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

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

Women of color and the hidden curriculum of school psychology doctoral programs.

Doctoral training programs educate future practitioners, scholars, and researchers. They therefore are an important site of inquiry for critical school psychologists interested in interrogating and confronting the inequities that exist within the field. We conducted four focus groups with 15 Black, Indigenous, and Women of Color (BIWOC) students in various APA-accredited school psychology Ph.D. programs to understand how they experienced their programs. We argue that the programs delivered a hidden curriculum to the participants through various socializing, vicarious, and disciplinary events, in addition to the official curriculum delivered to all students. Data analysis indicated that the hidden curriculum was delivered in four settings and consisted of six lessons, including (a) you do not belong here, (b) you cannot be trusted, (c) you are on your own, (d) you are not safe here, (e) you are a token, and (f) you will only get performative allyship from us. We discuss each of these lessons and contemplate on ways in which programs and faculty can combat their deleterious impact on the students.

The invisible work of persisting: BIWOC students in school psychology doctoral programs.

There are growing calls to diversify the ranks of school psychology practitioners, graduate educators, and researchers by recruiting more students of color in school psychology doctoral programs. Past research on retention across many fields in higher education indicates that Black, Indigenous, and women of color (BIWOC) students entering doctoral programs encounter isolation, lack of support, and microaggressions. Although this literature has illuminated how doctoral programs can push out BIWOC students, it has been critiqued for overlooking the creative and strategic means they use to stay in their programs. We analyzed 12 focus group interviews conducted with 15 BIWOC students in school psychology doctoral programs across the United States. Using the analytical construct of agency, we coded the transcripts to identify agentic actions of BIWOC that went beyond standard graduate school demands. We identified six types of actions that BIWOC carried out to combat the systemic barriers they encountered: teaching, protecting others, self-advocating, organizing, searching for community, and self-editing. Given that these actions were in addition to the basic program requirements, we argue that they are instances of the invisible work that BIWOC students did to persist in their doctoral programs. We discuss the implications of this invisible work and provide various recommendations for school psychology doctoral programs to reduce the burden of invisible work on BIWOC students. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Exploring New Functional Aspects of HTLV-1 RNA-Binding Protein Rex: How Does Rex Control Viral Replication?

After integration to the human genome as a provirus, human T-cell leukemia virus type 1 (HTLV-1) utilizes host T cell gene expression machinery for viral replication. The viral RNA-binding protein, Rex, is known to transport unspliced/incompletely spliced viral mRNAs encoding viral structural proteins out of the nucleus to enhance virus particle formation. However, the detailed mechanism of how Rex avoids extra splicing of unspliced/incompletely spliced viral mRNAs and stabilizes them for effective translation is still unclear. To elucidate the underlying molecular mechanism of Rex function, we comprehensively analyzed the changes in gene expression and splicing patterns in Rex-overexpressing T cells. In addition, we identified 81 human proteins interacting with Rex, involved in transcription, splicing, translation, and mRNA quality control. In particular, Rex interacts with NONO and SFPQ, which play important roles in the regulation of transcription and splicing. Accordingly, expression profiles and splicing patterns of a wide variety of genes are significantly changed in Rex-expressing T cells. Especially, the level of vPD-L1 mRNA that lacks the part of exon 4, thus encodes soluble PD-L1 was significantly increased in Rex-expressing cells. Overall, by integrated analysis of these three datasets, we showed for the first time that Rex intervenes the host gene expression machinery throughout the pathway, probably to escort viral unstable mRNAs from transcription (start) to translation (end). Upon exerting its function, Rex may alter the expression level and splicing patterns of various genes, thus influencing the phenotype of the host cell.

C-type lectin Mincle mediates cell death-triggered inflammation in acute kidney injury.

Accumulating evidence indicates that cell death triggers sterile inflammation and that impaired clearance of dead cells causes nonresolving inflammation; however, the underlying mechanisms are still unclear. Here, we show that macrophage-inducible C-type lectin (Mincle) senses renal tubular cell death to induce sustained inflammation after acute kidney injury in mice. Mincle-deficient mice were protected against tissue damage and subsequent atrophy of the kidney after ischemia-reperfusion injury. Using lipophilic extract from the injured kidney, we identified ß-glucosylceramide as an endogenous Mincle ligand. Notably, free cholesterol markedly enhanced the agonistic effect of ß-glucosylceramide on Mincle. Moreover, ß-glucosylceramide and free cholesterol accumulated in dead renal tubules in proximity to Mincle-expressing macrophages, where Mincle was supposed to inhibit clearance of dead cells and increase proinflammatory cytokine production. This study demonstrates that ß-glucosylceramide in combination with free cholesterol acts on Mincle as an endogenous ligand to induce cell death-triggered, sustained inflammation after acute kidney injury.

Effect of the natural compound trans­resveratrol on human MCM4 gene transcription.

trans­Resveratrol (Rsv) is a natural compound contained in red wine and grape skins that has various beneficial effects for organisms such as lengthening of their life span. Rsv induces expression of the human TP53 and HELB genes, which are involved in the regulation of DNA maintenance. In the present study, a luciferase expression vector containing 309 bp of the 5' upstream end of the human MCM4 gene was transfected into HeLa S3 cells. A luciferase assay revealed that Rsv treatment increased the minichromosome maintenance 4 (MCM4) gene promoter activity by GC­box and GGAA (TTCC) motifs. Electro phoretic mobility shift assay revealed that the specific binding factor (complex) contains PU.1 (SPI1). Quantitative reverse transcription­polymerase chain reaction analysis indicated that MCM4 gene expression was transiently induced by Rsv. Moreover, western blotting revealed that the SP1/PU.1 ratio markedly increased after Rsv treatment, indicating that a balance or profile of these transcription factors may control Rsv­inducible initiation of transcription. These observations indicated that the beneficial effects of Rsv can be attributed to induction of the chromosomal DNA maintenance factor encoding gene expression.

Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis-associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects.

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development.

[Determination of Nitrite in Meat and Fish Products using Dialysis Extraction].

A simple and useful method for the determination of nitrite in meat and fish products was developed. The sample (2.5 g) was extracted and cleaned up by dialysis in tris hydroxymethyl aminomethane solution with shaking at 80℃ for 2.5 hr. Nitrite in the dialysate was quantified by colorimetric analysis. Furthermore, the dialysate was cleaned up with SPE under cooling, and nitrite in the resulting solution was determined using LC-UV with an anion exchange column for confirmation. The recoveries of nitrite from frankfurter and fish sausage, fortified at the levels of 0.002 g/kg and the maximum usage dose (0.070 g/kg for meat products, 0.050 g/kg for fish sausage) ranged from 82.6 to 104.8% in colorimetric analysis and from 88.3 to 97.6% in LC-UV confirmation analysis. The values determined in various meat and fish products by the developed method and by the Japanese official method were approximately equivalent.

Assessment of in vitro antiovulatory activities of nonsteroidal anti-inflammatory drugs and comparison with in vivo reproductive toxicities of medaka (Oryzias latipes).

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used therapeutic agents; however, their pharmacological actions raise concerns about potential risks to the reproductive health of aquatic vertebrates. In the present study, a medaka ovulation assay was applied as an in vitro model to evaluate NSAID-induced antiovulatory activity. We first tested five NSAIDs, including diclofenac sodium (DCF), ketoprofen (KP), salicylic acid (SA), mefenamic acid (MA), and acetylsalicylic acid (ASA) for their antiovulatory activities toward the follicles isolated from the ovaries of spawning females. Of all the chemicals tested, DCF had the highest antiovulatory activity, with the concentration that caused 50% inhibition (IC50) (101 µM). MA was the second most potent inhibitor following DCF, but KP, SA, or ASA had little inhibitory effect on the ovulation of the follicles. The in vitro antiovulatory activity of five NSAIDs showed good correlation with data published on the inhibitory activity on human COX-2. Second, we selected DCF and SA as the most and least potent NSAIDs, respectively, and examined the effects on reproduction of intact fish in order to evaluate whether the ovulation assay was a reasonable predictor of potential reproductive effects in fish. Females exposed to DCF showed a concentration-dependent decrease in the number of spawned eggs and an increment in the gonadosomatic index (GSI), possibly due to an anovulation in the females. In contrast, neither fecundity nor the GSI of females decreased at up to 20 mg/L of SA, at which acute lethality to medaka was induced. In conclusion, the medaka ovulation assay reflected the potency of NSAID-induced antiovulatory activity and may thus serve as an in vitro model for the prediction of NSAID-induced reproductive toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1710-1719, 2016.

The cardioprotective effects of novel Na+/H+ exchanger inhibitor TY-51924 on ischemia/reperfusion injury.

The inhibitory effects of sodium 3-guanidinocarbonyl-2-methyl-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-9-ylmethyl sulfate monoethanolate (TY-51924) are selective for Na(+)/H(+) exchanger (NHE)-1 in PS120 cells expressing human NHE isoforms assayed by NH(4)Cl prepulse technique. The median inhibitory concentrations (micromolar) of TY-51924 were 0.095 ± 0.008 (NHE-1), 0.621 ± 0.093 (NHE-2), and >100 (NHE-3). In anesthetized dogs subjected to 90 minutes ischemia/300 minutes reperfusion, intravenous bolus TY-51924 at 5 and 10 mg/kg administered 5 minutes before reperfusion reduced infarct size. The infarct size reduction ratios of TY-51924 at 5 and 10 mg/kg versus vehicle were 32.8% and 52.4%, respectively. But TY-51924 at 10 mg/kg administered 10 minutes after reperfusion did not reduce infarct size. In 2-step intravenous infusion initiated 15 minutes before reperfusion, TY-51924 at low dose (3.8 mg/kg per 5 minutes, then 6.2 mg/kg per 20 minutes) and at high dose (7.6 mg/kg per 5 minutes, then 12.4 mg/kg per 20 minutes) reduced infarct size. The infarct size reduction ratios of TY-51924 at 10 and 20 mg/kg versus vehicle were 39.2% and 51.7%, respectively; plasma drug concentrations at reperfusion were 16.8 and 38.8 µg/mL, respectively. This indicates that maintaining a plasma drug concentration of >20 µg/mL at reperfusion enables TY-51924 to reduce infarct size by inhibiting the NHE, which is activated during the early period of reperfusion.

Microtubule disruption with BAPTA and dimethyl BAPTA by a calcium chelation-independent mechanism in 3T3-L1 adipocytes.

While the physiological role for calcium in the insulin action on glucose transport has been disputed, it was reassessed in a recent study by using a calcum chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). Although BAPTA has been widely used to study the role for calcium in a variety of cell functions, it has also been suggested to have properties unrelated to the calcium chelating activity. Here, we investigated the effects of BAPTA and dimethyl BAPTA on the cytoskeletons in 3T3-L1 adipocytes. Both calcium chelators were successfully loaded in 3T3-L1 adipocytes and inhibited endothelin-1-induced cytosolic calcium elevation. Confocal fluorescence microscopy revealed that BAPTA and dimethyl BAPTA caused profound depolymerization of the microtubules without affecting the cortical actin filaments in 3T3-L1 adipocytes. Biochemical quantification also showed that BAPTA and dimethyl BAPTA significantly decreased the amount of polymerized tubulin but had little effect on filamentous actin. Consistent with these results, GLUT4-positive perinuclear compartments were dispersed throughout the cytoplasm in BAPTA- or dimethyl BAPTA-loaded adipocytes. Intriguingly, these calcium chelators did not disrupt the microtubules in undifferentiated preadipocytes. The microtubule-depolymerizing property of BAPTA and dimethyl BAPTA is unrelated to calcium chelation, since the microtubules were resistant to depletion of cytosolic calcium by using a calcium ionophore A23187. Insulin-stimulated glucose transport was not affected by cytosolic calcium depletion with A23187, but significantly inhibited with BAPTA and dimethyl BAPTA to the extent similar to that with nocodazole. BAPTA and its derivatives should be used with caution in studies of cytoskeleton-related cell functions.