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.

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.

Lizhong decoction ameliorates pulmonary infection secondary to severe traumatic brain injury in rats by regulating the intestinal physical barrier and immune response.

ETHNOPHARMACOLOGICAL RELEVANCE: The pathogenesis of pulmonary infection secondary to severe traumatic brain injury (sTBI) is closely related to damage to the intestinal barrier. Lizhong decoction (LZD) is a prominent traditional Chinese medicine (TCM) that is widely used in clinical treatment to regulate gastrointestinal movement and enhance resistance. Nevertheless, the role and mechanism of LZD in lung infection secondary to sTBI have yet to be elucidated. AIM OF THE STUDY: Here, we evaluate the therapeutic effect of LZD on pulmonary infection secondary to sTBI in rats and discuss potential regulatory mechanisms. MATERIALS AND METHODS: The chemical constituents of LZD were analyzed by ultra-high performance liquid chromatography-Q Exactive-tandem mass spectrometry(UPLC-QE-MS/MS). The efficacy of LZD on rats with lung infection secondary to sTBI was examined by changes in brain morphology, coma time, brain water content, mNSS score, colony counts, 16S rRNA/RNaseP/MRP30 kDa(16S/RPP30), myeloperoxidase (MPO) content and pathology of lung tissue. The concentration of fluorescein isothiocyanate(FITC)-dextran in serum and the contents of secretory immunoglobulin A (SIgA) in colon tissue were detected by enzyme-linked immunosorbent assay (ELISA). Subsequently, Alcian Blue Periodic acid Schiff (AB-PAS) was used to detect colonic goblet cells. Immunofluorescence (IF) was used to detect the expression of tight junction proteins. The proportions of CD3+ cell, CD4+CD8+ T cells, CD45+ cell and CD103+ cells in the colon were analyzed by flow cytometry (FC). In addition, colon transcriptomics were analyzed by Illumina mRNA-Seq sequencing. Real-time quantitative polymerase chain reaction (qRT‒PCR) was used to verify the genes associated with LZD alleviation of intestinal barrier function. RESULTS: Twenty-nine chemical constituents of LZD were revealed with UPLC-QE-MS/MS analysis. Administration of LZD significantly reduced colony counts, 16S/RPP30 and MPO content in lung infection secondary to sTBI rats. In addition, LZD also reduced the serum FITC-glucan content and the SIgA content of the colon. Additionally, LZD significantly increased the number of colonic goblet cells and the expression of tight junction proteins. Furthermore, LZD significantly decreased the proportion of CD3+ cell, CD4+CD8+ T cells,CD45+ and CD103+ cells in colon tissue. Transcriptomic analysis identified 22 upregulated genes and 56 downregulated genes in sTBI compared to the sham group. The levels of seven genes were recovered after LZD treatment. qRT‒PCR successfully validated two genes (Jchain and IL-6) at the mRNA level. CONCLUSION: LZD can improves sTBI secondary lung infection by regulating the intestinal physical barrier and immune response. Thees results suggested that LZD may be a prospective treatment for pulmonary infection secondary to sTBI.

Comparison of adjuvant mechanisms of medium-chain triacylglycerol in a mouse FITC-induced contact hypersensitivity model.

The number of allergy sufferers has been increasing with the increase in chemicals to which we are potentially exposed. We have discovered that tributyrin, a short-chain triacylglycerol (TAG), enhanced fluorescein isothiocyanate (FITC)-induced contact hypersensitivity in a mouse model. Medium-chain triacylglycerols (MCTs) are used in cosmetics, with which we come into direct contact frequently, to maintain skin conditions and as a thickening agent for cosmetics. In this study, we examined whether MCTs with different side chain lengths enhanced skin sensitization to FITC in the mouse model. During skin sensitization to FITC, the presence of tributyrin (side chain carbon number, 4; C4) as well as that of each MCT, tricaproin (C6), tricaprylin (C8), or tricaprin (C10), resulted in enhanced skin sensitization, whereas that of trilaurin (C12) did not. As to the mechanism underlying the enhanced sensitization, three MCTs (C6, C8 and C10) facilitated migration of FTIC-presenting CD11c+ dendritic cells to draining lymph nodes. These results indicated that not only tributyrin but also MCTs, up to side chain carbon number 10, have an adjuvant effect on FITC-induced skin hypersensitivity in mice.

Dietary soy galactooligosaccharides affect the performance, intestinal function, and gut microbiota composition of growing chicks.

The objective of this experiment was to investigate the effects of the dietary soy galactooligosaccharides (GOS), raffinose and stachyose, on performance, gastrointestinal health, and systemic stress in young broilers. Birds were fed a GOS-devoid diet based on soy protein isolate (SPI) or the SPI diet with 0.9, 1.8, 2.7, or 3.6% added stachyose and raffinose in a ratio of 4:1 at the expense of corn starch. These 5 treatments were administered to 10 replicate cages of 8 birds. Performance was measured weekly and excreta moisture, N retention, apparent metabolizeable energy, and complete blood cell counts were determined at 14 and 21 d. At 21 d, 2 birds per cage were orally gavaged with fluorescein isothiocyanate-dextran (FITC-d) and serum samples were analyzed for FITC-d as a marker of gut leakage. Additionally, intestinal morphology, crop presumptive lactic acid bacteria (LAB) counts, crop and cecal pH, and cecal microbiota via16S rRNA microbial sequencing were evaluated at 21 d. From 0 to 21 d, feed intake increased linearly (P < 0.01) as dietary GOS increased, whereas BWG increased (P < 0.05) quadratically. Feed conversion ratio increased (P < 0.01) linearly as GOS increased. There were linear increases (P < 0.05) in excreta moisture as dietary GOS increased at 14 and 21 d, as well as dose-dependent responses (P < 0.05) in N retention, AME, and AMEn. There was a quadratic increase (P < 0.05) in crop LAB recovery and a linear decrease (P < 0.01) in ceca pH as GOS increased. At 14 d, a linear increase (P < 0.05) in blood heterophil to lymphocyte ratio was observed as dietary GOS increased. Serum concentrations of FITC-d increased quadratically (P < 0.01) to dietary GOS. Increasing levels of GOS influenced alpha and beta diversities and composition of gut microbiota, including the abundance of Ruminococcus and Bifidobacterium. Results from this trial indicate that soy-derived GOS exert dose-dependent effects on nutrient utilization and intestinal health in young broilers.

Self-emulsifying System Co-loaded with Paclitaxel and Coix Seed Oil Deeply Penetrated to Enhance Efficacy in Cervical Cancer.

BACKGROUND: Paclitaxel (PTX), voted as the promising natural medicine molecule, is widely used in the treatment of cancers. Nevertheless, its clinical application is strictly limited by its poor water solubility. OBJECTIVE: CP-MEs (Paclitaxel-coix seed oil coloaded microemulsion), a small-sized self-emulsifying nanoemulsion formed from a combination of PTX and coix seed oil (CSO), was developed in order to improve the solubility of paclitaxel and enhance anti-cervical cancer efficacy in vitro. CSO was selected as the oil phase to replace conventional organic solvents and achieve a synergistic anti-tumor effect with paclitaxel. METHODS: Pseudoternary phase diagram was applied to the study of CP-MEs formulation. CP-MEs were prepared and characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The encapsulation efficiency and drug loading efficiency (EE and LE) were detected by HPLC. MTT was adopted to evaluate the cytotoxicity of CP-MEs against HeLa cells. The cellular uptake and apoptotic ratio of CP-MEs were evaluated by flow cytometry. Notably, HeLa 3D tumor spheroid was adopted to evaluate tumor permeability of different size microemulsions as the model. RESULTS: The best self-emulsifying ability was exhibited by HS 15: PEG 400 combination. The appearance of CP-MEs was clear and transparent, which exhibited a small size (30.28 ± 0.36) and a slight negative surface charge (-4.40 ± 1.13) mV. The EE and LE of CP-MEs were 98.80% and 0.978%, respectively. The cumulative release rate within 48 h of the CP-MEs was 80.21%. In cellular studies, the uptake of fluorescein isothiocyanate (FITC) labeled CP-MEs (FITC/C-MEs) was 17.86-fold higher than the free FITC group, leading to significant synergistic anticancer activity in terms of cytotoxicity and apoptosis induction in vitro. The apoptotic rate of CP-MEs treated was 1.70-fold higher than PTXtreated. Notably, the penetration of CP-MEs in the HeLa 3D tumor sphere model was enhanced, which was related to deeply penetrated microemulsion of small size mediated at the tumor site. CONCLUSION: With the advantage of the small-sized self-emulsifying system, CP-MEs hold great potential to become an efficient nano drug delivery system for cervical cancer treatment in the clinic.

Development of Core@Shell γ-Fe2O3@MnxOy@SiO2 Nanoparticles for Hyperthermia, Targeting, and Imaging Applications.

Monodispersed core@shell γ-Fe2O3@MnxOy nanoparticles have been prepared through thermolysis of iron and manganese oleate. Further, these prepared nanoparticles are coated with biocompatible substances such as silica and polyethylene glycol. These particles are highly biocompatible for different cell lines such as normal and cancer cell lines. The nanoparticles are used as hyperthermia agents, and successful hyperthermia treatment in cancer cells is carried out. As compared to γ-Fe2O3@SiO2, γ-Fe2O3@MnxOy@SiO2 shows the enhanced killing of cancer cells through hyperthermia. In order to make them potential candidates for targeting to cancer cells, folic acid (FA) is tagged to the nanoparticles. Fluorescein isothiocyanate (FITC) is also tagged onto these nanoparticles for imaging. The developed γ-Fe2O3@MnxOy@SiO2 nanoparticle can act as a single entity for therapy through AC magnetic field, imaging through FITC and targeting through folic acid simultaneously. This is the first report on this material, which is highly biocompatible for hyperthermia, imaging, and targeting.

In Vitro Cell Death Mechanisms Induced by Dicoma anomala Root Extract in Combination with ZnPcS4 Mediated-Photodynamic Therapy in A549 Lung Cancer Cells.

Globally, lung cancer has remained the leading cause of morbidity and mortality in men and women. To enhance photodynamic therapeutic effects in vitro, the present study was designed to reduce dose-dependence in photodynamic therapy (PDT) and evaluate the anticancer effects of Dicoma anomala (D. anomala) root extracts (i.e., chloroform (Chl), ethyl acetate (EtOAc), and methanol (MeOH)) on A549 lung cancer cells. The most active extract of D. anomala (D.A) was used to establish the 50% inhibitory concentration (IC50), which was further used to evaluate the anticancer efficacy of D.A in combination with ZnPcS4-mediated PDT IC50. The study further evaluated cell death mechanisms by cell viability/ cytotoxicity (LIVE/DEADTM assay), flow cytometry (Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) staining), immunofluorescence (p38, p53, Bax, and caspase 3 expressions), and fluorometric multiplex assay (caspase 8 and 9) 24 h post-treatment with IC50 concentrations of ZnPcS4-mediated PDT and D.A MeOH root extract. Morphological changes were accompanied by a dose-dependent increase in cytotoxicity, decrease in viability, and proliferation in all experimental models. Apoptosis is the highly favored cell death mechanism observed in combination therapy groups. Apoptotic activities were supported by an increase in the number of dead cells in the LIVE/DEADTM assay, and the upregulation of p38, p53, Bax, caspase 3, 8, and 9 apoptotic proteins. In vitro experiments confirmed the cytotoxic and antiproliferative effects of D.A root extracts in monotherapy and in combination with ZnPcS4-mediated PDT. Taken together, our findings demonstrated that D.A could be a promising therapeutic candidate worth exploring in different types of cancer.

Research Note: Virulence gene downregulation and reduced intestinal colonization of Salmonella enterica serovar Typhimurium PHL2020 isolate in broilers by a natural antimicrobial (NeutraPath™).

The reduction in antibiotic growth promoter use in poultry, due to antibiotic resistance concerns, has created a need for natural solutions that control enteric pathogens like Salmonella. One of these natural feed additives, a select blend of essential oils, fatty acids, and an enterosorbent mineral (NeutraPath), was assessed for its effects on the intestinal colonization of Salmonella enterica serovar Typhimurium PHL2020 isolate (ST-PHL2020) in broiler chickens and ST-PHL2020 virulence gene expression. An in vitro digestion model simulating the pH and enzymatic conditions of 3 gastrointestinal compartments (crop, proventriculus, and intestine) was first used to evaluate the antibacterial effects of NeutraPath on ST-PHL2020. For the in vivo study, day-old male broilers (n = 90) were randomly allocated to 1 of 3 groups: control or NeutraPath supplemented at 0.25 or 0.5%. The dose rates were chosen to enable observable statistical effects during high Salmonella challenge. All groups were challenged with ST-PHL2020 (106 cfu/bird) via oral gavage on day 9. Bacterial load and prevalence of ST-PHL2020 were examined in ceca-cecal tonsils, and intestinal permeability was assessed via serum recovery of fluorescein isothiocyanate dextran (FITC-d) 24 h postchallenge. NeutraPath inhibited (P < 0.05) ST-PHL2020 growth in the in vitro digestion model compared to the control at all concentrations and in all compartments other than NeutraPath 0.25% in the crop. In vivo, NeutraPath 0.25 and 0.5% reduced (P < 0.05) the total cfu recovered and total prevalence of ST-PHL2020 in the ceca. The serum FITC-d levels were also reduced (P < 0.05) by NeutraPath. Further, NeutraPath's effects on ST-PHL2020's Salmonella pathogenicity island-1 virulence network development were explored via treating ST-PHL2020 at subinhibitory concentration (1 mg/mL) of NeutraPath in vitro. Compared to the control, NeutraPath downregulated ST-PHL2020 hilA and invF mRNA expression, which further blocked expression of key downstream effectors involved in ST-PHL2020 invasion. Collectively, NeutraPath has the potential to reduce ST-PHL2020 intestinal colonization in broilers and preserve intestinal barrier integrity.

Toxicological aspect of bioinsecticide pyrethrum extract and expressions of apoptotic gene levels in human hepotacellular carcinoma HepG2 cells.

Pyrethrum extract (PE), an important natural bioinsecticide, is extensively used across the world to control pest insects in homes and farms. The aim of this study was to evaluate the potential cytotoxic effect of PE using MTT assay and genotoxic effect using micronucleus (MN) assay. The changes in the expressions of the apoptosis genes in mRNA levels were also investigated using Real-Time qPCR analysis as well as the ratio of apoptotic/necrotic cells with AnnexinV-FITC/Propidium iodide (PI) assay in HepG2 cells. PE markedly suppressed the cell proliferation on HepG2 cells. It significantly increased the frequency of micronucleus (MN) at 500 and 1000 µg/mL. PE also induced the percentage of the cell population of late apoptotic/necrotic cells (FITC + PI+) and necrotic cells (FITC- PI+), especially at 4000 µg/mL analyzed by flow cytometry. PE caused significant fold changes in the expression of several apoptotic genes including APAF1, BIK, BAX, BAD, BID, MCL-1, CASP3, CASP1, CASP2, FAS, FADD and TNFRSF1A. In particular, the pro-apoptotic gene Hrk (Harakiri) remarkably and dose-dependently was overexpressed of the mRNA level. As a result, PE may exhibit cyto-genotoxic effects, especially at higher concentrations and lead to significant changes in the expression of mRNA levels in several apoptotic genes.HighlightsNatural bioinsecticide PE exhibited a cytotoxic effect in HepG2 cells.PE significantly induced the micronucleus (MN) frequency at 500 and 1000 µg/mL.This bioinsecticide induced cell death and it lead to significant fold changes in the expression of mRNA levels in several apoptotic genes in HepG2 cells.The highest increase of the expression of mRNA levels was determined in Hrk (Harakiri) at 4000 µg/mL.

Effects of a direct fed microbial (DFM) on broiler chickens exposed to acute and chronic cyclic heat stress in two consecutive experiments.

Two consecutive 35 d experiments were conducted to investigate the effects of a multistrain DFM fed continuously to broiler chickens exposed to HS from 28 to 35 d on broiler performance, body composition, ileal digestibility, and intestinal permeability using serum Fluorescein Isothiocyanate Dextran (FITC-d) concentration. The treatments were arranged as a 2 × 2 factorial with temperature: Elevated (HS: 33 ± 2°C for 6 h and 27.7°C for the remaining 18 h from 28 to 35 days of age) and Thermoneutral (TN: 22 to 24°C over the entire 24-h day from 28 to 35 days of age) and diet: corn-soybean meal based with and without DFM (3-strain Bacillus; Enviva PRO) fed over the entire 35-d period as the two factors. Experimental diets were formulated to meet all nutrient recommendations based on breed standards using a starter (0-10 d), grower (10-21 d), and finisher (21-35 d) period. For each of the 2 experiments, 648 Ross 708 broiler chicks were allotted among the treatments with 9 replicate pens of 18 broilers. Data were analyzed as a 2 × 2 factorial within each experiment in JMP 14. In both experiments, cloacal temperatures were increased (P ≤ 0.05) in the broilers subjected to the HS treatment at both 28 d (acute) and 35 d (chronic). Supplementing birds with DFM reduced cloacal temperatures in the Experiment 1 at 28 d, but not at the other time periods. The HS treatment reduced body weight gain and lean tissue accretion from 0 to 35 d in both experiments (P ≤ 0.05). In Experiment 2, when the litter was reused BWG was increased by 36 g/bird with supplementation of DFM (P ≤ 0.05). Ileal digestibility at 28 d (2 h post HS) was improved with DFM supplementation in both experiments (P ≤ 0.05). Serum FITC-d increased with HS at both 28 and 35 d. Serum FITC-d was generally decreased with DFM at 28 d but the response was inconsistent at 35 d. Overall, the results suggest that HS reduced broiler performance and DFM treatment improved intestinal permeability and nutrient digestibility responses to HS in both experiments but did not improve performance until built up litter was used in Experiment 2.

Bufalin Induces Programmed Necroptosis in Triple-Negative Breast Cancer Drug-Resistant Cell Lines through RIP1/ROS-Mediated Pathway.

OBJECTIVE: To explore the effect and mechanism of action of bufalin in triple-negative breast cancer (TNBC) drug-resistant cell lines. METHODS: The normal human mammary epithelial cell line, TNBC cell line, TNBC adriamycin-resistant cell line, and TNBC docetaxel-resistant cell line were treated with different doses of bufalin (0-1,000 nmol/L) at different time points (0-72 h). Propidium iodide staining, AV-FITC/PI double staining, Hoechst 33342/PI double staining and transmission electron microscopy (TEM) were used to evaluate the death patterns of the cell lines. RESULTS: Bufalin killed the TNBC cell line and its drug-resistant cell lines in a dose/time-dependent manner (all P<0.01). After treatment with bufalin for 24 h, the adriamycin-resistant cell line showed a co-existing pattern of necroptosis and apoptosis. However, at 48 h, necroptosis was the main manifestation. After treatment with bufalin, the expressions of tumor necrosis factor α, phospho-tumor necrosis factor receptor 1, phospho-receptor interacting protein 1 and c-caspase 3 increased (all P<0.01), the killing effect of bufalin could be mostly inhibited by NEC-1, and by z-VAD-fmk (both P<0.01). Besides, the intracellular reactive oxygen species (ROS) levels increased considerably (P<0.01), the antioxidant N-acetyl cysteine or Nec-1 could inhibit the increase of ROS level and the killing effect of bufalin (all P<0.01). The adriamycin-resistant cell line exhibited necroptosis characteristic after 48 h of bufalin treatment under TEM. CONCLUSIONS: Bufalin could induce necroptosis through RIP1/ROS-mediated pathway to kill the drug-resistant TNBC cell lines. This finding provides critical experimental data and theoretical basis for the clinical application of bufalin to overcome the difficulties in the treatment of TNBC.

Sulfated Extract of Abelmoschus Esculentus: A Potential Cancer Chemo-preventive Agent.

BACKGROUND: Abelmoschus esculentus (AE) (okra), is an edible plant used in many food applications. OBJECTIVE: This study explored whether sulfated AE (SAE) has promising cancer chemopreventive activities that may recommend it as a functional food supplement instead of (or in addition to) AE for the population at risk of cancer and in the health food industry. METHODS: Cytochrome P450-1A (CYP1A) was estimated by fluorescence enzymatic reaction, using ß-naphthoflavone-treated cells (CYP1A inducer). Peroxyl and hydroxyl radical scavenging was assayed by oxygen radical absorbance capacity assay. Flow cytometry was used to analyze apoptosis/necrosis in MCF-7 cells, cell cycle phases in MCF-7 cells, and macrophage binding to fluorescein isothiocyanate-lipopolysaccharide (FITC-LPS). Nitric oxide was determined by Griess assay in LPS-stimulated macrophages, and cytotoxicity was determined by MTT assay. Diethylnitrosamine (DEN) was used to induce hepatic tumor initiation in rats. Placental glutathione-S-transferase (GSTP; an initiation marker) was stained in a fluorescence immunohistochemical analysis of liver sections, and histopathological changes were examined. RESULTS: SAE exhibited strong antitumor initiation and antitumor promotion activities. It suppressed CYP1A, scavenged peroxyl and hydroxyl radicals, induced macrophage proliferation, suppressed macrophage binding to FITC-LPS, inhibited nitric oxide generation, showed specific cytotoxicity to human breast MCF-7 adenocarcinoma cells, and disturbed the cell cycle phases (S and G2/M phases) in association with an increased percentage of apoptotic/necrotic MCF-7 cells. Over a short time period, DEN stimulated liver cancer initiation, but SAE treatment reduced the DEN-induced histopathological alterations and inhibited CYP1A and GSTP. CONCLUSION: SAE extract has the potential for use as an alternative to AE in health foods to provide cancer chemoprevention in populations at risk for cancer.

Research Note: Modified serum fluorescein isothiocyanate dextran (FITC-d) assay procedure to determine intestinal permeability in poultry fed diets high in natural or synthetic pigments.

Oral administration of fluorescein isothiocyanate dextran (FITC-d) has been used as an indicator for intestinal permeability in poultry research for several years. Under healthy conditions, tight junctions in the intestinal wall will not allow the 4-6kDa FITC-d to enter the bloodstream. Detection of FITC-d in serum (1-hour post-oral administration of FITC-d) has proven to be a reliable indicator of leaky gut syndrome (increased intestinal inflammation and disruption of tight junctions). Administration of supplementary phytobiotics in feed, particularly products with high beta-carotene levels or other pigments, has resulted in strong serum background fluorescence, which can render this assay unreliable. To account for this increase in background autofluorescence, the FITC-d assay procedure has been modified to accommodate these particular serum samples by including pre-administration serum collection from each treatment group to remove background fluorescence. The modified FITC-d procedure detailed will allow for analysis of intestinal permeability in pigmented serum.

Switchable CAR-T Cells Outperformed Traditional Antibody-Redirected Therapeutics Targeting Breast Cancers.

Various antibody-redirected immunotherapeutic approaches, including antibody-drug conjugates (ADCs), bispecific antibodies (bsAbs), and chimeric antigen receptor-T (CAR-T) cells, have been devised to produce specific activity against various cancer types. Using genetically encoded unnatural amino acids, we generated a homogeneous Her2-targeted ADC, a T cell-redirected bsAb, and a FITC-modified antibody capable of redirecting anti-FITC CAR-T (switchable CAR-T; sCAR-T) cells to target different Her2-expressing breast cancers. sCAR-T cells showed activity against Her2-expressing tumor cells comparable to that of conventional anti-Her2 CAR-T cells and superior to that of ADC- and bsAb-based approaches. To prevent antigen escape, we designed bispecific sCAR-T cells targeting both the Her2 receptor and IGF1R, which showed an overall improved activity against cancer cells with low Her2 expression. This study increases our understanding of various explored cancer therapeutics and underscores the efficient application of sCAR-T cells as a promising therapeutic option for breast cancer patients with low or heterogeneous antigen expression.

Synergistic effect of the herbal mixture C5E on gemcitabine treatment in PANC­1 cells.

The present study aimed to determine the anticancer effect of the herbal mixture extract C5E in the pancreatic cancer cell line, PANC­1, in the absence or presence of gemcitabine treatment, a chemotherapeutic drug used for the treatment of pancreatic cancer. The anticancer effects of C5E, gemcitabine and C5E plus gemcitabine in PANC­1 cells following 72 h of treatment were investigated. The effect of each treatment on cell cycle arrest, apoptosis and the proportion of side population (SP) cells was determined using flow cytometric analysis following propidium iodide (PI), Annexin V­FITC/PI double staining and Hoechst 33342 staining, respectively. SP cells share similar characteristics to cancer stem­like cells, and a reduction in the SP is considered to be indicative of an anticancer effect. The percentage of SP cells and the cell viability of general PANC­1 cells were significantly decreased in response to all treatments. The percentage of SP cells was reduced from 8.2% (control) to 3.9, 7.2 and 5.1% following the treatment with C5E, gemcitabine and the co­treatment, respectively. All three treatments were discovered to inhibit cell viability by arresting the cell cycle at the S phase and promoted cell death by inducing early apoptosis, with the levels of apoptosis being increased from 1.9% (control) to 7.3, 2.5 and 12.0% following the treatment with C5E, gemcitabine and the co­treatment, respectively. The mRNA expression levels of sonic hedgehog, which is implicated in the development of certain types of cancer, were downregulated to a greater extent following the co­treatment with C5E and gemcitabine compared with the treatment with either C5E or gemcitabine alone. As the co­treatment with gemcitabine and C5E was more effective than each individual treatment, the present study suggested that the combined treatment may exhibit synergistic effects in PANC­1 cells.

MSNs-Based Nanocomposite for Biofilm Imaging and NIR-Activated Chem/Photothermal/Photodynamic Combination Therapy.

Bacterial infections caused by biofilms are severe clinical problems, resulting in high drug resistance by limiting the penetration of antibiotics. Herein, a near-infrared (NIR)-activated chem/photodynamic/photothermal combined therapeutic agent is proposed by loading fluorescein isothiocyanate (FITC), ultrasmall copper sulfide nanoparticles (Cu2-xSNPs), and ε-polylysine (PLL) onto mesoporous silica nanoparticles (MSNs) through a layer-by-layer self-assembly approach. FITC-doped MSNs are prepared to monitor the permeability and accumulation of nanocomposites into biofilms. MSNs can also act as hosts for the synthesis of ultrasmall Cu2-xSNPs, which has effective photodynamic and photothermal ablation against bacteria under NIR light irradiation. Moreover, biodegradable PLL introduced can not only enhance adhesion toward the bacterial surface to increase the effectiveness of phototherapy but also damage bacteria through electrostatic interaction. As a result, the prepared nanocomposites could not only penetrate biofilms but also ablate biofilms through combined chem/photodynamic/photothermal effects under NIR light irradiation. Furthermore, the nanocomposites could treat bacterial infections in vivo with negligible tissue toxicity. Overall, the finely designed nanocomposites are anticipated to display promising applications in imaging-guided chem/photodynamic/photothermal combined therapy for bacterial infections.

DNA-crosslinked alginate and layered microspheres to modulate the release of encapsulated FITC-dextran.

Alginate can be gently crosslinked by calcium into hydrogels and microspheres for the encapsulation and release of proteins and drugs. However, the release is often over short periods unless alginate is also covalently modified or crosslinked. This research aims to sustain the release of encapsulated model drug FITC-dextran by covalently crosslinking alginate with short oligomers DNA because evidence suggests that DNA may also interact with alginate to further increase effective crosslinking. Furthermore, modulating the release of drugs from alginate in response to specific proteins could tailor release profiles to improve patient treatment. This research develops a DNA-crosslinked alginate hydrogel and layered alginate microspheres to encapsulate and then sustain the release FITC-dextran (model drug). An aptamer sequence to hen egg-white lysozyme is included in one DNA strand to allow for the disruption of the crosslinks by interactions with human lysozyme. Alginate was covalently modified with complementary strands of DNA to crosslink the alginate into hydrogels, which had increased crosslinking density when re-swollen (in comparison to controls crosslinked with PEG) and could sustained the release of encapsulated FITC-dextran. When an aptamer sequence for hen lysozyme was included in the DNA crosslinks, the hydrogels decrosslinked when incubated in human lysozyme for 60 days. In addition, calcium alginate microspheres were coated with 3 alternating layers of poly-Lysine, DNA-crosslinked alginate, and poly-L-lysine. FITC-dextran loaded into the microspheres released in a sustained manner past 30 days (into PBS at 37 °C) and would likely continue to release for far longer had the studies continued. When incubated with 3 µM of human lysozyme, a burst release of FITC-dextran occurred from both the hydrogels and microspheres, with no changes in the controls. The increased release was in bursts followed by similar sustained release rates suggesting that the human lysozyme temporarily disrupted the DNA crosslinks which were then re-established or were influenced by interactions between DNA and alginate. Importantly, covalently bound complementary strands of DNA could crosslink the alginate and additional interactions appeared to further sustain the release of encapsulated therapeutics.

Microfluidic Fabrication of Structure-Controlled Chitosan Microcapsules via Interfacial Cross-Linking of Droplet Templates.

In this work, a simple and flexible method for the fabrication of chitosan microcapsules with controllable structures and functions via the interfacial cross-linking reaction of the water-in-oil (W/O) emulsion templates is developed. The interfacial cross-linking reactions of chitosan and terephthalaldehyde (TPA) in W/O emulsion templates are comprehensively studied. The interfacial cross-linking reactions of the droplet templates in both batchwise and continuous conditions are studied. A poly(dimethylsiloxane) (PDMS) droplet-capture microfluidic chip is fabricated to investigate the interfacial reaction in continuous conditions online. In this study, the size and shell thickness of the microcapsules are affected by the preparation condition, such as the template size, emulsifier concentration, TPA concentration, and cross-linking time. Moreover, the size and shell thickness changes of chitosan microcapsules prepared in continuous conditions are much faster than those prepared in batchwise conditions. By regulating the preparation parameters, the microcapsules with controllable structures are fabricated in both batchwise and continuous conditions. The drug release behaviors of the microcapsules with controllable structures are studied. Furthermore, by adding magnetic nanoparticles to the aqueous solution, magnetic-responsive microcapsules are fabricated easily. This work provides valuable guidance for the controllable fabrication of chitosan microcapsules with designed structures and functions via single emulsion templates.

An ex vivo multiparametric flow cytometry assay using human whole blood to simultaneously measure cytotoxicity and leishmanicidal activities.

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.