In vitro and vivo anti-tumor activity and mechanisms of the new cryptotanshinone derivative 11 against hepatocellular carcinoma.

Global burden of hepatocellular carcinoma (HCC) is increasing. Chemotherapy and immunotherapy are the prevailing options for therapy. Developing new therapeutic strategies for HCC patients is still highly desirable. Recent studies demonstrate that cryptotanshinone is capable of inhibiting tumor growth in HCC and induces antitumor immunity in vitro. In our previous research, we discovered a new cryptotanshinone derivative 11 as an effective immunoregulatory enzyme indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitor. This study aims to evaluate its in vitro and in vivo antitumor activity against hepatocellular carcinoma. 11 displayed robust anti-proliferative activity against HCC cell lines and promoted apoptosis of HCC cell line through the mitochondrial-mediated apoptotic pathway. In H22 tumor-bearing mice models, 11 exhibited significant in vivo anti-tumor activity with different administration routes. And no obvious toxicity was observed. RNA-seq analysis demonstrated the differential expressed genes and alteration of key pathways associated with immune responses after administration of 11. Up-regulation of anti-tumor cytokines and down-regulation of cytokines that promote tumor growth were indicated and further validated. Our study demonstrates that 11 exhibits promising anti-tumor activity both in vitro and in vivo against hepatocellular carcinoma cancer. It is a lead compound for HCC immunotherapy and is worthy for further development.

UvHOS3-mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in Ustilaginoidea virens.

Ustilaginoidea virens is the causal agent of rice false smut, which has recently become one of the most important rice diseases worldwide. Ustilaginoidins, a major type of mycotoxins produced in false smut balls, greatly deteriorates grain quality. Histone acetylation and deacetylation are involved in regulating secondary metabolism in fungi. However, little is yet known on the functions of histone deacetylases (HDACs) in virulence and mycotoxin biosynthesis in U. virens. Here, we characterized the functions of the HDAC UvHOS3 in U. virens. The ΔUvhos3 deletion mutant exhibited the phenotypes of retarded growth, increased mycelial branches and reduced conidiation and virulence. The ΔUvhos3 mutants were more sensitive to sorbitol, sodium dodecyl sulphate and oxidative stress/H2 O2 . ΔUvhos3 generated significantly more ustilaginoidins. RNA-Seq and metabolomics analyses also revealed that UvHOS3 is a key negative player in regulating secondary metabolism, especially mycotoxin biosynthesis. Notably, UvHOS3 mediates deacetylation of H3 and H4 at H3K9, H3K18, H3K27 and H4K8 residues. Chromatin immunoprecipitation assays indicated that UvHOS3 regulates mycotoxin biosynthesis, particularly for ustilaginoidin and sorbicillinoid production, by modulating the acetylation level of H3K18. Collectively, this study deepens the understanding of molecular mechanisms of the HDAC UvHOS3 in regulating virulence and mycotoxin biosynthesis in phytopathogenic fungi.

Nanoscale CuO charge and morphology control Fusarium suppression and nutrient biofortification in field-grown tomato and watermelon.

Limited data exist on how surface charge and morphology impact the effectiveness of nanoscale copper oxide (CuO) as an agricultural amendment under field conditions. This study investigated the impact of these factors on tomatoes and watermelons following foliar treatment with CuO nanosheets (NS-) or nanospikes (NP+ and NP-) exhibiting positive or negative surface charge. Results showed plant species-dependent benefits. Notably, tomatoes infected with Fusarium oxysporum had significantly reduced disease progression when treated with NS-. Watermelons benefited similarly from NP+. Although disease suppression was significant and trends indicated increased yield, the yield effects weren't statistically significant. However, several nanoscale treatments significantly enhanced the fruit's nutritional value, and this nano-enabled biofortification was a function of particle charge and morphology. Negatively charged nanospikes significantly increased the Fe content of healthy watermelon and tomato (20-28 %) and Ca in healthy tomato (66 %), compared to their positively charged counterpart. Negatively charged nanospikes also outperformed negatively charged nanosheets, leading to significant increases in the content of S and Mg in infected watermelon (37-38 %), Fe in healthy watermelon (58 %), and Ca (42 %) in healthy tomato. These findings highlight the potential of tuning nanoscale CuO chemistry for disease suppression and enhanced food quality under field conditions.

Histone Deacetylase UvHST2 Is a Global Regulator of Secondary Metabolism in Ustilaginoidea virens.

Ustilaginoidea virens, the causal agent of rice false smut, produces a large amount of mycotoxins, including ustilaginoidins and sorbicillinoids. However, little is known about the regulatory mechanism of mycotoxin biosynthesis inU. virens. Here, we demonstrate that the NAD+-dependent histone deacetylase UvHST2 negatively regulates ustilaginoidin biosynthesis. UvHst2 knockout caused retarded hypha growth and reduced conidiation and pathogenicity inU. virens. Transcriptome analysis revealed that the transcription factor genes, transporter genes, and other tailoring genes in eight biosynthetic gene clusters (BGCs) including ustilaginoidin and sorbicillinoid BGCs were upregulated in ΔUvhst2. Interestingly, the UvHst2 deletion affects alternative splicing. Metabolomics revealed that UvHST2 negatively regulates the biosynthesis of various mycotoxins including ustilaginoidins, sorbicillin, ochratoxin B, zearalenone, and O-M-sterigmatocystin. Combined transcriptome and metabolome analyses uncover that UvHST2 positively regulates pathogenicity but negatively modulates the expression of BGCs involved in secondary metabolism. Collectively, UvHST2 functions as a global regulator of secondary metabolism inU. virens.

Effect of nanopesticides (azoxystrobin and bifenthrin) on the phenolic content and metabolic profiles of strawberries (Fragaria × ananassa).

BACKGROUND: Nanoencapsulation has opened promising fields of innovation for pesticides. Conventional pesticides can cause side effects on plant metabolism. To date, the effect of nanoencapsulated pesticides on plant phenolic contents has not been reported. RESULTS: In this study, a comparative evaluation of the phenolic contents and metabolic profiles of strawberries was performed for plants grown under controlled field conditions and treated with two separate active ingredients, azoxystrobin and bifenthrin, loaded into two different types of nanocarriers (Allosperse® polymeric nanoparticles and SiO2 nanoparticles). There were small but significant decreases of the total phenolic content (9%) and pelargonidin 3-glucoside content (6%) in strawberries treated with the nanopesticides. An increase of 31% to 125% was observed in the levels of gallic acid, quercetin, and kaempferol in the strawberries treated with the nanoencapsulated pesticides compared with the conventional treatments. The effects of the nanocarriers on the metabolite and phenolic profiles was identified by principal component analysis. CONCLUSION: Overall, even though the effects of nanopesticides on the phenological parameters of strawberry plants were not obvious, there were significant changes to the plants at a molecular level. In particular, nanocarriers had some subtle effects on plant health and fruit quality through variations in total and individual phenolics in the fruits. Further research will be needed to assess the impact of diverse nanopesticides on other groups of plant metabolites. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Loss of MBNL1-mediated retrograde BDNF signaling in the myotonic dystrophy brain.

Reduced brain volume including atrophy in grey and white matter is commonly seen in myotonic dystrophy type 1 (DM1). DM1 is caused by an expansion of CTG trinucleotide repeats in the 3' untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Mutant DMPK mRNA containing expanded CUG RNA (DMPK-CUGexp) sequesters cytoplasmic MBNL1, resulting in morphological impairment. How DMPK-CUGexp and loss of MBNL1 cause histopathological phenotypes in the DM1 brain remains elusive. Here, we show that BDNF-TrkB retrograde transport is impaired in neurons expressing DMPK-CUGexp due to loss of cytoplasmic MBNL1 function. We reveal that mature BDNF protein levels are reduced in the brain of the DM1 mouse model EpA960/CaMKII-Cre. Exogenous BDNF treatment did not rescue impaired neurite outgrowth in neurons expressing DMPK-CUGexp, whereas overexpression of the cytoplasmic MBNL1 isoform in DMPK-CUGexp-expressing neurons improved their responsiveness to exogenous BDNF. We identify dynein light chain LC8-type 2, DYNLL2, as an MBNL1-interacting protein and demonstrate that their interaction is RNA-independent. Using time-lapse imaging, we show that overexpressed MBNL1 and DYNLL2 move along axonal processes together and that MBNL1-knockdown impairs the motility of mCherry-tagged DYNLL2, resulting in a reduced percentage of retrograde DYNLL2 movement. Examination of the distribution of DYNLL2 and activated phospho-TrkB (pTrkB) receptor in EpA960/CaMKII-Cre brains revealed an increase in the postsynaptic membrane fraction (LP1), indicating impaired retrograde transport. Finally, our neuropathological analysis of postmortem DM1 tissue reveals that reduced cytoplasmic MBNL1 expression is associated with an increase in DYNLL2 and activated pTrkB receptor levels in the synaptosomal fraction. Together, our results support that impaired MBNL1-mediated retrograde BDNF-TrkB signaling may contribute to the histopathological phenotypes of DM1.

Dynamics of dominant rhizospheric microbial communities responsible for trichlorfon absorption and translocation in maize seedlings.

In this study, the available phosphorus (AP) and TCF concentrations in soils and maize (Zea mays) seedling tissues were measured in response to escalating TCF concentrations during 216 hr of culture. Maize seedlings growth considerably enhanced soil TCF degradation, reaching the highest of 73.2% and 87.4% at 216 hr in 50 and 200 mg/kg TCF treatments, respectively, and increased AP contents in all the seedling tissues. Soil TCF was majorly accumulated in seedling roots, reaching maximum concentration of 0.017 and 0.076 mg/kg in TCF-50 and TCF-200, respectively. The hydrophilicity of TCF might hinder its translocation to the aboveground shoot and leaf. Using bacterial 16 S rRNA gene sequencing, we found that TCF addition drastically lessened bacterial community interactions and hindered the complexity of their biotic networks in rhizosphere than in bulk soils, leading to the homogeneity of bacterial communities that were resistant or prone to TCF biodegradation. Mantel test and redundancy analysis suggested a significant enrichment of dominant species Massilia belonging to Proteobacteria phyla, which in turn affecting TCF translocation and accumulation in maize seedling tissues. This study provided new insight into the biogeochemical fate of TCF in maize seedling and the responsible rhizobacterial community in soil TCF absorption and translocation.

Role of IL-22 in intestinal microenvironment and potential targeted therapy through diet.

IL-22 is a type 2 receptor cytokine in IL-10 family. IL-22 is usually secreted by innate and adaptive immune cells and takes its effects on non-hematopoietic cells. Through activate STAT3 pathway, IL-22 plays an important role in infection clearance and tissue regeneration, which is critical for barrier integrate and homeostasis. Abnormal activation of IL-22 signal was observed in inflammation diseases, autoimmune diseases, and cancers. We review the recent discoveries about the mechanism and regulation of IL-22 signal pathway from the perspective of intestinal micro-environment. Diet-based IL-22 target therapeutic strategies and their potential clinical significance will also be discussed.

Uptake and Translocation of a Silica Nanocarrier and an Encapsulated Organic Pesticide Following Foliar Application in Tomato Plants.

Pesticide nanoencapsulation and its foliar application are promising approaches for improving the efficiency of current pesticide application practices, whose losses can reach 99%. Here, we investigated the uptake and translocation of azoxystrobin, a systemic pesticide, encapsulated within porous hollow silica nanoparticles (PHSNs) of a mean diameter of 253 ± 73 nm, following foliar application on tomato plants. The PHSNs had 67% loading efficiency for azoxystrobin and enabled its controlled release over several days. Thus, the nanoencapsulated pesticide was taken up and distributed more slowly than the nonencapsulated pesticide. A total of 8.7 ± 1.3 µg of the azoxystrobin was quantified in different plant parts, 4 days after 20 µg of nanoencapsulated pesticide application on a single leaf of each plant. In parallel, the uptake and translocation of the PHSNs (as total Si and particulate SiO2) in the plant were characterized. The total Si translocated after 4 days was 15.5 ± 1.6 µg, and the uptake rate and translocation patterns for PHSNs were different from their pesticide load. Notably, PHSNs were translocated throughout the plant, although they were much larger than known size-exclusion limits (reportedly below 50 nm) in plant tissues, which points to knowledge gaps in the translocation mechanisms of nanoparticles in plants. The translocation patterns of azoxystrobin vary significantly following foliar uptake of the nanosilica-encapsulated and nonencapsulated pesticide formulations.

Correlation of bacterial community with phosphorus fraction drives discovery of Actinobacteria involved soil phosphorus transformation during the trichlorfon degradation.

Trichlorfon (TCF) is a broad-spectrum phosphorus (P)-containing pesticide, yet its effects on soil P fraction transformation and bacterial communities during the TCF degradation in soils is unknown. In this study, we investigated soil TCF degradation behavior at different contents of 50, 100 and 200 mg/kg, and analyzed residual TCF contents and metabolites by gas chromatography mass spectrometry after 216-h incubation. Our results suggested that TCF was gradually degraded in soils and was be initially hydrolyzed to dichlorvos via P-C bond cleavage and then other P-containing metabolites. By analyzing different P fractions and soil microbial community composition, we found significant increases of soil available phosphorus contents from 2.76 mg/kg (control) to 3.23 mg/kg (TCF-50), 5.12 mg/kg (TCF-100) and 5.72 mg/kg (TCF-200), respectively. Inorganic CaCl2-P was easily and instantly transformed to primary mineral inorganic P (Pi) forms of HCl-P and citrate-P, while the proportion of enzyme-P (a labile organic P) fluctuated throughout TCF degradation process. Soil available P contents and Pi fractions were significantly correlated with the relative abundance of Actinobacteria. These results highlighted that Actinobacteria is the dominant soil species utilizing TCF as P sources to increase its community richness, and subsequently affect the transformation of P fractions to regulate soil P cycle. Our study gives new understanding on the microorganisms can involve soil P transformation during organophosphorus pesticides degradation in soils, highlighting the importance of bacteria in P transformation and pesticides soil decontamination.

Risk factors of postoperative low cardiac output syndrome in children with congenital heart disease: A systematic review and meta-analysis.

Background: Low cardiac output syndrome (LCOS) is the most common complication after cardiac surgery, which is associated with the extension of postoperative hospital stay and postoperative death in children with congenital heart disease (CHD). Although there are some studies on the risk factors of LCOS in children with CHD, an unified conclusion is lack at present. Purposes: To synthesize the risk factors of LCOS after CHD in children, and to provide evidence-based insights into the early identification and early intervention of LCOS. Methods: The databases of the China National Knowledge Infrastructure (CNKI), Wanfang Database, China Science and Technology Journal Database (VIP), PubMed, Cochrane Library, Embase and Web of Science were searched for relevant articles that were published between the establishing time of each database and January 2022. Based on retrospective records or cohort studies, the influencing factors of postoperative low cardiac output in children with congenital heart disease were included in Meta analysis.This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The risk of bias was evaluated according to the Newcastle-Ottawa Scale (NOS). RevMan 5.4 software was used to conduct the meta-analysis. Results: A total of 1,886 records were screened, of which 18 were included in the final review. In total, 37 risk factors were identified in the systematic review. Meta- analysis showed that age, type of CHD, cardiac reoperation, biventricular shunt before operation, CPB duration, ACC duration, postoperative residual shunt, cTn-1 level 2 h after CPB > 14 ng/ml and postoperative 24 h MR-ProADM level > 1.5 nmol/l were independent risk factors of LCOS. Additionally, the level of blood oxygen saturation before the operation was found to have no statistically significant relationship with LOCS. Conclusion: The risk factors of postoperative LCOS in children with CHD are related to disease condition, intraoperative time and postoperative related indexes, so early prevention should be aimed at high-risk children. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/, identifier: CRD42022323043.

Development of an LC-MS-based method to study the fate of nanoencapsulated pesticides in soils and strawberry plant.

The increased production and use of nanopesticides will increase the likelihood of their exposure to humans and the environment. In order to properly evaluate their risk, it will be necessary to rigorously quantify their concentrations in major environmental compartments including water, soil and food. Due to major differences in the characteristics of their formulation, it is unclear whether analytical techniques that have been developed for conventional pesticides will allow quantification of the nano-forms. Therefore, it is necessary to develop and validate analytical techniques for the quantification of nanopesticides in foods and the environment. The goal of this study was to validate a method for analyzing the active ingredients of two pesticides with different physicochemical properties: azoxystrobin (AZOX, a fungicide, log Kow 3.7) and bifenthrin (BFT, an insecticide, log Kow 6.6) that were applied to agricultural soils, either as a conventional formulation or encapsulated in nanoparticles (either Allosperse® or porous hollow nSiO2). Pesticide-free strawberry plants (Fragaria × ananassa) and three different agricultural soils were spiked with the active ingredients (azoxystrobin and bifenthrin), in either conventional or nano formulations. A modified QuEChERS approach was used to extract the pesticides from the strawberry plants (roots, leaves and fruits) and a solvent extraction (1:2 acetonitrile) was employed for the soils. Samples were analyzed by liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry in order to determine method detection limits, recoveries, precision and matrix effects for both the "conventional" and nanoencapsulated pesticides. Results for the modified method indicated good recoveries and precision for the analysis of the nanoencapsulated pesticides from strawberries and agricultural soils, with recoveries ranging from 85 to 127% (AZOX) and 68-138% (BFT). The results indicated that the presence of the nanoencapsulants had significant effects on the efficiency of extraction and the quantification of the active ingredients. The modified analytical methods were successfully used to measure strawberry and soil samples from a field experiment, providing the means to explore the fate of nanoencapsulated pesticides in food and environmental matrices.

Enhanced Lead (Pb) immobilization in red soil by phosphate solubilizing fungi associated with tricalcium phosphate influencing microbial community composition and Pb translocation in Lactuca sativa L.

Phosphate (P) minerals and phosphate solubilizing fungi (PSF) play essential roles in lead (Pb) immobilization, but their roles in driving Pb bioavailability and ecological risks in red soil remains poorly understood. In this study, the inoculation of P. oxalicum and TCP successfully enhanced available P (AP) and urease concentrations in artificially Pb contaminated red soil. Combined P. oxalicum and TCP inoculation significantly reduced Pb bioavailability, bioaccessibility, leachability and mobility by increasing soil AP concentration and forming stable Pb-P compounds during the 21-day experiment. Soil AP and Pb bioavailability play an important role in shifting soil microbial communities induced by co-occurrence of P. oxalicum and TCP. Combined P. oxalicum and TCP could notably promote the relative abundances of predominant soil genus to enhance microbial resistance to soil Pb. Likewise, coexistence of P. oxalicum and TCP showed the highest biomass and better branch root development of Pb-stressed in lettuces (Lactuca sativa L.) in pot experiment, and significantly reduced up to 88.1% of Pb translocation from soil to root over control. The reductions of Pb translocation and accumulation in root in P. oxalicum + TCP treatment could enhance the oxidase activities and alleviate the oxidative damages of H2O2 and O2.- in shoot tissues. Our study provided strong evidence to use PSF associated with P materials for the stable and eco-friendly soil Pb remediation.

Combined effects of Penicillium oxalicum and tricalcium phosphate on lead immobilization: Performance, mechanisms and stabilities.

Phosphorus (P) containing minerals are identified as effective Pb stabilizers in soil, while their low solubility limit the Pb immobilization efficiency. In this work, the combination of phosphate solubilizing fungi (PSF) Penicillium oxalicum and tricalcium phosphate (TCP) was constructed and applied to improve Pb immobilization stabilities in medium and soils. P. oxalicum+ TCP could significantly improve Pb2+ removal to above 99% under different TCP/Pb2+ and pH values. TCP and P. oxalicum could remarkably immobilize Pb by ion exchange, and PbC2O4 precipitation or surface adsorption, respectively. While the enhanced Pb immobilization in P. oxalicum+ TCP was explained by stronger Pb2+ interaction with tryptophan protein-like substances in extracellular polymeric substance, and the formation of the most stable Pb-phosphate compound hydroxypyromorphite (Pb5(PO4)3OH). Toxicity characteristic leaching procedure test showed that only 0.91% of Pb2+ was leachable in P. oxalicum+ TCP treatment, significantly lower than that in P. oxalicum (2.90%) and TCP (7.52%) treatments. In addition, the lowest soil exchangeable Pb fraction (37.1%) and the highest available soil P (88.0 mg/kg) were both found in P. oxalicum+ TCP treatment. By synergistically forming stable Pb-containing products, thus the combination of PSF and P minerals could significantly improve Pb2+ immobilization and stability in soils.

Effects of pyrolysis temperature, feedstock type and compaction on water retention of biochar amended soil.

Recent studies on water retention behaviour of biochar amended soil rarely considers the effect of pyrolysis temperature and also feedstock type into account. It is well known that pyrolysis temperature and feedstock type influences the physical and chemical properties of biochar due to stagewise decomposition of structure and chemical bonds. Further, soil density, which is in a loose state (in agricultural applications) and dense (in geo-environmental engineering applications) can also influence water retention behaviour of biochar amended soils. The major objective of this study is to investigate the water retention properties of soil amended with three different biochars in both loose and dense state. The biochars, i.e. water hyacinth biochar (WHB), chicken manure biochar (CMB) and wood biochar (WB) were produced in-house at different pyrolysis temperature. After then, biochars at 5% and 10% (w/w%) were amended to the soil. Water retention behaviour (soil suction and gravimetric water content) was studied under drying and wetting cycle simulated by varying relative humidity (RH, 50-90%). Results show that 10% WHB produced at 300 °C were found to possess highest water retention. CMB is found to possess higher water retention than WB for 10% amendment ratio. In general, the addition of three biochars (at both 300 °C and 600 °C) at 10% (w/w) significantly improved the water retention at all suction ranges in both loose and dense compaction state as compared to that of the bare soil. The adsorption (wetting) and desorption (drying) capacity of biochar amended soils is constant at corresponding RH.

Time-varying serum albumin levels and all-cause mortality in prevalent peritoneal dialysis patients: a 5-year observational study.

BACKGROUND: In this study, we investigated the association of time-varying serum albumin levels with mortality over a 5-year period in one cohort of patients undergoing long-term peritoneal dialysis (PD) therapy. METHODS: The participants in this study enrolled 302 patients who underwent long-term PD at a single PD center in Taiwan. We reviewed medical records from 2011 to 2015 retrospectively. Time-averaged albumin level and serum albumin reach rate (defined as the percentage of serum albumin measurements that reached ≥3.5 g/dL) were applied as the predictor variables in the first 2 years (2011-2012). All-cause mortality was used as the outcome variable in the subsequent 3 years (2013-2015). Hazard function of all-cause mortality in the study participants was examined by using Cox proportional hazard regression models . RESULTS: Patients with different albumin reach rates (75-< 100%, 50-< 75%, 1-< 50%) did not exhibit a significantly increased risk for all-cause mortality. Patients with a 0% albumin reach rate exhibited a significantly increased risk for all-cause mortality (hazard ratio [HR] 7.59, 95% confidence interval [CI], 2.38-24.21) by fully adjusted analysis. Patients with time-averaged albumin levels of < 3.5 g/dL (HR 15.49, 95% CI 1.74-137.72) exhibited a higher risk for all-cause mortality than those with serum albumin levels ≥4.0 g/dL. CONCLUSIONS: This study demonstrated that higher serum albumin reach rates and higher time-averaged serum albumin levels are associated with a lower mortality rate over a 5-year period among patients undergoing long-term PD.

Ubiquitination of MBNL1 Is Required for Its Cytoplasmic Localization and Function in Promoting Neurite Outgrowth.

The Muscleblind-like protein family (MBNL) plays an important role in regulating the transition between differentiation and pluripotency and in the pathogenesis of myotonic dystrophy type 1 (DM1), a CTG expansion disorder. How different MBNL isoforms contribute to the differentiation and are affected in DM1 has not been investigated. Here, we show that the MBNL1 cytoplasmic, but not nuclear, isoform promotes neurite morphogenesis and reverses the morphological defects caused by expanded CUG RNA. Cytoplasmic MBNL1 is polyubiquitinated by lysine 63 (K63). Reduced cytoplasmic MBNL1 in the DM1 mouse brain is consistent with the reduced extent of K63 ubiquitination. Expanded CUG RNA induced the deubiqutination of cytoplasmic MBNL1, which resulted in nuclear translocation and morphological impairment that could be ameliorated by inhibiting K63-linked polyubiquitin chain degradation. Our results suggest that K63-linked ubiquitination of MBNL1 is required for its cytoplasmic localization and that deubiquitination of cytoplasmic MBNL1 is pathogenic in the DM1 brain.

Reduced cytoplasmic MBNL1 is an early event in a brain-specific mouse model of myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats in the 3' untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Cognitive impairment associated with structural change in the brain is prevalent in DM1. How this histopathological abnormality during disease progression develops remains elusive. Nuclear accumulation of mutant DMPK mRNA containing expanded CUG RNA disrupting the cytoplasmic and nuclear activities of muscleblind-like (MBNL) protein has been implicated in DM1 neural pathogenesis. The association between MBNL dysfunction and morphological changes has not been investigated. We generated a mouse model for postnatal expression of expanded CUG RNA in the brain that recapitulates the features of the DM1 brain, including the formation of nuclear RNA and MBNL foci, learning disability, brain atrophy and misregulated alternative splicing. Characterization of the pathological abnormalities by a time-course study revealed that hippocampus-related learning and synaptic potentiation were impaired before structural changes in the brain, followed by brain atrophy associated with progressive reduction of axon and dendrite integrity. Moreover, cytoplasmic MBNL1 distribution on dendrites decreased before dendrite degeneration, whereas reduced MBNL2 expression and altered MBNL-regulated alternative splicing was evident after degeneration. These results suggest that the expression of expanded CUG RNA in the DM1 brain results in neurodegenerative processes, with reduced cytoplasmic MBNL1 as an early event response to expanded CUG RNA.

Implication of cerebral dopamine-beta hydroxylase for cardiovascular and mood regulation in rats.

The essentiality of the role of norepinephrine (NE) in the central nervous system has recently been reconsidered. NE exerts many effects and mediates a number of functions in living organisms. Dopamine-beta-hydroxylase (DBH) is the crucial enzyme for NE and epinephrine biosynthesis. Removal of this enzyme causes deficient NE at sympathetic terminals characterized by orthostatic hypotension in humans. The hypothesis tested in this study was that NE deficiency in the central nervous system caused autonomic failure in cardiovascular regulation. The immunotoxin anti-DBH-saporin (DSAP) was used to examine the putative role of cerebral NE. Male Sprague-Dawley rats were injected, intracerebroventricularly (icv), with DSAP and cardiovascular reactivity, as well as behavioral variables in the open-field locomotion test (OLT), sucrose intake test (SIT) and forced swim test (FST), were monitored for changes. The results indicated that treatment with DSAP caused significant reductions in spontaneous blood pressure (BP) and heart rate (HR), and a decrease in the rearing position on the OLT, in the same group of rats. In addition, a significant increase in mobility with low concurrent immobility frequencies was observed on the FST. However, there was no variation on the SIT. In conclusion, a deficiency in the cerebral DBH might dysregulate the autonomic outflows and, thus, leads to lower BP and HR. However, there was no mood change such as despair or anhedonia observed in the experiments.

Estimation of labeling efficiency in pseudocontinuous arterial spin labeling.

Pseudocontinuous arterial spin labeling MRI is a new arterial spin labeling technique that has the potential of combining advantages of continuous arterial spin labeling and pulsed arterial spin labeling. However, unlike continuous arterial spin labeling, the labeling process of pseudocontinuous arterial spin labeling is not strictly an adiabatic inversion and the efficiency of labeling may be subject specific. Here, three experiments were performed to study the labeling efficiency in pseudocontinuous arterial spin labeling MRI. First, the optimal labeling position was determined empirically to be approximately 84 mm below the anterior commissure-posterior commissure line in order to achieve the highest sensitivity. Second, an experimental method was developed to utilize phase-contrast velocity MRI as a normalization factor and to estimate the labeling efficiency in vivo, which was founded to be 0.86 +/- 0.06 (n = 10, mean +/- standard deviation). Third, we compared the labeling efficiency of pseudocontinuous arterial spin labeling MRI under normocapnic and hypercapnic (inhalation of 5% CO(2)) conditions and showed that a higher flow velocity in the feeding arteries resulted in a reduction in the labeling efficiency. In summary, our results suggest that labeling efficiency is a critical parameter in pseudocontinuous arterial spin labeling MRI not only in terms of achieving highest sensitivity but also in quantification of absolute cerebral blood flow in milliliters per minute per 100 g. We propose that the labeling efficiency should be estimated using phase-contrast velocity MRI on a subject-specific basis.