Average Fluorescence Intensity Research Articles

Hederagenin Induces Apoptosis of Human Hepatoma HepG2 Cells via the Mitochondrial Pathway.

The objective of this study is to assess the antitumor effects of hederagenin (HDG) in liver cancer (LC) cells and explore the related mechanisms. HepG2 cells were treated with HDG and cisplatin, respectively. The CCK8 assay was used to detect cell activity, DAPI staining was used to detect the proportion of living cells, TUNEL assay to detect the proportion of apoptotic cells, flow cytometry to detect the membrane potential, fluoroscopic electron microscopy to detect microstructural changes to the mitochondrial, and western blot analysis and high-content screening to detect apoptosisrelated proteins. Treatment with HDG inhibited the growth of HepG2 cells, decreased the proportion of viable cells, increased the proportion of apoptotic cells, and significantly increased the proportion of cells in the G1 phase. Fluorescence staining showed that HDG damaged the mitochondria of HepG2 cells and significantly decreased the number of mitochondria. Flow cytometry showed that HDG decreased the mitochondrial membrane potential of HepG2 cells. Observations by electron microscopy showed that HDG caused swelling and vacuole formation of the mitochondria of HepG2 cells. HDG significantly reduced the average fluorescence intensity of Bcl-2 in HepG2 cells and significantly increased that of the pro-apoptosis proteins Bax, Cytochrome-c, and Caspase-3. HDG induced apoptosis of HepG2 cells via the mitochondrial pathway.

Carnosine-copper chelator-modified small-diameter vascular grafts for the promotion of anticoagulation and endothelial regeneration

Thrombosis and poor endothelialization of small-diameter vascular grafts (SDVGs) remain the leading cause of frequent failure, despite current tissue-engineered vascular grafts providing a feasible strategy in clinical treatment strategies for the small-diameter (<6 mm) blood vessels in management of cardiovascular disease. Copper ions are normally used to achieve the purpose of antithrombotic because they can produce nitric oxide (NO) with the catalysis of NO donors. However, copper ions also participate in the Fenton reaction to produce reactive oxygen species (ROS), which would adversely affect vascular graft remodeling. Here carnosine, an ion-chelating agent with anti-inflammatory and antioxidant properties, was used to chelate with copper ions to synthesize carnosine-Cu (II) chelate which showed effective activity of antithrombotic, anti-inflammatory, and antioxidant. Besides, our research further demonstrated that copper ions still retain the catalytic activities for producing NO after chelating with carnosine accompanied by the production of lower levels of ROS (the average fluorescence intensity of ROS decreased from 79.69 to 7.13). Furthermore, the grafts achieved excellent patency with satisfactory endothelialization (the endothelial coverage reached 80.64 %) and significantly improved deposition of collagen and glycosaminoglycan for up to 12 weeks. In general, Car-Cu (II) modified SDVGs provided a facile approach to addressing the problems of thrombus and poor endothelialization in SDVGs for the clinic, which is significant in cardiovascular regenerative medicine.

Mechanism of acupotomy on chondrocyte ferroptosis in rabbits with knee osteoarthritis based on HSPA5/GPX4 signaling pathway

To observe the effect of acupotomy on heat shock protein A family member 5 (HSPA5)/glutathione peroxidase 4 (GPX4) signaling pathway in the chondrocytes of the rabbits with knee osteoarthritis (KOA) and explore the mechanism of acupotomy on chondrocyte ferroptosis in KOA. Twenty-seven New Zealand rabbits were randomly divided into a normal group, a model group and an acupotomy group, with 9 rabbits in each group. The left hind limb was fixed by the modified Videman method for 6 weeks to establish KOA model. After modeling, acupotomy was given in the acupotomy group, once a week and for consecutive 3 weeks. Using Lequesne MG score, the local symptoms, physical signs and functions of knee joint were evaluated. With HE staining and saffrane-solid green staining adopted, the morphology of chondrocytes and cartilage tissue was observed. Under transmission electron microscope, the mitochondrial structure of chondrocytes was observed. The iron content of cartilage tissue was detected by iron ion kit. The mitochondrial membrane potential (Δψm) and the reactive oxygen species (ROS) level in cartilage tissue were determined by flow cytometry, and the mitochondrial damage rate was calculated. The mRNA expression of HSPA5, GPX4, type Ⅱ collagen α1 chain (COL2A1), matrix metalloproteinases (MMP) 3 and MMP13 was detected by the real-time quantitative PCR; and the protein expression of HSPA5, GPX4, type Ⅱ collagen (COL-Ⅱ), MMP3 and MMP13 was detected by Western blot. The mean flourscence intensity of HSPA5 and GPX4 in cartilage tissue was determined by immunofluorescence. Before intervention, compared with the normal group, the Lequesne MG scores were increased in the model group and the acupotomy group (P<0.01). After intervention, the Lequesne MG score in the acupotomy group was decreased when compared with that in the model group. In comparison with that in the normal group, the number of chondrocytes was reduced and the cells were disarranged; the layers of cartilage structure were unclear, the tide lines disordered and blurred; the mitochondria were wrinkled and the mitochondrial crista decreased or even disappeared in the model group. Compared with the model group, the number of chondrocytes was increased, the layers of cartilage structure were clear, the tide lines recovered, the number of mitochondria elevated, with normal structure and more crista in the acupotomy group. The iron content of cartilage tissue was increased (P<0.01), the Δψm of chondrocytes was declined, the mitochondrial damage rate was increased (P<0.01), the average fluorescence intensity of ROS was increased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was decreased (P<0.01), the mRNA and protein expression of MMP3 and MMP13 was increased (P<0.01) and the average fluorescence intensity of HSPA5, GPX4 was decreased (P<0.01) in the model group when compared with those in the normal group. Compared with the model group, the iron content in cartilage tissue was reduced (P<0.01), the Δψm of chondrocytes was increased, the mitochondrial damage rate was decreased (P<0.01), and the average fluorescence intensity of ROS was decreased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was higher (P<0.01), and the mRNA and protein expression of MMP3 and MMP13 was lower, and the average fluorescence intensity of HSPA5, GPX4 was increased (P<0.01) in the acupotomy group. Acupotomy can alleviate cartilage injury of KOA rabbits, and its mechanism may be related to the regulation of HSPA5/GPX4 signaling pathway to maintain iron homeostasis in articular cartilage, thus inhibiting chondrocyte ferroptosis and relieving extracellular matrix degradation.

A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

Propionic acid ameliorates cognitive function through immunomodulatory effects on Th17 cells in perioperative neurocognitive disorders

BackgroundElderly patients undergoing surgery are prone to cognitive decline known as perioperative neurocognitive disorders (PND). Several studies have shown that the microglial activation and the decrease of short-chain fatty acids (SCFAs) in gut induced by surgery may be related to the pathogenesis of PND. The purpose of this study was to determine whether microglia and short-chain fatty acids were involved in cognitive dysfunction in aged rats. MethodsMale wild-type Wistar rats aged 11–12 months were randomly divided into control group (Ctrl: Veh group), propionic acid group (Ctrl: PA group), exploratory laparotomy group (LP: Veh group) and propionic acid + exploratory laparotomy group (LP: PA group) according to whether exploratory laparotomy (LP) or PA pretreatment for 21 days was performed. The motor ability of the rats was evaluated by open field test on postoperative day 3 (POD3), and then the cognitive function was evaluated by Y-maze test and fear conditioning test. The expression of IL-1β, IL-6, RORγt and IL-17A mRNA in hippocampus was detected by RT-qPCR, the expression of IL-17A and IL-17RA in hippocampus was detected by Western blot, and the activation of microglia was detected by immunofluorescence. ResultsThe PND rat model was successfully established by laparotomy. Compared with Ctrl: Veh group, the body weight of LP: Veh group decreased, the percentage of spontaneous alternations in Y maze decreased (P < 0.001), and the percentage of freezing time in contextual fear test decreased (P < 0.001). Surgery triggers neuroinflammation, manifested as the elevated levels of the inflammatory cytokines IL-1β (P < 0.001) and IL-6 (P < 0.001), the increased expression of the transcription factor RORγt (P = 0.0181, POD1; P = 0.0073, POD5)and major inflammatory cytokines IL-17A (P = 0.0215, POD1; P = 0.0071, POD5), and the increased average fluorescence intensity of Iba1 (P < 0.001, POD1; P < 0.001, POD5). After PA preconditioning, the recovery of rats in LP: PA group was faster than that in LP: Veh group as the body weight lost on POD1 (P = 0.0148) was close to the baseline level on POD5 (P = 0.1846), and they performed better in behavioral tests. The levels of IL-1β (P < 0.001) and IL-6 (P = 0.0035) inflammatory factors in hippocampus decreased on POD1 and the average fluorescence intensity of Iba1 decreased (P = 0.0024, POD1; P < 0.001, POD5), representing the neuroinflammation was significantly improved. Besides, the levels of RORγt mRNA (P = 0.0231, POD1; P = 0.0251, POD5) and IL-17A mRNA (P = 0.0208, POD1; P = 0.0071, POD5) in hippocampus as well as the expression of IL-17A (P = 0.0057, POD1; P < 0.001, POD5) and IL-17RA (P = 0.0388) decreased. ConclusionPA pretreatment results in reduced postoperative neuroinflammation and improved cognitive function, potentially attributed to the regulatory effects of PA on Th17-mediated immune responses.

Inhibitory effect and action mechanism of citral against black rot in pitaya fruit

Blackspot, caused by Alternaria alternata, is a significant postharvest disease in pitaya. Currently, there are limited effective fungicides to manage this disease. This study aimed to evaluate the antifungal activity of citral against the blackspot pathogen and to determine the protective effect of citral on pitaya. Our results demonstrated that citral effectively curtailed the mycelial growth and spore germination of Alternaria alternata, with a minimum inhibitory concentration (MIC) of 0.6 μL/mL. Observations under a light microscope revealed that citral-treated hyphae displayed characteristics such as sparseness, roughness, twisting, shriveling, and irregular internode separation, along with prominent vacuolation. Propidium iodide and 2,7-Dichlorodi-hydrofluorescein diacetate staining results revealed that the mycelia from the MIC citral-treated group emitted robust fluorescence, with the average fluorescence intensities being 4.03 and 8.09 times higher than the control group, respectively. Furthermore, citral treatment resulted in a decline in the lipid content and dry weight of the hyphae, increased nucleic acid and protein leakage, enhanced exosporial conductivity, and augmented superoxide dismutase (SOD) and catalase (CAT) enzyme activities. Additionally, it fostered the accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Fruit damage inoculation assays indicated that an appropriate concentration of citral (1/2 MIC, MIC) could notably diminish the fruit spot diameter expansion. Thus, citral shows promise as a potential alternative fungicide for managing postharvest black rot in pitaya.

CD4+ CD11b+ T cells infiltrate and aggravate the traumatic brain injury depending on brain-to-cervical lymph node signaling.

We aim to identify the specific CD4+ T-cell subtype influenced by brain-to-CLN signaling and explore their role during the acute phase of traumatic brain injury (TBI). Cervical lymphadenectomy or cervical afferent lymphatic ligation was performed before TBI. Cytokine array and western blot were used to detect cytokines, while the motor function was assessed using mNss and rotarod test. CD4+ T-cell subtypes in blood, brain, and CLNs were analyzed with Cytometry by time-of-flight analysis (CyTOF) or fluorescence-activated cell sorting (FACS). Brain edema and volume changes were measured by 9.4T MRI. Neuronal apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Cervical lymphadenectomy and ligation of cervical lymphatic vessels resulted in a decreased infiltration of CD4+ T cells, specifically CD11b-positive CD4+ T cells, within the affected region. The population of CD4+ CD11b+ T cells increased in ligated CLNs, accompanied by a decrease in the average fluorescence intensity of sphingosine-1-phosphate receptor-1 (S1PR1) on these cells. Administration of CD4+ CD11b+ T cells sorted from CLNs into the lateral ventricle reversed the attenuated neurologic deficits, brain edema, and lesion volume following cervical lymphadenectomy. The infiltration of CD4+ CD11b+ T cells exacerbates secondary brain damage in TBI, and this process is modulated by brain-to-CLN signaling.

The Role of β1 Integrin/CD29 as a Potential Prognostic Factor for the Risk of Progression to Cervical Carcinoma in HPV-Associated Lesions.

Background and Objectives: Available evidence reports the overexpression of β1 integrin in dysplastic rather than normal cervical tissue. We aimed to evaluate the involvement of β1 (CD29) integrin in the progressive pathogenesis of cervical intraepithelial neoplasia (CIN). Materials and Methods: From January 2019 to December 2021, we prospectively enrolled women undergoing a colposcopy with a cervical biopsy for abnormal cervical cytology and/or undefined cytology with a positive HPV DNA test and women with relapsing cervical inflammatory disorders. Based on the histopathological results, women were divided into four groups: group A (CIN1), group B (CIN2), group C (CIN3), and group D (no CIN diagnosis) as a control group. Subsequently, cytofluorimetry and immunohistochemical analysis (based on the identified positive cell ratios as follows: ≤10%, negative; 10-25%, 1+ (weak); 25-50%, 2+ (medium); ≥50%, and 3+ (high)) for β1 integrin were carried out. Results: In total, 154 women were included. The average fluorescence intensity in the four groups was 2.35 ± 1.37, 2.73 ± 1.56, 3.09 ± 1.56, and 2.13 ± 1.25 UA from groups A to D, respectively; this figure was significantly different for CIN3 (group C) women relative to the other groups (p = 0.0132). Higher β1 integrin/CD29 concentrations in the CIN groups with HR-HPV 16 and 18 were also detected (p = 0.0292, 0.0367, and 0.0357 respectively for CIN3, CIN2, and CIN1). Immunohistochemistry analysis showed higher results for the CIN3 group compared to controls and all the other groups (p < 0.001). Conclusions: β1/CD29 integrin expression increased with CIN grade, and it was significantly higher in CIN3 lesions. This could be used as a promising screening tool to identify women prone to developing high-grade cervical lesions. However, additional evidence is needed to strengthen these findings.

Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage

To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism. The LPS induced type II alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting. Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2 438.95±64.89, 3 036.41±96.69 vs. 1 736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67 205.60±3 430.40 vs. 56 272.67±7 650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

Construction of a near infrared fluorescence system for imaging of biological tissues.

Surgical procedures often rely on unaided visual observation or endoscopic assistance, which may pose challenges in cases involving intricate anatomical relationships. Real-time imaging technologies capable of intraoperative visualization of target organs have the potential to enhance the precision of surgical procedures by facilitating accurate identification, separation, and protection of vital tissues or organs. Despite these advantages, the widespread adoption of such technologies has been hindered by factors such as the prohibitive cost of equipment. This study aims to optimize and develop a device based on Indocyanine Green (ICG) for fluorescence imaging. The objective is to monitor changes in the average fluorescence intensity of ICG in the bladder, offering valuable guidance for surgeries involving the bladder. 1. Male rabbits were administered 0.01mg/ml ICG via the renal pelvis and ear vein to obtain fluorescence images of the ureter, bladder, and small intestine. 2. After ligating the bilateral ureters of male rabbits, a retrograde bladder perfusion of 5ml 0.01mg/ml ICG was conducted to capture fluorescence images of the bladder over time. The average fluorescence intensity was computed using Image Pro Plus 6.0, and the corresponding curve was generated using Prism 8.0. Using a similar methodology, the average fluorescence intensity of male rabbits without ureteral ligation was measured and plotted over time. 1. The developed device facilitated imaging of the ureter, bladder, and small intestine. 2. The bladder's average fluorescence intensity exhibited changes over time in response to urine production and ureteral ligation, contrasting with observations without ureteral ligation. We have successfully constructed and optimized a modular fluorescence imaging system for organs and tissues. This system proves effective in imaging experiments involving hollow organs in animals and offers valuable insights for relevant surgical procedures.

Features of Fluorescence Profiles and Species Composition of Phytoplankton in the Black Sea and the Sea of Azov

The aim of this work was to study the properties of vertical fluorescence intensity (FI) profiles of phytoplankton pigments (chlorophyll-a, phycocyanin, phycoerythrin, and beta-carotene) using data on the phytoplankton species composition obtained during the cruise 114 of the R/V ‘Professor Vodyanitsky’. The analysis of covariance matrices of phytoplankton pigment FI profiles in the upper 50-meter layer of the Black Sea indicates that the pigment composition of phytoplankton changes with depth, which may be associated with changes in its species composition. At the same time, 80 % variability of phytoplankton pigment FI profiles in the upper 20-meter layer is described by the first eigenvector. It agrees well with direct observations of the phytoplankton species composition, indicating the dominance of one phytoplankton division in the 20-meter layer. In addition, there are regional peculiarities: for example, the average FI values of the phytoplankton pigments in the Sea of Azov are significantly higher than those in the Black Sea, which is associated with a higher concentration of phytoplankton in the Sea of Azov.

Utilization and transformation of Chrysotila dentata-derived dissolved organic matter by phycosphere bacteria Marinobacter hydrocarbonoclasticus and Bacillus firmus.

The dissolved organic matter (DOM) released from the cocoolithophores (Chrysotila dentata) was studied in laboratory experiments after co-culturing C. dentata with bacteria. Marinobacter hydrocarbonoclasticus (CA6)-γ-Proteobacteria and Bacillus firmus (CF2) were used to investigate the utilization and processing of the DOM derived from C. dentata, utilizing fluorescence excitation-emission matrix (EEM) combined with parallel factor analysis (EEM-PARAFAC), while measuring algal abundance and photosynthetic parameters. The experimental groups consisted of axenic C. dentata groups, filter cultured with bacteria (CA6 or CF2) groups, C. dentata co-cultured with bacteria (CA6 or CF2) groups and axenic bacteria (CA6 or CF2) groups. We then evaluated the processing of DOM by determining four fluorescence indices. The number of C. dentata cells and the photosynthetic capacity of microalgae were enhanced by CA6 and CF2. The main known fluorophores, including humic-like components and protein-like components, were present in all sample. The protein-like component of algal-bacterial co-cultures was effectively utilized by CA6 and CF2. The humic-like components increased at the end of the culture time for all cultures. Meanwhile, the average fluorescence intensity of protein-like in CA6 co-culture with algae was lower than that in CF2 co-culture with algae over time. On the other hand, the average fluorescence intensity of humic-like in CA6 was higher than CF2. However, the total change in fluorescence in humic-like and protein-like of axenic CF2 cultures was lower than that of CA6. Hence, the ability of CA6 to transform microalgal-derived DOM was superior to that of CF2, and CF2's ability to consume bacterial-derived DOM was superior to that of CA6.

Nanoliposome-Mediated Encapsulation of Chlorella Oil for the Development of a Controlled-Release Lipid-Lowering Formulation.

Chlorella oil nanoliposomes (CO-NLP) were synthesized through ultrasonic injection with ethanol, and their physicochemical properties and hypolipidemic efficacy were systematically investigated. The results revealed that the mean particle size of CO-NLP was 86.90 nm and the encapsulation efficiency (EE) was 92.84%. Storage conditions at 4 °C were conducive to the stability of CO-NLP, maintaining an EE of approximately 90% even after 10 days of storage. The release profile of CO-NLP adhered more closely to the first-order kinetic model during in vitro assessments, exhibiting a slower release rate compared to free microalgae oil. In simulated in vitro digestion experiments, lipolytic reactions of CO-NLP were observed during intestinal digestion subsequent to nanoliposome administration. Notably, the inhibitory effect of CO-NLP on cholesterol esterase activity was measured at 85.42%. Additionally, the average fluorescence intensity of nematodes in the CO-NLP group was 52.17% lower than in the control group at a CO-NLP concentration of 500 μg/mL, which suggests a pronounced lipid-lowering effect of CO-NLP. Therefore, the CO-NLP exhibited characteristics of small and uniform particle size, elevated storage stability, gradual release during intestinal digestion, and a noteworthy hypolipidemic effect. These findings designate CO-NLP as a novel lipid-lowering active product, demonstrating potential for the development of functional foods.

A one-step process for multi-gradient wettability modification on a polymer surface.

The surface modification technique is applied in microfluidic devices to modify wettability and achieve different flow velocities. Currently available methods for poly(dimethylsiloxane) (PDMS) surfaces may reliably induce wettability changes, but only one area can be altered at a time. This work introduces the controlled gradient oxygen plasma modification (CGPM) technique, which layers several resin masks with varying porosities on top of the PDMS surface. Selective wettability of the PDMS surface can be achieved by varying the oxygen plasma density above the modified material's surface by manipulation of the porosity value. Through the implementation of the COMSOL plasma module, the impact of the mask's porosity, through-hole size, distribution, and distance from the PDMS surface on wettability was studied. The suggested CGPM approach was characterized by contact angle measurements. During the 25-second CGPM procedure, the PDMS surface's contact angle continually changed from 8.77° to 76.98°. An integrated microfluidic device was created and manufactured to identify D-dimers to illustrate this method. In comparison with standard oxygen plasma treatment, the D-dimer assay was finished in 10 minutes and had a dynamic range of 1-1000 ng mL-1, with a peak fluorescence signal augmentation of 78.3% and an average fluorescence intensity enhancement of 31.1%.

Yigong Powder regulates CXCL12/CXCR4 signaling to reduce glutamate release and prevent cognitive decline in mouse model of aging

This study aims to explore the effect of preventive administration of Yigong Powder on the learning and memory abilities of the mouse model of aging induced by D-galactose and decipher the underlying mechanism, so as to provide a basis for the application of Yigong Powder in the prevention and treatment of cognitive decline. Forty KM mice were randomized into control, model, donepezil(1.5 mg·kg~(-1)), and high-dose(7.5 g·kg~(-1)) and low-dose(3.75 g·kg~(-1)) Yigong Powder groups. The mice in other groups except the control group were injected with D-galactose(200 g·kg~(-1)) at the back of the neck for the modeling of aging. At the same time, the mice were administrated with corresponding drugs by gavage for one month. Morris water maze was used to examine the learning and memory abilities of the mice. Hematoxylin-eosin staining was employed to observe the pathological and morphological changes of the hippocampus. The immunofluorescence assay was employed to detect the expression of ionized calcium-binding adapter molecule 1(IBA1), glial fibrillary acidic protein(GFAP), chemokine C-X-C-motif ligand 12(CXCL12), chemokine C-X-C-motif receptor 4(CXCR4) in the hippocampus and observe the positional relationship between IBA1, GFAP, and CXCR4. Western blot was employed to determine the protein levels of extracellular regulated kinase(ERK), p-ERK, and tumor necrosis factor receptor 1(TNFR1). Enzyme-linked immunosorbent assay was employed to measure the levels of glutamate and tumor necrosis factor(TNF-α) in the brain tissue and the level of TNF-α in the serum and spleen. Yigong Powder significantly shortened the escape latency, increased the times crossing platforms, and prolonged the cumulative time in quadrants of the aging mice. It alleviated the nerve cell disarrangement, increased intercellular space, and cell degeneration or death in the hippocampus and reduced the pathology score of the damaged nerve. Moreover, Yigong Powder reduced the positive area of IBA1 and GFAP, reduced the levels of TNF-α in the brain tissue, serum, and spleen, and decreased spleen index. Furthermore, Yigong Powder decreased the average fluorescence intensity of CXCL12 and CXCR4, reduced CXCR4-positive astrocytes and microglia, down-regulated the protein levels of p-ERK/ERK and TNFR1, and lowered the level of glutamate in the brain tissue. This study showed that the preventive administration of Yigong Powder can ameliorate the learning and memory decline of the D-galactose-induced aging mice by regulating the immune function of the spleen and the CXCL12/CXCR4 signaling in the brain to reduce glutamate release. However, the mechanism of Yigong San in preventing and treating dementia via regulating spleen and stomach function remains to be studied.

Identification of Autoantibodies Associated with Connective Tissue Disorders in Idiopathic Multicentric Castleman Disease

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare, atypical lymphoproliferative disorder with significant morbidity and mortality and unknown etiology. While anti-interleukin-6 (IL-6) therapy with siltuximab is effective in 34-50% of iMCD patients, disease pathophysiology remains largely unknown. A fundamental question is whether iMCD is most appropriately considered an infectious disease caused by an as-yet-unknown pathogen, an autoimmune disease caused by autoantibodies, an autoinflammatory disease caused by germline mutations, or a neoplastic disease caused by somatic mutations in a clonal cell population. While limited evidence exists for each of these hypotheses, the pathogen hypothesis has been the most systematically evaluated to date. In this study, we quantified the levels of autoantibodies that may be involved in iMCD pathology. Methods: We constructed two custom bead-based protein arrays to screen serum samples for IgG autoantibodies from iMCD patients (n = 101) and healthy controls (HC, n = 30). The Connective Tissue Disease (CTD) array consisted of 52 antigens associated with traditional CTDs including in Scleroderma, Myositis/Overlap Syndromes, Systemic Lupus Erythematosus (SLE)/Sjögren's, GI/Endocrine, DNA-Associated, and Inflammation/Stress. The Anti-Cytokine Autoantibody (ACA) array included 38 cytokines, chemokines, and cell surface proteins. Serum samples came from the University of Pennsylvania (n = 38), University of Arkansas for Medical Sciences (n = 45), and Osaka University (Japan, n = 18). A “positive” autoantibody result in any sample was defined as greater than five standard deviations (SD) above the average mean fluorescent intensity (MFI) of the HC and at least 3,000 MFI. Fisher's exact tests were used to determine associations between autoantibody positivity and disease status. Results: To evaluate assay precision, we investigated anti-IL-6 antibodies given that a large portion (38%) of iMCD patients were on anti-IL-6 therapy. As expected, 40 iMCD samples were positive for anti-IL-6 antibodies, the most common autoantibody detected in iMCD subjects. IL-6 was then removed from further analyses. Next, we evaluated the proportion of CTD autoantibody positive iMCD patients. Overall, 46 (46%) iMCD subjects were positive for a CTD autoantibody compared to five (17%) in HCs (OR 4.1, P = 0.005) (Figure 1A). When investigating autoantibody prevalence in CTD subcategories, Myositis/Overlap Syndromes (OR 7.5, P = 0.026) and SLE/Sjögren's (OR 6.2, P = 0.073) autoantibodies trended higher in iMCD patients, but these associations did not survive multiple testing correction. The most common Myositis/Overlap Syndrome autoantibodies were anti-Mi2 (iMCD: 7/101 vs HC: 0/30), and anti-SRP54 (7/101 vs 0/30). The most common SLE/Sjögren's autoantibodies were anti-La/SSB (9/101 vs 0/30) and anti-Ro60 (5/101 vs 0/30). For the custom ACA array, there were significantly more ACA positive iMCD subjects (n = 40, 40%) than HC (n = 3, 10%; OR 5.8, P = 0.002) (Figure 1B). The most common ACAs in iMCD were anti-OSM (9/101 vs 0/30), anti-TNF (6/101 vs 0/30), anti-ITM2B (6/101 vs 0/30), and IFNe (5/101 vs 1/30). Conclusions: IgG autoantibodies associated with CTDs, such as anti-Mi2, anti-SRP54, anti-La, anti-Ro, and anti-Histone 3; and ACAs, such as anti-OSM, anti-TNF, and anti-ITM2B were common in iMCD patients. While autoantibodies suggest autoimmune involvement, the presence of autoantibodies does not directly implicate autoimmunity as a pathological mechanism. For example, Hodgkin's disease and other post-infectious etiologies can present with elevated autoantibodies. Although not surviving multiple hypothesis correction, myositis and SLE autoantibodies trended higher in iMCD patients. While most of these autoantibodies have not been reported, anti-Ro and and anti-La antibodies have been reported previously in iMCD case reports. The presence of anti-OSM is interesting, as OSM is a member of the IL-6 family. Two hypothesized mechanisms include that autoantibodies may be non-neutralizing leading to increased half-life and cytokine activity or autoantibodies are neutralizing and lack of negative feedback from OSM may lead to increased signaling via other IL-6-family cytokines. More research is needed to understand the role of these autoantibodies and, if pathogenic, whether iMCD should be considered an autoimmune disease.

Interaction and mechanism between arginine functionalized hydroxyapatite nanoparticles and human umbilical vein endothelial cells

The interaction between terbium-doped arginine functionalized hydroxyapatite nanoparticles (Arg@HAPTb NPs) and cells, and the hindered endocytosis mechanisms of human umbilical vein endothelial cells (HUVECs) were studied by observing the cell uptake of Arg@HAPTb NPs with laser scanning confocal microscopy. Average fluorescence intensity of cells after uptaking different concentrations of NPs was determined by flow cytometer. The results indicate that internalized Arg@HAPTb NPs mainly exist in the cytoplasm of HUVECs, and most of them distribute around the cell nuclei. The entrapment of Arg@HAPTb NPs shows time-dependent and concentration- dependent manners with optimal time of 4 h and concentration of 50 mg/L. Arginine modification significantly improves the uptake efficiency of HAPTb NPs. The entry of Arg@HAPTb NPs into HUVECs is mainly through concave protein-mediated endocytosis, an energy-dependent active transport process.

Photo-physical characterization of high triplet yield brominated fluoresceins by transient state (TRAST) spectroscopy

Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy. However, their typically long lifetimes also make them highly environment sensitive, suggesting fluorophores with prominent dark-state formation yields to be used as microenvironmental sensors in bio-molecular spectroscopy and imaging. In this work, we analyzed the singlet–triplet transitions of fluorescein and three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone. Using transient state (TRAST) spectroscopy, we found a prominent internal heavy atom (IHA) enhancement of the intersystem crossing (ISC) rates upon bromination, inferred by density functional theory calculations to take place via a higher triplet state, followed by relaxation to the lowest triplet state. A corresponding external heavy atom (EHA) enhancement was found upon adding potassium iodide (KI). Notably, increased KI concentrations still resulted in lowered triplet state buildup in the brominated fluorophores, due to relatively lower enhancements in ISC, than in the triplet decay. Together with an antioxidative effect on the fluorophores, adding KI thus generated a fluorescence enhancement of the brominated fluorophores. By TRAST measurements, analyzing the average fluorescence intensity of fluorescent molecules subject to a systematically varied excitation modulation, dark state transitions within very high triplet yield (>90%) fluorophores can be directly analyzed under biologically relevant conditions. These measurements, not possible by other techniques such as fluorescence correlation spectroscopy, opens for bio-sensing applications based on high triplet yield fluorophores, and for characterization of high triplet yield photodynamic therapy agents, and how they are influenced by IHA and EHA effects.

Effect of acupotomy on mitophagy mediated by PINK1/Parkin pathway in cartilage of rabbits with knee osteoarthritis

To observe the effect of acupotomy on mitophagy mediated by PINK1/Parkin pathway in cartilage of rabbits with knee osteoarthritis (KOA), so as to explore its mechanism in inhibiting cartilage damage. Twenty-one New Zealand rabbits were randomly divided into normal, model, and acupotomy groups, with 7 rabbits in each group. The KOA rabbit model was established by using the Videman method. Rabbits in the acupotomy group received regular acupotomy treatment around the knee joint nodules or tendons once a week for 3 consecutive weeks. HE staining and transmission electron microscopy were used to observe the morphological and ultrastructural changes in knee joint cartilage of rabbits. Flow cytometry was used to measure the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) average fluorescence intensity in chondrocytes. Immunofluorescence was performed to detect the fluorescence intensity of LC3B, PINK1 and Parkin in cartilage tissue. Western blot was conducted to measure the protein expression levels of p62, LC3Ⅱ/Ⅰ, PINK1, and Parkin in cartilage tissue. Compared to the normal group, the model group showed fissures and tissue fibrosis on the surface of rabbit knee joint cartilages, loose distribution of chondrocytes, decreased autophagosomes, and abnormal mitochondrial morphology. The fluorescence intensity of LC3B, PINK1 and Parkin, the expression levels of LC3Ⅱ/Ⅰ, PINK1 and Parkin proteins in cartilage tissue were significantly decreased (P<0.01), while the percentage of chondrocytes with low Δψm, the average fluorescence intensity of ROS, and the expression of p62 protein in cartilage tissue were significantly increased (P<0.01). Compared to the model group, the acupotomy group showed no obvious defects on the surface of rabbit knee joint cartilage, relatively dense distribution of chondrocytes, increased autophagosomes, and relatively normal mitochondrial morphology. The fluorescence intensity of LC3B, PINK1 and Parkin, the expression of LC3Ⅱ/Ⅰ, PINK1 and Parkin proteins in cartilage tissue were significantly increased (P<0.01, P<0.05), while the percentage of chondrocytes with low Δψm, the average fluorescence intensity of ROS, and the expression of p62 protein in cartilage tissue were significantly decreased (P<0.01). Acupotomy may promote mitophagy by regulating the PINK1/Parkin pathway, thereby improving cartilage damage in rabbits with KOA.

Aralia saponin A isolated from Achyranthes bidentata Bl. ameliorates LPS/D-GalN induced acute liver injury via SPHK1/S1P/S1PR1 pathway in vivo and in vitro

ObjectiveAcute liver injury (ALI) refers to a disease in which the liver is affected by factors such as chemical substances, alcohol, and virus infection in a short time, resulting in damage to liver cells. Achyranthes bidentata Bl. with the hepatoprotective activity has attracted great attention. In this study, a pentacyclic triterpenoid (Aralia saponin A, AsA) was isolated from roots of Achyranthes bidentata Bl. and its anti-ALI activity, as well as the mechanisms, were investigated for the first time. MethodsAsA (10 or 20 mg/kg, i.g.) was administered over a period of 1 weeks, following which liver injury was induced by LPS (10 µg/kg)/D-GalN (700 mg/kg). H&E staining of liver, Aspartate amino transferase (AST), Alanine transaminase (ALT) and cytokines was employed to investigate ALI relevant features. The mitochondrial morphology and levels of mitochondrial membrane potential (MMP), oxidative stress balance, apoptosis, average fluorescence intensity of 2-DG, natural killer (NK) cells in liver tissues were determined to assess the oxidative stress damage and inflammatory injury. Transcriptomics and metabonomics analysis were employed to clarify the mechanisms. Additionally, the mRNA and protein expression levels of Sphingosine 1-phosphate (S1P), Sphingosine kinase-1 (SPKH1), Sphingosine 1 phosphate receptor 1 (S1PR1), Sphingosine 1 phosphate receptor 3 (S1PR3), TNF receptor associated factor 2 (TRAF-2), Phospho-NF- kappaB p65 (p-P65), NF- kappaB p65 (P65), Proto-oncogene ras (Ras), Ras-related C3 botulinum toxin substrate (Rac), Phospholipase C (PLC), Interleukin 6 (IL-6), Tumor necrosis factor α (TNF-α), Interleukin 1β (IL-1β), Vascular cell adhesion molecule 1 (Vcam1), CC chemokine ligand-2 (Ccl2) were analyzed. The mediating role of SPHK1 in the observed effects caused by AsA was assessed by investigatin SPHK1 transfection silencing/overexpression against AsA in AML12 cells induced by LPS/D-GalN. ResultsAsA can ameliorate liver function, inflammation, mitochondrial structure and oxidative stress in the ALI model. Meanwhile, AsA led to downregulated expression of proteins associated with sphingolipid signaling pathway. Silencing of SPHK1 led to enhanced protective effects of AsA, while over-expression of SPHK1 led to degraded protective effects of AsA in LPS/D-GalN-induced AML12 cells, suggesting that ALI is regulated by active molecules of AsA by means of SPHK1 mediation. ConclusionsAsA can ameliorate LPS/D-GalN-induced ALI by inhibiting inflammation and oxidative stress via the SPHK1/S1P/S1PR1 pathway. In this way, a molecular justification is provided for AsA application in ALI treatment.