Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury. Acute kidney injury (AKI) is responsible for approximately 5% of all hospitalizations, is associated with increased morbidity and mortality, as well as progression to chronic kidney disease (CKD) and end-stage renal disease (ESRD) . Nearly The early phase of IRI (up to 48 h post-IRI) is associated with inflammation, particularly, recruitment of circulating neutrophils to the kidney ,4. NeutrNuclear factor-κB (NFκB)-mediated gene expression is a hallmark of tissue inflammation . The NFκThe effect of NFκB on gene expression is further altered by post translational modification, including acetylation . SpecifiAccordingly we sought to study the effect of BRD4 inhibition on the early phase of inflammation that characterizes renal IRI. We first performed an in silico analysis of NFκB-mediated gene expression in early IRI, Next, we examined the effect of BRD4 inhibition on neutrophil adhesion to cultured endothelial cells and NFκB-mediated gene expression in kidney tubule cells. Subsequently, we studied the effect of BRD4 inhibition in a murine model of IRI. In vitro, we observed that BRD4 inhibition reduces neutrophil adhesion to the endothelium following hypoxia re-oxygenation and decreases inflammatory gene expression in primary tubule cells. In vivo, BRD4 treatment attenuates NFκB-mediated gene expression, reduces neutrophil recruitment and activation, and decreases tubular injury.The activation of canonical NFκB signaling is one of the primary mechanisms regulating gene expression in the setting of tissue inflammation, and has been implicated in the renal response to changes in the redox state that accompanies IRI . To asseCanonical NFκB signaling pathway is regulated by the BET protein BRD4 . We chosRecent studies suggested that BRD4 inhibition can also affect cell proliferation through c-Fos/activator protein 1 (AP-1) signaling and fibrosis through transforming growth factor (TGF)-β signaling ,24,25. TWe initially performed cell viability assays to confirm MS417 has no cytotoxic effects on either endothelial cells or neutrophils. Isolated neutrophils were treated with varying concentrations of MS417 ranging from 1 nM to 1 mM A. NeutroNeutrophil recruitment is one of the first steps in the innate immune response to tissue injury. To transmigrate to the site of injury, neutrophils must first be recruited at the inner surface of the vascular endothelium . Therefop < 0.001). Taken together, these studies show that both neutrophils and endothelial cells were affected by MS417 treatment. To better represent IRI in an in vitro setting, HUVEC were subjected to hypoxia-re-oxygenation. Following two hours of hypoxia, HUVEC were co-incubated with neutrophils treated with either 100 nM or 1 μM of MS417 and re-oxygenated for three hours. Neutrophil adhesion to endothelial cells was attenuated when neutrophils were treated with MS417 in a dose-dependent manner .To better understand the effects of BRD4 inhibition on NFκB-mediated gene expression, an in vivo time course experiment was performed. Mice were treated with MS417 or saline (control) by oral gavage daily for seven days prior to unilateral IRI A. Expres+ve/F4/80−ve .Finally, we examined whether BRD4 inhibition with MS417 treatment was able to reduce tubular injury following IRI. PAS-stained sections taken at 6, 24 and 48 h post-IRI were scored in a blinded manner for standard morphologic changes of tubular damage A–D. As ep < 0.01) and LCN2 (p = 0.0516) 24 h after IRI.We also assessed kidney injury by measuring gene expression of two classic markers of tubular injury, namely HAVCR1  and LCN2  A–C 42,4,442,43. p = 0.004)  . CollectAKI is associated with increased hospital morbidity and mortality and leads to an increased risk of developing chronic kidney disease (CKD) end-stage renal disease (ESRD) . IRI is The first goal of the present study was to better understand the cellular response during the early phase of IRI-induced inflammation, of which NFκB is a major regulator. Previous studies demonstrating NFκB in the tissue response to IRI have focused on NFκB target genes and/or direct interference with NFκB activity ,47,48. WOur approach to block NFκB signaling focused on BET proteins. The BET protein family consists of BRD2, BRD3, BRD4 and BRDT, all of which have two conserved bromodomains at the N-terminal and an extra-terminal recruitment domain at the C-terminal ,52. BromIn cancer cells, the BRD4/RelA complex avoids degradation, resulting in continuous activation of NFκB, which promotes cell proliferation . Thus, sWe first confirmed the inhibitory effect of MS417 on NFκB-mediated luciferase activity. As expected, MS417 reduced NFκB-mediated luciferase in response to TNFα. Recent studies have suggested a broader role of BRD4 outside of NFκB signaling, including effects on c-Fos/AP-1-mediated gene expression and TGF-β-mediated gene expression ,24,25. GTaken together, these finding suggest that MS417 may have broader effects on IRI beyond the inhibition of NFκB. While we characterized the effects of BRD4 inhibition on the initial inflammatory phase, we did not assess the effects on the subsequent regeneration/repair and fibrotic phases. The regenerative phase is necessary to repair the damaged epithelium through proliferation that is primarily mediated by growth factors . It is tWe hypothesized that BRD4 inhibition would reduce neutrophil adhesion to endothelial cells. The up-regulation of endothelial adhesion molecules, including vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), is crucial for leukocyte adhesion following IRI ,63. TNFαWhile we did not address the mechanisms responsible for the in vitro effect of BRD4 inhibition on neutrophil adhesion, it was previously shown that anti-CD66 monoclonal antibody treatment can inhibit neutrophil adhesion to endothelial cells . Also, bWe next studied the effect of BRD4 inhibition on the early accumulation of neutrophils in murine kidneys following IRI as well as on bone marrow and blood neutrophils. While we did not measure BRD4 expression following IRI, previous investigators have shown as increase in BRD4 expression following IRI and a reduction with JQ1 treatment . NeutropSeveral studies have looked at inhibiting neutrophil accumulation in tissue by targeting different steps in recruitment including chemotaxis, activation, and transmigration ,74,75. IRecent studies have shown that in response to a variety of stimuli, neutrophils become primed or activated leading to an increase in surface expression of adhesion receptors including LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and L-selectin (CD62L) ,78,79,80We therefore assessed activation markers in neutrophils in the bone marrow, circulation and kidney tissue following IRI. This analysis of neutrophil cell surface marker was performed as previously described ,82,83. AWhile it has previously been thought that neutrophils undergo apoptosis at the site of inflammation and are phagocytosed by macrophages, it is now understood that neutrophils undergo a process termed reverse migration, and re-enter the vasculature ,85. PrevInterestingly, BRD4 inhibition suppressed neutrophil up-regulation of CD66a in response to IRI. CD66 has been shown to activate β2-integrins and play a role in neutrophil recruitment and arrest, while treatment with anti-CD66a monoclonal antibodies significantly decreased neutrophil adhesion to stimulated HUVEC ,37. ConsOur final observation was that BRD4 inhibition reduces the severity of kidney injury following IRI, and this injury correlated with neutrophil infiltration. BRD4 inhibition reduced the expression of both kidney injury markers, HAVCR1 and LCN2, and decreased injury to the tubules. While HAVCR1 and NGAL are not directly mediated by NFκB, previous studies targeting NFκB signaling have demonstrated a subsequent down-regulation in expression of these genes ,90,91. JOur study had some key strengths. Our study confirmed previous observations, albeit in different systems, around BRD4 inhibition affecting neutrophil adhesion and infiltration ,57,76. IOur study has some important limitations. We did not relate the impact of early treatment on longer term kidney outcomes. Interestingly, we observed that MS417 inhibits TGF-β-induced gene expression, suggesting that kidney fibrosis following IRI may also be attenuated by MS417 treatment. Furthermore, the current study focused on the relationship between inflammation injury and neutrophil accumulation. Other cell types, including monocytes, play a key role in inflammation following IRI; however, we did not assess the impact of BRD4 inhibition on these cell types ,93. FromIn summary, we report that BRD4 inhibition with MS417 attenuates neutrophil adhesion to endothelial cells in vitro, and reduces neutrophil counts in the bone marrow and kidney, attenuates activated neutrophils in the kidney, and reduces tubular injury following IRI, in vivo. Taken together, these findings suggest that targeting NFκB by inhibiting BRD4 may be a potential therapeutic approach to IRI.p < 0.05) were performed by comparing the individual time points  to sham mice. False discovery rate was corrected with Benjamini and Hochberg multiple testing correction (q < 0.05). Significantly enriched biological processes were identified using Biological Networks Gene Ontology with Benjamini and Hochberg multiple testing correction (p < 0.05) and then run on Enrichment Map with Jaccard coefficient of 0.5 .NFκB-mediated genes identified by Pahl et al. were compared to genes differentially expressed in kidney tissue at 4, 24 and 48 h following bilateral IRI in mice identified by Liu et al. to discover 34 NFκB-mediated genes expressed following IRI ,20. Heat2 as previously described [Immortalized proximal tubular HK-2 cells were cultured in equal parts Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F12 media supplemented with 10% fetal bovine serum, 10ng/mL epidermal growth factor, 5 μg/mL transferrin, 5 μg/mL insulin, 0.05 μM hydrocortisone, 50 units/mL penicillin, and 50 μg/mL streptomycin and incubated at 37 °C with 5% COescribed ,103.2 as previously described [Human primary renal proximal tubular epithelial cells (PTECs) were purchased from Lonza, Basel, Switzerland. The cells were cultured in DMEM/F12 media supplemented with 10% fetal bovine serum, 10 ng/mL epidermal growth factor, 5 μg/mL transferrin, 5 μg/mL insulin, 0.05 μM hydrocortisone, 50 units/mL penicillin, and 50 μg/mL streptomycin and incubated at 37 °C with 5% COescribed ,104® PCS100013™) were purchased from the American Type Culture Collection . HUVEC were grown to 80% confluency in Endothelial Cell Growth Medium  and maintained at 37 °C and at room air oxygen tension (21% O2) and 5% CO2 as previously described [Primary Umbilical Vein Endothelial Cells; Normal, Human, Pooled (HUVEC)  vector and incubated with fresh growth medium (DMEM/F-12) for 24 h, and then serum starved (serum-free DMEM/F-12) for 24 h. Cells were then treated with 10 ng/mL TNFα (NFκB), 50 ng/mL EGF (AP-1), or 5 ng/mL TGF-β (SBE) as a positive control for 6 h, 1 μM MS417 for 6 h, or a combination of 1 μM MS417 for 1 h then the varying positive control for 6 h. Control group was treated with serum free DMEM/F-12 for 6 h. Reporter activities were measured using the Promega, Madison Wisconsin dual-luciferase assay kit. The luciferase activity was normalized to the Renilla luciferase activity.2O2 in DMEM/F12 for 0 (control), 1, 6, or 24 h. Media was removed; cells were washed twice with PBS and then collected using a cell scraper. Cells were spun down at 14,000 rpm for 2 min and the pellet was stored at −80 °C. In a subsequent set of experiments, cells were pre-treated with 1 μM MS417 for 2 h, then 200 μM H2O2 for 6 h.Human primary proximal tubular epithelial cells were cultured until passage 6, serum starved for 24 h and then treated with 200 μM H2 and 1 mM MgCl2. All experiments were performed immediately after neutrophil isolation to minimize their in vitro activation.The protocol for human participation for research was reviewed and approved by The Hospital for Sick Children Research Ethics Board : Ethical Conduct for Research Involving Humans (2018)”. Written, informed consent was obtained from all participants before participation and experiments were performed as previously described . Neutrop2 and balanced N2 at 37 °C. PO2 within the hypoxic chamber was calibrated and monitored during experiments using a Proox 110 oxygen controller system . Cells were exposed to 2 h of hypoxia followed by treatment with 1 μM MS417 and varying periods of re-oxygenation ranging from 3 to 24 h, as indicated.Hypoxia/re-oxygenation was performed as previously described . Hypoxic5 cells/well) were then incubated with confluent HUVEC monolayers stimulated with TNFα and allowed to adhere for 30 min. Following incubation, non-adherent cells were removed by centrifuging the plate upside down. Adhesion was measured using a fluorescent plate reader at excitation and emission wavelengths of 494 and 517 nm, respectively. In another set of experiments, either neutrophils or HUVEC alone were treated with MS417 for 1 h, and unbound MS417 was removed prior to performing adhesion assays.Neutrophil-endothelial adhesion assays were performed as previously described . Freshly5 neutrophils per well) as per manufacturers’ instructions. Neutrophils were treated with varying concentrations of MS417 ranging from 1 nM to 1 mM for 1 hr at 37 °C. At endpoint, MTT labeling reagent -2,5-diphenyltetrazolium bromide or MTT) was added to each well at a final concentration 0.5 mg/mL. Cells were incubated for 4 h at 37 °C before adding 100 μL per well of the solubilization solution and incubated over night at 37 °C. Cell viability was measured by reading the absorbance on a microplate reader at 600 nm. The reference wavelength of 660 nm was used for background reduction. Cell viability following hypoxia and varying periods of re-oxygenation with 1 μM MS417 treatment was also measured. HUVEC were exposed to 2 h of hypoxia followed by treatment with 1 μM MS417 and 0, 6, 12 and 24 h of re-oxygenation with cell viability being measured as previously explained.Cell viability was assessed using a MTT assay kit I . The calorimetric MTT Assay was performed on freshly isolated human neutrophils in a 96-well format  following manufacturers’ instructions. Isolated RNA samples were reverse-transcribed using QuantiTect Reverse Transcription (Qiagen) following manufacturers’ instructions. Quantitative polymerase chain reaction was run on ViiA 7 Real-Time PCR System  using Taqman Fast Advanced Master Mix (Life Technologies). Human primers (Life Technologies) were purchased for the following genes: CCL2 (MCP1), Hs00234140_m1; CXCl2, Hs00601975_m1; IL6, Hs00174131_m1; and 18s, Hs03003631_g1. Mouse primers (Life Technologies) were purchased for the following genes: CCL2 (MCP1), Mm0044124_m1; TNFα, Mm00443260_g1; HAVCR1 (KIM1), Mm00506686_m1; LCN2 (NGAL), Mm01324470_m1; ICAM1, Mm00516023_m1; VCAM1, Mm01320970_m1; and GAPDH, Mm99999915_g1. Reaction mixtures were subjected to the amplification procedure as per the manufacturers’ instructions.n = 7–11/group). Mice were treated with 1 μM MS417 or saline (control) by oral gavage daily for 7 days prior to IRI. The core temperature of the mice was maintained between 34 °C and 36 °C with a heating pad, and a midline incision was made under anesthesia (isoflurane 2–3%). The renal pedicle was exposed and clamped for 45 min. The clamp was removed, allowing reperfusion of the kidneys for 6, 24, and 48 h. Mice were maintained at 30–32 °C until fully recovered. Sham-treated mice underwent the same procedure without clamping of the renal pedicle. At the time of sacrifice, mice were euthanized and kidneys, blood and bone marrow (femur) were collected.All animal experiments were approved by the University of Toronto Faculty of Medicine Animal Care Committee and the Research Ethics Board (protocol #20011495) and conducted in accordance to Canadian Council on Animal Care (CCAC) guidelines. C57BL/6 male mice  were randomized into groups and the groups were randomized on the day of surgery  for immunohistochemistry analysis. Fixed kidney tissue was then paraffin-embedded, sectioned, stained and scanned. Histopathological injury to the tubules was assessed by PAS-staining three-μm sections. Neutrophil infiltration was assessed by Ly6B.2 clone 7/4 antibody staining . Stained sections were visualized using the OlympusIX81 microscope and analyzed with ImageJ software. All sections were analyzed and quantified in a blinded manner by an experienced pathologist. Tubular injury was assessed over the entire outer medulla (corticomedullary border) based on tubular necrosis or luminal slough. Scores were assigned from 0 to 4 according to the percentage of involvement: 0—none; 0.5—<5%; 1—5 to 25%; 2—25 to 50%; 3—50 to 75%; 4—75 to 100%; 0.5 is added for any inner medullary tubular injury, and 0.5 is added for any cortical tubular injury. Semi-quantitative of neutrophil infiltration was assessed over the entire outer medulla (corticomedullary border) based on the proportion of area occupied by neutrophils. Scores were assigned from 0 to 4 according to the percentage of involvement: 0—none; 0.5—<5%; 1—5 to 25%; 2—25 to 50%; 3—50 to 75%; 4—75 to 100%; 0.5 is added for any inner medullary infiltration, and 0.5 is added for any cortical infiltration.−/−, 1% bovine serum albumin, 2 mM EDTA). Cells were blocked with rat serum  and mouse immunoglobulin IgG  and then labeled with an antibody master mix consisting of specific Cluster of Differentiation (CD) markers . Sample acquisition was performed using an LSR Fortessa or X-20  flow cytometer. Minimum of 2 × 105 gated events were acquired in each sample. Mouse neutrophils were gated as L Ly6G+ve/F4/80−ve. Negative staining for each antibody was established with appropriate fluorescence minus one (FMO) controls. Absolute neutrophils counts were determined as the product of total cell counts and the percentage gated neutrophils. Absolute neutrophil counts were per femur for BM, per 200 μL for blood, and per half kidney.After IRI, flow cytometry was used to quantify the infiltrating neutrophils and their subsets in the bone marrow, blood and kidney tissue. Sample fixation, labeling and flow cytometric analysis of mouse samples were performed as previously described . Kidneysp-value of less than 0.05 was considered significant with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.All results are expressed as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used unless otherwise stated. All statistical analysis was performed with GraphPad Prism 7 Software . A


Lignosus rhinocerus (LR). The aims of this study were to investigate the neuroprotective effect of three extracts of LR, including ethanol extract (LRE), cold water extract (LRC) and hot water extract (LRH), against glutamate-induced oxidative stress in mouse hippocampal (HT22) cells (in vitro model) as well as to determine their effect in Caenorhabditis elegans (in vivo model). We found that only LRE exhibited neuroprotective effects both in vitro  and in vivo (prevention of neurotoxicity in C. elegans). Therefore, active chemical constituents in LRE may serve as neuroprotectant candidates. Nevertheless, LRE extracts should be extensively studied for their neuroprotective activity in the future.Neurodegenerative diseases are recognized as one of the major public health issues in aging populations worldwide. High reactive oxygen species (ROS) cause oxidative stress, leading to cellular injury and neuronal cell death. While it has been used as traditional medicine, little is known about the neuroprotective effect of the Tiger Milk Mushroom Lignosus rhinocerus (LR) having been used as a traditional medicine, little is known about the neuroprotective effects of LR extracts. This study aims to investigate the neuroprotective effect of three extracts of LR against glutamate-induced oxidative stress in mouse hippocampal (HT22) cells as well as to determine their effect in Caenorhabditis elegans. In vitro, we assessed the toxicity of three LR extracts (ethanol extract (LRE), cold-water extract (LRC) and hot-water extract (LRH)) and their protective activity by MTT assay, Annexin V-FITC/propidium iodide staining, Mitochondrial Membrane Potential (MMP) and intracellular ROS accumulation. Furthermore, we determined the expression of antioxidant genes , superoxide dismutase (SOD1 and SOD2) and glutathione peroxidase (GPx)) by qRT-PCR. In vivo, we investigated the neuroprotective effect of LRE, not only against an Aβ-induced deficit in chemotaxis behavior (Alzheimer model) but also against PolyQ40 formation (model for Morbus Huntington) in transgenic C. elegans. Only LRE significantly reduced both apoptosis and intracellular ROS levels and significantly increased the expression of antioxidant genes after glutamate-induced oxidative stress in HT22 cells. In addition, LRE significantly improved the Chemotaxis Index (CI) in C. elegans and significantly decreased PolyQ40 aggregation. Altogether, the LRE exhibited neuroprotective properties both in vitro and in vivo.Despite the Tiger Milk Mushroom  Neurodegenerative disorders including Alzheimer’s diseases (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) involve degeneration or death of nerve cells ,2. One oLignosus rhinocerus (LR), found in Southeast Asia and China, has been used in folk medicine for asthma treatment [The Tiger Milk Mushroom reatment . Furtherreatment ,11,12. Treatment . Besidesreatment ,14.Mouse hippocampal (HT22) cells were used to study glutamate-induced oxidative stress. Glutamate can enhance intracellular ROS levels and lead to oxidative stress in HT22 because of their lack of an ionotrophic glutamate receptor . In consC. elegans, a free-living nematode, contains many genes, which are homologues to those of humans [C. elegans is widely used as an animal model for the study of oxidative stress, aging, longevity and neurodegenerative diseases [f humans . This nediseases ,18.C. elegans as an in vivo model for Morbus Alzheimer and Huntington.However, no or few studies have reported the protective effects of extracts from the Tiger Milk Mushroom in brain cells. Thus, in this study, we set out to investigate the potential neuroprotective effect of LR extracts against glutamate-induced oxidative stress, and also determined their effect on the expression of antioxidant genes in HT22 cells. In addition, we investigated potential neuroprotective effects of LR extracts in 2DCFDA) from Sigma-Aldrich GmbH . Dulbecco’s modified Eagle medium (DMEM)/low glucose, fetal bovine serum (FBS) and penicillin-streptomycin solution  were purchased from HyClone . Chloroform, diethyl pyrocarbonate (DEPC), isopropanol and L-glutamic acid were from Sigma-Aldrich ; primers and RT Premix were purchased from Bioneer . Phosphate Buffered Saline (PBS) was from Hyclone. Trypan Blue Stain and Trizol Reagent were from Invitrogen . Sodium azide was purchased from AppliChem GmbH , and EGCG was purchased from Sigma–Aldrich .Analytical grade ethanol was purchased from Merck . Dimethyl sulfoxide (DMSO), 2’, 7’-dichlorodihydrofluorescein diacetate (HL. rhinocerus (LR) was obtained from LiGNO Biotech™ Sdn Bhd, Selangor, Malaysia. This powder was extracted into three fractions with ethanol, cold water and hot water using the maceration technique. Briefly, 100 g of LR powder was macerated with 1 L of ethanol and the extract was placed on the shaker at 4 °C for 24 h. After that, it was filtered by using Whatman® No.2 filter paper and ethanol was removed by rotary evaporation  to yield the crude ethanol extract (LRE). Cold water extraction: 100 g of LR powder was suspended in sterile water and placed on the shaker at 4 °C for 24 h. For hot water extraction, sclerotial powder was extracted with water at 95–100 °C for 2 h. After that, the mixture was filtered and freeze dried by lyophilizer  to give a crude cold-water extract (LRC) and a crude hot-water extract (LRH). Yields of LRE, LRC and LRH were 0.73 g, 11.07 g and 10.13 g, respectively. Before starting the experiment, the crude extract of LRE was dissolved in DMSO, while the crude extracts of LRC and LRH were dissolved in sterile water to make the 100 mg/mL stock solution.A powder of cultivated strain TM02 of 2 at 37 °C. For treatment, The HT22 cells were divided into 10 groups including control group; DMSO-treated group; three control extract groups ; 0.25 mM N-acetylcysteine (NAC) as a positive control; four co-treatment groups with 5 mM glutamate including three extract-treated groups  and 0.25 mM NAC + 5 mM glutamate. All groups were incubated for 14 h in 5% CO2 in a 37 °C incubator.Mouse hippocampal HT22 cells  were cultured in a DMEM medium (Hyclone), supplied with 10% fetal bovine serum, in a humidified atmosphere containing 5% CO2 at 37 °C. Next, the cells were treated with the group of treatments as mentioned above and incubated at 5% CO2 at 37 °C for 14 h. After incubation, an MTT solution was added to each well and incubated for 3 h more in the incubator. After that, the insoluble formazan was dissolved with 10% SDS and incubated in the incubator overnight. The MTT product was measured at 570 nm using a microplate reader. The percent of cell viability was calculated by the following formular.MTT assay, a colorimetric assay, was used to assess metabolic activity in the cells. Briefly, HT22 cells were seeded into 96-well plates and incubated overnight at 5% CO5 cells) were seeded in a 12-well plate and incubated overnight at 5% CO2 at 37 °C. Next, the cells were treated with the group of treatments as mentioned before and incubated in the incubator for 14 h more. After incubation, cells were harvested, washed and stained with annexin V/PI solution for 15 min in the dark. Live and dead cells were determined by using a BD FACSCalibur™ flow cytometer . Data were collected for groups of at least 10,000 cells and results are shown as the percentage of apoptotic cells.A fluorescein isothiocyanate (FITC) conjugated form of Annexin V is used to detect apoptotic cells. HT22 cells  for 20 min. Next, the solution was removed and the cells were washed with 1X PBS and then 100 µL/well 1X PBS was added to the plate. The samples were measured with a microplate reader at an excitation of about 550 nm and emission of about 580 nm.The MMP was determined by using a commercial kit  including the cationic dye TMRE (tetramethylrhodamine ethyl ester perchlorate) and a mitochondrial membrane potential disruptor CCCP (carbonylcyanide 3-chlorophenylhydrazone) as a positive control for the test. TMRE, a cell membrane permeable fluorescent dye, was accumulated in intact mitochondria. Depolarized or inactive mitochondria exhibit decreased membrane potential, resulting in reduced TMRE accumulation. Briefly, cells were seeded in a 96-well plate and incubated overnight at 5% COIntracellular ROS were determined using the CM-H2DCFDA . After 14 h treatment, 10 μM of H2DCFDA was added to HT22 cells and incubated for 30 min at 37 °C, followed by washing three times with PBS. The fluorescence intensity  was measured using an EnSpire® Multimode Plate Reader  and the photographs were obtained using an Axio Observer A1 fluorescence microscope .In brief, total RNA was isolated from specific treatment cells using Trizol reagent). Using Accupower RT Premix (Bioneer), 1 μg of total RNA was converted to cDNA. Quantitative real-time PCR reaction was performed by using the Green Star PCR Master Mix where SYBR Green was included (Bioneer). Then, the specific genes CAT, SOD1, SOD2 and GPx were determined by the Exicycler Real Time Quantitative Thermal Block (Bioneer). The specific primers were previously reported by our research group . The relEscherichia coli OP50 as a food source and kept at 16 °C, except for AM141 which were kept at 20 °C.Strains CL2355 (smg-1(cc546) dvIs50 (pCL45 (snb-1::Abeta 1–42:3’ UTR (long) + mtl-2::GFP) I), CL2122 (dvIs15 ((pPD30.38)unc-54(vector) + (pCL26)mtl-2::GFP)) and AM141 (rmls133 (unc-54::54p::Q40::YFP)) were obtained from Caenorhabditis Genetics Center . All worms were maintained on a Nematode Growth Medium (NGM) agar containing Concerning age-synchronized worms, the eggs were isolated by a bleaching reagent (5M NaOH and 5% NaOCl). They were then vortexed for 10 min and centrifuged for 40 s at 1300 rpm. Next, the supernatant was discarded and the eggs washed with sterile water twice. Then, as much water as possible was removed. An 8 mL M9 buffer was added to a 60 × 15 mm petri dish and the eggs were transferred into the dish and placed in a 20 °C incubator for 16 h. The eggs in the M9 buffer were hatched and remained at the L1 larvae stage.E. coli OP 50 as a food source. Each strain of worms was divided into a control group, a DMSO-treated group and an LRE-treated group at concentrations of 50, 100 and 200 µg/mL, and 50 µg/mL EGCG (dissolved in DMSO) as a positive control. All worms were kept at 16 °C for 36 h, and then shifted to 23 °C and incubated for a further 36 h. Increased temperature is required for the pan-neuronal Aβ1–42 expression in the strain CL2355. After the incubation, the worms were washed three times with an M9 buffer to completely remove E. coli OP50. Finally, around 40 worms were placed in the center of a chemotaxis agar plate (94 mm). Before placing the worms there, 1 μL of the attractant odorant  together with 1 μL of 1 M sodium azide, used as a worm paralyser, were added to one side of the plate. On the opposite side, 1 μL of the control odorant (99.8% ethanol) along with 1 μL of 1 M sodium azide were added. Then, all plates were kept at 23 °C for 1 h. After that, the worms were counted from both sides and the chemotaxis index (CI) was calculated by the following formular:Na: Number of worms at the attractant positionNc: Number of worms at the control positionNt: Total number of worms on the plateTransgenic worms, including CL2355 with a pan-neuronal expression of the Human Abeta peptide, and CL2122, a control worm, were used. This assay was slightly modified from Wu Y. et al. . Both thThe synchronized transgenic AM141, expressing PolyQ40::YFP as a reporter gene, were treated the same as with the chemotaxis assay, Then, the worms were incubated at 20 °C for 72 h. After the incubation period, the worms were mounted on a glass slide with a drop of 10 mM sodium azide for paralysis and images of at least thirty worms per group were collected. Fluorescent expression was detected by BIOREVO BZ-9000 fluorescence microscope  using 10 × objective lens at constant exposure time. The number in the PolyQ40 aggregation, located in the muscle cells of the worms, was counted manually. The mean ± SEM was analyzed for three independent replications.p < 0.05 were considered statistically significant.Data are presented as the mean of three independent experiments (the mean ± SEM) and analyzed with GraphPad Prism 6. Statistical comparison between the control and treatments was performed using one-way ANOVA following Bonferroni’s method (post-hoc). Lifespan data were determined by log-rank (Mantel-Cox) tests followed by the Gehan–Breslow–Wilcoxon test. All the experiments were performed at least three times. Differences with p < 0.001 compared to control) , a cold-water extract (LRC) and a hot-water extract (LRH). The results showed that 5 mM glutamate significantly reduced the cell viability of HT22 cells by 44.71 ± 3.15% (control) . In addicontrol) a. Howevecontrol) b,c.In a second step, we investigated the role of apoptosis in glutamate-induced cell death in HT22 cells. In accordance with the MTT results, we selected concentrations of 100 and 200 µg/mL of LR and 5 mM glutamate. We found that 5 mM glutamate increased the percentage of apoptotic cells from 10.71 ± 2.91% in controls to 62.64 ± 8.64%. Apoptosis in LRE- and NAC-treated cells was similar to that of controls. However, co-treatment cells with LRE and glutamate reduced the percentage of apoptotic cells to 10.18 ± 4.266% and 9.796 ± 4.416% (LRE) and 10.73 ± 4.175% (NAC) a. HoweveGlutamate reduced the MMP of HT22 cells to 8.00 ± 9.23 compared to control (56.81 ± 11.28). Similarly, the cells, treated with CCCP as a positive control for MMP, significantly decreased the fluorescence intensity to 15.29 ± 7.947. LRE and 0.25 mM NAC alone had no influence on MMP. However, a co-treatment of the cells with 100 and 200 μg/mL of LRE and 5 mM glutamate significantly improved the TMRE fluorescence intensity in a dose-dependent manner compared to glutamate-treated cells at 47.19 ± 9.774, and 58.62 ± 14.36, respectively. Moreover, the co-treatment with NAC had a positive effect . Whereas, co-treatment of the cells with LRE (100 and 200 μg/mL) and 5 mM glutamate significantly reduced intracellular ROS accumulation in a dose-dependent manner compared to control to 126.6 ± 8.39%, and 116.1 ± 7.99% a; moreovTo better understand the antioxidant effects of LRE in previous experiments, we analysed the expression of genes that are potentially relevant in the antioxidant response of cells, such as the genes for catalase (CAT), superoxide dismutase (SOD1 and SOD2) and glutathione peroxidase (GPx).Glutamate treatment reduced the expression of CAT and GPx in cells. The expression of CAT, SOD1, SOD2 and GPx was significantly enhanced in cells co-treated with 100 and 200 μg/mL LRE or NAC and 5 mM glutamate a–d.The transgenic CL2355 worms express Aβ in nerve cells (Alzheimer model). As a physiological response, these nematodes show a reduced ability for chemotaxis. In order to determine potential neuroprotective effects, we selected three concentrations of LRE  that gave good effects in HT22 cells.The Chemotaxis Index (CI) increased in a dose-dependent manner when treated with 50, 100 and 200 μg/mL LRE at 0.37 ± 0.04, 0.45 ± 0.03 and 0.49 ± 0.03, respectively, compared to 0.48 ± 0.06 in the EGCG positive control. However, there was no difference detected in CI in transgenic CL2122 worms (without Aβ) when treated with LRE and 50 μg/mL EGCG .The accumulation of PolyQ40 proteins in transgenic AM141 worms was used as a model for Huntigton. When AM141 worms were treated with 50, 100 and 200 μg/mL LRE, the number of PolyQ40 aggregates was reduced in a dose-dependent manner to 30.18 ± 0.41, 26.38 ± 0.28 and 14.21 ± 0.36, respectively, compared to the DMSO control (49.40 ± 0.65). Similarly, the worms treated with 50 μg/mL EGCG showed a significantly reduced PolyQ40 aggregate accumulation .L. rhinocerus (LR) or Tiger Milk Mushroom have been used as a folk medicinal mushroom for treatment of asthma [Neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s Disease (HD) are related to an increase of intracellular reactive oxygen species (ROS) accumulation ,23,24,25f asthma . Severalf asthma ,30. Theyf asthma . In addif asthma .Glutamate, an excitatory neurotransmitter, has positive effects on several brain functions such as cognition, memory and learning . HoweverC. elegans. In this cell line, we induced oxidative stress by using 5 mM glutamate which has been reported to reduce approximately 50% of the cells [In the present study, we report, for the first time to our knowledge, the neuroprotection of LR extracts against glutamate-induced oxidative stress in HT22 cells and neurotoxicity in he cells ,43. Intehe cells . The prohe cells ,46,47,48he cells ,13,30. Hhe cells ,50,51. Uhe cells . In addihe cells . TherefoC. elegans, leading to an increase in the CI of the CL 2355 transgenic strain, containing the human Aβ peptide that is also known as a hallmark of AD [Furthermore, LRE exerted a neuroprotective effect against Aβ-induced deficits in chemotaxis behavior in rk of AD . Similarrk of AD ,65,66,67C. elegans in an Alzheimer and a Huntington model. However, further studies to identify the bioactive components and exact mechanism of LRE are required to support their ability for the development of a neuroprotective supplement in the future.From three extracts of the Tiger Milk Mushroom, only LRE showed the neuroprotective effect in hippocampal neuronal cells which is apparently mediated via inhibition of intracellular ROS accumulation and increases in both MMP and expression of antioxidant genes. In addition, LRE also had a neuroprotective effect in


Clear cell renal cell carcinoma (ccRCC) is the predominant cause of kidney cancer death attributed to its prevalence (70%) and its nature being the most aggressive form of kidney cancer. Most ccRCC deaths are resulted from metastasis. It is essential to know which ccRCCs are at risk of metastasis and the development to lethal disease; however, our capacity for such analysis remains poor. To improve this diagnostic capacity, we have examined a comprehensive ccRCC dataset containing 512 patients and have produced a 9-gene signature. This signature is novel; all its 9 components genes are unknown to be related to ccRCC. Importantly, all 9 individual genes possess significant ability in diagnosis of ccRCC metastasis and fatality; the combination of these genes or this signature predicts deadly ccRCCs at an impressive efficiency. This research will open new avenues in ccRCC research and will have a major impact in reducing ccRCC-associated deaths.n = 611) were derived from ccRCCs with (n = 111) and without IQGAP1 (n = 397) reduction using the TCGA PanCancer Atlas ccRCC dataset. These DEGs exhibit downregulations of immune response and upregulations of DNA damage repair pathways. Through randomization of the TCGA dataset into a training and testing subpopulation, a 9-gene panel (SigIQGAP1NW) was derived; it predicts poor OS in training, testing, and the full population at a hazard ratio (HR) 2.718, p < 2 × 10−16, p = 1.08 × 10−5, and p < 2 × 10−16, respectively. SigIQGAP1NW independently associates with poor OS  after adjusting for a set of clinical features, and it discriminates ccRCC mortality at time-dependent AUC values of 70% at 13.8 months, 69%/31M, 69%/49M, and 75.3%/71M. All nine component genes of SigIQGAP1NW are novel to ccRCC. The inclusion of RECQL4 (a DNA helicase) in SigIQGAP1NW agrees with IQGAP1 DEGs enhancing DNA repair. THSD7A affects kidney function; its presence in SigIQGAP1NW is consistent with our observed THSD7A downregulation in ccRCC (n = 523) compared to non-tumor kidney tissues (n = 100). Collectively, we report a novel multigene panel that robustly predicts poor OS in ccRCC.We observed associations of IQGAP1 downregulation with poor overall survival (OS) in clear cell renal cell carcinoma (ccRCC). Differentially expressed genes (DEGs,  Renal ctistics) . Clear ctistics) ,5 with atistics)  or a comtistics) . Inhibittistics) , mutatiotistics) , and protistics) , as the tistics) ,14. Losstistics) ,14. The tistics) ,8,9. HowA variety of serum and urine biomarkers have been actively investigated, including circulating tumor cells, cell-free tumor DNA, circulating RNA, and proteins. These biomarkers need substantial validation . Most reIQGAP1 belongs to the IQ motif GTPase-activating scaffold proteins (IQGAPs). IQGAP1 facilitates ERK activation, binds Cdc42 and Rac1, and stabilizes their GTP association, thereby induces cytoskeleton reorganization . With thn = 512), which can be used for survival analysis [n = 611) have been identified relative to IQGAP1 downregulation. Using the Elastic-net regression with 10-fold cross-validation, a 9-gene signature was constructed from a training population obtained by random division of the PanCancer cohort. This signature is novel; among the nine genes, three have not been reported for oncogenic involvement, and none has been reported in ccRCC. Importantly, this signature and its component genes robustly associate with reductions in OS, disease-specific survival (DSS), progression-free survival (PFS), and the adverse features  of ccRCC. Collectively, this research has generated a novel and robust gene signature with promising clinical applications in managing ccRCC patients.We performed a systemic study on the potential biomarker values of IQGAP1 in ccRCC. Using the TCGA PanCancer Atlas ccRCC dataset  [p < 0.05) (n = 111) and a low-risk group without the reduction (n = 399) using −0.8 SD cutoff point. From these two groups, a set of DEGs were obtained; at q < 0.0001, n = 6563 DEGs were identified. As IQGAP1 is reduced at the log2 value of −0.95 (fold 1.93 reduction) in the high-risk group (q = 2.54 × 10−57), we have thus defined DEGs at q < 0.0001 and fold change ≥ |1.93| or log2 value ≥ |0.95|, which resulted in n = 611 genes  , we show < 0.05) . We furt < 0.05) . To furt < 0.05) , we divi11 genes  that arehttps://metascape.org/gp/index.html#/main/step1) [To analyze the pathway or processes affected by the IQGAP1 network (DEGs), enrichment analysis was performed using the Metascape network (n/step1) . A set on/step1) . Other cThe above concept is further supported by gene set enrichment analyses. A robust downregulation of immune responses is observed, including downregulations of interferon γ response, IL-2-STAT5 signaling, allograft rejection, TNFα signaling, complement, inflammatory response, and others , Table 1n = 300) and testing (n = 208) population at the ratio of 6:4. The effectiveness of randomization was confirmed based on the distributions of age and other clinical features , which includes nine genes  The TCGA PanCancer ccRCC cohort contains comprehensive genetic and gene expression data obtained from primary or localized ccRCCs following resection; the cohort has been curated to support survival outcome analyses, including OS, disease-specific survival (DSS), and progression-free survival (PFS) . (2) As features . From thfeatures  for impane genes .i)n (fi: Cox coefficient (coef) of genei × genei expression, n = 9). Individual Cox coefs were obtained by multivariate Cox analysis. The scores significantly predict the fatality risk at HR = 2.72, 95% CI = 2.16–3.44, and p < 2 × 10−16  at HR = 1.82, 95% CI = 1.31–2.57, and p = 0.000421. Collectively, evidence supports SigIQGAP1 in assessing the risk of primary ccRCCs progression to a metastasis disease. Furthermore, all nine component genes of SigIQGAP1NW are individually associated with OS shortening (p = 6.57 × 10−9), SPACA6 (p = 4.18 × 10−6), LOC155060 (p = 2.62 × 10−5), LOC100128288 (p = 6.82 × 10−6), SNHG10 (p = 2.76 × 10−6), RECQL4 (p = 0.00661), HERC2P2 (p = 4.15 × 10−6), and ATXN7L2 (p = 4.01 × 10−5) are all independent risk factors of poor OS following the adjustment for age at diagnosis, sex, stage, tumor grade, and Winter Hypoxia Score. Taken together, evidence supports SigIQGAP1NW being a novel and robust multigene panel in predicting OS of ccRCC.To further examine SigIQGAP1NW biomarker potential, we investigated its relationship in predicting the fatality risk of ccRCC with other established clinical features. As expected, age at diagnosis, stage, tumor grade, and Winter Hypoxia Score are all clear risk factors of poor OS . ActivatC cohort . ConsistC cohort . Since aC cohort  supportsortening . Except In view of all nine component genes being individual predictors of OS shortening , we haveThe nine component genes identified to consist of long non-coding RNA (lncRNA) LINC01089, lncRNA LOC100128288, AI894139 pseudogene LOC155060, hect domain and RLD 2 pseudogene 2 HERC2P2, a non-protein-coding RNA SNHG10, and four protein-coding genes . LncRNAs are known to regulate gene expression in part via their sponge actions towards miRNAs ,28, and n = 2, PubMed) for a negative association with breast cancer metastasis in part via inhibition of the Wnt/β-catenin signaling [LINC01089 has been reported in a limited number of articles  relative to IQGAP1 downregulation affect pathways regulating the typical functions of IQGAP1 [We approach this clinical need through the investigation of a novel ccRCC factor IQGAP1. We demonstrate for the first time that downregulation of IQGAP1 is significantly associated with reductions of OS in ccRCC patients. The DEGs , we constructed a novel and robust multigene panel (SigIQGAP1NW) in assessing poor OS of ccRCC. We took advantage of two approaches in the signature construction: Random division of cohort into a training and testing population and the involvement of cross-validation in covariate selections from the training group. Although cross-validation can be equivalent to the conventional validation by splitting a dataset into a training set and a validation (testing) set, we trust that the inclusion of both in our study contributed to producing a robust gene signature. Additional favorable factors include the TCGA PanCancer Atlas ccRCC dataset being an excellent resource supporting OS biomarker studies .An attractive feature of SigIQGAP1NW is its novelty, in which none of the nine component genes has been previously reported in ccRCC . The impAnother feature of SigIQGAP1NW is the inclusion of five non-protein-coding genes among its nine component genes . With cuAmong the four protein-coding component genes, they likely influence different aspects of oncogenesis. SPACA6 (Sperm Acrosome Associated 6) is an oocyte factor and contributes to sperm-egg fusion , with itn = 1647) from the ASSURE cohort  revealed their modest prediction effectiveness with SSIGN being the best performer (C-index 0.688) which modestly outperformed the 2002 TNM system (C-index 0.603) [Taken together, we have constructed a novel and robust multigene panel SigIQGAP1NW in predicting poor prognosis in ccRCC. While its potential in clinical applications is appealing, further investigations into SigIQGAP1NW potential in ccRCC need to be performed both retrospectively and prospectively. The latter aspect is likely more critical. Assessment of RCC recurrence is a major event of patient management; clinically, this risk has been evaluated using a set of clinical features, including stage, grade, particularly the TNM system, and others . Eight cx 0.603) . Both UIx 0.603) . While ix 0.603) , were non = 7) panel was very recently constructed with a property to significantly predict ccRCC OS [The modest performance of these widely used clinical models clearly calls for reliable molecular events-based biomarkers. Together with our report here, the search for ccRCC biomarkers has shown promising progress lately. Based on the metabolic feature of ccRCC, a 10-gene set was recently reported, which predicts ccRCC OS . A set occRCC OS . FinallyccRCC OS . While thttps://www.cbioportal.org/) contains the most well-organized and comprehensive cancer genetic data for different cancer types. The TCGA PanCancer Atlas ccRCC dataset consists of 512 patients with primary ccRCC. All tumors have been surgically removed and profiled for RNA expression using RNA sequencing. The dataset has been well-demonstrated for its suitability in ccRCC OS biomarker studies [cBioPortal ,23 (http studies .https://metascape.org/gp/index.html#/main/step1) and Galaxy  for gene set enrichment.Enrichment analyses were performed using Metascape   regression analyses were performed using the R https://www.cbioportal.org/) was used to derive DEGs relative to IQGAP1 downregulation (n = 611). The random split of the dataset into a training and testing at 6:4 was performed using R. DEGs were selected for impact on OS using Elastic-net logistic regression within the glmnet package in R with 10-fold cross-validation. The mixing parameter of α was used at 0.5. At α = 0, Elastic-net operates as Ridge regression, which shrinks the coefficients of correlated predictors without performing covariate selection; at α = 1, it runs as Lasso, which tends to select one covariate among a group of related variables; this will reduce signature’s biomarker potential. Six rounds of selection at the setting were performed, and all unique genes obtained were combined into the final multigene panel SigIQGAP1NW.The TCGA PanCancer Atlas ccRCC dataset within the cBioPortal database ,23 (http1 × gene1exp + coef2 × gene2exp + … …+ coefn × genenexp), where coef1 … coefn are the coefs of individual genes and gene1exp … … genenexp are the expression of individual genes.All component genes were examined for an association with OS shortening using multivariate Cox PH regression with the R “survival” package. The signature scores for individual patients were given using Sum (coefMaxstat package) in R.Cutoff point to separate tumors with a high risk of mortality was estimated using Maximally Selected Rank Statistics  analysis was performed using the R timeROC package. A value of p < 0.05 is considered statistically significant.Kaplan-Meier surviving curves and log-rank test were carried out using the R We provide the first evidence for a significant association of IQGAP1 reductions with ccRCC mortality. IQGAP1 downregulation correlates with network changes consisting of 611 DEGs; these DEGs are enriched in pathways important to ccRCC and the typical features of the disease. Furthermore, from the IQGAP1-associated network, a novel 9-gene signature (SigIQGAP1NW) has been constructed; it robustly predicts ccRCC fatality and recurrence after nephrectomy with a high level of certainty. Furthermore, all nine component genes of SigIQGAP1NW are novel to ccRCC, and 5/9 are novel to oncogenic functions in general. We also provide the first evidence for ccRCC-associated alterations in two component genes, HERC2P2 and THSD7A. This research may have a profound impact on ccRCC with respect to research and patient management.A USA provisional patent for results reported in this manuscript was recently filed.


Previously, we have shown that AA enhances hyperoxic macrophage functions by reducing hyperoxia-induced HMGB1 release. Using a mouse model of HALI, we determined the effects of AA on hyperoxia-induced inflammatory lung injury. The i.p. administration of 50 mg/kg of AA to mice exposed to 72 h of ≥98% O2 significantly decreased hyperoxia-induced oxidative and nitrosative stress in mouse lungs. There was a significant decrease in the levels of airway HMGB1 , leukocyte infiltration  and improved lung integrity in mice treated with AA. Our study is the first to report that the dietary antioxidants, ascorbic acid and sulforaphane, ameliorate HALI and attenuate hyperoxia-induced macrophage dysfunction through an HMGB1-mediated pathway. Thus, dietary antioxidants could be used as potential treatments for oxidative-stress-induced acute inflammatory lung injury in patients receiving mechanical ventilation.Mechanical ventilation with hyperoxia is the major supportive measure to treat patients with acute lung injury and acute respiratory distress syndrome (ARDS). However, prolonged exposure to hyperoxia can induce oxidative inflammatory lung injury. Previously, we have shown that high levels of airway high-mobility group box 1 protein (HMGB1) mediate hyperoxia-induced acute lung injury (HALI). Using both ascorbic acid  and sulforaphane (SFN), an inducer of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), we tested the hypothesis that dietary antioxidants can mitigate HALI by ameliorating HMGB1-compromised macrophage function in phagocytosis by attenuating hyperoxia-induced extracellular HMGB1 accumulation. Our results indicated that SFN, which has been shown to attenute HALI in mice exposed to hyperoxia, dose-dependently restored hyperoxia-compromised macrophage function in phagocytosis (75.9 ± 3.5% in 0.33 µM SFN versus 50.7 ± 1.8% in dimethyl sulfoxide (DMSO) control,  Globally, acute respiratory distress syndrome (ARDS) accounts for 10% of intensive care unit admissions and results in 3 million deaths annually ,3, resulThe prolonged exposure to hyperoxia can induce excessive generation of reactive oxygen species (ROS), particularly superoxide, by both the mitochondrial electron transport chain and NADPH oxidases ,9,11. ThAlthough lung damage in HALI can result from direct damage caused by excessive ROS, it may also be mediated by the airway accumulation of high-mobility group box 1 protein (HMGB1), a damage-associated molecular pattern (DAMP) protein . Extrace2 with a site-directed mutation in the Nrf2 gene had an exacerbation of pulmonary hyper-permeability, neutrophilic inflammation and epithelial injury compared to wild type mice [Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the antioxidant response element (ARE)-mediated transcriptional activation of several endogenous phase 2 detoxifying and antioxidant enzymes ,22. Nrf2ype mice . FurtherAscorbic acid (AA) is a dietary supplement and an essential vitamin that scavenges ROS by donating electrons to produce a more reductive environment ,25,26,27In this study, we also tested whether SFN can attenuate hyperoxia-compromised macrophage phagocytosis and inhibit hyperoxia-induced extracellular accumulation using the established phagocytosis assay and Western blot analysis for HMGB1 in cultured macrophages as described in previously published studies ,16,17. Ip < 0.05, p < 0.05, p < 0.05; p < 0.05, Hyperoxia impairs macrophage function in phagocytosis, thereby diminishing the host defense in clearing apoptotic neutrophils, resulting in pronounced accumulation of leukocytes in the airways ,18,28,294 versus 2.2 ± 0.025 × 104 AU, p < 0.05, 4 AU in the 0.11 µM group, 2.4 ± 0.05 × 104 AU in the 0.33 µM group, and 1.93 ± 0.09 × 104 AU in the 1 µM group, compared to 2.9 ± 0.06 × 104 AU in the vehicle control group, p < 0.05, Nrf2 has been reported to have a prophylactic effect in animals model of ALI induced by hyperoxia, cigarette smoke, and oleic acid ,30,31,32p < 0.05, p < 0.05, To determine whether SFN-enhanced macrophage function under hyperoxic conditions is attributable to HMGB1-mediated mechanisms, we determined the extracellular levels of HMGB1 in the cell culture supernatant of RAW 264.7 cells exposed to hyperoxia. HMGB1 levels were significantly greater in the cell culture media under hyperoxic conditions compared to that of the room air control group  indicated that the lungs of hyperoxia-exposed mice were infiltrated by a significantly greater number of leukocytes compared to the lungs of mice exposed to room air  of AA did not significantly decrease the number of airway leukocytes in mice exposed to hyperoxia compared to saline-treated controls  for 72 h. Consistent with the data from cultured macrophages, there was a significant increase in the ORP in the airways of mice exposed to hyperoxia compared to mice exposed to room air  in macrophages . ORP is p < 0.05; i.p. of AA significantly reduced nitrite levels in mice exposed to hyperoxia, compared to the saline control , and seeded into tissue culture plates for experimental analysis. For all experiments, cells were allowed to adhere for 6 h, and then exposed to room air (21% O2) or 95% O2 in the presence or absence of R,S-sulforaphane  (0–1 µM) or sodium L-ascorbate for 24 h. Hyperoxic exposure was performed in sealed, humidified plexiglass chambers  flushed with a mixture of 95% O2 and 5% CO2. The MiniOx oxygen analyzer  was used to monitor O2 levels.Murine macrophage-like RAW 264.7 cells  were cultured in Dulbecco’s Modified Eagle Medium , supplemented with 10% fetal bovine serum . Cells were maintained at 37°C in 21% O2, and seeded into tissue culture plates for experimental analysis.BMDM were harvested as previously described (28), with minor modifications. Femurs from 8 to 12 weeks-old male C57BL/6 mice were removed with both heads intact and transferred to a petri dish containing RPMI-1640 media supplemented with L-glutamine , containing 10% fetal bovine serum, and 1% penicillin/streptomycin . Bones were placed in 70% ethanol for 1 min, washed with sterile phosphate buffered saline (PBS) (pH = 7.4) for 1 min, and the hip and knee joints were removed. The bone marrow was flushed into a sterile 15 mL centrifuge tube by passing PBS through the knee side of the bone until all the bone marrow was flushed. The marrow was dispersed by aspirating through a 19-gauge needle twice, then centrifuged at 1500 rpm for 5 min at 4 °C. Cells were resuspended, cultured, and differentiated in RPMI-1640 media containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 15% L929-cell conditioned media  as previously described . The cel2. Subsequently, the cells were incubated at 37 °C with fluorescin isothiocyanate (FITC)-labeled minibeads  opsonized in fetal bovine serum (FBS) at a ratio of 100:1 beads per cell. Cells were immediately placed on ice and washed with ice-cold PBS to stop phagocytosis, and extracellular beads were quenched with 0.4% trypan blue . Cells were fixed with 3.7% paraformaldehyde  at room temperature and nuclei were stained with DAPI , and the cytoplasm stained with rhodamine phalloidin  overnight to visualize the cytoskeleton. Cells were immersed in immersion oil  and imaged using the EVOS FL Auto Imaging System . Phagocytic activity was assessed by quantification of FITC minibeads per cell for approximately 200 cells per well using ImageJ Software .The phagocytosis assay was performed as previously described (29), with minor modifications. Cells were seeded in 24-well plates, allowed to adhere for 6 h, and incubated with SFN for 24 h in 95% OThe levels of intracellular ROS were determined using the OxiSelect Intracellular ROS Assay Kit , according to manufacturer’s protocol. Briefly, cells were plated in clear 96-well culture plates, allowed to adhere for 6 h, and incubated with SFN in the presence of hyperoxia for 24 h. Following incubation, the cells were washed 3 times with PBS, loaded with 100 µL 1X DCFH-DA and incubated at 37 °C for 30 min. Cells were again washed 3 times with PBS before adding 100 µL media and 100 µL 2X lysis buffer (1% phenymethanesulfonyl fluoride (PMSF)  and 1% protease inhibitor cocktail (PI)  in PBS) to each well for 5 min. Finally, 150 µL of the supernatant was transferred to a black, clear-bottomed 96-well culture plate and fluorescence was measured using the FilterMax F5 Multi-Mode Microplate Reader  at 480 nm/530 nm.2 in the presence or absence of SFN  in reduced-serum Opti-Mem media for 24 h. After incubation, the cell culture media was collected and concentrated in Centricon 10K  centrifuge tubes for 40 min at 4200 RPM and the concentrated samples were used for experiments.RAW 264.7 cells were seeded in 6-well plates and allowed to adhere for 6 h. Cells were placed in 95% OThe protein concentration in cell lysates was determined using the Pierce bicinchoninic protein assay kit , according to manufacturer’s protocol. Forty percent of the concentrated supernatants were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes . Nonspecific binding sites were blocked by incubating membranes for 1 h at room temperature in Pierce Clear Milk Blocking Buffer , diluted in Tris-buffered saline containing 1% Tween 20 (TBST). Membranes were washed 3 times with TBST and incubated overnight at 4 °C with anti-HMGB1 rabbit polyclonal primary antibody . The membranes were washed three times with TBST and incubated for 1 h with anti-rabbit horseradish peroxide-coupled secondary antibody . The membranes were again washed 3 times in TBST and immunoreactive proteins were visualized using the SuperSignal West Pico chemiluminescent substrate kit , according to manufacturer’s instructions. Images were developed using the ChemiDoc MP Imaging System  and the bands were quantified using ImageJ Software .ad libitum access to standard rodent food and water. Mice were randomized blindly to receive either sodium L-ascorbate   or saline (200 µL), administered by intraperitoneal injection, every 12 h, starting 24 h after the onset of hyperoxia exposure. A 2-sample t-test at the 0.05 significance level indicated that with 12 mice per treatment group, there is an 80% power to detect an effect size of 1.2. Thus, all animal experiments were performed three separate times with 3 to 4 mice each per treatment group. Mice were exposed to hyperoxia as previously described [i.p.) and BALF and lung tissues were collected for further analysis. The lungs were gently lavaged twice with 1 mL of sterile, nonpyrogenic PBS solution . BALF samples were centrifuged for 10 min at 1100 RPM and the supernatants were collected in Eppendorf tubes and stored at –80 °C.C57BL/6 mice  were used in this study, in accordance with the Institutional Animal Care and Use Committees of St. John’s University (protocol #1853 was approved on 7 April 2017). Mice were housed in a pathogen-free environment and kept in a 12 h light/dark cycle. All mice had Histopathological evaluation was performed on paraffin-embedded tissues as described previously . BrieflyOxidative stress was measured by assessing the oxidation-reduction potential (ORP) using the RedoxSYS Diagnostic System . Mice were exposed to hyperoxia and treated with AA as indicated. BALF was collected after treatment. A sensor strip was used to measure the static ORP (sORP) or redox status at room temperature. Briefly, the sensor was inserted into a replaceable sensor connector module, and 30 μL of sample was applied at the sample aperture on the disposable sensor strip. After sample application, the measurements were automated by the RedoxSYS Diagnostic System , which detected when sufficient sample equilibrium on the sensor strip was achieved.A Griess assay kit  was used to measure nitrate and nitrite species in BALF samples. All assays were performed at room temperature. A nitrate standard solution (100 µL) was serially diluted (from 100–1.6 µM) in duplicate in a 96-well, flat-bottomed, polystyrene microtiter plate. The diluting medium (PBS) was used as the standard blank. After loading the plate with samples (100 µL), the addition of vanadium (III) chloride (100 µL) to each well was rapidly followed by addition of the freshly mixed Griess reagents, sulfanilamide (50 µL) and N-(1-naphthyl)ethylenediamine dihydrochloride (50 µL). Absorbance was measured at 540 nm using a plate reader Synergy LX multi-mode reader  following a 30 min incubation period.a priori significance level was p > 0.05.The data are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. The data were analyzed using Student’s unpaired t test or ANOVA. The post hoc analyses were conducted using Dunnett’s or Sidak’s multiple comparison test. The In this study, we showed that the dietary antioxidants, AA and SFN, are prophylactic in hyperoxia-induced lung injury or hyperoxia-compromised macrophage function in phagocytosis. Congruent with the findings in published studies ,28, the


Arabidopsis thaliana are encoded by a gene family. Two mutants of one gene family member, AtCuAOδ, showed delayed seed germination, leaf emergence, and flowering time. The height of the primary inflorescence shoot was reduced, and developmental leaf senescence was delayed. Siliques were significantly longer in mutant lines and contained more seeds. The phenotype of AtCuAOδ over-expressors was less affected. Before flowering, there was a significant increase in putrescine in AtCuAOδ mutant leaves compared to wild type (WT), while after flowering both spermidine and spermine concentrations were significantly higher than in WT leaves. The expression of GA (gibberellic acid) biosynthetic genes was repressed and the content of GA1, GA7, GA8, GA9, and GA20 was reduced in the mutants. The inhibitor of copper-containing amine oxidases, aminoguanidine hydrochloride, mimicked the effect of AtCuAOδ mutation on WT seed germination. Delayed germination, reduced shoot height, and delayed flowering in the mutants were rescued by GA3 treatment. These data strongly suggest AtCuAOδ is an important gene regulating PA homeostasis, and that a perturbation of PAs affects plant development through a reduction in GA biosynthesis.Polyamines (PAs) are essential metabolites in plants performing multiple functions during growth and development. Copper-containing amine oxidases (CuAOs) catalyse the catabolism of PAs and in  Polyamines (PAs) are low-molecular-weight organic cations. They are present at relatively high concentrations in all eukaryotic cells and interact with a range of macromolecules including DNA, RNA, proteins, and phospholipids to stabilise them. In plants, PAs are involved in a wide range of crucial cellular processes, for example, controlling cell division and elongation, plant growth and development, reproduction, and senescence . Owing tThe most common PAs in plants are the tri-amine spermidine (Spd), the tetra-amine spermine (Spm), and their di-amine precursor putrescine (Put). In most plant species, the first PA produced from the biosynthetic pathway of PAs is Put, which is an important intermediate precursor in the synthesis of the higher amines Spd and Spm. In Arabidopsis, Put forms indirectly from L-arginine by arginine decarboxylase (ADC) . The con2O2, ammonia (NH4+), and 4-aminobutyraldehyde, which then directly cyclizes to ∆1-pyrroline that is further converted to GABA by the influence of aldehyde dehydrogenase  . cDNA synthesis was tested by PCR using ) method . All priv/v) cold methanol  using a microdevice. Deuterated GAs  and 13C6-IAA, 75 ng each  were added as internal standards to account for purification losses.Approximately 3 g of rosettes were extracted and purified as previously described ,44,45,46Methanol was evaporated at 35 °C under a vacuum and then the pH was adjusted to 2.8 and the aqueous phase was extracted with ethyl acetate. Extracts were dried and resuspended in 0.3–0.5 mL of water containing 0.01% acetic acid and 10% methanol. Samples were analysed by HPLC analysis using a Kontron instrument  with a UV detector (operating at 214 nm). The flow rate used to separate samples was 1 mL min−1 over 150 × 4.6 mm ID ODS Hypersil  with a particle size of 5 μm. Elution was isocratic, at 10% MeOH, for 4 min. Then, a double gradient from 10% to 30% MeOH was applied over 20 min, followed by 30% to 100% MeOH over 40 min. Fractions that corresponded to elution volumes of standard GAs were collected separately. Fractions were then separated on a 250 × 4.6 mm ID, Nucleosil 100-5 N(CH3)2 column  and eluted isocratically using 100% methanol that contained 0.01% acetic acid and a flow rate of 1 mL min-1. Fractions corresponding to elution volumes of standard GAs were collected, and then they were dried and silylated with N,O bis (trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane  for 1 h at 70 °C.−1. Oven temperature was kept at 80 °C for 2 min, then increased to 300 °C at 20 °C min-1. The injector and transfer line were at 250 °C and the ion source temperature was at 200 °C [−1. Data were the means of three biological replicates. Gibberellins were identified by comparison to standards using full mass spectra. Quantification was performed with reference to standard plots of concentration ratios versus ion ratios, obtained by analysing known mixtures of unlabelled and labelled GAs.A Saturn 2200 quadrupole ion trap mass spectrometer coupled to a CP-3800 gas chromatograph  equipped with a MEGA 1MS capillary column  was used for chromatography tandem mass spectrometry (GC-MS/MS) analysis . Dried at 200 °C . Full-sc


The only way out is to develop efficient, safe, and economical biomass processing protocols that can lead toward biofuels and fine chemicals. This research is one of such consequences involving the deconstruction and conversion of wheat straw carbohydrate constituents into reducing sugars via one-pot reaction promoted by Lewis acidic pyridinium-based ionic liquids (PyILs) mixed with different metal salts (MCl). Various parameters such as the type of metal salt, loading amount of metal salt, time, temperature, particle size of biomass, and water content which affect the deconstruction of wheat straw have been evaluated and optimized. Among the studied ionic liquid (IL) and metal salt systems, the best results were obtained with [BMPy] Solvents such as ethyl acetate and c et al. .254 Merck) and visualized under a UV lamp (UVGL-25 minor light multiband UV-254/366). TRS content produced was quantified by DNS assay using a UV–spectrophotometer . Spectroscopic analyses by FTIR and 1HNMR were recorded via Agilent Cary 630 FTIR with a scanning range of 4,000–400 cm−1 and Advance AV at 400 MHz, respectively. SEM was done via SEM Vega Tescon with variable pressure. Samples were made conductive by gold sputtering, and images were taken at different magnifications. The change in crystallinity due to delignification was recorded using PAN analytical X′pert PRO diffractometer using CuKα radiation (λ = 1.54 Å) at 40 kV and 30 mA with a scanning rate of 0.01°/s and scanning speed of 1°/min in a 2θ range of 10–80°.Progress of IL synthetic reaction was monitored via TLC , MP (95–97°C), literature MP (95°C) : 3,058 (Ar–H stretching), 2,962 , 1,633 (C = N), 1,505 (C = C), 1,157 (C–N).FTIR  δ (ppm): 0.85 , 1.24–1.29 , 1.88–1.93 , 2.46 , 4.83 , 7.85 , 8.27 , 8.56 , 8.62   .0). Reaction mixture was heated with constant stirring at a specified reaction temperature for a considerable time. After that, a 20 mg sample was withdrawn for sugar analysis.[BMPy]Cl (0.9g) was taken in a glass vial immersed in an oil bath and stirred constantly at 100°C for 10 min to ensure the removal of all possible moisture content. To this molten salt, metal chloride (10 wt%) was added and stirred for 30 min followed by the addition of wheat straw  is the concentration of TRS, M2 (g) is the mass of sample withdrawn from the reaction mixture, Mb (g) is the mass of wheat straw added to the reaction, M0(g) is the total mass of the reaction mixture, 150 is in multiples of diluted sample, and 1.11 is the ratio of molecular weight of sugar C6H12O6 to that of cellulosic content C6H10O5 of wheat straw.where CAfter saccharification reaction, antisolvents   on the response  was analyzed statistically to determine their mean ± SD. Analysis of average, standard deviation, and standard error was performed using IBM SPSS Statistics 21. One-way analysis of variance (ANOVA) was also performed by IBM SPSS Statistics 21 and OriginPro 8.5 at 0.05 level to determine the significance of differences between the response and independent variables  also predict the goodness of fit on the data.All the experiments were performed in triplicates, and the effects of independent variables, type of metal chloride and their different catalytic loadings, biomass loading into IL, time, temperature, biomass particle size, and water content were studied. From all these independent variables, effect of quantitative ones  associated with CoCl2·6H2O.Experiments were carried out at 100°C, and samples for TRS analysis were collected at two different time intervals: after 1 and 2 h. Percentage of TRS was calculated by using DNS assay while measuring the absorbance at 540 nm. Actually, the absorbance is due to the brown-colored 3-amino-5-nitrosalicylic acid (ANS) that is formed by redox reaction of 3,5-DNS with sugars , while the lignin was recovered by acetone evaporation from the filtrate followed by centrifugation. The retrieved lignin fraction was approximately 75 wt% of the original lignin content or 13 wt% in terms of original biomass content. As lignin is the main obstacle for utilization of carbohydrate content of biomass ; methyl, methylene, and methyne groups ; and diaryl C–O stretch and alkyl–aryl (R–O) stretch  . The untreated wheat straw appeared as rough flakes exhibiting highly rigid structure due to the presence of lignin and its compact binding with that of cellulose due to the cohesiveness imparted by hemicellulose. On the other hand, delignified wheat straw showed different morphology, having a highly porous flat sheet with a rough surface and thus making the enzymatic saccharification more favorable by allowing deeper penetration of enzymes.The effect of IL pretreatment on morphology and structure of wheat straw was noted in SEM images taken at different magnifications –C. The u+1HNMR : 3,359, 3,041 (Ar–H stretching), 2,958, 2,871 , 1,631 (C=N), 1,503 (C=C), 1,154 (C-N).FTIR  δ (ppm): 0.85 , 1.33 , 1.9 , 2.46 , 4.46 , 7.9 , 8.27 , 8.62 , 8.67 .2·6H2O was used with PyIL at 100°C for 2 h with 100 μm wheat straw particle size. In addition, 67 wt% lignin was separated from wheat straw by the same catalytic system. In this system, the percentage conversion of wheat straw carbohydrates content is somehow comparable to that of pure cellulose, indicating that other components, especially lignin, have little effect on carbohydrate conversion. Delignification and refinement of cellulose in regenerated material were confirmed by FTIR, SEM, and XRD analyses. The IL–metal catalytic systems showed greater regeneration (86% recovery for [BMPy]+IL-based catalysts containing [BMPy]Cl coupled with nine different metal chlorides have been applied for effective deconstruction of wheat straw biomass. The generation of TRS was 70%, which accounted for 32% conversion of carbohydrate contents of wheat straw when 10 wt% CoClThe raw data supporting the conclusions are provided in this article and its supplementary information.SN performed the experimental work and wrote the paper. MU supervised the work and corrected the manuscript. AA, NM, and FS have partially written the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate whether high glucose (HG) alters Rab20 expression and compromises gap junction intercellular communication (GJIC) and cell survival, retinal cells were studied for altered intracellular trafficking of connexin 43 (Cx43). Retinal endothelial cells (RRECs) and retinal Müller cells (rMCs) were grown in normal  or HG (30 mM glucose) medium for seven days. In parallel, cells grown in HG medium were transfected with either Rab20 siRNA or scrambled siRNA as a control. Rab20 and Cx43 expression and their localization and distribution were assessed using Western Blot and immunostaining, respectively. Changes in GJIC activity were assessed using scrape load dye transfer, and apoptosis was identified using differential dye staining assay. In RRECs or rMCs grown in HG medium, Rab20 expression was significantly increased concomitant with a decreased number of Cx43 plaques. Importantly, a significant increase in the number of Cx43 plaques and GJIC activity was observed in cells transfected with Rab20 siRNA. Additionally, Rab20 downregulation inhibited HG-induced apoptosis in RRECs and rMCs. Results indicate HG-mediated Rab20 upregulation decreases Cx43 localization at the cell surface, resulting in compromised GJIC activity. Reducing Rab20 expression could be a useful strategy in preventing HG-induced vascular and Müller cell death associated with diabetic retinopathy. Diabetic retinopathy is the leading cause of blindness in the working-age population in Western countries . The ear2+, and other molecules among them essential for cell survival, growth, proliferation, and homeostasis [GJIC enables the exchange of small molecules through gap junction channels granting passage of ions, nutrients, and other signaling molecules (up to 1 kD) between neighboring cells and is essential for the maintenance of retinal homeostasis . Connexieostasis ,30. Impoeostasis ,18,31,32eostasis . A clinieostasis .Studies indicate that Müller glial cells are in close apposition with each other, making frequent contact with the capillaries in the retina . Cx43 arMigration of Cx43 gap junction proteins to the cell surface is essential for GJIC activity. GTPases play an important role in regulating intracellular trafficking and facilitating membrane fusion and transport of proteins to the cell surface. Intracellular trafficking pathways comprise overlapping relays of GTPases acting sequentially, coordinating transport and membrane fusion by cycling between the membrane-bound, active GTP, and inactive, cytosolic GDP-states. One GTPase, Rab20, has been identified as a potential regulator of Cx43 trafficking . AlthougAlthough much has been elucidated in the deleterious effects of HG on GJIC activity and retinal cell apoptosis, the exact mechanism by which intracellular transport of Cx43 is reduced remains unclear. GTPases likely play a critical part in this pathway and warrant further exploration. Therefore, the current study was undertaken to investigate whether HG alters Rab20 expression and subsequently affects Cx43 localization, GJIC activity, and cell survival in RRECs and rMC-1.Endothelial cells exhibiting von Willebrand factor (vWF) were isolated from rat retinal capillaries (RRECs) as previously described  and usedTo assess whether reducing Rab20 overexpression influences Cx43 localization, GJIC activity, and cell viability, RREC or rMC-1 grown under HG condition were subjected to transfection with 40 nM Rab20 siRNA  or scrambled siRNA  as a negative control using 8 μM Lipofectin  diluted with Opti-MEM reduced serum medium (Invitrogen). Transfected cells were harvested and subjected to scrape load dye transfer (SLDT) analysis, immunostaining, immunoprecipitation, Western blot (WB) analysis, or differential dye staining after 7 days of HG exposure.To determine whether changes in Rab20 levels alter Cx43 distribution and localization, coimmunostaining was performed. Briefly, RRECs or rMC-1 were plated on glass coverslips and subjected to 4% paraformaldehyde fixation for 15 min at room temperature, then exposed to ice-cold methanol and incubated with 2% bovine serum albumin (BSA) for 60 min to block nonspecific antibody binding. Incubation with rabbit-Cx43 antibody solution  and mouse-Rab20 antibody solution  occurred overnight in a moisture chamber at 4 °C. The next day, the coverslips were incubated with antirabbit secondary antibody conjugated with FITC  and antimouse secondary antibody conjugated with Rhodamine Red (Jackson ImmunoResearch Laboratories) for 1 h at room temperature in the dark. Cells were photographed under a confocal microscope , and Cx43/Rab20 immunofluorescence staining data were obtained by counting Cx43 plaques on adjacent cell bodies and analyzing Rab20 immunofluorescence values normalized by the total number of cells per field using NIH ImageJ software, respectively. Specifically, Cx43 punctate “dots” in adjacent cells were counted in at least ten random fields from each experimental group, which were then normalized by the total number of cells per field to quantify Cx43 immunostaining .Total protein was isolated from RRECs or rMC-1, and IP for Rab20 was performed with the use of agarose beads as previously described . A totalTo investigate the effects of altered Rab20 expression on cell viability, differential dye staining assay was performed to identify apoptotic cells . The pri2+ and Mg2+ several times. Random cuts were then made in the monolayer using a razor blade. A solution containing PBS with 0.05% Lucifer Yellow  was applied to the cells and incubated at room temperature for 5 min. Following incubation, cells were rinsed with PBS containing 0.01% Ca2+ and Mg2+ three times. Cells were then fixed with 4% paraformaldehyde, visualized using a FITC filter, and photographed using a fluorescence microscope (Nikon). To evaluate GJIC activity, dye-coupled cell layers were counted and analyzed in at least ten random fields.SLDT assay is a technique to assess cell–cell coupling. Cells grown to confluent monolayer are subjected to random cuts allowing small molecules <1 kD in size to transverse between cells through gap junctions ,43. In pp < 0.05 represented statistical significance.Data are expressed as means ± SD. One-way ANOVA followed by Bonferroni post-hoc test was performed to assess differences between multiple groups. Six replicates were performed for each experiment, and the data were analyzed for statistical significance. p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; Following immunoprecipitation for Rab20, the expression level for Rab20 was significantly elevated in RRECs or rMC-1 grown under HG condition compared to those grown in normal glucose (NG) condition (RRECs: 130 ± 3% of NG vs. 100 ± 1% of NG; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; To investigate whether Rab20 upregulation influences Cx43 distribution and localization in RRECs or rMC-1, Cx43 immunostaining was performed. As expected, RRECs or rMC-1 grown in HG medium exhibited reduced Cx43 immunostaining (RRECs: 62 ± 3% of NG vs. 100 ± 5% of NG; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; p < 0.05; n = 6; To better understand the association between increased Rab20 expression and GJIC activity, gap-junction-mediated dye coupling between cells was evaluated through the SLDT assay in the context of Rab20 levels. The number of cells that the Lucifer Yellow dye has traversed from the point of “cut” represents the number of dye-coupled cell layers. In RRECs and rMC-1 grown under HG condition, the number of dye-coupled cell layers was significantly reduced compared to those grown in NG condition (RRECs: 59 ± 1% of NG vs. 100 ± 3% of NG; p < 0.01, n = 6; p < 0.05; n = 6; p < 0.01; n = 6; p < 0.05; n = 6; Differential dye staining assay was performed to investigate whether changes in Rab20 expression impact cell survival. As expected, a significant increase in the number of apoptotic cells was observed in RRECs and rMC-1 grown under HG condition compared to cells grown in NG condition (RRECs: 483 ± 18% of NG vs. 100 ± 32% of NG; To the best of our knowledge, this is the first study demonstrating that HG upregulates Rab20 expression in retinal endothelial cells and Müller cells. Moreover, this aberrant Rab20 upregulation effectively inhibits Cx43 intracellular trafficking to the cell surface, thereby compromising GJIC activity and promoting loss of retinal endothelial cells and retinal Müller cells associated with diabetic retinopathy. Interestingly, when HG-mediated Rab20 upregulation was reduced using Rab20 siRNA, it facilitated Cx43 localization at the cell surface, which is indicative of improved Cx43 intracellular trafficking. The increase in Cx43 localization at the cell surface was concomitant with effective GJIC activity and cell survival. Overall, these results indicate that a HG-induced increase in Rab20 levels may interfere with Cx43 intracellular trafficking and compromise cell–cell communication in retinal endothelial cells and Müller cells.Although the role of Rab20 in mediating Cx43 intracellular trafficking was examined in the current study, the exact functions of Rab20 are not well understood. Rab20 has been found to regulate phagosome maturation and contribute to the macropinocytic pathway ,45. ProvFindings from this study indicate that HG-mediated Rab20 upregulation in retinal endothelial cells and Müller cells impedes Cx43 localization to the cell surface, and that inhibiting Rab20 overexpression using a siRNA strategy could confer protection to these cells by improving GJIC and ultimately rescuing retinal vascular and glial cells from HG-induced apoptosis. Therefore, targeting Rab20 overexpression could be useful in improving cell–cell communication in retinal endothelial cells and Müller cells, and preventing neurovascular disruption associated with diabetic retinopathy.


Tenebrio molitor) are edible insects consumed in feed and food. In the current study, the optimal harvesting time of mealworm larvae for use as aquafeed was investigated during the ages of 30–90 days after hatching (DAH). Development of digestive enzymes, proximate composition, and in vitro protein digestibility using digestive enzymes from African catfish (Clarias gariepinus) and Nile tilapia (Oreochromis niloticus), were used as criteria. The specific activities of pepsin and trypsin significantly decreased with age (p < 0.05) from the first harvesting time until 50 and 45 DAH, respectively, while steadiness in these enzyme activities was observed onwards. Chymotrypsin specific activity appeared constant across all harvesting times. The specific activity of amylase significantly decreased in the later stages of development, while cellulase exhibited a different pattern suggesting it has a major role in dietary fiber utilization relative to starch. Regarding proximate compositions of the mealworm larvae, the moisture and ash contents decreased significantly with age, while the protein content exhibited the opposite trend with the highest contents from 60 to 90 DAH. Crude lipid was generally fairly constant, but its lowest value was observed in the earliest stage. In vitro protein digestibility was not significantly different across all harvesting times for both fish species, except for the significantly decreased digestibility value at 65 DAH relative to 30 and 35 DAH for Nile tilapia. However, based on the economic benefits of time for growth increment and proximate chemical composition, approximately 60 DAH is proposed as suitable for harvesting mealworm larvae to be used in fish feed.Mealworm larvae ( Tenebrio molitor) are edible insects consumed by people in many Asian countries, particularly in Southeast Asia [Clarias gariepinus [Salmo salar [Pagellus bogaraveo [Sparus aurata [Oreochromis niloticus [Pagrus major [Mealworm larvae  technique has been used to screen feedstuff or feed for animal rearing ,16,17. IMusa acuminata × Musa balbisiana) were placed on wheat bran to serve as a source of water and additives. The mealworm larvae were reared at a stocking density of 300 individuals per container, under a natural diurnal cycle (12-h dark/12-h light). Feces and dead mealworm larvae were removed from the container, and then new wheat bran was added to maintain 1 kg per container. The mealworm larvae were harvested at 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90 days after hatching (DAH). They were fasted for 12 h prior to sampling. All the samples were packed in polyethylene bags and chilled on ice for 30 min. At each harvesting time, twenty mealworm larvae were sampled while ten mealworm larvae (n = 10 per container) were weighed on a digital microbalance , and their lengths were measured to the nearest 0.01 mm using a digital Vernier caliper .Preparation, rearing and harvesting of all animals in the current study conformed to the “Ethical Principles and Guidelines for the Use of Animals for Scientific Purposes”, National Research Council, Thailand (Application No. U1-06514-2560). Ten-day-old mealworm larvae were purchased from a private farm in the Nakhon Si Thammarat province of Thailand. They were reared in three plastic containers (30 cm width × 40 cm length × 10 cm height) containing 1 kg of wheat bran each. Two equal pieces of a ripe banana (−1 of KMnO4 for 30 min [w/v) using a micro-homogenizer . The homogenate was centrifuged at 15,000× g for 30 min at 4 °C, and then the supernatant was collected, aliquoted, and kept at −20 °C until use. The concentration of protein in the crude enzyme extract was assayed using the method of Lowry et al. [−1) was used to quantify the enzyme-specific activities (U mg protein−1). All assays were performed within one month after extraction.Undesirable contamination was removed carefully from the mealworm larvae, and then they were soaked in 5 mg Lr 30 min ,19, rinsN-benzoyl-L-Arg-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrates, respectively. The assay conditions were pH 8.5 and 55 °C [p-nitroanilide at 410 nm was used to quantify the activities of both enzymes. The amylase activity was determined based on the method of Bernfeld [Pepsin activity was determined based on the method of Worthington  using hend 55 °C , and pH nd 55 °C , for theBernfeld , using sBernfeld . CellulaBernfeld , using cBernfeld . One unin = 3 pooled sample per treatment) were minced and analyzed for moisture and ash contents according to standard methods of the AOAC [TM reagent. Crude lipid was determined by extracting dried mealworm larval samples with ethyl acetate for 2 h in a rotary mixer, as described in Supannapong et al. [Whole larval mealworm carcasses  at a ratio of 1: 6 (w/v), using a micro-homogenizer . Centrifugation was performed at 15,000× g for 30 min at 4 °C prior to collecting the supernatant. The aliquots were kept at −20 °C until used for the IVD assay.For the IVD test, thirty Nile tilapia (1.39 ± 0.28 g body weight) and African catfish (5.45 ± 0.24 g body weight) were purchased from a private farm in Songkhla province of Thailand. The fish were fed a commercial diet containing 18 and 25% crude protein , respectively, and they were fasted for 12 h prior to sampling. The fish were sacrificed by chilling in ice, and only the intestine was carefully removed, weighed, and then used for digestive enzyme extraction. Three pooled intestinal samples (ten fish per sample) were extracted in 0.2 M Na2HPO4-NaH2PO4 buffer (pH 8.2) was added to perform the alkaline condition, contributing the near-optimal pH for digesting by fish trypsin. These mixtures were incubated at 30 °C under 200 rpm agitation for 24 h, against blank samples in which the enzyme volumes were replaced by extraction buffer. This condition provides a linear range of digested products  after performing substrate: enzyme ratio (200: 1 w/v) for 24 h. The reaction was terminated by boiling at 100 °C for 10 min and then cooled down at room temperature for 30 min. The liberated amino acids in the digested solution were determined based on the trinitrobenzene sulfonic (TNBS) acid method, as described by Rungruangsak-Torrissen et al. [2HPO4-NaH2PO4 buffer (pH 8.2), and 1 mL of 0.1% TNBS. These mixtures were incubated in the dark at 60 °C for 1 h and then stopped by adding 1 mL of 1 M HCl. The colorimetric detection was measured spectrophotometrically at 420 nm against a linear range of standard DL-alanine. Trypsin activity in dialyzed crude enzyme extract from triplicate samples was analyzed [DL-alanine equivalent g mealworm larvae−1.Fresh mealworm larvae were freeze-dried for 24 h using a freeze dryer  to eliminate moisture, and then they were minced to a homogeneous powder. The crude digestive enzymes from fish were dialyzed overnight against the extraction buffer. The in vitro protein digestibility procedure was performed as described by Thongprajukaew et al. . The rean et al. . The reaanalyzed  before sp < 0.05 in all statistical analyses. Relationships between each pair of digestive enzymes were assessed from Pearson correlations.The experiment followed a completely randomized design (13 treatments × 3 replications). All the data were analyzed using the Statistical Package for Social Sciences, Version 14  and were reported as mean ± standard error of the mean (SEM). The normality and homogeneity of variance were checked. Means were compared using One-Way ANOVA, and differences were statistically analyzed with Duncan’s multiple range test and regarded as significant at y = (−6 × 10−10)x6 + (2 × 10−7)x5 − (3 × 10−5)x4 + 0.002x3 − 0.106x2 + 2.375x − 20.44 and y = (−2 × 10−7)x6 + (6 × 10−5) − 0.007x4 + 0.569x3 − 22.16x2 + 449.2x − 3697, respectively. The larval body lengths and weights at 30 to 90 DAH are illustrated in The length and weight of the mealworm larvae increased progressively from 30 DAH on to 50 DAH and to 55 DAH, respectively a. A stabp < 0.05). High specific activity of both enzymes was observed from the first sampling time until 45 and 40 DAH, respectively, and then a steady level was maintained until 90 DAH. On the other hand, chymotrypsin specific activity appeared constant across all sampled times  of protein, using digestive enzymes from Nile tilapia was generally similar across thirteen sampled times, but significantly decreased IVD was observed at 65 DAH relative to 30 and 35 DAH a. There Thrips palmi), soybean looper (Pseudoplusia includens), Mexican bean beetle (Epilachna varivestis), and velvetbean caterpillar  [The experiment was terminated at 90 DAH since some mealworm larvae were transitioning into the pupal stage. Weight and length of mealworm larvae fitted the polynomial regression models well, providing correlations of 0.962–0.985. These morphometric parameters were relatively constant from 65 and 80 DAH, respectively. These findings indicate that the growth rate was slower than linear, as the larvae spend their time eating and growing for the next transformation, concurrently with molting. This polynomial growth pattern has also been reported in the same species by Özsoy , as wellmatalis) ,34.r = 0.761, p < 0.01, n = 39) between these enzymes throughout sampling. As regards chymotrypsin, this enzyme is activated by trypsin. Its relatively constant activity suggests a minor role of this enzyme in digesting protein, relative to pepsin-like enzymes or trypsin.Fly enzymes appear functionally to be similar to vertebrate enzymes . In the r = 0.417, p < 0.01, n = 39) between the specific activities of pepsin and amylase. Over the sampled times, significant changes in carbohydrate-digesting enzymes (amylase and cellulase) were observed 10 days prior to the end of sampling, as compared to the youngest stage. These findings suggest that sources high in cellulosic materials might be more suitable than starch-based materials. This matches well with the proximate composition of wheat bran from the current study and the typical feed for this species  supplemented with protein sources such as soybean flour, skimmed milk powder, or yeast [Physiological changes between protein and carbohydrate catabolism occur concurrently, as indicated by the significant positive relationship  [Barbonymus gonionotus) [Hyriopsis (Hyriopsis) bialatus) [Nauphoeta cinerea) meal, superworm (Zophobas morio) larvae meal, Madagascar hissing cockroach (Gromphadorhina portentosa) meal, and Jamaican field cricket (Gryllus assimilis) meal [IVD values depend on the quality of feedstuffs rather than their quantity ,45,46. Ilendens) , Nile tiialatus) ,48. Baseis) meal .Digestive enzymes in mealworm larvae were genetically programed across thirteen developmental stages since only one type of feed was used throughout the experiment. Of the five digestive enzymes studied, carbohydrate-digesting enzymes (cellulase and amylase) play a major role in utilizing nutrients relative to protein-digesting enzymes . The proximate composition of the mealworm larvae varied with the developmental stage, with protein being the major constituent, which is why they could be an excellent source of protein. However, mealworm larvae are not suitable as the main feed ingredient due to their high amounts of lipids, mainly unsaturated fatty acids, but they could be used in a mixture of different insects to obtain an ideal nutritional medium ,42. For