibronectin (FN) and collagen (COL)1 and 3 deposition by HIMF. RNAseq showed TAb-treated cells expressed 432 downregulated and 402 upregulated genes. Functional enrichment analysis showed pathways associated with fibroblast activation/proliferation, ECM, and fibrosis-associated signaling (e.g. AKT) pathways to be downregulated in TAb-treated HIMF. Associated genes included nuclear protein (NUPR1) and sphingolipid signaling pathway proteins (SMPD1 and CTSD), which may be potential targets for TG2-mediated activity in HIMF. Deletion of TGM2 prior to inducing murine DSS or TNBS experimental fibrosis did not lead to any difference in inflammation, but decreased fibrosis (picrosirius red intensity, COL1/FN expression, wall thickness) in TG2 cKO mice vs. wild type (P≤0.05). Conclusion TG2 is involved in inflammation-independent progression of intestinal fibrosis and may represent a future anti-fibrotic target.

Red light intensity modulation, temperature sensing and bioimaging of NaLuF4:Er3+/Tm3+/Yb3+ microcrystals under 1532 nm laser excitation

This paper reports on microcrystals with deep tissue penetration depth, strong red light emission, and thermometric function. The red light emission of NaLuF4:20 % Er3+/1 % Tm3+/10 % Yb3+ microcrystals is exceptionally strong, thanks to the introduction of Tm3+ and Yb3+ ions energy capture centers. In bioimaging, the 1532 nm laser exhibits deeper imaging depth due to weak photon scattering and autofluorescence. Furthermore, red light emission has the greatest tissue penetration depth in the visible region. The luminescence intensity and tissue penetration depth of both the excitation and emission light make them suitable for deep tissue imaging. We investigated temperature sensing performance of NaLuF4:20 % Er3+/1 % Tm3+/10 % Yb3+ microcrystals, and found that the maximum relative sensitivity of LIR (I521/I540) reaches 0.92 %K−1. The microcrystals NaLuF4:20 % Er3+/1 % Tm3+/10 % Yb3+ are anticipated to have application in deep tissue imaging and temperature sensing.

Red luminescence properties of Na5Y(WO4)4 doped with Eu3+ and alkali metal ions

Abstract Na5Y(WO4)4: 6 % Eu3+, 6 % R+ (R = Li, Na, K) phosphors were synthesized using a high-temperature solid-state reaction method. Through a series of characterization methods, the crystal structure, morphology, composition, and luminescence properties of the sample are obtained. At the same time, the properties of Na5Y(WO4)4: Eu3+ phosphor added with alkali metal ions such as Li+, Na+, K+ were also studied. The experiment showed the red light emission and fluorescence lifetime of Na5Y(WO4)4: 6 % Eu3+ are improved through the doping of low concentration alkali metal agents, and Na5Y(WO4)4: 6 % Eu3+, 6 % Li+ obtained the strongest red light emission intensity. The results indicate that the red emissions intensity of Na5Y(WO4)4: Eu3+ can be enhanced through the Li+ alkali metal agents, which may lead to less use of Y3+ and hence lower cost.

Red fluorescence intensity as a criterion for assessing remineralization efficacy in early carious lesions

ObjectivesEvaluating early carious lesion activity with an objective and clinically valid approach is crucial for developing effective treatment plans. Therefore, we here assessed the activity of non-cavitated carious lesions using a quantitative light-induced fluorescence-digital (QLF-D) camera and compared the remineralization efficiency after fluoride treatment according to the lesion's activity level. MethodsRed fluorescence emission rate (ΔR) and fluorescence loss (ΔF) were evaluated in 44 non-cavitated carious lesions by using a QLF-D camera. Based on the ΔR level, the lesions were classified into 22 active (ΔR ≥37.55) and 22 inactive carious lesions (ΔR <37.55). Each lesion was treated with 1.23 % fluoride gel for 60 s and then immersed into artificial saliva for 7 days. Subsequently, ΔR and ΔF changes in the lesions were measured. ResultsSignificant interactions between lesion activity and time were found for both ΔR and ΔF (p < 0.001). ΔR of active lesions declined faster and ΔF increased more steeply than did inactive lesions. Specifically, on day 7 post-fluoride treatment, the ΔR reduction rate was 1.40-times higher in active lesions, and the ΔF recovery rate was 2.50-times higher, indicating that active lesions respond more markedly to fluoride application. ConclusionsThis study highlighted the significance of ΔR in predicting remineralization efficiency in non-cavitated carious lesions after fluoride application. It underscored the importance of accurately assessing caries activity when formulating effective treatment plans. Lesion activity, as determined by ΔR, not only influences the outcome of remineralization treatments but also provides a more objective measure for tailoring caries management strategies.

Meat quality of pigs fed grape pomace in different production systems.

The main objective of this study was to evaluate the effects of supplementation the diet of pigs with grape pomace preserved in silage form (GPS) and its interaction with indoor and outdoor production systems, with and without access to vegetation, on the attributes of meat quality produced. Analyzes of proximal composition, cholesterol content, fatty acid profile, shear force, texture profile and sensory analysis were performed. During cold storage, oxidative stability and objective color were analyzed. Statistical analysis was performed in a 3x2 factorial design (production systems (S) x GPS-feed (F)) and the interaction between them (S*F). The results showed that there was no interaction between the production system and GPS feeding for the attributes evaluated. The proximate composition and fatty acid profile of the muscle remained unchanged. Additionally, it provides higher subjective and objective tenderness, higher red color intensity, and reduces lipid oxidation under refrigeration. The supplementation of pig feed with GPS improve the quality of the meat and constitute a sustainable alternative for the winemaking residue.

Molecularly imprinted polymer on cotton materials as substrates for smartphone-based image and distance-based analysis of Cu(II) in water samples.

Cotton fabric was used as a substrate for smartphone-based image analysis of Cu(II) in drinking water. To enhance its selective and specific binding sites on the cotton surface, a molecularly imprinted polymer (MIP) was introduced using color complexes of 4-(2-pyridylazo)resorcinol-Cu(II) (PAR-Cu(II)) as the template molecule, 3-aminopropyltriethoxysilane (APTES) as the functional monomer, tetraethoxysilane (TEOS) as the crosslinker and NH3 as the catalyst. After achieving optimum conditions, the obtained CF-MIP/PAR-Cu(II) presented a red color, which was changed to yellow upon the removal of Cu(II) with 1.5 M HCl. After using CF-MIP/PAR to detect Cu(II), the red, green and blue intensities of the images captured using a smartphone were analyzed using the ImageJ program. For the calibration curve plotted between Δgreen intensity and Cu concentration, the linear range was 0.10-1.0 mg L-1 with the best correlation coefficient (R2) of 0.999. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 0.038 and 0.11 mg L-1, respectively. To obtain a distance-based device, MIP-modified cotton thread (CT-MIP/PAR) with a four-channel design was used as an alternative device. The distance of red color development was measured after using CT-MIP/PAR to detect Cu(II). The linear range was 0.50-3.0 mg L-1 with an R2 of 0.997. The LOD and LOQ were 0.18 and 0.56 mg L-1, respectively. The proposed methods provide simple, portable and inexpensive devices with high accuracy and precision for the detection of Cu(II) in drinking water.

Red urine syndrome in dromedary camels: Clinical, etiological, hematobiochemical sonographic and pathologic findings

Background: Although blood urine is frequently observed in dromedary camels, little attention is gained and only it was reported as case reports. Aim: This study was carried out to examine dromedary camels suffering from red urine syndrome from the points of the clinical, etiological, hematobiochemical, ultrasonographic and pathological characteristics. Methods: Thirty-one camels with red urine and fifteen controls were enrolled. With a duration ranging from five days to nine months, clinical manifestations included weakness, red discoloration of the urine, dribbling of urine, straining during urination and abdominal pain. Blood was sampled in EDTA and plain tubes. Results: The urine red color intensity was marked in 23 camels. In 5 camels discolored red urine was moderate while red urine was voided intermittently in the remaining 3 camels. Wide stance of the hind legs and pain reactions during urination were recorded in 18 camels. In all 31 camels, urine samples were centrifuged and sedimentation of red deposits was found. Nephrolithiasis was detected in 3 animals. one female camel had bilateral hydronephrosis. Hyperechoic urine was imaged within the renal pelvis in 7 camels. In addition, hypoechoic fluid was imaged within the peritoneal cavity in 8 animals. Ruptured and collapsed urinary bladder was found in 2 male camels. In addition, bilateral pyelonephritis was found in another male camel. Abscessation of the left and right kidneys confirmed by ultrasound-guided aspiration was confirmed in 3 and 2 females, respectively. Peri-renal abscessation of the right kidney was detected in a female camel. Large, misshaped hypoechoic mass involving the right kidney was found in 1 female. A large mass king neoplasia was also imaged in a female camel distal to and compressing the left kidney, which proved histologically to be leiomyoma. Moderate to severe thickening and corrugation of the urinary bladder mucosa was detected in 18 of the diseased camels. Conclusion: This study's syndrome of red urine in camels resulted mainly from hematuria. The existing etiologies were the nephrolithiasis, cystitis, pyelonephritis, peri-renal and renal abscessation, and renal neoplasia. Ultrasonography was superior in assessing the renal parenchyma and urinary bladder for the verification of the existing nephrolithiasis, hydronephrosis, pyelonephritis, peri-renal and renal abscessation, cystitis and ruptured or perforated bladder.

Regression models from portable NIR spectra for predicting the carcass traits and meat quality of beef cattle.

The aims of this study were to predict carcass and meat traits, as well as the chemical composition of the 9th to 11th rib sections of beef cattle from portable NIR spectra. The 9th to 11th rib section was obtained from 60 Nellore bulls and cull cows. NIR spectra were acquired at: P1 -center of Longissimus muscle; and P2 -subcutaneous fat cap. The models accurately estimated (P ≥ 0.083) all carcass and meat quality traits, except those for predicting red (a*) and yellow (b*) intensity from P1, and 12th-rib fat from P2. However, precision was highly variable among the models; those for the prediction of carcass pHu, 12th rib fat, toughness from P1, and those for 12th rib fat, a* and b* from P2 presented high precision (R2 ≥ 0.65 or CCC ≥ 0.63), whereas all other models evaluated presented moderate to low precision (R2 ≤ 0.39). Models built from P1 and P2 accurately estimated (P ≥ 0.066) the chemical composition of the meat plus fat, bones and, meat plus fat plus bones, except those for predicting the ether extract (EE) and crude protein (CP) of bones and the EE of Meat plus bones fraction from P2. However, precision was highly variable among the models (-0.08 ≤ R2 ≤ 0.86) of the 9th and 11th rib section. Those models for the prediction of dry matter (DM) and EE of the bones from P1; of EE from P1; and of EE, mineral matter (MM), CP from P2 of meat plus fat plus bones presented high precision (R2 ≥ 0.76 or CCC ≥ 0.62), whereas all other models evaluated presented moderate to low precision (R2 ≤ 0.45). Thus, models built from portable NIR spectra acquired at different points of the 9th to 11th rib section were recommended for predicting carcass and muscle quality traits as well as for predicting the chemical composition of this section of beef cattle. However, it is noteworthy, that the small sample size was one of the limitations of this study.

Early Diagnosis of Neonatal Sepsis Using the Leukocyte Alkaline Phosphatase Score: A Cohort Study

Introduction: The clinical features of sepsis in neonates are subtle and non specific, requiring a high index of suspicion for early diagnosis. Blood culture is the gold standard for diagnosis, but it is time consuming. Therefore, there is a need for a costeffective and reliable screening tool. The Leukocyte Alkaline Phosphatase (LAP) activity of neutrophils is known to increase during bacterial infections in adults. Aim: To determine the activity of LAP in Neonatal Sepsis (NS) and compare the results with blood culture. Materials and Methods: This is a prospective cohort study conducted from January 2018 to June 2019 at Mandya Institute of Medical Sciences, Karnataka, India. Total of 200 neonates by their haematological profile were clinically suspected of having sepsis. A peripheral smear was prepared from a drop of blood and stained to assess the LAP activity. In each smear, a total of 100 consecutive segmented neutrophils were examined and were rated from 0 to 4 according to the red granular precipitate intensity within their cytoplasm. The possible range of 0-400. The blood culture report was obtained from the case sheet. The neonates were divided into two groups: Group 1 consisted of culture-proven sepsis cases, and Group 2 consisted of neonates with clinical suspicion of sepsis but negative blood culture results. Descriptive analysis was performed using mean and standard deviation for quantitative variables, and frequency and proportion for categorical variables. Chi-square test was conducted, and a p-value &lt;0.05 was considered statistically significant. Results: Out of the 200 cases studied, 64 neonates showed a positive blood culture. The most common causative organism observed was Klebsiella pneumoniae, which was seen in 56 neonates. The age of the neonates ranged from newborn to 28 days old, with 115 being male. Furthermore, 116 neonates were term neonates, and 146 presented with early onset sepsis. The study revealed a wide range of LAP activity in both groups. In the culture-positive sepsis group, the LAP activity ranged from 36 to 350, while in the clinical sepsis group, it ranged from 10 to 354. Due to the broad range of LAP scores observed, it is concluded that LAP activity is not useful as a screening test. Conclusion: Findings of the present study indicate that LAP activity exhibited a wide range in both the culture-proven and clinical sepsis groups. However, due to this variability, LAP activity does not appear to be a reliable screening test for NS. While LAP activity assessment may still hold diagnostic value, further research is needed to refine its utility and explore its clinical relevance.

Excellent red upconversion luminescence in GdLaO3:Er3+/Yb3+/Sc3+ under 980 nm laser excitation

Er3+/Yb3+ are distributed in a double-layer structure, blocked by a single layer of Sc3+/La3+. Red UCL intensity of (Gd0.8Er0.1Yb0.1)(La0.9Sc0.1)O3 is comparable to β-NaYF4:Er3+/Yb3+.

Impact of storage conditions on the instrumental colour of buckwheat products for meat industry applications

Buckwheat is one of the functional pseudocereals rich in antioxidants, nutrients, bioactive compounds, and phytochemicals. Colour represents one of the most important sensory parameters in the quality assessment of the meat products. The aim of this study was to determine the possible effect of 9-month artificial aging storage at 40 ? 2?C on the change in instrumental colour of buckwheat products used in the meat industry. In the CIE L*a*b* system, L*, a*, and b* coordinates are used to specify the data of instrumental colour properties. The results were statistically processed by two-way ANOVA (P&lt;0.001) and Tukey?s Honestly Significant Difference post-hoc tests (P&lt;0.05). Storage and product type (and their interaction) factors significantly influenced instrumental colour. During storage, significant differences were observed between the time points (0, 3, 6, and 9 months) in flour, with trends of decreasing lightness (L*) and increasing red (a*) and yellow (b*) colour intensity values. Regarding product type, significantly higher L* values were observed in flour, while a* values were higher in grains. Except for the 3rd month, b* values did not show significant differences. The insights gained in this study may indicate the further application of stored buckwheat flour and grains in obtaining technologically justified and colour-sensory acceptable meat end-products for consumers.

A portable fluorescence detection device based on a smartphone employing carbon nanodots for Mn2+ sensing.

The measurement of fluorescence emission for quantitative analysis is typically based on a traditional spectrofluorometer, which limits an onsite detection approach. Thus, an alternative device should be developed for fulfilling this analysis outside of the laboratory. Therefore, a low-cost, portable, and low-energy consumption fluorescence reader-based smartphone device was developed. An ultraviolet light-emitting diodes (UV-LED) was used to construct the fluorescence device-based smartphone as a low-power excitation light source. The smartphone camera was used as a detector for detecting photons from the fluorescence emission process of the fluorescence probe and was connected to a digital image platform. Transparent acrylic with orange and yellow colors was employed as a filter for reducing the interference from light source intensity. The obtained digital image was converted to red, green and blue (RGB) intensity using a custom-designed smartphone application. N,S-doped carbon nanodots (N,S-CDs) were demonstrated to be a good fluorescence indicator for determining trace quantities of Mn2+ in cosmetics. The approach exhibited high selectivity and sensitivity, detecting and quantifying analytes at 1-5 μM concentrations. Furthermore, the method's detection limit of 0.5 μM reflects its capacity to detect trace amounts of a target analyte. Mn2+ in cosmetic products was successfully analyzed using this device with high accuracy comparable with the results from inductively coupled plasma-optical emission spectroscopy (ICP-OES).

Ddx5 participates in regulation of Col10a1 expression and chondrocyte hypertrophic differentiation in vitro.

The type X collagen gene (Col10a1), is a specific molecular marker of hypertrophic chondrocytes during endochondral ossification. Col10a1 expression is known to be influenced by many regulators. In this study, we aim to investigate how DEAD-box helicase 5 (DDX5), a potential binding factor for Col10a1 enhancer, may play a role in Col10a1 expression and chondrocyte hypertrophic differentiation in vitro. The potential binding factors of the 150-bp Col10a1 cis-enhancer were identified with the hTFtarget database. The expression of DDX5 and COL10A1 was detected by quantitative real-time PCR (qRT-PCR) and Western blot in chondrogenic ATDC5 and MCT cell models with or without Ddx5 knockdown or overexpression. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were performed to determine the interaction between DDX5 and the Col10a1 enhancer. The effect and mechanism of DDX5 on chondrocyte differentiation and maturation was evaluated by alcian blue, alkaline phosphatase (ALP), and alizarin red staining in ATDC5 cell lines with stable knockdown of Ddx5. DDX5 was identified as a potential binding factor for the Col10a1 enhancer. The expression of DDX5 in hypertrophic chondrocytes was higher than that in proliferative chondrocytes. Knockdown of Ddx5 decreased, while overexpression of Ddx5 slightly increased COL10A1 expression. DDX5 promotes the enhancer activity of Col10a1 as demonstrated by dual-luciferase reporter assay, and the ChIP experiment suggests a direct interaction between DDX5 and the Col10a1 enhancer. Compared to the control (NC) group, we observed weaker alcian blue and ALP staining intensity in the Ddx5 knockdown group of ATDC5 cells cultured both for 7 and 14 days. Whereas weaker alizarin red staining intensity was only found in the Ddx5 knockdown group of cells cultured for 7 days. Meanwhile, knockdown of Ddx5 significantly reduced the level of runt-related transcription factor 2 (RUNX2) in related ATDC5 cells examined. Our results suggest that DDX5 acts as a positive regulator for Col10a1 expression and may cooperate with RUNX2 together to control Col10a1 expression and promote the proliferation and maturation of chondrocytes.

Study of the Oxidative and Microbiological Stability of Nitrite-Reduced, Vacuum-Packed, Refrigerated Lamb Sausages Supplemented with Red Propolis Extract.

Vacuum-packed lamb sausages with or without red propolis extract and a reduced sodium nitrite content were evaluated for oxidative and microbiological stability during storage for 21 days at 2 °C. The following treatments were evaluated: EN150 (control, base formulation (BF) + 500 mg/kg sodium erythorbate and 150 mg/kg sodium nitrite); EN75 (BF + 500 mg/kg sodium erythorbate and 75 mg/kg sodium nitrite); P1N75 (without the addition of erythorbate, BF + 1800 mg/kg propolis extract and 75 mg/kg sodium nitrite); and P2N75 (without the addition of erythorbate, BF + 3600 mg/kg propolis extract and 75 mg/kg sodium nitrite). Analyses were conducted to characterize the samples on day 0 with respect to the proximate composition (moisture, protein, fat, and ash) and sensory acceptance. Stability during refrigerated storage was evaluated on days 0, 7, 14 and 21 for the parameters pH, color profile (L*, a*, and b*), TBARs index (oxidative stability) and microbiological count of aerobic psychrotrophic microorganisms. Texture profile, cooking weight loss (WLC), peroxide index and free fatty acids were evaluated on days 0 and 21. The treatments with propolis and reduced nitrite (EN150 and P1N75) showed a red color intensity (a*) similar to the treatment with erythorbate and the same nitrite content (EN75) at the end of storage, maintaining the characteristic reddish color of the sausages. The extract slowed down lipid oxidation during storage, especially P2N75, which showed the lowest level of TBARS (0.39 mg MDA/kg) and the peroxide index (2.13 mEq g O2) on day 21. The residual nitrite value in EN75 was the lowest (p < 0.05) on day 21, showing that synthetic antioxidants are more efficient than the extract in nitrite reduction reactions. The results for the counts of psychrotrophic microorganisms showed that the extract did not have the expected antimicrobial effect on the growth of this microorganisms, and leveling the results revealed no differences (p < 0.05) between the treatments. Despite the red propolis extract not showing a significant antimicrobial improvement in lamb sausages, it can be considered a healthy option with good prospects for replacing synthetic antioxidants with a natural product.

An opportunity for acerola pulp (Malpighia emarginata DC) valorization evaluating its performance during the block cryoconcentration by physicochemical, bioactive compounds, HPLC–ESI-MS/MS, and multi-elemental profile analysis

The present study evaluated the effect of cryoconcentration of pulp blocks of acerola (Malpighia emarginata DC). The study evaluated cryoconcentration in three stages. The cryoconcentrated samples, the ice fractions, and the initial pulp were evaluated for physicochemical composition, bioactive composition, and multielement profile. The cryoconcentrated sample obtained in the third stage of cryoconcentration showed the best results for the concentration factor, process efficiency, total soluble solids content, red color intensity, and increasing of the macro and micronutrients: Cu, Ca, S, Sr, K, Mn, Na, P, Mg, Fe. All stages presented good performance in the total soluble solids content, increase in the titratable acidity of the concentrates, and progressive increase in the intensity of the red color. Generally, higher levels of total phenolic and antioxidant activity were found for the 2nd and 3rd concentrates. The phenolic activity showed an increase of 166.90% in the 3rd stage concentrate compared to fresh pulp, and the antioxidant activity was 112.10% by the ABTS method and 131.60% by the DPPH method, both in the 3rd stage concentrate. The major individual polyphenols were Ferulic acid, Protocatechuic acid, and Taxifolin, with significant increases in the concentration of the compounds in the 2nd and 3rd stage concentrates. In addition, the contents of potentially toxic metals were below detection limits. During the cryoconcentration process, there was a decrease in the values ​​of vitamin C content, moisture content, density, and elements Cu, Sr, and Zn.

New insights into micro-algal astaxanthin's effect on deoxynivalenol-induced spleen lymphocytes pyroptosis in Cyprinus carpio: Involving mitophagy and mtROS-NF-κB-dependent NLRP3 inflammasome

Deoxynivalenol (DON) is one of the most common sources of fungal toxins in fish feed, posing a significant risk to the immune and reproductive systems of fish. Microalgal astaxanthin (MIA), a potent antioxidant derived from microalgae, confers multifarious advantages upon piscine organisms, notably encompassing its anti-inflammatory and antioxidant prowess. Herein, we investigated the potential of MIA in ameliorating the immunotoxicity of DON on carp (Cyprinus carpio L.) based on spleen lymphocytes treated with DON (1.5 ng/ml) and/or MIA (96 μM). Firstly, CCK8 results showed that DON resulted in excessive death of spleen lymphocytes. Secondly, spleen lymphocytes treated with DON had a higher proportion of pyroptosis, and the mRNA and protein levels of pyroptosis (NLRP3, IL-1β and ASC) in spleen lymphocytes were increased. Thirdly, the relative red fluorescence intensity of JC-1 and DCFH-DA showed decreased mitochondrial membrane potential and increased ROS in spleen lymphocytes treated with DON. Mitochondrial ATP, DNA and NADPH/NADP+ analysis revealed decreased mitochondrial ATP, DNA and NADPH/NADP+ levels in DON-treated lymphocytes, corroborating the association between DON exposure and elevated intracellular ATP, DNA and NADPH/NADP+ in lymphocytes. DON exposure resulted in the downregulation of mitophagy-related genes and proteins (PINK1, Parkin and LC3) in lymphocytes. Notably, these effects were counteracted by treatment with MIA. Furthermore, DON led to the elevated secretion of inflammatory factors (TNF-α, IL-4 and IFN-γ), thereby inducing immune dysfunction in spleen lymphocytes. Encouragingly, MIA treatment effectively mitigated the immunotoxic effects induced by DON, demonstrating its potential in ameliorating pyroptosis, mitochondrial dysfunction and mitophagy impairment via regulating the mtROS-NF-κB axis in lymphocytes. This study sheds light on safeguarding farmed fish against agrobiological threats posed by DON, highlighting the valuable applications of MIA in aquaculture.

Induction of Browning in White Adipocytes: Fucoidan Characterization and Gold Nanoparticle Synthesis from Undaria pinnatifida Sporophyll Extract.

Seaweed extracts and their specific polysaccharides are widely known for their ability to act as reducing and capping agents during nanoparticle synthesis. Their application is highly favored in green synthesis methods, owing to their eco-friendliness, cost-effectiveness, and remarkable time and energy efficiency. In this study, fucoidan extracted from Undaria pinnatifida sporophyll (UPS) is introduced as a polysaccharide that effectively serves as a dual-function reducing and capping agent for the synthesis of gold nanoparticles (AuNPs). Results from various analyses indicate that AuNPs derived from UPS extract display a uniform spherical shape with an average size of 28.34 ± 1.15 nm and a zeta potential of -37.49 ± 2.13 mV, conclusively confirming the presence of Au. The FT-IR spectra distinctly revealed the characteristic fucoidan bands on the stabilized UPS-AuNPs surface. A 1H-NMR analysis provided additional confirmation by revealing the presence of specific fucoidan protons on the UPS-AuNPs surface. To comprehensively evaluate the impact of UPS extract, UPS-AuNPs, and fucoidan on the biological properties of adipocytes, a rigorous comparative analysis of lipid droplet formation and morphology was conducted. Our findings revealed that adipocytes treated with UPS extract, fucoidan, and UPS-AuNPs, in that order, exhibited a reduction in the total lipid droplet surface area, maximum Ferret diameter, and overall Nile red staining intensity when compared to mature white adipocytes. Furthermore, our analysis of the effects of UPS extracts, UPS-AuNPs, and fucoidan on the expression of key markers associated with white adipose tissue browning, such as UCP1, PGC1a, and PRDM16, demonstrated increased mRNA and protein expression levels in the following order: UPS-AuNPs > fucoidan > UPS extracts. Notably, the production of active mitochondria, which play a crucial role in enhancing energy expenditure in beige adipocytes, also increased in the following order: UPS-AuNPs > fucoidan > UPS extract. These findings underscore the pivotal role of UPS extract, fucoidan, and UPS-AuNPs in promoting adipocyte browning and subsequently enhancing energy expenditure.

Development of a Cre-recombination-based color-switching reporter system for cell fusion detection

Cell fusion plays a key role in the development and formation of tissues and organs in several organisms. Skeletal myogenesis is assessed in vitro by cell shape and gene and protein expression using immunofluorescence and immunoblotting assays. However, these conventional methods are complex and do not allow for easy time-course observation in living cells. Therefore, this study aimed to develop a Cre recombination-based fluorescent reporter system to monitor cell-cell fusion. We combined green and red fluorescent proteins with a Cre-loxP system to detect syncytium formation using a fluorescent binary switch. This allowed us to visualize mononucleated cells with green fluorescence before fusion and multinucleated syncytia with red fluorescence by conditional expression after cell fusion. The formation of multinuclear myotubes during myogenic differentiation was detected by the change in fluorescence from green to red after Cre-mediated recombination. The distribution of the fluorescence signal correlated with the expression of myogenic differentiation markers. Moreover, red reporter fluorescence intensity was correlated with the number of nuclei contained in the red fluorescent-positive myotubes. We also successfully demonstrated that our fusion monitoring system is applicable to the formation of skeletal muscle myotube and placental syncytiotrophoblast. These results suggest that the color-switching fluorescent reporter system, using Cre-mediated recombination, could be a robust tool used to facilitate the study of cell-to-cell fusion.

Experimentally determined leaflet-leaflet phase diagram of an asymmetric lipid bilayer.

We have determined the partial leaflet-leaflet phase diagram of an asymmetric lipid bilayer at ambient temperature using asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs with varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were hemifused to a supported lipid bilayer (SLB) composed of DOPC, resulting in lipid exchange between their outer leaflets. The GUVs and SLB contained a red and green lipid fluorophore, respectively, thus enabling the use of confocal fluorescence imaging to determine both the extent of lipid exchange (quantified for individual vesicles by the loss of red intensity and gain of green intensity) and the presence or absence of phase separation in aGUVs. Consistent with previous reports, we found that hemifusion results in large variation in outer leaflet exchange for individual GUVs, which allowed us to interrogate the phase behavior at multiple points within the asymmetric composition space of the binary mixture. When initially symmetric GUVs showed coexisting gel and fluid domains, aGUVs with less than ~50% outer leaflet exchange were also phase-separated. In contrast, aGUVs with greater than 50% outer leaflet exchange were uniform and fluid. In some cases, we also observed three coexisting bilayer-spanning phases: two registered phases and an anti-registered phase. These results suggest that a relatively large unfavorable midplane interaction between ordered and disordered phases in opposing leaflets (i.e., a midplane surface tension) can overwhelm the driving force for lateral phase separation within one of the leaflets, resulting in an asymmetric bilayer with two uniformly mixed leaflets that is poised to phase-separate upon leaflet scrambling.

Abstract 11891: Pulmonary Delivery of Novel BEPI/NO-Microparticle for Treatment of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is an incurable disease characterized by elevated blood pressure in the pulmonary artery causing right heart failure and even sudden cardiac death. Nitric oxide (NO) is a strong potent regulator of PAH. However, the therapeutic effects of exogenous NO depend on its concentration and its half-life is very short. Hypothesis: we prepared NO-releasing inhalers with controlled porous structures to modulate the deposition in lung. Methods and Results: NO-releasing inhalers with micro-sized pores showed improved nebulization efficiency. Furthermore, the cGMP expression and macrophage polarization were evaluated. The fluorescence intensity of Nile red was analyzed using photoluminescence spectroscopy to confirm the amounts of NO-MPs on the slide glass. As with confocal images, 0.8k BPEI/NO-MPs could arrive the lower region than 25k BPEI/NO-MPs. NO produced by endothelial nitric oxide synthase (eNOS) induce vasodilation by increasing cGMP in vascular smooth muscle cells. Hence, the delivery of NO mimicking the physical production of the endothelium in range of 0.05-0.4 nmol cm -2 min -1 is required to restore vascular homeostasis. Therefore, BPEI0.8k/NO-MPs release 0.277 nmol cm -2 min -1 of NO that mimics the environment in healthy endothelium, while BPEI25k/NO-MPs showed a higher concentration as 0.548 nmol cm -2 min -1 . BPEI0.8k/NO-MPs increased the synthesis of cGMP in cells, whereas BPEI25k/NO-MPs presented adverse effects due to an initial burst release. When cell populations were quantified, BPEI0.8k/NO-MPs increased the number of M2-like cells while decreasing the number of M1-like cells. Furthermore, using fluorescence activated cell sorting analysis, proteins and gene levels of cells, anti-inflammatory factors increased in BPEI0.8k/NO-MPs treatment groups Conclusions: novel BPEI/NO-releasing inhalers could deliver NO to deep lungs and has the potential for treatment of PAH