Retinoic Acid Prevents the Neuronal Damage Through the Regulation of Parvalbumin in an Ischemic Stroke Model.

Ischemic stroke is a neurological disease that causes brain damage by increasing oxidative stress and ion imbalance. Retinoic acid is a major metabolite of vitamin A and regulates oxidative stress, calcium homeostasis, and cell death. Intracellular calcium is involved in neuronal growth and synaptic plasticity. Parvalbumin is a calcium-binding protein that is mainly expressed in brain. In this study, we investigated whether retinoic acid has neuroprotective effects by controlling intracellular calcium concentration and parvalbumin expression in ischemic brain damage. Middle cerebral artery occlusion (MCAO) was performed to induce cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected into the abdominal cavity for four days before surgery and cerebral cortices were collected 24 h after MCAO for further studies. MCAO damage induced neurological deficits and histopathological changes and decreased parvalbumin expression. However, retinoic acid treatment alleviated these changes. In cultured neurons, glutamate (5 mM) exposure induced neuronal cell death, increased intracellular calcium concentration, and decreased parvalbumin expression. Retinoic acid treatment attenuated these changes against glutamate toxicity in a dose-dependent manner. It also regulates glutamate induced change in bcl-2 and bax expression. The mitigation effects of retinoic acid were greater under non-transfection conditions than under parvalbumin siRNA transfection conditions. Our findings showed that retinoic acid modulates intracellular calcium concentration and parvalbumin expression and prevents apoptosis in ischemic brain injury. In conclusion, retinoic acid contributes to the preservation of neurons from ischemic stroke by controlling parvalbumin expression and apoptosis-related proteins.

Wwox Binding to the Murine Brca1-BRCT Domain Regulates Timing of Brip1 and CtIP Phospho-Protein Interactions with This Domain at DNA Double-Strand Breaks, and Repair Pathway Choice.

Wwox-deficient human cells show elevated homologous recombination, leading to resistance to killing by double-strand break-inducing agents. Human Wwox binds to the Brca1 981-PPLF-984 Wwox-binding motif, likely blocking the pChk2 phosphorylation site at Brca1-S988. This phosphorylation site is conserved across mammalian species; the PPLF motif is conserved in primates but not in rodents. We now show that murine Wwox does not bind Brca1 near the conserved mouse Brca1 phospho-S971 site, leaving it open for Chk2 phosphorylation and Brca1 activation. Instead, murine Wwox binds to Brca1 through its BRCT domain, where pAbraxas, pBrip1, and pCtIP, of the A, B, and C binding complexes, interact to regulate double-strand break repair pathway response. In Wwox-deficient mouse cells, the Brca1-BRCT domain is thus accessible for immediate binding of these phospho-proteins. We confirm elevated homologous recombination in Wwox-silenced murine cells, as in human cells. Wwox-deficient murine cells showed increased ionizing radiation-induced Abraxas, Brca1, and CtIP foci and long resected single-strand DNA, early after ionizing radiation. Wwox deletion increased the basal level of Brca1-CtIP interaction and the expression level of the MRN-CtIP protein complex, key players in end-resection, and facilitated Brca1 release from foci. Inhibition of phospho-Chk2 phosphorylation of Brca1-S971 delays the end-resection; the delay of premature end-resection by combining Chk2 inhibition with ionizing radiation or carboplatin treatment restored ionizing radiation and platinum sensitivity in Wwox-deficient murine cells, as in human cells, supporting the use of murine in vitro and in vivo models in preclinical cancer treatment research.

Histone acetyltransferase 1 is required for DNA replication fork function and stability.

The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.

Epigallocatechin Gallate Alleviates Down-Regulation of Thioredoxin in Ischemic Brain Damage and Glutamate-Exposed Neuron.

Epigallocatechin gallate (EGCG) is one of polyphenol that is abundant in green tea. It has anti-oxidative activity and exerts neuroprotective effects in ischemic brain damage. Ischemic conditions induce oxidative stress and result in cell death. Thioredoxin is a small redox protein that plays an important role in the regulation of oxidation and reduction. This study was designed to investigate the regulation of thioredoxin by EGCG in ischemic brain damage. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia in male Sprague-Dawley rats. The EGCG (50 mg/kg) or was administered before MCAO surgical operation. Neurological behavior test, reactive oxygen species (ROS), and lipid peroxidation (LPO) measurement were performed 24 h after MCAO. The cerebral cortex was isolated for further experiments. EGCG alleviated MCAO-induced neurological deficits and increases in ROS and LPO levels. EGCG also ameliorated the decrease in thioredoxin expression by MCAO. This finding was confirmed using various techniques such as Western blot analysis, reverse transcription PCR, and immunofluorescence staining. Results of immunoprecipitation showed that MCAO decreases the interaction between apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin, while EGCG treatment attenuates this decrease. EGCG also attenuated decrease of cell viability and thioredoxin expression in glutamate-exposed neuron in a dose-dependent manner. It alleviated the increase of caspase-3 by glutamate exposure. However, this effect of EGCG on caspase-3 change was weakened in thioredoxin siRNA-transfected neurons. These findings suggest that EGCG exerts a neuroprotective effect by regulating thioredoxin expression and modulating ASK1 and thioredoxin binding in ischemic brain damage.

Compressive Sensing Spectroscopy Using a Residual Convolutional Neural Network.

Compressive sensing (CS) spectroscopy is well known for developing a compact spectrometer which consists of two parts: compressively measuring an input spectrum and recovering the spectrum using reconstruction techniques. Our goal here is to propose a novel residual convolutional neural network (ResCNN) for reconstructing the spectrum from the compressed measurements. The proposed ResCNN comprises learnable layers and a residual connection between the input and the output of these learnable layers. The ResCNN is trained using both synthetic and measured spectral datasets. The results demonstrate that ResCNN shows better spectral recovery performance in terms of average root mean squared errors (RMSEs) and peak signal to noise ratios (PSNRs) than existing approaches such as the sparse recovery methods and the spectral recovery using CNN. Unlike sparse recovery methods, ResCNN does not require a priori knowledge of a sparsifying basis nor prior information on the spectral features of the dataset. Moreover, ResCNN produces stable reconstructions under noisy conditions. Finally, ResCNN is converged faster than CNN.

Mechanisms shaping the mutational landscape of the FRA3B/FHIT-deficient cancer genome.

Genome instability is an enabling characteristic of cancer that facilitates the acquisition of oncogenic mutations that drive tumorigenesis. Underlying much of the instability in cancer is DNA replication stress, which causes both chromosome structural changes and single base-pair mutations. Common fragile sites are some of the earliest and most frequently altered loci in tumors. Notably, the fragile locus, FRA3B, lies within the fragile histidine triad (FHIT) gene, and consequently deletions within FHIT are common in cancer. We review the evidence in support of FHIT as a DNA caretaker and discuss the mechanism by which FHIT promotes genome stability. FHIT increases thymidine kinase 1 (TK1) translation to balance the deoxyribonucleotide triphosphates (dNTPs) for efficient DNA replication. Consequently, FHIT-loss causes replication stress, DNA breaks, aneuploidy, copy-number changes (CNCs), small insertions and deletions, and point mutations. Moreover, FHIT-loss-induced replication stress and DNA breaks cooperate with APOBEC3B overexpression to catalyze DNA hypermutation in cancer, as APOBEC family enzymes prefer single-stranded DNA (ssDNA) as substrates and ssDNA is enriched at sites of both replication stress and DNA breaks. Consistent with the frequent loss of FHIT across a broad spectrum of cancer types, FHIT-deficiency is highly associated with the ubiquitous, clock-like mutation signature 5 occurring in all cancer types thus far examined. The ongoing destabilization of the genome caused by FHIT loss underlies recurrent inactivation of tumor suppressors and activation of oncogenes. Considering that more than 50% of cancers are FHIT-deficient, we propose that FRA3B/FHIT fragility shapes the mutational landscape of cancer genomes.

Identification of Proteins Differentially Expressed by Quercetin Treatment in a Middle Cerebral Artery Occlusion Model: A Proteomics Approach.

Cerebral ischemia is a major cause of death and neurological disability. It also leads to severe brain tissue damage by excessive generation of oxidative stress. Quercetin is a bioflavonoid substance that acts an antioxidant agent and exerts a neuroprotective effect against cerebral ischemia. The aim of this study was to detect specific proteins that are differentially expressed in response to quercetin treatment in focal cerebral ischemia. Adult male rats were intraperitoneally injected with vehicle or quercetin (10 mg/kg) 30 min prior to right middle cerebral artery occlusion (MCAO). Brain tissues were collected 24 h after MCAO surgery and right cerebral cortices proteins were identified by two-dimensional gel electrophoresis and mass spectrometry. MCAO leads to neurological behavior disorders, infarction, and histopathological change. However, quercetin treatment alleviated MCAO-induced neuronal deficits and damages. We identified specific proteins differentially expressed between vehicle- and quercetin-treated animals. Among these detected proteins, isocitrate dehydrogenase [NAD+], adenosylhomocysteinase, pyruvate kinase, and ubiquitin carboxy terminal hydrolase L1 were decreased in vehicle-treated animals, while quercetin administration alleviated the MCAO-induced decreases in these proteins. However, 60 kDa heat shock protein and collapsin response mediator protein 2 were increased in the vehicle-treated animals, and quercetin treatment attenuated increases in these proteins. The expression changes in these proteins were confirmed by Western blot and reverse transcription-PCR analyses. These proteins are associated with cellular differentiation, metabolism, and oxidative stress related proteins. These results suggest that quercetin reduces ischemic injury by modulating the expression of various proteins in focal cerebral ischemia.

Characterization of maltotriose production by hydrolyzing of soluble starch with α-amylase from Microbulbifer thermotolerans DAU221.

A maltotriose-producing α-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli, using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified α-amylase with a molecular mass of 80 kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50 °C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76 h, 44.95 °C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10 mM Cu(2+), Fe(3+), Hg(2+), Zn(2+), and EDTA, but exhibited extreme stability toward hexane. The K m and V max values for the hydrolysis of soluble starch were 1.08 mg/mL and 1.736 mmol maltotriose/mg protein/min, respectively.

Epigallocatechin gallate improves neuronal damage in animal model of ischemic stroke and glutamate-exposed neurons via modulation of hippocalcin expression.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Chlorogenic acid regulates the expression of protein phosphatase 2A subunit B in the cerebral cortex of a rat stroke model and glutamate-exposed neurons.

BACKGROUND: Ischemic stroke is a serious neurological disorder caused by blockages in cerebral artery. Protein phosphatase 2A (PP2A) is a phosphatase that performs a critical role in cell signaling and growth. PP2A subunit B acts as a neuroprotective agent in the nerve system. Chlorogenic acid, which is mainly found in roasted coffee, has antioxidant, anti-inflammatory, and anti-apoptotic effects. We hypothesized that chlorogenic acid modulates PP2A subunit B expression in ischemic stroke models and glutamate-mediated neurons. Middle artery occlusion (MCAO) surgery was operated and chlorogenic acid (30 mg/kg) or phosphate buffer saline was treated 2 h after MCAO. The cerebral cortex was collected 24 h after surgery and the change of PP2A subunit B expression was analyzed. Glutamate and/or chlorogenic acid were treated in cultured neurons, further study was performed. RESULTS: A decrease in PP2A subunit B expression in MCAO animals was identified. Chlorogenic acid alleviated this decrease due to ischemic injury. Moreover, the number of PP2A subunit B-positive cells in the ischemic cerebral cortex was significantly decreased, chlorogenic acid alleviated this decrease. We also found protective effects of chlorogenic acid in neurons exposed to glutamate. Glutamate decreased the expression of PP2A subunit B and chlorogenic acid mitigated this decrease. Our results elucidated that chlorogenic acid performs neuroprotective functions and attenuates the reduction of PP2A subunit B by brain damage and glutamate-mediated excitotoxicity. CONCLUSIONS: We showed that chlorogenic acid attenuated the decrease of PP2A subunit B in ischemic injury and neurons exposed to glutamate. Since PP2A subunit B contributes to the protection of brain tissue, we can suggest that chlorogenic acid preserves neurons by modulating PP2A subunit B during ischemic damage.

Modulation of thioredoxin by chlorogenic acid in an ischemic stroke model and glutamate-exposed neurons.

Ischemic stroke increases the production of reactive oxygen species (ROS), which can eventually lead to neuronal death. Thioredoxin is a small reductase protein that acts as an eliminator of ROS and protects neurons from brain damage. Chlorogenic acid is known as a phenolic compound that has a neuroprotective effect. We investigated the change of thioredoxin expression by chlorogenic acid in a middle cerebral artery occlusion (MCAO) animal model. Adult rats were injected intraperitoneally with phosphate buffered saline or chlorogenic acid (30 mg/kg) 2 h after MCAO. MCAO damage induced neurological defects and increased ROS and lipid peroxidation levels, however, chlorogenic acid mitigated these changes. MCAO damage reduced thioredoxin expression, which was mitigated by chlorogenic acid treatment. The interaction between thioredoxin and apoptosis signal-regulating kinase 1 (ASK1) was decreased in MCAO animals, chlorogenic acid treatment prevented this decrease. In cultured neurons, chlorogenic acid dose-dependently attenuated glutamate-induced decreases in cell viability and thioredoxin expression. Glutamate toxicity downregulated bcl-2 and upregulated bax, cytochrome c, and caspase-3, however, chlorogenic acid attenuated these changes. The mitigating effect of chlorogenic acid was lower in thioredoxin siRNA-transfected cells than in non-transfected cells. These results provide evidence that chlorogenic acid exerts potent antioxidant and neuroprotective effects through regulation of thioredoxin and modulation of ASK1 and thioredoxin binding in ischemic brain injury. These findings indicate that chlorogenic acid exerts a neuroprotective effect by regulating thioredoxin expression in cerebral ischemia and glutamate exposure conditions.

Assessing systemic, developmental, and reproductive toxicity and estrogenicity of Korean red ginseng extract G1899 in juvenile Sprague-Dawley Rats.

Background: Korean red ginseng (KRG) is a product from ginseng roots, which is enriched with ginsenosides and has been utilized for a long time as an adaptogen to alleviate various physiological or disease conditions. While KRG is generally considered safe, conducting a thorough toxicological assessment of the spray-dried powder G1899 during the juvenile period is essential to establish its safety profile. This study aimed to assess the safety of G1899 during the juvenile period using Sprague-Dawley rats. Methods: Two studies were conducted separately: a juvenile toxicity study and a uterotrophic bioassay. To assess the potential toxicity at systemic, postnatal developmental, and reproductive levels, G1899 was orally gavaged once a day in post-weaning juvenile Sprague-Dawley (SD) rats at 0, 1250, 2500, or 5000 mg/kg/day. Estrogenicity was assessed by orally gavaging G1899 in immature female SD rats at 0, 2500, or 5000 mg/kg/day on postnatal days (PND) 19-21, followed by a uterotrophic bioassay. These studies were conducted in accordance with the Good Laboratory Practice (GLP) regulations and regulatory test guidelines. Results: Regarding juvenile toxicity, no abnormalities related to the G1899 treatment were observed in any group during the experiment. Moreover, no uterotrophic responses were observed in the dosed female group. Based on these results, the no observed adverse effect level (NOAEL) of G1899 was determined to be at least 5000 mg/kg/day for general systemic function, developmental/reproductive function, and estrogenic activity. Conclusion: Our results suggest that G1899 is not toxic to juveniles at doses of up to 5000 mg/kg/day.

ISG15 mediates the function of extracellular vesicles in promoting ovarian cancer progression and metastasis.

The interferon stimulated gene 15 (ISG15), a ubiquitin like protein and its conjugates have been implicated in various human malignancies. However, its role in ovarian cancer progression and metastasis is largely unknown. In high grade serous ovarian cancer (HGSOC), ascites is the major contributor to peritoneal metastasis. In this study, we identified significantly elevated ISG15 protein expression in HGSOC patient ascites, ascites derived primary ovarian cancer cells (POCCs), POCC small extracellular vesicles (sEVs) as well as metastatic tissue. Our results demonstrates that ISG15 increases exocytosis in ascites-derived POCCs by decreasing the endosome-lysosomal fusion, indicating a key role in sEV secretion. Further, knockdown (KD) of ISG15 resulted in a significant decrease in vesicles secretion from HGSOC cells and in vivo mouse models, leading to reduced HGSOC cell migration and invasion. Furthermore, our pre-clinical mouse model studies revealed the influence of vesicular ISG15 on disease progression and metastasis. In addition, knockdown of ISG15 or using the ISG15 inhibitor, DAP5, in combination therapy with carboplatin showed to improve the platinum sensitivity in-vitro and reduce tumour burden in-vivo. We also found that ISG15 expression within sEV represents a promising prognostic marker for HGSOC patients. Our findings suggest that ISG15 is a potential therapeutic target for inhibiting progression and metastasis in HGSOC and that vesicular ISG15 expression could be a promising biomarker in the clinical management of ovarian cancer. Significance: High-grade serous ovarian cancer (HGSOC) has high morbidity and mortality rates, but its progression and metastasis are still poorly understood, and there is an urgent need for early detection and targeted therapies. Our study presents novel findings that implicate ISG15-mediated vesicular proteins in the advancement and spread of HGSOC. These results offer pre-clinical evidence of potential new molecular targets, prognostic markers and therapeutic strategies for HGSOC that could ultimately enhance patient survival.

ß-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties.

The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, cloned, sequenced and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34-residues. The deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99 %) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca(2+) binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74 kDa. The optimum temperature and pH of the enzyme were 40 °C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca(2+) and inhibited by Ba(2+), Cu(2+), and Hg(2+). The K m and V max values calculated were 0.48 mg/ml and 51.38 mg of ß-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to ß-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to ß-cyclodextrin.

Comparison of RT-qPCR and RT-ddPCR with Rift valley fever virus (RVFV) RNA.

Rift valley fever (RVF) is an important zoonotic disease caused by the Rift valley fever virus (RVFV) which can affect ruminants and humans. In this study, a comparison was done of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital PCR (RT-ddPCR) assays with synthesized RVFV RNA, cultured viral RNA, and mock clinical RVFV RNA samples. The genomic segments (L, M, and S) of three RVFV strains (BIME01, Kenya56, and ZH548) were synthesized and used as templates for in vitro transcription (IVT). Both the RT-qPCR and RT-ddPCR assays for RVFV did not react with any of the negative reference viral genomes. Thus, both the RT-qPCR and RT-ddPCR assays are specific to RVFV. The comparison of both the RT-qPCR and RT-ddPCR assays with serially diluted templates showed that the LoD of both assays are similar, and a concordant of the results was observed. The LoD of both assays reached the practical measurable minimum concentration. Taken altogether, the sensitivity of the RT-qPCR and RT-ddPCR assays is similar, and the material measured by RT-ddPCR can be used as a reference material for RT-qPCR.

Epigallocatechin gallate restores the reduction of protein phosphatase 2 A subunit B caused by middle cerebral artery occlusion.

BACKGROUND: Epigallocatechin gallate (EGCG) is a flavonoid compound commonly found in green tea. It exhibits antioxidant, anti-inflammatory, and neuroprotective effects in cerebral ischemia. Protein phosphatase 2 A (PP2A) is an important serine/threonine phosphatase enzyme involved in various cellular activities. PP2A subunit B is present abundantly in the brain and plays an important role in the nervous system. We investigated the effect of EGCG on the expression level of PP2A subunit B in cerebral ischemia caused by middle cerebral artery occlusion (MCAO). EGCG (50 mg/kg) or vehicle was injected into the peritoneal cavity prior to MCAO surgery. Neurological behavior tests were performed 24 h after MCAO, and right cerebral cortex tissue was collected. Cerebral ischemia caused serious neurological abnormalities, which were alleviated by EGCG administration. We screened the expression of PP2A subunits containing A, B, and C using reverse-transcription PCR. We confirmed that PP2A subunit B exhibited significant changes in MCAO animals compared to subunits A and C. We continuously examined the expression of PP2A subunit B protein in MCAO animals using Western blot analysis. RESULTS: EGCG alleviated the reduction of PP2A subunit B protein by MCAO damage. In addition, immunohistochemistry demonstrated a decrease in the number of PP2A subunit B-positive cells in the cerebral cortex, and EGCG attenuated this decrease. Maintenance of PP2A subunit B is important for normal brain function. CONCLUSION: Therefore, our findings suggest that EGCG exerts neuroprotective effects against cerebral ischemia through modulation of PP2A subunit B expression.

Development of Human Rhinovirus RNA Reference Material Using Digital PCR.

The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at -80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 105 copies/µL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and -20 °C and for 12 months at a temperature of -80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.

Retinoic acid regulates the ubiquitin-proteasome system in a middle cerebral artery occlusion animal model.

BACKGROUND: Retinoic acid is a major metabolite of vitamin A and exerts beneficial effects including anti-oxidant and anti-inflammatory activities in neurons. The ubiquitin-proteasome system is an important biological system that regulates cell survival. Ubiquitination regulates protein degradation and plays an important role in oxidative stress. Deubiquitinating enzymes cleave ubiquitin from proteins and control ubiquitination-induced degradation. We detected decreases in ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in cerebral ischemic damage. In this study, we investigated whether retinoic acid regulates the expression of deubiquitinating enzymes ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in cerebral ischemic injury. Right middle cerebral artery occlusion (MCAO) was performed to induce cerebral ischemic damage in male rats. Retinoic acid (5 mg/kg) or vehicle was intraperitoneally injected every day from 4 days before surgery. Neurological behavioral tests were performed 24 h after MCAO, and right cerebral cortical tissues were collected. RESULTS: MCAO damage caused neurological behavioral dysfunction, and retinoic acid alleviated these deficits. The identified proteins decreased in MCAO animals with vehicle, while retinoic acid treatment attenuated these decreases. The results of proteomic study were confirmed by a reverse transcription-PCR technique. Expressions of ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 were decreased in MCAO animals treated with vehicle. Retinoic acid treatment alleviated these MCAO-induced reductions. The ubiquitin-proteasome system plays an essential role in maintaining cell function and preserving cell shape against ischemic damage. CONCLUSIONS: These findings suggest that retinoic acid regulates ubiquitin- and proteasome-related proteins including ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in a brain ischemia model. Changes in these proteins are involved in the neuroprotective effects of retinoic acid.

Chlorogenic acid alleviates cerebral ischemia-induced neuroinflammation via attenuating nuclear factor kappa B activation.

Ischemic stroke is the most common type of stroke and is caused by vascular closure. Chlorogenic acid is a polyphenolic compound that is present in various plants. It is used as a traditional oriental medicine because of its anti-oxidant and anti-inflammatory properties. We investigated whether chlorogenic acid mediates neuroprotective effects by regulating pro-inflammatory proteins. Focal cerebral ischemia was induced through middle cerebral artery occlusion (MCAO) surgery in adult rats. Chlorogenic acid (30 mg/kg) or vehicle was injected into the abdominal cavity 2 h after MCAO. Rats were sacrificed 24 h after MCAO surgery and brain tissues were isolated immediately. MCAO caused histopathological changes in the ischemic cerebral cortex, and chlorogenic acid attenuated these changes. Chlorogenic acid reduced MCAO-induced reactive oxygen species generation and oxidative stress increase in the cerebral cortex. Furthermore, cerebral ischemia increased the expression of ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP), which are microglia and astrocyte activation markers, respectively. However, chlorogenic acid prevented MCAO-induced these increases. MCAO damage also increased the expression of nuclear factor-κB (NF-κB), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α). Chlorogenic acid treatment attenuated these increases caused by MCAO. These proteins are representative pro-inflammatory markers. This study confirmed that chlorogenic acid exerts an anti-oxidative effect and elucidated anti-inflammatory effect through regulating NF-κB, IL-1ß, and TNF-α on cerebral ischemia. Thus, we can suggest that chlorogenic acid has neuroprotective effects by reducing oxidative stress and controlling pro-inflammatory proteins against cerebral ischemic damage.

Quercetin ameliorates glutamate toxicity-induced neuronal cell death by controlling calcium-binding protein parvalbumin.

BACKGROUND: Glutamate is the main excitatory neurotransmitter. Excessive glutamate causes excitatory toxicity and increases intracellular calcium, leading to neuronal death. Parvalbumin is a calcium-binding protein that regulates calcium homeostasis. Quercetin is a polyphenol found in plant and has neuroprotective effects against neurodegenerative diseases. OBJECTIVES: We investigated whether quercetin regulates apoptosis by modulating parvalbumin expression in glutamate induced neuronal damage. METHODS: Glutamate was treated in hippocampal-derived cell line, and quercetin or vehicle was treated 1 h before glutamate exposure. Cells were collected for experimental procedure 24 h after glutamate treatment and intracellular calcium concentration and parvalbumin expression were examined. Parvalbumin small interfering RNA (siRNA) transfection was performed to detect the relation between parvalbumin and apoptosis. RESULTS: Glutamate reduced cell viability and increased intracellular calcium concentration, while quercetin preserved calcium concentration and neuronal damage. Moreover, glutamate reduced parvalbumin expression and quercetin alleviated this reduction. Glutamate increased caspase-3 expression, and quercetin attenuated this increase in both parvalbumin siRNA transfected and non-transfected cells. The alleviative effect of quercetin was statistically significant in non-transfected cells. Moreover, glutamate decreased bcl-2 and increased bax expressions, while quercetin alleviated these changes. The alleviative effect of quercetin in bcl-2 family protein expression was more remarkable in non-transfected cells. CONCLUSIONS: These results demonstrate that parvalbumin contributes to the maintainace of intracellular calcium concentration and the prevention of apoptosis, and quercetin modulates parvalbumin expression in glutamate-exposed cells. Thus, these findings suggest that quercetin performs neuroprotective function against glutamate toxicity by regulating parvalbumin expression.