Characterization of Organic Anion and Cation Transport in Three Human Renal Proximal Tubular Epithelial Models.

The polarised expression of specific transporters in proximal tubular epithelial cells is important for the renal clearance of many endogenous and exogenous compounds. Thus, ideally, the in vitro tools utilised for predictions would have a similar expression of apical and basolateral xenobiotic transporters as in vivo. Here, we assessed the functionality of organic cation and anion transporters in proximal tubular-like cells (PTL) differentiated from human induced pluripotent stem cells (iPSC), primary human proximal tubular epithelial cells (PTEC), and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1). Organic cation and anion transport were studied using the fluorescent substrates 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) and 6-carboxyfluorescein (6-CF), respectively. The level and rate of intracellular ASP accumulation in PTL following basolateral application were slightly lower but within a 3-fold range compared to primary PTEC and RPTEC/TERT1 cells. The basolateral uptake of ASP and its subsequent apical efflux could be inhibited by basolateral exposure to quinidine in all models. Of the three models, only PTL showed a modest preferential basolateral-to-apical 6-CF transfer. These results show that organic cation transport could be demonstrated in all three models, but more research is needed to improve and optimise organic anion transporter expression and functionality.

Fine-tuning of membrane permeability by reversible photoisomerization of aryl-azo derivatives of thymol embedded in lipid nanoparticles.

Responsiveness of liposomes to external stimuli, such as light, should allow a precise spatial and temporal control of release of therapeutic agents or ion transmembrane transport. Here, some aryl-azo derivatives of thymol are synthesized and embedded into liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to obtain light-sensitive membranes whose photo-responsiveness, release behaviour, and permeability towards Cl- ions are investigated. The hybrid systems are in-depth characterized by dynamic light scattering, atomic force microscopy and Raman spectroscopy. In liposomal bilayer the selected guests undergo reversible photoinduced isomerization upon irradiation with UV and visible light, alternately. Non-irradiated hybrid liposomes retain entrapped 5(6)-carboxyfluorescein (CF), slowing its spontaneous leakage, whereas UV-irradiation promotes CF release, due to guest trans-to-cis isomerization. Photoisomerization also influences membrane permeability towards Cl- ions. Data processing, according to first-order kinetics, demonstrates that Cl- transmembrane transport is enhanced by switching the guest from trans to cis but restored by back-switching the guest from cis to trans upon illumination with blue light. Finally, the passage of Cl- ions across the bilayer can be fine-tuned by irradiation with light of longer λ and different light-exposure times. Fine-tuning the photo-induced structural response of the liposomal membrane upon isomerization is a promising step towards effective photo-dynamic therapy.

Simultaneous detection of breast cancer biomarkers circROBO1 and BRCA1 based on a CRISPR-Cas13a/Cas12a system.

Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.

Malignance-restriction activity exhibited by bioactive compounds of selected actinobacteria as silver nanoparticles against A549 lung cancer cell lines.

This article deals with the antibacterial and anticancer potential of secondary metabolites produced by actinomycetes also reported as actinobacteria, Microbacterium proteolyticum (MN560041), and Streptomycetes rochei, where preliminary studies were done with the well diffusion method. These actinobacteria's silver nanoparticles were synthesized and characterized using transmission electron microscopy (TEM) and UV-Visible spectroscopy. Anticancer was measured using the MTT test, reactive oxygen species (ROS) generation measured with DCFDA, mitochondrial membrane potential (MMP) measurement, and DAPI fluorescence intensity activity was measured in treated and non-treated cancerous cells. The IC50 value for 5-FU (a), LA2(O) (b), LA2(R) (c), LA2(ON) (d), and LA2(RN) (e) was obtained at 3.91 µg/mL (52.73% cell viability), 56.12 µg/mL (52.35% cell viability), 44.90 µg/mL (52.3% cell viability), 3.45 µg/mL (50.25% cell viability), and 8.05 µg/mL (48.72% cell viability), respectively. TEM micrographs revealed discrete, well-separated AgNPs particles of size 7.88 ± 2 to 12.86 ± 0.24 nm. Gas chromatography-mass spectrometry was also performed to detect the compounds in bioactive metabolites where n-hexadecanoic acid was obtained as the most significant one. MTT test showed a substantial decline in A549 cell viability (up to 48.72%), 2.75-fold increase in ROS generation was noticed in comparison to untreated A549 lung cancer cells when measured with DCFDA. A total of 0.31-fold decrease in MMP and 1.74-fold increase in DAPI fluorescence intensity compared to untreated A549 lung cancer cells suggests that the synthesized nanoparticles promote apoptosis in cancerous cells. Our findings suggests that the secondary metabolites of M. proteolyticum and S. rochei in nanoparticle form can be used as a significant compound against lung cancers.

Analysis and Development of Antigen-specific T Cells Derived from Peripheral Blood Mononuclear Cells of Healthy Donors.

BACKGROUND/AIM: Adoptive cell therapy using antigen-specific T cells is a promising treatment modality for cancer patients. Various methods to isolate specific T cells and identify corresponding T cell receptor (TCR) sequences are known. This study aimed to identify antigen-specific TCR from T cells isolated using carboxyfluorescein succinimidyl ester (CFSE), which marks proliferating activated T cells. MATERIALS AND METHODS: CFSE stained healthy donor peripheral blood mononuclear cells (PBMCs) were treated with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptides for seven days. Then, proliferating T cells with decreased CFSE staining were isolated and single cell VDJ sequencing was performed on isolated T cells to identify antigen-specific TCRs. RESULTS: As antigen-specific TCR candidates, ten TCR clones were selected for the CMV antigen and five for the EBV antigen. The reactivity of ten CMV TCR-transduced T cells and one EBV TCR-transduced T cell toward T2 cells pulsed with CMV or EBV peptide was confirmed via NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assays. CONCLUSION: Identification of antigen-specific TCRs with CFSE staining is a valid method for the development of effective immunotherapy. The identified CMV- or EBV-specific TCRs can be used for adoptive cell therapy to treat cancer.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Mitigation effect of hesperidin on X-ray radiation-induced intestinal barrier dysfunction in Caco-2 cell monolayers.

The tight junctions (TJs) and barrier function of the intestinal epithelium are highly sensitive to radiation. However, polyphenols can be used to reverse the effects of radiation. Here, we investigated the effects of hesperidin (hesperetin-7-rhamnoglucoside) on X-ray-induced intestinal barrier dysfunction in human epithelial Caco-2 monolayers. To examine whether hesperidin mitigated the effects of X-ray exposure (2 Gy), cell survival was evaluated and intestinal barrier function was assessed by measuring the transepithelial flux, apparent permeability coefficient (Papp), and barrier integrity. Hesperidin improved the survival of Caco-2 cell monolayers and attenuated X-ray exposure-induced intestinal barrier dysfunction. For fluorescein transport experiments, transepithelial flux and Papp of fluorescein in control group were significantly elevated by X-ray, but were restored to near control by 10 µM hesperidin pretreatment. Further, X-ray exposure decreased the barrier integrity and TJ interruption by reducing TJ-related proteins occludin and claudin-4, whereas cell monolayers pretreated with hesperidin before X-ray exposure were reinstated to control level. It was concluded that hesperidin treatment before X-ray exposure alleviated X-ray-induced intestinal barrier dysfunction through regulation of TJ-related proteins. These results indicate that hesperidin prevents and mitigates X-ray-induced intestinal barrier dysfunction.

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Detection of Nanoparticle-Mediated Total Oxidative Stress in T Cells Using CM-H2DCFDA Dye.

Oxidative stress is commonly observed in cells following exposure to nanoparticles. Both negative (e.g., cytotoxicity and inflammation) and beneficial (e.g., anti-inflammatory and tumor growth inhibiting) responses have been linked in the literature to oxidative stress, emphasizing the importance of developing methodologies to study this phenomenon in cells following their exposure to nanoparticles. In the protocol described herein, primary human T cells isolated from the peripheral blood of healthy donor volunteers are treated with nanoparticles and controls, and the generation of reactive oxygen species is detected by flow cytometry using CM-H2DCFDA reagent.

Visualization of bone formation in sheep's middle ear by using fluorochrome sequential labelling (FSL).

One factor for the lacking integration of the middle ear stapes footplate prosthesis or the missing healing of stapes footplate fractures could be the known osteogenic inactivity. In contrast, it was recently demonstrated that titanium prostheses with an applied collagen matrix and immobilised growth factors stimulate osteoblastic activation and differentiation on the stapes footplate. Regarding those findings, the aim of this study was to evaluate the potential of bone regeneration including bone remodeling in the middle ear. Ten one-year-old female merino sheep underwent a middle ear surgery without implantation of middle ear prostheses or any other component for activating bone formation. Post-operatively, four fluorochromes (tetracycline, alizarin complexion, calcein green and xylenol orange) were administered by subcutaneous injection at different time points after surgery (1 day: tetracycline, 7 days: alizarin, 14 days: calcein, 28 days: xylenol). After 12 weeks, the temporal bones including the lateral skull base were extracted and histologically analyzed. Fluorescence microscopy analysis of the entire stapes with the oval niche, but in particular stapes footplate and the Crura stapedis revealed evidence of new bone formation. Calcein was detected in all and xylenol in 60% of the animals. In contrast, tetracycline and alizarin could only be verified in two animals. The authors were able to demonstrate the osseoregenerative potential of the middle ear, in particular of the stapes footplate, using fluorescence sequence labelling.

Comparative Analysis of Fluorescent Labeling Techniques for Tracking Canine Amniotic Stem Cells.

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.

Human amniotic MSCs-mediated anti-inflammation of CD206hiIL-10hi macrophages alleviates isoproterenol-induced ventricular remodeling in mice.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammation which makes them suitable for the treatment of various diseases. OBJECTIVE: This study aimed to explore the therapeutic effect and molecular mechanism of hAMSCs in ventricular remodeling (VR). METHODS: hAMSCs were characterized by a series of experiments such as flow cytometric analysis, immunofluorescence, differentiative induction and tumorigenicity. Mouse VR model was induced by isoproterenol (ISO) peritoneally, and the therapeutic effects and the potential mechanisms of hAMSCs transplantation were evaluated by echocardiography, carboxy fluorescein diacetate succinimidyl ester (CFSE) labeled cell tracing, histochemistry, qRT-PCR and western blot analysis. The co-culturing experiments were carried out for further exploring the mechanisms of hAMSCs-derived conditioned medium (CM) on macrophage polarization and fibroblast fibrosis in vitro. RESULTS: hAMSCs transplantation significantly alleviated ISO-induced VR including cardiac hypertrophy and fibrosis with the improvements of cardiac functions. CFSE labeled hAMSCs kept an undifferentiated state in heart, indicating that hAMSCs-mediated the improvement of ISO-induced VR might be related to their paracrine effects. hAMSCs markedly inhibited ISO-induced inflammation and fibrosis, seen as the increase of M2 macrophage infiltration and the expressions of CD206 and IL-10, and the decreases of CD86, iNOS, COL3 and αSMA expressions in heart, suggesting that hAMSCs transplantation promoted the polarization of M2 macrophages and inhibited the polarization of M1 macrophages. Mechanically, hAMSCs-derived CM significantly increased the expressions of CD206, IL-10, Arg-1 and reduced the expressions of iNOS and IL-6 in RAW264.7 macrophages in vitro. Interestingly, RAW264.7-CM remarkably promoted the expressions of anti-inflammatory factors such as IL-10, IDO, and COX2 in hAMSCs. Furthermore, the CM derived from hAMSCs pretreated with RAW264.7-CM markedly inhibited the expressions of fibrogenesis genes such as αSMA and COL3 in 3T3 cells. CONCLUSION: Our results demonstrated that hAMSCs effectively alleviated ISO-induced cardiac hypertrophy and fibrosis, and improved the cardiac functions in mice, and the underlying mechanisms might be related to inhibiting the inflammation and fibrosis during the ventricular remodeling through promoting the polarization of CD206hiIL-10hi macrophages in heart tissues. Our study strongly suggested that by taking the advantages of the potent immunosuppressive and anti-inflammatory effects, hAMSCs may provide an alternative therapeutic approach for prevention and treatment of VR clinically.

Molecular mechanism for the unusual enhancement of the second-step chemiluminescence production from the carcinogenic tetrabromohydroquinone and H2O2.

We have found recently that two-step intrinsic hydroxyl radical (·OH)-dependent chemiluminescence (CL) could be produced by carcinogenic tetrahaloquinone and H2O2. However, the first-step CL was too fast to clearly detect the stepwise generation of ·OH and CL, and to distinguish the exact dividing point between the first-step and second-step CL. Here we found that, extremely clear two-step intrinsic CL could be produced by the relative slow reaction of tetrabromohydroquinone (TBHQ) with H2O2, which was directly dependent on the two-step ·OH generation. Interestingly, the second-step, but not the first-step CL production of TBHQ/H2O2 (CRET donor) was markedly enhanced by fluorescein (a typical xanthene dye, CRET acceptor) through a unique chemiluminescence resonance energy transfer (CRET) process. The novel CRET system of TBHQ/H2O2/fluorescein was successfully applied for the sensitive detection of TBHQ with the detection limit as low as 2.5 µmol/L. These findings will help to develop more sensitive and highly efficient CL or CRET systems and specific CL sensor to detect the carcinogenic haloquinones, which may have broad environmental applications.

The Long-Term Effect on Dry Eye of Posterior Approach Ptosis Surgery Vs. Upper Eyelid Blepharoplasty.

PURPOSE: To examine the long-term effect of combined blepharoplasty and Müller muscle-conjunctival resection (MMCR) compared to an upper blepharoplasty procedure on dry eye syndrome. METHODS: This is a Prospective comparative case series. Two groups of patients participated in this study: the blepharoplasty group included adult patients that underwent blepharoplasty at least 3 years earlier and the ptosis group consisting of adult patients that underwent MMCR with blepharoplasty at least 3 years earlier. The parameters that were compared for all patients before the procedure, on postoperative day 90, and at the long-term follow-up were: Schirmer-test 2, tear break-up time (TBUT), fluorescein staining, and lissamine green (LG) staining. RESULTS: The participants included 25 post-MMCR patients with a mean follow-up of 4.94 ± 0.64 years and 15 post-blepharoplasty patients with a mean follow-up of 4.22 ± 0.32 years. There was a significant increase in the postoperative LG and fluorescein staining scores compared to the preoperative scores in the ptosis group (p < .01 and p < .01, respectively) as well as a decrease in postoperative TBUT compared to the preoperative values (p = .044). Those parameters were not significant in the blepharoplasty group. CONCLUSIONS: Patients who underwent MMCR, but not those following upper blepharoplasty, showed signs of dry eye compared to the preoperative status after long-term follow-up. Dry eye signs should be examined before MMCR surgery, and patients should be aware of the high risk of developing dry eye and the need for long-term treatment. Surgeons should carefully consider performing MMCR for patients with severe dry eye.

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.

Pan-EGFR Inhibitor Dacomitinib Resensitizes Paclitaxel and Induces Apoptosis via Elevating Intracellular ROS Levels in Ovarian Cancer SKOV3-TR Cells.

Paclitaxel is still used as a standard first-line treatment for ovarian cancer. Although paclitaxel is effective for many types of cancer, the emergence of chemoresistant cells represents a major challenge in chemotherapy. Our study aimed to analyze the cellular mechanism of dacomitinib, a pan-epidermal growth factor receptor (EGFR) inhibitor, which resensitized paclitaxel and induced cell cytotoxicity in paclitaxel-resistant ovarian cancer SKOV3-TR cells. We investigated the significant reduction in cell viability cotreated with dacomitinib and paclitaxel by WST-1 assay and flow cytometry analysis. Dacomitinib inhibited EGFR family proteins, including EGFR and HER2, as well as its downstream signaling proteins, including AKT, STAT3, ERK, and p38. In addition, dacomitinib inhibited the phosphorylation of Bad, and combination treatment with paclitaxel effectively suppressed the expression of Mcl-1. A 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay revealed a substantial elevation in cellular reactive oxygen species (ROS) levels in SKOV3-TR cells cotreated with dacomitinib and paclitaxel, which subsequently mediated cell cytotoxicity. Additionally, we confirmed that dacomitinib inhibits chemoresistance in paclitaxel-resistant ovarian cancer HeyA8-MDR cells. Collectively, our research indicated that dacomitinib effectively resensitized paclitaxel in SKOV3-TR cells by inhibiting EGFR signaling and elevating intracellular ROS levels.

Alternative Approach for the Treatment of Conjunctivochalasis: Plasma-Based Conjunctivoplasty.

PURPOSE: The aim of this study was to evaluate the efficacy of plasma-based conjunctivoplasty as a new surgical approach for conjunctivochalasis. METHODS: This prospective, noncomparative, interventional study included research on 42 eyes of 33 patients who underwent plasma therapy because of conjunctivochalasis between February 2020 and December 2021. Maintaining a 2-mm distance from the limbus, at least 3 lines (approximately 2 mm deep) of plasma therapy were applied to the conjunctiva from the temporal quadrant to the nasal quadrant. Patient symptoms, the fluorescein clearance test, ocular surface integrity with fluorescein staining, the Ocular Surface Disease Index questionnaire, tear breakup time (TBUT), tear meniscus height, and complications were evaluated. RESULTS: The mean patient age at the time of surgery was 67.3 ± 7.2 (range: 54-81) years. After surgery, the grades of conjunctivochalasis decreased in all patients. Although epiphora was present in 28 eyes (66.6%) preoperatively, none of the cases had epiphora during the follow-up period. The Ocular Surface Disease Index score improved significantly from 34.7 ± 10.3 preoperatively to 5.0 ± 4.2 3 months postoperatively ( P <0.001). Although the preoperative TBUT was 5.1 ± 2.2 seconds, the TBUT increased to 10.0 ± 2.3 seconds 3 months postoperatively ( P <0.001). Chemosis developed in only 1 eye (2.3%) and completely regressed with topical steroid treatment in the first postoperative week. CONCLUSIONS: Plasma-based conjunctivoplasty is a minimally invasive and simple surgery with less intraoperative and postoperative discomfort, a fast recovery, and can be suggested as an alternative approach to other treatment methods.

The Use of Carboxyfluorescein Reveals the Transport Function of MCT6/SLC16A5 Associated with CD147 as a Chloride-Sensitive Organic Anion Transporter in Mammalian Cells.

Oral drug absorption involves drug permeation across the apical and basolateral membranes of enterocytes. Although transporters mediating the influx of anionic drugs in the apical membranes have been identified, transporters responsible for efflux in the basolateral membranes remain unclear. Monocarboxylate transporter 6 (MCT6/SLC16A5) has been reported to localize to the apical and basolateral membranes of human enterocytes and to transport organic anions such as bumetanide and nateglinide in the Xenopus oocyte expression system; however, its transport functions have not been elucidated in detail. In this study, we characterized the function of MCT6 expressed in HEK293T cells and explored fluorescent probes to more easily evaluate MCT6 function. The results illustrated that MCT6 interacts with CD147 to localize at the plasma membrane. When the uptake of various fluorescein derivatives was examined in NaCl-free uptake buffer (pH 5.5), the uptake of 5-carboxyfluorescein (5-CF) was significantly greater in MCT6 and CD147-expressing cells. MCT6-mediated 5-CF uptake was saturable with a Km of 1.07 mM and inhibited by several substrates/inhibitors of organic anion transporters and extracellular Cl ion with an IC50 of 53.7 mM. These results suggest that MCT6 is a chloride-sensitive organic anion transporter that can be characterized using 5-CF as a fluorescent probe.

Network pharmacology analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer's disease. / çŸ¥æ¯æ²»ç–—é˜¿å°”èŒ¨æµ·é»˜ç— ç½‘ç»œè¯ç†å­¦ç ”ç©¶åŠå®žéªŒéªŒè¯.

OBJECTIVES: To explore the mechanism of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD). METHODS: The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aß1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aß deposition in the pharynx was detected by Thioflavin S staining. RESULTS: Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aß1-42 (both P<0.01), inhibit the expression of ß-secretase 1 (BACE1), APP and Aß1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3ß/GSK3ß in SKNMC cells (all P<0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aß deposition. CONCLUSIONS: Anemarrhenae Rhizoma may reduce the production of Aß in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3ß pathway.

OUTER RETINOPATHY AND MICROANGIOPATHY IN ACUTE MYELOGENOUS LEUKEMIA.

PURPOSE: To describe a patient with acute myelogenous leukemia who presented with a recurrent, bilateral, outer retinopathy, before and after consolidative peripheral blood stem cell transplantation complicated by chronic graft-versus-host disease. METHODS: This is a retrospective review of records from a 23-year-old woman with acute myelogenous leukemia who underwent comprehensive ophthalmic evaluations for over a year including chromatic perimetry and multifocal electroretinograms, imaging with spectral domain optical coherence tomography, near-infrared and short-wavelength fundus reflectance and autofluorescence, fluorescein and optical coherence tomography angiography. RESULTS: The patient presented with recurrent, unilateral paracentral scotomas. There was localized loss of inner segment ellipsoid (EZ) and photoreceptor outer segment signals (IZ) in the pericentral retina of both eyes co-localizing with hyperreflective lesions on near-infrared reflectance. She subsequently lost vision (visual acuity = 20/200) in the right eye a year after consolidative peripheral blood stem cell transplantation complicated by steroid-resistant-chronic graft-versus-host disease. There was loss of the EZ and IZ signals corresponding to a dense central cone scotoma and multifocal electroretinograms depression. Near-infrared autofluorescence, fluorescein and optical coherence tomography angiography were within normal limits. Visual acuity (20/20) and retinal sensitivities improved with restoration of the EZ/IZ signals after oral prednisone and intravenous rituximab, but left a residual photoreceptor loss and paracentral scotoma. CONCLUSION: We propose that an immune-mediated microangiopathy may explain the protracted, recurrent course of primary photoreceptor abnormalities in our patient, which was further complicated by manifestations of chronic graft-versus-host disease following consolidative peripheral blood stem cell transplantation. Outer retinal findings previously documented in leukemia may be explained by a similar mechanism.