27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways.

The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV- and chemotherapeutic drug-induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU-based chemotherapy.

Diagnostic Utility of TUNEL Staining for Degenerative Keratoacanthoma Requiring Pathologic Differentiation from Seborrheic Keratosis.

Tumors developed in 2 old women presented with pathological findings similar to seborrheic keratosis, although the clinical feature of tumor showed typical keratoacanthoma. In addition to these two cases, we compared the pathological findings of a total of four cases, one case each of keratoacanthoma and seborrheic keratosis, which were clinically and histopathological typical. These two cases and the typical keratoacanthoma showed cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and infiltration of cytotoxic T cells. The keratoacanthoma in the decompensated stage may be histologically similar to seborrheic keratosis. TUNEL staining can help in the diagnosis of fading keratoacanthoma.

C9orf72-Derived Proline:Arginine Poly-Dipeptides Modulate Cytoskeleton and Mechanical Stress Response.

Proline:arginine (PR) poly-dipeptides from the GGGGCC repeat expansion in C9orf72 have cytotoxicity and bind intermediate filaments (IFs). However, it remains unknown how PR poly-dipeptides affect cytoskeletal organization and focal adhesion (FA) formation. Here, we show that changes to the cytoskeleton and FA by PR poly-dipeptides result in the alteration of cell stiffness and mechanical stress response. PR poly-dipeptides increased the junctions and branches of the IF network and increased cell stiffness. They also changed the distribution of actin filaments and increased the size of FA and intracellular calcium concentration. PR poly-dipeptides or an inhibitor of IF organization prevented cell detachment. Furthermore, PR poly-dipeptides induced upregulation of mechanical stress response factors and led to a maladaptive response to cyclic stretch. These results suggest that the effects of PR poly-dipeptides on mechanical properties and mechanical stress response may serve as a pathogenesis of C9orf72-related neurodegeneration.

Gene Expression Profiles of Human Cerebral Organoids Identify PPAR Pathway and PKM2 as Key Markers for Oxygen-Glucose Deprivation and Reoxygenation.

Ischemic stroke is one of the most common neurological diseases. However, the impact of ischemic stroke on human cerebral tissue remains largely unknown due to a lack of ischemic human brain samples. In this study, we applied cerebral organoids derived from human induced pluripotent stem cells to evaluate the effect of oxygen-glucose deprivation/reoxygenation (OGD/R). Pathway analysis showed the relationships between vitamin digestion and absorption, fat digestion and absorption, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and complement and coagulation cascades. Combinational verification with transcriptome and gene expression analysis of different cell types revealed fatty acids-related PPAR signaling pathway and pyruvate kinase isoform M2 (PKM2) as key markers of neuronal cells in response to OGD/R. These findings suggest that, although there remain some limitations to be improved, our ischemic stroke model using human cerebral organoids would be a potentially useful tool when combined with other conventional two-dimensional (2D) mono-culture systems.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

Brainstem Organoids From Human Pluripotent Stem Cells.

The brainstem is a posterior region of the brain, composed of three parts, midbrain, pons, and medulla oblongata. It is critical in controlling heartbeat, blood pressure, and respiration, all of which are life-sustaining functions, and therefore, damages to or disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells (hPSCs) recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models are limited in the extent hPSCs recapitulate human brain development and hence are not able to fully elucidate the diseases affecting various components of the brain such as brainstem. Here, we developed a method to generate human brainstem organoids (hBSOs), containing midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, dopaminergic neurons, and neural crest lineage cells. Single-cell RNA sequence (scRNA-seq) analysis, together with evidence from proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem, which raises the possibility of making use of hBSOs in investigating central nervous system disorders affecting brainstem and in efficient drug screenings.

Soluble HLA-G is absent from human embryo cultures: a reassessment of sHLA-G detection methods.

In recent years, the number of patients receiving in vitro fertilization (IVF) has been increasing, though the rate of successful implantations has remained at 10-20%. A major goal of this procedure is to afford the ability to select embryos with the most potential for implantation and development. Previous studies claimed to have detected soluble HLA-G (sHLA-G) protein in culture supernatant from 2 to 3-day embryos using ELISA methods, and concluded that sHLA-G protein levels were associated with successful implantation. This result, if substantiated could provide an important tool for IVF. In this study, we have re-examined these experiments by attempting to detect sHLA-G in the medium from 2 to 3-day embryos (84 samples) and 4 to 6-day embryos (25 samples) in which a part of blastocyst has started to differentiate into trophoblasts. Using a highly specific and sensitive ELISA, no sHLA-G protein was detectable in any sample, despite the fact that 27 of the 109 samples were from successfully implanted embryos. These results indicate that 2-6-day embryos do not secrete sHLA-G detectable by ELISA, and therefore that sHLA-G in culture medium is not a useful for successful implantation at this stage of development.

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli.

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.

Natural reduced water suppressed anxiety and protected the heightened oxidative stress in rats.

BACKGROUND: In Japan, the effects of reduced water, such as hydrogen-rich electrolyzed reduced water and natural reduced water, like Hita Tenryosui water®, have been examined. The purpose of the present study was to identify the role of natural reduced water in anxiety and blood biochemical analysis. MATERIALS AND METHODS: Natural reduced water and distilled water were administered to rats for 180 consecutive days, and their effect on anxiety-like behavior and depression was examined by using elevated plus maze, light/dark, forced swimming, and conditioned fear tests. Before and after administration of natural reduced or distilled water, we performed blood and urine analyses. RESULTS: Natural reduced water exhibited anxiolytic-like effects in the conditioned fear and elevated plus maze tests. The mean levels of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the natural reduced water were significantly lower than the distilled water group. Natural reduced water group also showed decrease in blood-urea nitrogen levels compared with the distilled water group. CONCLUSION: These results indicate that natural reduced water may decrease anxiety-related behaviors and prevent heightened oxidative stress.

Different functioning of prefrontal cortex predicts treatment response after a selective serotonin reuptake inhibitor treatment in patients with major depression.

BACKGROUND: Major depressive disorder (MDD) is often resistant to treatment with usual approaches. Patients with MDD have shown hypofunction of the frontotemporal cortex in verbal fluency test (VFT)-related near-infrared spectroscopy (NIRS). METHODS: We examined whether the reactions to drug treatment in treatment-naive patients with MDD could be predicted by NIRS outcomes at the initial investigation. All subjects underwent psychological testing to determine levels of anxiety and depression. VFT was used to examine the functioning of the frontotemporal lobes. We administered selective serotonin reuptake inhibitors (SSRIs) for 12 weeks. Subjects included 28 patients with MDD with response to SSRIs (Response group), 19 with no response (Non-Response group), and 63 age-, sex-, and education years-matched healthy controls (HC). RESULTS: We found in the frontotemporal region that hemodynamic responses were significantly smaller in patients with Response and Non-Response groups than in HC before treatment. We also found in the medial frontal region that hemodynamic responses were significantly larger in patients with Response groups than in patients with Non-Response group before treatment. Patients with MDD scored significantly higher anxiety and depressive states than those in HC on several measures. The Response and Non-Response groups also had higher scores in future denial, threat prediction, self-denial, past denial, and interpersonal threat sections of Anxiety Cognition Scale (DACS). According to the stepwise regression analysis, one variable was determined as independent predictors of response: confusion (Post-POMS). LIMITATIONS: The number of patients and healthy controls was relatively small, and we will increase the number of participants in future studies. NIRS has reduced spatial resolution, which confuses the identification of the measurement position when using NIRS alone. CONCLUSION: Cognitive vulnerabilities are associated with predictors of SSRI treatment response. Different hemodynamic activities in the frontotemporal cortex predict response to SSRI treatment in MDD.

Gender differences in automatic thoughts and cortisol and alpha-amylase responses to acute psychosocial stress in patients with obsessive-compulsive personality disorder.

INTRODUCTION: Obsessive-compulsive personality disorder (OCPD) has a pervasive pattern of preoccupation with orderliness, perfection, and mental and interpersonal control at the expense of flexibility, openness, and efficiency. The aims of the present study were to explore the relationship between OCPD and psychological stress and psychological tests. METHODS: We evaluated 63 OCPD patients and 107 healthy controls (HCs). We collected saliva samples from patients and controls before and after a social stress procedure, the Trier Social Stress Test (TSST), to measure the concentrations of salivary alpha-amylase (sAA) and salivary cortisol. The Childhood Trauma Questionnaire (CTQ), Profile of Mood State (POMS), State-Trait Anxiety Inventory (STAI), Beck Depression Inventory (BDI), Social Adaptation Self-Evaluation Scale (SASS), and Depression and Anxiety Cognition Scale (DACS) were administered to patients and HCs. RESULTS: Following TSST exposure, the salivary amylase and cortisol levels were significantly decreased in male patients compared with controls. Additionally, OCPD patients had higher CTQ, POMS, STAI, and BDI scores than HCs and exhibited significantly higher anxiety and depressive states. OCPD patients scored higher on future denial and threat prediction as per the DACS tool. According to a stepwise regression analysis, STAI, POMS, and salivary cortisol responses were independent predictors of OCPD. CONCLUSIONS: Our results suggested that attenuated sympathetic and parasympathetic reactivity in male OCPD patients occurs along with attenuated salivary amylase and cortisol responses to the TSST. In addition, there was a significant difference between OCPD patients and HCs in child trauma, mood, anxiety, and cognition. The finding support the modeling role of cortisol (20min) on the relationships between STAI trait and depression among OCPD.

High-throughput screening of cell death inducible short peptides from TNF-related apoptosis-inducing ligand sequence.

Therapeutic peptides and small molecules, rationally designed to trigger cell death have attracted strong attention. Cell death inducible peptides were screened from amino acid sequence of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using Fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of TRAIL scan was prepared and cell viability assay was directly performed on peptide disk with Jurkat cells. Six peptide sequences that could induce cell death were found. Peptide sequence with RNSCWSKD (TRAIL(227-234)) that exist in the zinc-binding site revealed high cell death inducible activity. Apoptotic cell death was observed when cells were treated with soluble synthesized peptide.

The surface expression of HLA-F on decidual trophoblasts increases from mid to term gestation.

HLA-F has recently only begun to be studied in earnest, and has been thought not to be expressed on the cell surface. However, in our previous report, we demonstrated surface expression of HLA-F on extravillous trophoblasts (EVTs) invading the decidua in term placental tissues. To better understand its function, we attempted to determine when surface expression of HLA-F begins during normal pregnancy, and whether there is a difference in expression between normal and preeclamptic placentas, by comparing the expression of HLA-G and -E by immunohistochemical staining with anti-HLA-E, -F and -G antibodies (3D12, 3D11 and 87G, respectively). In EVTs, HLA-F was expressed only in the cytoplasm weakly during the first trimester, after which expression increased and moved to the cell surface with the progression of pregnancy from the second trimester, which was confirmed by the results of double-labeled immunofluorescence staining with anti-HLA-F and anti-HLA-G antibodies, and by flow cytometry using trophoblasts isolated from the decidua. HLA-E showed similar expression as HLA-F, though it was expressed on the cell surface from the first trimester, while HLA-G was expressed strongly in the cytoplasm and on the cell surface during all stages of pregnancy. The expressions of HLA-E, -F and -G in preeclamptic placentas were not different from those in normal placentas, though there were a greater number of necrotic EVTs in preeclampsia. The increase in expression of HLA-E and HLA-F from the second trimester to full term was coincident with the timing of rapid growth of the fetus. Our results suggest that these may function together to prepare an environment that supports fetal growth.

sHLA-G and sHLA-I levels in follicular fluid are not associated with successful implantation.

In the field of in vitro fertilization (IVF), useful markers for the prediction of successful implantation for oocyte or embryo selection are essential. It has been reported that sHLA-G (sHLA-G1/HLA-G5) could be detected in the supernatant of the fertilized embryo and in follicular fluid samples (FFs), and that the presence of sHLA-G was related to successful implantation. If sHLA-G could be used as a marker of oocyte selection from multiple FFs, oocytes could be selected without physical contact, thus reducing the likelihood of damage. To investigate the potential for sHLA-G as a marker of oocyte selection from multiple FFs in one patient, protein levels of total protein, sHLA-G, and sHLA-I (sHLA-A, B, and C) were examined in FFs. The variation among multiple FFs in total protein level and sHLA-G level was not related to successful pregnancy. The average sHLA-I levels did not differ in the successful implantation and unsuccessful implantation groups, indicating that sHLA-I levels were not related to successful pregnancy. Furthermore, sHLA-G in FFs was not detected by western blotting, despite being detected by ELISA, while sHLA-I was detected by both ELISA and western blot. These data suggest that sHLA-G in FF might not be a useful marker for oocyte selection as measurements of sHLA-G were inconsistent and there was no association with successful pregnancy. Further, more rigorously tested ELISA systems for detecting sHLA-G in body fluids are necessary before the utility of sHLA-G for diagnosis can be established.

Sex determines cortisol and alpha-amylase responses to acute physical and psychosocial stress in patients with avoidant personality disorder.

INTRODUCTIONS: Avoidant personality disorder (AVPD) has excessive and pervasive anxiety and discomfort in social situations. The aims of this study were to explore the relationship between AVPD and physical and psychological stress and psychological tests. METHODS: We evaluated 93 AVPD patients and 355 nonpatient controls by salivary amylase and cortisol responses during exposure to the Trier Social Stress Test (TSST) and electrical stimulation stress. Spielberger state-trait anxiety inventory (STAI), Profile of Mood State (POMS), Beck Depression Inventory (BDI), Depression and Anxiety Cognition Scale (DACS), and Childhood Trauma Questionnaire (CTQ) were administered. RESULTS: Following electrical stimulation, salivary cortisol levels in female AVPD decreased significantly less than that in female's controls, but salivary cortisol levels did not show a difference between male AVPD patients and controls. Salivary alpha-amylase (sAA) levels did not show a difference between females or male AVPD patients and controls. Following TSST exposure, sAA levels did not show a difference between females or male AVPD patients and controls. Salivary cortisol levels did not show a difference between females or male AVPD patients and controls. In the AVPD patients, POMS scores were significantly higher compared with the controls. STAI, BDI, DACS scores, and CTQ significantly increased in the AVPD patients compared with the controls. LF in heart rate variability in AVPD significantly increased more compared with controls. CONCLUSIONS: These results suggest that heightened sympathetic reactivity in female AVPD co-occurs with attenuated salivary cortisol responses to electric stimulation stress and there is a significant difference between AVPD and controls in mood, anxiety, social cognition, and automatic nerve systems.