Ketogenic Diet Protects from Experimental Colitis in a Mouse Model Regardless of Dietary Fat Source.

While ketogenic diets (KDs) may have potential as adjunct treatments for gastrointestinal diseases, there is little knowledge on how the fat source of these diets impacts intestinal health. The objective of this study was to investigate how the source of dietary fat of KD influences experimental colitis. We fed nine-week-old male C57BL/6J mice (n = 36) with a low-fat control diet or KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) for four weeks and then induced colitis with dextran sodium sulfate (DSS). To compare the diets, we analyzed macroscopic and histological changes in the colon, intestinal permeability to fluorescein isothiocyanate-dextran (FITC-dextran), and the colonic expression of tight junction proteins and inflammatory markers. While the effects were more pronounced with LA-KD, both KDs markedly alleviated DSS-induced histological lesions. LA-KD prevented inflammation-related weight loss and the shortening of the colon, as well as preserved Il1b and Tnf expression at a healthy level. Despite no significant between-group differences in permeability to FITC-dextran, LA-KD mitigated changes in tight junction protein expression. Thus, KDs may have preventive potential against intestinal inflammation, with the level of the effect being dependent on the dietary fat source.

Preventative Effects of Milk Fat Globule Membrane Ingredients on DSS-Induced Mucosal Injury in Intestinal Epithelial Cells.

The goblet cells of the gastrointestinal tract (GIT) produce glycoproteins called mucins that form a protective barrier from digestive contents and external stimuli. Recent evidence suggests that the milk fat globule membrane (MFGM) and its milk phospholipid component (MPL) can benefit the GIT through improving barrier function. Our objective was to compare the effects of two digested MFGM ingredients with or without dextran sodium sulfate (DSS)-induced barrier stress on mucin proteins. Co-cultured Caco-2/HT29-MTX intestinal cells were treated with in vitro digests of 2%, 5%, and 10% (w/v) MFGM or MPL alone for 6 h or followed by challenge with 2.5% DSS (6 h). Transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran (FD4) permeability measurements were used to measure changes in barrier integrity. Mucin characterization was performed using a combination of slot blotting techniques for secreted (MUC5AC, MUC2) and transmembrane (MUC3A, MUC1) mucins, scanning electron microscopy (SEM), and periodic acid Schiff (PAS)/Alcian blue staining. Digested MFGM and MPL prevented a DSS-induced reduction in secreted mucins, which corresponded to the prevention of DSS-induced increases in FD4 permeability. SEM and PAS/Alcian blue staining showed similar visual trends for secreted mucin production. A predictive bioinformatic approach was also used to identify potential KEGG pathways involved in MFGM-mediated mucosal maintenance under colitis conditions. This preliminary in silico evidence, combined with our in vitro findings, suggests the role of MFGM in inducing repair and maintenance of the mucosal barrier.

Choosing T-cell sources determines CAR-T cell activity in neuroblastoma.

Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.

Confocal laser endomicroscopy as predictive biomarker of clinical and endoscopic efficacy of vedolizumab in ulcerative colitis: The DETECT study.

AIMS: In patients with ulcerative colitis (UC), no biomarker is available to help the physician to choose the most suitable biotherapy. The primary objective of this pilot study was to assess the feasibility of identification of α4ß7- and TNF-expressing cells, to predict the response to vedolizumab using confocal laser endoscopy (CLE). METHODS: Patients with moderate-to-severe UC, naïve of biotherapy, received vedolizumab. Clinical evaluation was performed at each infusion. Endoscopic evaluation was performed before inclusion and at week 22. Fresh colonic biopsies were stained using FITC-labelled vedolizumab and Alexa fluor-labelled adalimumab and ex vivo dual-band CLE images were acquired. Blood samples were collected to measure trough concentrations of vedolizumab and to determine absolute counts of T and B cells subpopulations, NK cells and monocytes. RESULTS: Nineteen patients were enrolled in the study and received at least one dose of vedolizumab. Clinical remission and endoscopic improvement were observed in 58% of whom 5 patients (45%) had an endoscopic subscore of 0. In terms of clinical response and remission, endoscopic improvement and histologic response, FITC-conjugated vedolizumab staining tended to be higher in responder patients compared to non-responders at week 22. A threshold value of 6 positive FITC-vedolizumab staining areas detected by CLE seemed informative to discriminate the responders and non-responders. The results were similar in terms of clinical remission and endoscopic improvement with a sensitivity of 78% and a specificity of 85% (p = 0.05). Trough concentrations and blood immune cells were not associated with responses to vedolizumab. CONCLUSION: This pilot study demonstrate that dual-band CLE is feasible to detect α4ß7- and TNF-expressing cells. Positive α4ß7 staining seems to be associated with clinical and endoscopic remission in UC patients treated by anti-α4ß7-integrin, subject to validation by larger-scale studies. Clinical-trial.gov: NCT02878083.

High biocompatible FITC-conjugated silica nanoparticles for cell labeling in both in vitro and in vivo models.

Fluorescence nanosilica-based cell tracker has been explored and applied in cell biological research. However, the aggregation of these nanoparticles at physiological pH is still the main limitation. In this research, we introduced a novel fluorescence nano-based cell tracker suitable for application in live cells. The silica-coated fluorescein isothiocyanate isomer (FITC-SiO2) nanoparticles (NPs) were modified with carboxymethylsilanetriol disodium salt (FITC-SiO2-COOH), integrating the dianion form of FITC molecules. This nanosystem exhibited superior dispersion in aqueous solutions and effectively mitigated dye leakage. These labeled NPs displayed notable biocompatibility and minimal cytotoxicity in both in vitro and in vivo conditions. Significantly, the NPs did not have negative implications on cell migration or angiogenesis. They successfully penetrated primary fibroblasts, human umbilical vein endothelial cells and HeLa cells in both 2D and 3D cultures, with the fluorescence signal enduring for over 72 h. Furthermore, the NP signals were consistently observed in the developing gastrointestinal tract of live medaka fish larvae for extended periods during phases of subdued digestive activity, without manifesting any apparent acute toxicity. These results underscore the promising utility of FITC-SiO2-COOH NPs as advanced live cell trackers in biological research.

Endari treatment ameliorates sickle cell-related disruption in intestinal barrier functions and is associated with prolonged survival in sickle cell mice.

BACKGROUND: Endari (L-glutamine) is a conditional amino acid that reduces the frequency of vaso-occlusive crisis (VOC) in sickle cell disease (SCD). AIM: To investigate whether Endari could ameliorate intestinal barrier function and improve survival outcomes in SCD. METHODS: We treated female Townes SCD mice with Endari and evaluated their intestinal barrier functions by measuring the recovery of orally administered fluorescein isothiocyanate (FITC)-conjugated dextran 4 kDa in serum, and serum intestinal fatty acid binding proteins (iFABP) and lipopolysaccharide (LPS) concentrations by ELISA. We also explored the impact the Endari has on the survival of the SCD mice that underwent repeated experimentally-induced VOC. RESULTS: Compared to SCD mice treated with water only, Endari-treated mice showed improved intestinal barrier functions, with decrease in the barrier permeability and reduction in the translocation of lipopolysaccharides from the intestinal lumen into the circulation. These changes occurred after only 4 weeks of Endari treatment. Improved intestinal barrier function was also associated with prolonged survival in Endari-treated SCD mice after repeated experimentally-induced VOC. CONCLUSION: Our findings provide the evidence supporting the beneficial effects of Enadri in improving intestinal barrier function and associated survival outcomes in SCD.

Integration of lanthanide MOFs/methylcellulose-based fluorescent sensor arrays and deep learning for fish freshness monitoring.

Preserving fish meat poses a significant challenge due to its high protein and low fat content. This study introduces a novel approach that utilizes a common type of lanthanide metal-organic frameworks (Ln-MOFs), EuMOFs, in combination with 5-fluorescein isothiocyanate (FITC) and methylcellulose (MC) to develop fluorescent sensor arrays for real-time monitoring the freshness of fish meat. The EuMOF-FITC/MC fluorescence films were characterized with excellent fluorescence response, ideal morphology, good mechanical properties, and improved hydrophobicity. The efficacy of the fluorescence sensor array was evaluated by testing various concentrations of spoilage gases (such as ammonia, dimethylamine, and trimethylamine) within a 20-min timeframe using a smartphone-based camera obscura device. This sensor array enables the real-time monitoring of fish freshness, with the ability to preliminarily identify the freshness status of mackerel meat with the naked eye. Furthermore, the study employed four convolutional neural network (CNN) models to enhance the performance of freshness assessment, all of which achieved accuracy levels exceeding 93 %. Notably, the ResNext-101 model demonstrated a particularly high accuracy of 98.97 %. These results highlight the potential of the EuMOF-based fluorescence sensor array, in conjunction with the CNN model, as a reliable and accurate method for real-time monitoring the freshness of fish meat.

Ameliorative Effects of Larazotide Acetate on Intestinal Permeability and Bacterial Translocation in Acute Pancreatitis Model in Rats.

BACKGROUND: Intestinal barrier dysfunction in acute pancreatitis (AP) may progress to systemic inflammatory response syndrome (SIRS) and multi-organ failures by causing bacterial translocation. Larazotide acetate (LA) is a molecule that acts as a tight junction (TJ) regulator by blocking zonulin (Zo) receptors in the intestine. AIMS: In our study, we aimed to investigate the effects of LA on intestinal barrier dysfunction and bacterial translocation in the AP model in rats. METHODS: Thirty-two male Sprague-Dawley rats were divided into 4 groups; control, larazotide (LAR), AP, and AP + LAR. The AP model was created by administering 250 mg/100 g bm L-Arginine intraperitoneally 2 times with an hour interval. AP + LAR group received prophylactic 0.01 mg/mL LA orally for 7 days before the first dose of L-Arginine. For intestinal permeability analysis, fluorescein isothiocyanate-dextran (FITC-Dextran) was applied to rats by gavage. The positivity of any of the liver, small intestine mesentery, and spleen cultures were defined as bacterial translocation. Histopathologically damage and zonulin immunoreactivity in the intestine were investigated. RESULTS: Compared to the control group, the intestinal damage scores, anti-Zo-1 immunoreactivity H-Score, serum FITC-Dextran levels and bacterial translocation frequency (100% versus 0%) in the AP group were significantly higher (all p < 0.01). Intestinal damage scores, anti-Zo-1 immunoreactivity H-score, serum FITC-Dextran levels, and bacterial translocation frequency (50% versus 100%) were significantly lower in the AP + LAR group compared to the AP group (all p < 0.01). CONCLUSIONS: Our findings show that LA reduces the increased intestinal permeability and intestinal damage by its effect on Zo in the AP model in rats, and decreases the frequency of bacterial translocation as a result of these positive effects.

Hyaluronan-based nano-formulation with mesoporous silica enhances the anticancer efficacy of phloroglucinol against gastrointestinal cancers.

Gastrointestinal cancers are one among the most frequently reported cancers where colorectal and gastric cancers ranks third leading cause of cancer related death worldwide. Phloroglucinol, a well-known therapeutic agent for cancer, where its usage has been limited due to its poor water solubility and bioavailability. Hence, our study aims to synthesize and characterize Hyaluronan grafted phloroglucinol loaded Mesoporous silica nanoparticles (MSN-PG-HA). Our nano-formulation hasn't shown any teratogenic effect on Zebrafish embryos, no hemolysis and toxic effect with normal fibroblast cells with a maximum concentration of 300 µg/mL. The cumulative drug release profile of MSN-PG-HA showed a maximum drug release of 96.9 % with 5 mM GSH under redox responsive drug release, which is crucial for targeting cancer cells. In addition, the MSN-PG-HA nanoparticles showed significant a cytotoxic effect against HCT-116, AGS and SW-620 with IC50 values of 86.5 µg/mL, 80.65 µg/mL and 109.255 µg/mL respectively. Also, the cellular uptake assay has shown an increased uptake of FITC-labeled-MSN-PG-HA by HA-receptor mediated endocytosis than FITC-labeled-MSN-PG without HA modification in CD44+ gastrointestinal cancer cell lines. The ability of MSN-PG-HA to target CD44+ cells was further exploited for its application in cancer stem cell research utilizing in silico analysis with various stem cell pathway related targets, in which PG showed higher binding affinity with Gli 1 and the simulation studies proving its effectiveness in disrupting the protein structure. Thus, the findings of our study with nano-formulation are safe and non-toxic to recommend for targeted drug delivery against gastrointestinal cancers as well as its affinity towards cancer stem cell pathway related proteins proving to be a significant formulation for cancer stem cell research.

Gelatin but not type I collagen promotes bacteria phagocytosis in PMA-treated U937 human lymphoma cells.

PURPOSE: Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. MATERIALS AND METHODS: Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. RESULTS: Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. CONCLUSION: This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Diffusion Mediates Molecular Transport through the Perivascular Space in the Brain.

The perivascular space has been proposed as a clearance pathway for degradation products in the brain, including amyloid ß, the accumulation of which may induce Alzheimer's disease. Live images were acquired using a two-photon microscope through a closed cranial window in mice. In topical application experiments, the dynamics of FITC-dextran were evaluated from 30 to 150 min after the application and closure of the window. In continuous injection experiments, image acquisition began before the continuous injection of FITC-dextran. The transport of dextran molecules of different sizes was evaluated. In topical application experiments, circumferential accumulation around the penetrating arteries, veins, and capillaries was observed, even at the beginning of the observation period. No further increases were detected. In continuous injection experiments, a time-dependent increase in the fluorescence intensity was observed around the penetrating arteries and veins. Lower-molecular-weight dextran was transported more rapidly than higher-molecular-weight dextran, especially around the arteries. The largest dextran molecules were not transported significantly during the observation period. The size-dependent transport of dextran observed in the present study strongly suggests that diffusion is the main mechanism mediating substance transport in the perivascular space.

Specificity and sensitivity of an indirect fluorescent antibody test to detect antibodies against bovine viral diarrhea virus.

Since being reported in 1979 and 2006, indirect fluorescent antibody (IFA) tests have not been reported to detect bovine viral diarrhea virus (BVDV) antibodies to our knowledge. Thus, we re-evaluated the efficacy and usefulness of IFA tests for BVDV serology. We tested 4 combinations of 2 antibody conjugates (fluorescein isothiocyanate [FITC]-conjugated rabbit IgG anti-bovine IgG; rabbit IgG F(ab')2 fragment anti-bovine IgG [F(ab')2 FITC-IgG]) and 2 washing solutions (PBS; carbonate-bicarbonate-buffered saline [CBBS]) to evaluate the specificity of an IFA test for BVDV. We compared the sensitivity of the optimal combination with virus neutralization (VN) tests and an ELISA, and compared IFA with VN titers against different genotype (subgenotype) strains. For the F(ab')2 FITC-IgG/CBBS combination, only 1 of the 156 (0.6%) 4-fold diluted cattle sera resulted in a nonspecific reaction; other combinations led to a much higher incidence (22.9-37.2%). For the F(ab')2 FITC-IgG/CBBS combination, IFA detection rates were identical (36 of 59) for BVDV1 and BVDV2 genotypes, and IFA titers against them were strongly correlated (r = 0.99). The antibody-detection rates of the IFA tests were almost identical to those of VN tests and the ELISA (κ: 0.96 and 0.89, respectively). The IFA titers against 4 strains (BVDV1a, BVDV1j, BVDV2a, and an unidentified strain) were similar, 1,024 to ≥4,096, although the VN titers were different. Thus, our IFA tests were specific and sensitive, and more useful than VN tests given that the IFA tests could evaluate the immune status of cattle using a representative strain, regardless of genotype (subgenotype).

Effects of phytase supplementation on broilers fed with calcium and phosphorus-reduced diets, challenged with Eimeria maxima and Eimeria acervulina: influence on growth performance, body composition, bone health, and intestinal integrity.

An experiment was conducted to evaluate the effects of phytase in calcium (Ca) and available phosphorous (avP)-reduced diet on growth performance, body composition, bone health, and intestinal integrity of broilers challenged with Eimeria maxima and Eimeria acervulina. A total of 672 14-day-old male broilers were allocated to a 2 × 4 factorial arrangement with 6 replicates per treatment and 14 birds per replicate. Two factors were Eimeria challenge and 4 dietary treatments: 1) a positive control (PC; 0.84% Ca and 0.42% avP); 2) a negative control (NC; 0.74% Ca and 0.27% avP); 3) NC + 500 FTU/Kg of phytase (NC + 500PHY); and 4) NC + 1,500 FTU/Kg of phytase (NC + 1500PHY). On d 14, birds in the Eimeria-challenged groups received a solution containing 15,000 sporulated oocysts of E. maxima and 75,000 sporulated oocysts of E. acervulina via oral gavage. At 5 d postinoculation (DPI), the challenged birds showed a higher (P < 0.01) FITC-d level than the unchallenged birds. While the permeability of the NC group did not differ from the PC group, the phytase supplementation groups (NC + 500PHY and NC + 1500PHY) showed lower (P < 0.05) serum FITC-d levels compared to the NC group. Interaction effects (P < 0.05) of Eimeria challenge and dietary treatments on feed intake (FI), mucin-2 (MUC2) gene expression, bone ash concentration, and mineral apposition rate (MAR) were observed. On 0 to 6 and 0 to 9 DPI, Eimeria challenge decreased (P < 0.01) body weight (BW), body weight gain (BWG), FI, bone mineral density (BMD), bone mineral content (BMC), bone area, fat free bone weight (FFBW), bone ash weight, bone ash percentage and bone ash concentration; and it showed a higher FCR (P < 0.01) compared to the unchallenged group. The reduction Ca and avP in the diet (NC) did not exert adverse effects on all parameters in birds, and supplementing phytase at levels of 500 or 1,500 FTU/Kg improved body composition, bone mineralization, and intestinal permeability, with the higher dose of 1,500 FTU/Kg showing more pronounced enhancements. There was an observed increase in FI (P < 0.01) when phytase was supplemented at 1,500 FTU/Kg during 0 to 6 DPI. In conclusion, results from the current study suggest that dietary nutrients, such as Ca and avP, can be moderately reduced with the supplementation of phytase, particularly in birds infected with Eimeria spp., which has the potential to save feed cost without compromising growth performance, bone health, and intestinal integrity of broilers.

Combination of self-assembling system and N,O-carboxymethyl chitosan improves ocular residence of anti-glaucoma drug.

Glaucoma is known to be one of the principal causes of vision loss due to elevated intraocular pressure. Currently, latanoprost eye drops is used as first-line treatment for glaucoma; however, it possesses low bioavailability due to rapid precorneal clearance. A novel delivery system with a mucoadhesive property could overcome this problem. Therefore, we attempt to develop a combination of self-assembling latanoprost nanomicelles (Latcel) and a mucoadhesive polymer (N,O-carboxymethyl chitosan: N,O-CMC) to improve the corneal residence time. Latcel was developed using Poloxamer-407 by thin film hydration method, followed by the addition of N,O-CMC using simple solvation to obtain Latcel-CMC and characterized using various physicochemical characterization techniques. The particle size of Latcel-CMC was 94.07 ± 2.48 nm and a zeta potential of -16.03 ± 0.66 mV, with a sustained release for 24h whereas marketed latanoprost drops released 90 % of the drug within 1h. In vitro cytotoxicity studies, HET-CAM, and in vivo Draize test showed the biocompatibility of Latcel-CMC. Cellular uptake studies performed using fluorescein isothiocyanate (FITC) loaded nanomicelles in human corneal epithelial cells indicates the increased cellular uptake as compare to plain FITC solution. In vivo ocular residence time was evaluated in Wistar rats using Indocyanine green (ICG) loaded nanomicelles by an in vivo imaging system (IVIS), indicating Latcel-CMC (8h) has better residence time than plain ICG solution (2h). The Latcel-CMC showed improved corneal residence time and sustained release of latanoprost due to increased mucoadhesion. Thus, the developed N,O-Carboxymethyl chitosan based nanomicelles eye drop could be a better strategy than conventional eye drops for topical delivery of latanoprost to treat glaucoma.

Glucose-lightened upconversion nanoprobes for accurate cellular-discrimination based on Warburg effect.

Accurate cellular-recognition based disease therapy is of significance for precision medicine. However, except of specific antibody-coupling strategy, very few probes have been reported to efficiently discriminate normal cells and lesion cells through cellular microenvironment. Herein, we proposed a glucose selectively-lightened upconversion nanoprobe to recognize cancer cells from a pile of normal cells based on Warburg effect, that indicated a heightened demand for glucose intake for cancer cells. The nanoprobes were constructed by mesoporous silica-coated upconversion nanoparticles (UCNP@mSiO2) with the crucial incorporation of a glucose-responsive modality, benzoboric acid (BA)-modified fluorescein molecules (FITC-BA). In cancer cells, the presence of elevated glucose concentrations triggered the transformation of FITC-BA to FITC-Glucose to recover nanoprobes' luminescence, however, the nanoprobes exhibited a shielded luminescent effect in healthy cells. To validate the hypothesis of accurate cellular-discrimination, a photodynamic therapy modality, riboflavin, with a specific ratio were also loaded into above UCNP@mSiO2 nanoprobes for effective production of reactive oxygen species to kill cells. It was found that 97.8% of cancer cells were cleaned up, but normal cells retained a nearly 100% viability after 10 min laser illumination. By leveraging the metabolic disparity from Warburg effect, the nanoprobes offer a highly accurate cellular discrimination, and significantly mitigate "off-target" damage commonly associated with conventional therapies.

Liver and spleen predominantly mediate calciprotein particle clearance in a rat model of chronic kidney disease.

Calciprotein particles (CPPs) provide an efficient mineral buffering system to prevent the complexation of phosphate and calcium in the circulation. However, in chronic kidney disease (CKD), the phosphate load exceeds the mineral buffering capacity, resulting in the formation of crystalline CPP2 particles. CPP2 have been associated with cardiovascular events and mortality. Moreover, CPP2 have been demonstrated to induce calcification in vitro. In this study, we examined the fate of CPP2 in a rat model of CKD. Calcification was induced in Sprague-Dawley rats by 5/6 nephrectomy (5/6-Nx) combined with a high-phosphate diet. Control rats received sham surgery and high-phosphate diet. Twelve weeks after surgery, kidney failure was significantly induced in 5/6-Nx rats as determined by enhanced creatinine and urea plasma levels and abnormal kidney histological architecture. Subsequently, radioactive and fluorescent (FITC)-labeled CPP2 ([89Zr]Zr-CPP2-FITC) were injected intravenously to determine clearance in vivo. Using positron emission tomography scans and radioactive biodistribution measurements, it was demonstrated that [89Zr]Zr-CPP2-FITC are mainly present in the liver and spleen in both 5/6-Nx and sham rats. Immunohistochemistry showed that [89Zr]Zr-CPP2-FITC are predominantly taken up by Kupffer cells and macrophages. However, [89Zr]Zr-CPP2-FITC could also be detected in hepatocytes. In the different parts of the aorta and in the blood, low values of [89Zr]Zr-CPP2-FITC were detectable, independent of the presence of calcification. CPP2 are cleared rapidly from the circulation by the liver and spleen in a rat model of CKD. In the liver, Kupffer cells, macrophages, and hepatocytes contribute to CPP2 clearance.NEW & NOTEWORTHY Calciprotein particles (CPPs) buffer calcium and phosphate in the blood to prevent formation of crystals. In CKD, increased phosphate levels may exceed the buffering capacity of CPPs, resulting in crystalline CPPs that induce calcification. This study demonstrates that labeled CPPs are predominantly cleared from the circulation in the liver by Kupffer cells, macrophages, and hepatocytes. Our results suggest that targeting liver CPP clearance may reduce the burden of crystalline CPP in the development of vascular calcification.

Intracellular presence of Helicobacter pylori antigen and genes within gastric and vaginal Candida.

BACKGROUND: Helicobacter pylori infections are generally acquired during childhood and affect half of the global population, but its transmission route remains unclear. It is reported that H. pylori can be internalized into Candida, but more evidence is needed for the internalization of H. pylori in human gastrointestinal Candida and vaginal Candida. METHODS: Candida was isolated from vaginal discharge and gastric mucosa biopsies. We PCR-amplified and sequenced H. pylori-specific genes from Candida genomic DNA. Using optical and immunofluorescence microscopy, we identified and observed bacteria-like bodies (BLBs) in Candida isolates and subcultures. Intracellular H. pylori antigen were detected by immunofluorescence using Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies. Urease activity in H. pylori internalized by Candida was detected by inoculating with urea-based Sabouraud dextrose agar, which changed the agar color from yellow to pink, indicating urease activity. RESULTS: A total of 59 vaginal Candida and two gastric Candida strains were isolated from vaginal discharge and gastric mucosa. Twenty-three isolates were positive for H. pylori 16S rDNA, 12 were positive for cagA and 21 were positive for ureA. The BLBs could be observed in Candida cells, which were positive for H. pylori 16S rDNA, and were viable determined by the LIVE/DEAD BacLight Bacterial Viability kit. Fluorescein isothiocyanate (FITC)-conjugated antibodies could be reacted specifically with H. pylori antigen inside Candida cells by immunofluorescence. Finally, H. pylori-positive Candida remained positive for H. pylori 16S rDNA even after ten subcultures. Urease activity of H. pylori internalized by Candida was positive. CONCLUSION: In the form of BLBs, H. pylori can internalize into gastric Candida and even vaginal Candida, which might have great significance in its transmission and pathogenicity.

Unraveling Interaction of Rhenium-188 Microspheres with Primary Hepatic Cancer Cell: A Breakthrough Study.

Introduction: Hepatocellular carcinoma is a prevalent contributor to global mortality rates. The main palliative treatments are trans-arterial chemoembolization and selective intra-arterial radionuclide therapy. Methods: A novel freeze-dried nonradioactive microsphere kit formulation has been developed, and the behavior and therapeutic potential of 188Re microspheres have been assessed. The microspheres were labeled with fluorescein isothiocyanate (FITC) and 188ReO4-. The uptake of FITC microspheres by HepG2 cells was examined at various time intervals. The impact of 188Re microspheres on cell viability and the mode of cell death were investigated with HepG2 cells using MTT and Annexin FITC-V/propidium iodide (PI) apoptosis assay. Results: The labeling efficiency of microspheres was more than 99% with FITC and 188ReO4-. The maximum uptake of FITC microspheres by HepG2 cells was achieved at 6 h. The exposure to 188Re microspheres has shown a decrease in cellular viability from 77.81% ± 0.015% to 42.03% ± 0.148% at 192 h of incubation (∼11 half-lives). The cellular uptake of 188Re microspheres was 0.255-0.901 MBq. These values were concordant with Annexin FITC-V/PI apoptosis assay. At 192 h, 53.28% ± 0.01% of cells entered the apoptotic phase after treatment with 188Re microspheres, and only 39.34% ± 0.02% of cells remained viable. However, in the cells treated with 188ReO4- alone, 74.86% ± 0.005% of cells were viable, and only 24.75% ± 0.577% of cells were in the early apoptotic phase at 192 h. Conclusion: The data revealed that 188Re microspheres treatment led to significant growth inhibition in HepG2 cells compared with 188ReO4-.

Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

BACKGROUND: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. METHODS AND RESULTS: Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. CONCLUSIONS: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

Enhanced detection of acrylamide using a versatile solid-state upconversion sensor through spectral and visual analysis.

Acrylamide (AM) generally forms in high-temperature processes and has been classified as a potential carcinogen. In this study, we put forward a maneuverable solid-state luminescence sensor using polydimethylsiloxane (PDMS) as the matrix coupled with upconversion nanoparticles as the indicator. The core-shell upconversion nanoparticles emitting cyan light were uniformly encapsulated in PDMS. Then it was further modified with complementary DNA of AM aptamer. The nanocrystalline fluorescein isothiocyanate isomer (FITC), coupled with AM aptamer, was attached to the surface of PDMS. FITC effectively quenched the upconversion luminescence through fluorescence resonance energy transfer (FRET). The introduction of AM resulted in preferentially bound to aptamer caused the separation of the quencher and the donor, and led to luminescence recovery. The developed sensor was applied for both spectral and visual monitoring, demonstrating a detection limit (LOD) of 1.00 nM and 1.07 nM, respectively. Importantly, in the actual foodstuffs detection, there is no obvious difference between the results of this study and the standard method, which indicates the developed method has good accuracy. Therefore, this solid-state sensor has the potential for on-site detection using a smartphone device and an Android application.