ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

Elucidating the callus-to-shoot-forming mechanism in Capsicum annuum 'Dempsey' through comparative transcriptome analyses.

BACKGROUND: The formation of shoots plays a pivotal role in plant organogenesis and productivity. Despite its significance, the underlying molecular mechanism of de novo regeneration has not been extensively elucidated in Capsicum annuum 'Dempsey', a bell pepper cultivar. To address this, we performed a comparative transcriptome analysis focusing on the differential expression in C. annuum 'Dempsey' shoot, callus, and leaf tissue. We further investigated phytohormone-related biological processes and their interacting genes in the C. annuum 'Dempsey' transcriptome based on comparative transcriptomic analysis across five species. RESULTS: We provided a comprehensive view of the gene networks regulating shoot formation on the callus, revealing a strong involvement of hypoxia responses and oxidative stress. Our comparative transcriptome analysis revealed a significant conservation in the increase of gene expression patterns related to auxin and defense mechanisms in both callus and shoot tissues. Consequently, hypoxia response and defense mechanism emerged as critical regulators in callus and shoot formation in C. annuum 'Dempsey'. Current transcriptome data also indicated a substantial decline in gene expression linked to photosynthesis within regenerative tissues, implying a deactivation of the regulatory system governing photosynthesis in C. annuum 'Dempsey'. CONCLUSION: Coupled with defense mechanisms, we thus considered spatial redistribution of auxin to play a critical role in the shoot morphogenesis via primordia outgrowth. Our findings shed light on shoot formation mechanisms in C. annuum 'Dempsey' explants, important information for regeneration programs, and have broader implications for precise molecular breeding in recalcitrant crops.

Anti-Diabetic Potential of Sargassum horneri and Ulva australis Extracts In Vitro and In Vivo.

Sargassum horneri (SH) and Ulva australis (UA) are marine waste resources that cause environmental and economic problems when entering or multiplying the coastal waters of Jeju Island. We analyzed their anti-diabetic efficacy to assess their reusability as functional additives. The alpha-glucosidase inhibitory activity of SH and UA extracts was confirmed, and the effect of UA extract was higher than that of SH. After the induction of insulin-resistant HepG2 cells, the effects of the two marine extracts on oxidative stress, intracellular glucose uptake, and glycogen content were compared to the positive control, metformin. Treatment of insulin-resistant HepG2 cells with SH and UA resulted in a concentration-dependent decrease in oxidative stress and increased intracellular glucose uptake and glycogen content. Moreover, SH and UA treatment upregulated the expression of IRS-1, AKT, and GLUT4, which are suppressed in insulin resistance, to a similar degree to metformin, and suppressed the expression of FoxO1, PEPCK involved in gluconeogenesis, and GSK-3ß involved in glycogen metabolism. The oral administration of these extracts to rats with streptozotocin-induced diabetes led to a higher weight gain than that in the diabetic group. Insulin resistance and oral glucose tolerance are alleviated by the regulation of blood glucose. Thus, the SH and UA extracts may be used in the development of therapeutic agents or supplements to improve insulin resistance.

NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development.

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

Harnessing CRISPR/Cas9 for Enhanced Disease Resistance in Hot Peppers: A Comparative Study on CaMLO2-Gene-Editing Efficiency across Six Cultivars.

The Capsicum annuum Mildew Locus O (CaMLO2) gene is vital for plant defense responses against fungal pathogens like powdery mildew, a significant threat to greenhouse pepper crops. Recent advancements in genome editing, particularly using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, have unlocked unprecedented opportunities for modifying disease-resistant genes and improving crop characteristics. However, the application of CRISPR technology in pepper cultivars has been limited, and the regeneration process remains challenging. This study addresses these limitations by investigating the feasibility of using the validated CaMLO2 genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/CaMLO2single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/CaMLO2sgRNA2-RNP systems by extending their application from the bell pepper 'Dempsey' and the hot pepper 'CM334' to six commercial hot pepper cultivars. Across the six cultivars, CaMLO2sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas CaMLO2sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/CaMLO2sgRNA1-RNP system to achieve efficient gene editing at the CaMLO2 locus in different Capsicum annuum cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with CaMLO2-mediated enhanced disease resistance.

Zinc Finger E-Box Binding Homeobox 2 as a Prognostic Biomarker in Various Cancers and Its Correlation with Infiltrating Immune Cells in Ovarian Cancer.

This study investigated the expression of zinc finger E-box binding homeobox 2 (ZEB2), its prognostic significance in various cancers, and the correlation between ZEB2 and infiltrating immune cells and ZEB2-related proteins in ovarian cancer (OV). The Gene Expression Profiling Interactive Analysis tool was used to analyze RNA sequencing data and cancer survival rates, based on normal and tumor tissue data available in The Cancer Genome Atlas (TCGA) database. The Kaplan-Meier plotter and PrognoScan databases were used to analyze the prognostic value of ZEB2 in OV (n = 1144). The Tumor Immune Estimation Resource was used to investigate the correlation between ZEB2 and infiltrating immune cells in various cancers, including OV. High ZEB2 expression was associated with a poorer prognosis in OV. In OV, ZEB2 is positively correlated with CD8+T cells, neutrophils, macrophages, and dendritic cell invasion; and ZEB2 is negatively correlated with tumor-infiltrating B cells. The STRING database was used to investigate the correlations with ZEB2-related proteins. The results reveal that ZEB2 was positively correlated with SMAD1 and SMAD2 in OV. Our findings may serve as a potential prognostic biomarker, and provide novel insights into the tumor immunology in OV. Thus, ZEB2 may be a potential diagnostic and therapeutic target in OV.

A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins.

KEY MESSAGE: Calli protoplasts isolated from three soybean cultivars are useful tools to evaluate guide RNAs for clustered regularly interspaced short palindromic repeats (CRISPR)-based precise gene editing. A type V CRISPR effector, LbCpf1(Cas12a) from Lachnospiraceae bacterium ND 2006, has been used for precision editing of the plant genome. We report that callus-derived protoplasts from three soybeans, including Glycine Max var. Williams 82 and two Korean cultivars (Kwangan and Daewon) represent efficient systems for the screening of active crRNA for CRISPR/LbCpf1. CRISPR/LbCpf1 ribonucleoproteins (RNPs) were delivered as complexes of purified endonucleases mixed with designed crRNA to simultaneously edit target genes of GlymaFAD2-1A and GlymaFAD2-1B transfected into three soybean protoplasts including genome-sequenced Williams 82 with cultivars, Kwangan and Daewon. Previously, we reported that nine crRNAs designed for LbCpf1 exhibited varying degrees of editing efficacy for two FAD2 genes. Among the nine crRNAs, the LbCpf1-crRNA3 complexes showed the highest efficiency in soybean cotyledon protoplasts. The new screening systems of callus protoplasts from three soybeans have been successfully used to transfect GFP-tagged markers and CRISPR/LbCpf1 RNPs. The callus protoplasts confirm that the LbCpf1-crRNA3 complex is an active crRNA for LbCpf1 to edit two FAD2 genes similar to cotyledon protoplasts. These results demonstrate that soybean callus protoplast-based CRISPR/crRNA selection is a new and practical tool to screen the efficacy of crRNAs and a prerequisite for progressive regeneration of the edited soybean.

Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey.

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains-AGL1, EHA101, and GV3101-to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum.

BACKGROUND: DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops. RESULTS: This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum 'CM334' and C. annuum 'Dempsey'. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs. CONCLUSIONS: Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.

CRISPR/Cas9-mediated editing of 1-aminocyclopropane-1-carboxylate oxidase1 enhances Petunia flower longevity.

The genes that encode the ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) are thought to be involved in flower senescence. Hence, we investigated whether the transcript levels of PhACO genes (PhACO1, PhACO3 and PhACO4) in Petunia cv. Mirage Rose are associated with ethylene production at different flowering stages. High transcript levels were detected in the late flowering stage and linked to high ethylene levels. PhACO1 was subsequently edited using the CRISPR/Cas9 system, and its role in ethylene production was investigated. PhACO1-edited T0 mutant lines, regardless of mutant type (homozygous or monoallelic), exhibited significantly reduced ethylene production and enhanced flower longevity compared with wild-type. Flower longevity and the reduction in ethylene production were observed to be stronger in homozygous plants than in their monoallelic counterparts. Additionally, the transmission of the edited gene to the T1 (lines 6 and 36) generation was also confirmed, with the results for flower longevity and ethylene production proving to be identical to those of the T0 mutant lines. Overall, this study increases the understanding of the role of PhACO1 in petunia flower longevity and also points to the CRISPR/Cas9 system being a powerful tool in the improvement of floricultural quality.

SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation.

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.

Can patient and family education prevent medical errors? A descriptive study.

BACKGROUND: This study aims to increase understanding of how patient and family education affects the prevention of medical errors, thereby providing basic data for developing educational contents. METHODS: This descriptive study surveyed patients, families, and Patient Safety Officers to investigate the relationship between educational contents and medical error prevention. The Chi-square test and ANOVA were used to derive the results of this study. The educational contents used in this study consisted of health information (1. current medicines, 2. allergies, 3. health history, 4. previous treatments/tests and complications associated with them) and Speak Up (1. handwashing, 2. patient identification, 3. asking about medical conditions, 4. asking about test results, 5. asking about behaviour and changes in lifestyle, 6. asking about the care plan, 7. asking about medicines, and 8. asking about medicine interactions). RESULTS: In this study, the first criterion for choosing a hospital for treatment in Korea was 'Hospital with a famous doctor' (58.6% patient; 57.7% families). Of the patients and their families surveyed, 82.2% responded that hospitals in Korea were safe. The most common education in hospitals is 'Describe your medical condition', given to 69.0% of patients, and 'Hospitalisation orientation', given to 63.4% of families. The most important factors in preventing patient safety events were statistically significant differences among patients, family members, and Patient Safety Officers (p = 0.001). Patients and families had the highest 'Patient and family participation' (31.0% of patients; 39.4% of families) and Patient Safety Officers had the highest 'Patient safety culture' (47.8%). CONCLUSIONS: Participants thought that educational contents developed through this study could prevent medical errors. The results of this study are expected to provide basic data for national patient safety campaigns and standardised educational content development to prevent medical errors.

Bioinformatic analysis of regulation of natural antisense transcripts by transposable elements in human mRNA.

Non-coding RNA is no longer considered to be "junk" DNA, based on evidence uncovered in recent decades. In particular, the important role played by natural antisense transcripts (NATs) in regulating the expression of genes is receiving increasing attention. However, the regulatory mechanisms of NATs remain incompletely understood. It is well-known that the insertion of transposable elements (TEs) can affect gene transcription. Using a bioinformatics approach, we identified NATs using human mRNA sequences from the UCSC Genome Browser Database. Our in silico analysis identified 1079 NATs and 700 sense-antisense gene pairs. We identified 179 NATs that showed evidence of having been affected by TEs during cellular gene expression. These findings may provide an understanding of the complex regulation mechanisms of NATs. If our understanding of NATs as modulators of gene expression is further enhanced, we can develop ways to control gene expression.

Effects of Queso Blanco cheese containing Bifidobacterium longum KACC 91563 on fecal microbiota, metabolite and serum cytokine in healthy beagle dogs.

The study describes the effects of administration of Queso Blanco cheese containing Bifidobacterium longum on the fecal microbiota, metabolite and serum cytokine in healthy beagle dogs. Twelve healthy beagle dogs were randomly divided in three groups of four dogs each:a control group, not fed with any cheese, and groups fed with Queso Blanco cheese with B. longum KACC 91563 (QCB) or without B. longum (QC) for 8 weeks. Fecal microbiota was analyzed using a culture-based method and 16s rRNA gene sequencing. Serum cytokine levels, activation of natural killer cells, and proliferation of peripheral blood mononuclear cells were determined. SPME-GC-MS method was used to determine the concentrations of short chain fatty acids and indole in dog feces. Administration of QCB for 4 weeks significantly increased the Bifidobacterium. QCB supplementation for 8 weeks reduceds Enterobacteriaceae and Clostridium perfringens (p < 0.05). The abundance of Fusobacterium, Blautia and Collinesella in QCB group were reduced as compared with the control group. Serum TNF-α and IL-6 levels at 8 weeks significantly increased in QCB group as compared with QC group. There was no change in the concentrations of total short chain fatty acids by B. longum at 0 and 4 weeks. At week 8, the acetic acid, propionic acid and butyric acid of the QCB and QC groups were significantly decreased compared to the control group. In conclusion, our results demonstrate that administration of QCB had positive effects on fecal microbiota and immune response in beagle dogs. We suggest that Queso Blanco cheese containing B. longum KACC 91563 could be used as a functional food for companion animals.

Histone H3K9 Demethylase JMJD2B Plays a Role in LXRα-Dependent Lipogenesis.

Ligand-activated liver X receptor α (LXRα) upregulates the expression of hepatic lipogenic genes, which leads to triglyceride (TG) accumulation, resulting in nonalcoholic fatty liver disease (NAFLD). Thus, LXRα regulation may provide a novel therapeutic target against NAFLD. However, histone methylation-mediated epigenetic regulation involved in LXRα-dependent lipogenesis is poorly understood. In this study, we investigated the functional role of the histone demethylase Jumonji domain-containing protein 2B (JMJD2B) in LXRα-dependent lipogenesis. JMJD2B expression level was upregulated in HepG2 cells treated with LXRα agonist T0901317 or palmitate and the liver of mice administered with T0901317 or fed a high-fat diet. Knockdown of JMJD2B using siRNA abrogated T0901317-induced LXRα-dependent lipogenic gene expression and lowered intracellular TG accumulation. Conversely, overexpression of JMJD2B in HepG2 cells upregulated the expression of LXRα-dependent lipogenic genes, in line with increased intracellular TG levels. JMJD2B overexpression or T0901317 treatment induced the recruitment of JMJD2B and LXRα to LXR response elements (LXRE) in the promoter region of LXRα-target gene and reduced the enrichment of H3K9me2 and H3K9me3 in the vicinity of the LXRE. Furthermore, JMJD2B enhanced T0901317 or LXRα-induced transcriptional activities of reporters containing LXRE. A co-immunoprecipitation assay revealed that JMJD2B interacted with activated LXRα. Moreover, overexpression of JMJD2B in mice resulted in upregulation of hepatic LXRα-dependent lipogenic genes, consistent with development of hepatic steatosis. Taken together, these results indicate that JMJD2B plays a role in LXRα-mediated lipogenesis via removing the repressive histone marks, H3K9me2 and H3K9me3, at LXRE, which might contribute to hepatic steatosis.

The Synergistic Effect of Co-Treatment of Methyl Jasmonate and Cyclodextrins on Pterocarpan Production in Sophora flavescens Cell Cultures.

Pterocarpans are derivatives of isoflavonoids, found in many species of the family Fabaceae. Sophora flavescens Aiton is a promising traditional Asian medicinal plant. Plant cell suspension cultures represent an excellent source for the production of valuable secondary metabolites. Herein, we found that methyl jasmonate (MJ) elicited the activation of pterocarpan biosynthetic genes in cell suspension cultures of S. flavescens and enhanced the accumulation of pterocarpans, producing mainly trifolirhizin, trifolirhizin malonate, and maackiain. MJ application stimulated the expression of structural genes (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3'H, and IFR) of the pterocarpan biosynthetic pathway. In addition, the co-treatment of MJ and methyl-ß-cyclodextrin (MeßCD) as a solubilizer exhibited a synergistic effect on the activation of the pterocarpan biosynthetic genes. The maximum level of total pterocarpan production (37.2 mg/g dry weight (DW)) was obtained on day 17 after the application of 50 µM MJ on cells. We also found that the combined treatment of cells for seven days with MJ and MeßCD synergistically induced the pterocarpan production (trifolirhizin, trifolirhizin malonate, and maackiain) in the cells (58 mg/g DW) and culture medium (222.7 mg/L). Noteworthy, the co-treatment only stimulated the elevated extracellular production of maackiain in the culture medium, indicating its extracellular secretion; however, its glycosides (trifolirhizin and trifolirhizin malonate) were not detected in any significant amounts in the culture medium. This work provides new strategies for the pterocarpan production in plant cell suspension cultures, and shows MeßCD to be an effective solubilizer for the extracellular production of maackiain in the cell cultures of S. flavescens.

Blood Pressure Drop and Penumbral Tissue Loss in Nonrecanalized Emergent Large Vessel Occlusion.

Background and Purpose- For patients with emergent large vessel occlusion who may not benefit from timely recanalization treatment, maintaining adequate cerebral perfusion to prevent penumbral tissue loss is a key therapeutic strategy. Cerebral perfusion should be proportional to systemic blood pressure (BP) due to the loss of autoregulation properties in ischemic brain tissue. We hypothesized that acute fluctuations in BP would lead to aggravated penumbral tissue loss in persistent large vessel occlusion. Methods- A total of 80 patients with persistent large vessel occlusion of internal carotid artery or middle cerebral artery admitted within 24 hours after onset, and with a baseline, National Institutes of Health Stroke Scale score ≥4-point were included. Baseline and follow-up (median 88 hours) magnetic resonance images were analyzed, and penumbra was defined as the Tmax>6 s region excluding baseline infarction. The hypoperfusion intensity ratio (Tmax>10 s/Tmax>6 s) was calculated within the penumbra. Penumbral tissue loss (%) was defined as the proportion of follow-up infarct in the penumbra. With serial BP measurements in the first 24 hours (median 29, interquartile range 26-35), BP and BP variability parameters, including BPdropmax (change from local maxima to minima), were calculated and compared. Generalized linear models were applied to examine the association between BP parameters and the penumbral tissue loss. Results- The median penumbral volume was 79.3 mL (interquartile range, 38.2-129.6) and median penumbral tissue loss was 36.7% (interquartile range, 12.0-56.1). In a multivariable analysis, systolic BP (SBP) SBPdropmax (ß±SE of fourth quartile, 17.82±6.58; P value, 0.01) and diastolic BP (DBP) DBPdropmax (ß±SE of fourth quartile, 14.04±6.38; P value, 0.01) were associated with increasing penumbral tissue loss, independently of age, baseline infarction and hypoperfusion intensity ratio. DBPincmax, SBPmax, DBPmax, SBPmax-min, DBPmax-min, and most of the DBP variability indices were associated with penumbral tissue loss. Conclusions- BP fluctuations, even a brief and drastic BP drop in the first 24 hours, significantly contributed to penumbral tissue loss irrespective of baseline hypoperfusion.

Spatial Regulation of ABCG25, an ABA Exporter, Is an Important Component of the Mechanism Controlling Cellular ABA Levels.

The phytohormone abscisic acid (ABA) plays crucial roles in various physiological processes, including responses to abiotic stresses, in plants. Recently, multiple ABA transporters were identified. The loss-of-function and gain-of-function mutants of these transporters show altered ABA sensitivity and stomata regulation, highlighting the importance of ABA transporters in ABA-mediated processes. However, how the activity of these transporters is regulated remains elusive. Here, we show that spatial regulation of ATP BINDING CASETTE G25 (ABCG25), an ABA exporter, is an important mechanism controlling its activity. ABCG25, as a soluble green fluorescent protein (sGFP) fusion, was subject to posttranslational regulation via clathrin-dependent and adaptor protein complex-2-dependent endocytosis followed by trafficking to the vacuole. The levels of sGFP:ABCG25 at the plasma membrane (PM) were regulated by abiotic stresses and exogenously applied ABA; PM-localized sGFP:ABCG25 decreased under abiotic stress conditions via activation of endocytosis in an ABA-independent manner, but increased upon application of exogenous ABA via activation of recycling from early endosomes in an ABA-dependent manner. Based on these findings, we propose that the spatial regulation of ABCG25 is an important component of the mechanism by which plants fine-tune cellular ABA levels according to cellular and environmental conditions.

A longitudinal study of the associations of BDNF genotype and methylation with poststroke anxiety.

BACKGROUND: Although the precise etiology of poststroke anxiety (PSA) has yet to be fully elucidated, it is known that brain-derived neurotrophic factor (BDNF) is important for neural plasticity and long-term potentiation, associated with the pathophysiology of anxiety. The expression of BDNF is regulated by epigenetic and genetic profiles. Thus, we investigated the association between BDNF methylation status and PSA at 2 weeks and 1 year after stroke while accounting for interactions with the BDNF Val66Met polymorphism. METHODS: The baseline sample comprised 286 patients who were assessed at 2 weeks after stroke; of these patients, 222 (78%) were followed up with at 1 year after stroke. The presence of PSA was determined using the anxiety subscale of the Hospital Anxiety and Depression Scale (HADS), and the effects of BDNF methylation status and polymorphisms on PSA status were assessed with multivariate logistic regression models. RESULTS: The prevalence of PSA was slightly lower (27 [9.4%]) at baseline, and 35 (15.8%) patients were identified as having PSA at the 1-year follow-up. Stroke patients with a higher average methylation status were more likely to have PSA at 1 year. The BDNF Val66Met polymorphism was not independently associated with PSA during either the acute or chronic phase after stroke, but there was a significant interactive effect between BDNF methylation and genotype on PSA at 2 weeks. CONCLUSIONS: In this study, BDNF methylation in combination with the met/met BDNF polymorphism (Val66Met polymorphism) was associated with PSA. These findings may help identify patients at higher risk for PSA.

Crystal structure of Arabidopsis thaliana RabA1a.

RabGTPase is a member of the Ras superfamily of small GTPases, which share a GTP-binding pocket containing highly conserved motifs that promote GTP hydrolysis. In Arabidopsis, the RabA group, which corresponds to the Rab11 group in animals, functions in the recycling of endosomes that control docking and fusion during vesicle transport. However, their molecular mechanisms remain unknown. In this study, we determined the crystal structures of the GDP-bound inactive form and both GppNHp- and GTP-bound active forms of RabA1a, at resolutions of 2.8, 2.6, and 2.6 Å, respectively. A bound sulfate ion in the active site of the GDP-bound structure stabilized Switch II by bridging the interaction between a magnesium ion and Arg74. Comparisons of the two states of RabA1a with Rab11 proteins revealed clear differences in the Switch I and II loops. These results suggested that conformational change of the Switch regions of RabA1a, derived by GTP or GDP binding, could maintain subcellular membrane traffic through the specific interaction of effector molecules.