RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.

Association between Genetic Polymorphism of SCN1A, GABRA1 and ABCB1 and Drug Responsiveness in Vietnamese Epileptic Children.

Background and Objectives: Drug resistant epilepsy (DRE) is a major hurdle in epilepsy, which hinders clinical care, patients' management and treatment outcomes. DRE may partially result from genetic variants that alter proteins responsible for drug targets and drug transporters in the brain. We aimed to examine the relationship between SCN1A, GABRA1 and ABCB1 polymorphism and drug response in epilepsy children in Vietnam. Materials and Methods: In total, 213 children diagnosed with epilepsy were recruited in this study (101 were drug responsive and 112 were drug resistant). Sanger sequencing had been performed in order to detect six single nucleotide polymorphisms (SNPs) belonging to SCN1A (rs2298771, rs3812718, rs10188577), GABRA1 (rs2279020) and ABCB1 (rs1128503, rs1045642) in study group. The link between SNPs and drug response status was examined by the Chi-squared test or the Fisher's exact test. Results: Among six investigated SNPs, two SNPs showed significant difference between the responsive and the resistant group. Among those, heterozygous genotype of SCN1A rs2298771 (AG) were at higher frequency in the resistant patients compared with responsive patients, playing as risk factor of refractory epilepsy. Conversely, the heterozygous genotype of SCN1A rs3812718 (CT) was significantly lower in the resistant compared with the responsive group. No significant association was found between the remaining four SNPs and drug response. Conclusions: Our study demonstrated a significant association between the SCN1A genetic polymorphism which increased risk of drug-resistant epilepsy in Vietnamese epileptic children. This important finding further supports the underlying molecular mechanisms of SCN1A genetic variants in the pathogenesis of drug-resistant epilepsy in children.

Soluble Immune Checkpoint Protein CD27 Is a Novel Prognostic Biomarker of Hepatocellular Carcinoma Development in Hepatitis C Virus-Sustained Virological Response Patients.

Factors affecting the probability of hepatocellular carcinoma (HCC) development even after sustained virological response (SVR) following anti-hepatitis C virus (HCV) therapy remain unelucidated. This study characterized the role of 16 soluble (s) immune checkpoint proteins in 168 HCV-SVR patients, with 47 developing HCC at the study end point. At baseline, high concentrations of 10 immune checkpoint proteins were found in the sera of the HCC group. At the study end point, levels of sCD27, sCD28, sCD40, and sCD86 in the HCC group, which were depleted following SVR, returned to higher levels than those in the non-HCC group. More importantly, patients with baseline levels of sCD27 ≥ 4104 pg/mL, sCD28 ≥ 1530 pg/mL, and sCD40 ≥ 688 pg/mL predicted a significantly greater HCC cumulative rate. Although sCD27 was elevated in patient sera, its membrane-bound form, mCD27, accumulated in the tumor and peritumor area, mainly localized in T cells. Interestingly, T-cell activation time dependently induced sCD27. Furthermore, CD70, the ligand of CD27, was robustly expressed in HCC area in which CD70 promoter methylation analysis indicated the hypomethylation compared with the nontumor pairs. Recombinant human CD27 treatment induced the proliferation of CD70-bearing HepG2 cells via the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase pathway, but not NF-κB or p38 pathway. In conclusion, these data indicate that baseline sCD27, sCD28, and sCD40 levels could be used as HCC prognostic markers in HCV-SVR patients. sCD27 likely promotes HepG2 cell growth via the CD27-CD70 axis.

Hexa Histidine-Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species.

BACKGROUND AND AIMS: Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH AND RESULTS: Cygb-deficient mice that had bile duct ligation-induced liver cholestasis or choline-deficient amino acid-defined diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb-overexpressing mice. We produced hexa histidine-tagged recombinant human CYGB (His-CYGB), traced its biodistribution, and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes through a clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type 1 alpha 1 production and α-smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-ß secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA-sequencing analysis revealed the down-regulation of inflammation- and fibrosis-related genes and the up-regulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in chimeric mice with humanized livers. CONCLUSIONS: His-CYGB could have antifibrotic clinical applications for human chronic liver diseases.

Molecular diversity of Rice grassy stunt virus in Vietnam.

Rice grassy stunt virus (RGSV, Tenuivirus) recently emerged on rice in Vietnam, causing high yield losses during 2006-2009. The genetic diversity of RGSV is poorly documented. In this study, the two genes encoded by each ambisense segment RNA3 and RNA5 of RGSV isolates from six provinces of South Vietnam were sequenced. P3 and Pc3 (RNA3) have unknown function, P5 (RNA5) encodes the putative silencing suppressor, and Pc5 (RNA5) encodes the nucleocapsid protein (N). The sequences of 17 Vietnamese isolates were compared with reference isolates from North and South Philippines. The average nucleotide diversity among the isolates was low. We confirmed a higher variability of RNA3 than RNA5 and Pc3 than P3. No relationships between the genetic diversity and the geographic distribution of RGSV isolates could be ascertained, likely because of the long-distance migration of the insect vector. This data will contribute to a better understanding on the RGSV epidemiology in South Vietnam, a prerequisite for further management of the disease and rice breeding for resistance.

Super-Resolution Imaging Reveals the Nanoscale Distributions of Dystroglycan and Integrin Itga7 in Zebrafish Muscle Fibers.

Cell signaling is determined partially by the localization and abundance of proteins. Dystroglycan and integrin are both transmembrane receptors that connect the cytoskeleton inside muscle cells to the extracellular matrix outside muscle cells, maintaining proper adhesion and function of muscle. The position and abundance of Dystroglycan relative to integrins is thought to be important for muscle adhesion and function. The subcellular localization and quantification of these receptor proteins can be determined at the nanometer scale by FPALM super-resolution microscopy. We used FPALM to determine localizations of Dystroglycan and integrin proteins in muscle fibers of intact zebrafish (Danio rerio). Results were consistent with confocal imaging data, but illuminate further details at the nanoscale and show the feasibility of using FPALM to quantify interactions of two proteins in a whole organism.

The Origins of the Dark-Hyperactivity and Negative Peer Relationships, an Objectively Lower Sleep Efficiency, and a Longer Sleep Onset Latency at Age Five Were Associated with Callous-Unemotional Traits and Low Empathy at Age 14.

BACKGROUND: Within the spectrum of emotional competencies, callous-unemotional traits are socially discouraged, while empathy is considered a socially much more accepted emotional trait. This holds particularly true for adolescents, who are still building up their social and emotional competencies. The aims of the present study were two-fold: First, longitudinally, to identify traits of behavioral problems and objective sleep dimensions at the age of 5 years to predict callous-unemotional traits and empathy at the age of 14 years. Second, cross-sectionally, to associate callous-unemotional traits and empathy with current insomnia, stress, and mental toughness. METHODS: Preschoolers at the age of 5 years were contacted nine years later at the age of 14 years. At 5 years, parents rated their children's behavior (Strength and Difficulties Questionnaire, SDQ); in parallel, children underwent a one-night sleep-EEG assessment. At the age of 14 years, adolescents completed a series of questionnaires covering callous-unemotional traits, insomnia, empathy, stress, and mental toughness. RESULTS: A total of 77 adolescents (38.1% females) took part in the present study. Longitudinally, higher scores for hyperactivity at age 5 significantly predicted higher callous-unemotional traits at age 14. A higher score for negative peer relationships at age 5 significantly predicted lower scores for cognitive empathy at age 14. Further, objective sleep-EEG measures showed that a higher sleep efficiency and a shorter sleep latency was associated with lower scores for callousness. Cross-sectionally, higher scores for callous-unemotional traits were associated with higher insomnia and stress, while lower insomnia was associated with higher empathy. Mental toughness was unrelated to callous-unemotional traits and empathy. CONCLUSIONS: It appears that hyperactivity traits and negative peer relationships and more unfavorable objective sleep patterns at 5 years predicted socially discouraged callous-unemotional traits and low empathy during adolescence. Further, cross-sectionally at the age of 14, callous-unemotional traits, subjective poor sleep, and higher stress were associated.

Mediating Effects of Self-Esteem on the Relationship Between Self-Compassion and Empathy Among Vietnamese University Students.

Empathy is a critical element of subjective well-being and an important personality trait among undergraduate students. To improve empathy among undergraduate students, the current study examined the relationship between self-compassion and empathy and the mediating role of self-esteem in this relationship. Participants were six hundred and twenty-two (320 males and 302 females) students from five Vietnamese universities, aged 18-21 years (M age = 19.5; SD age = 0.95 years), who completed the self-compassion scale (SCS), empathy scale in adults (BES-A), and self-esteem scale of Toulouse (ETES). The results indicated that (1) self-compassion was positively associated with empathy; (2) self-esteem mediated the relationship between the two variables. Therefore, enhancing undergraduate students' self-compassion may be an effective way to improve their empathy. However, additional studies are required to elucidate the role of self-compassion in the educational context.

New Angled Twin-layer Porous Substrate Photobioreactors for Cultivation of Nannochloropsis oculata.

An angled twin-layer porous substrate photobioreactor (TL-PSBR) using LED light was designed to cultivate Nannochloropsis oculata. Flocculation and sedimentation by modification of pH to 11 were determined as the optimal method for harvesting the N. oculata inoculum. The following optimised parameters were found: tilt angle 15°, Kraft 220 g m-2 paper as substrate material, initial inoculum density of 12.5 g m-2, 140 µmol photons m-2 s-1 light intensity, and a light/dark cycle of 6:6 (h). Test cultivation for 14 days was performed under optimised conditions. The total dried biomass standing crop was 75.5 g m-2 growth area with an average productivity of 6.3 g m-2 d-1, the productivity per volume of used culture medium was 126.2 mg/L d-1, total lipid content 21.9% (w/w), and the highest productivity of total lipids was 1.33 g m-2 d-1. The dry algal biomass contained 3% eicosapentaenoic acid (w/w), 3.7% palmitoleic acid (w/w), and 513 mg kg-1 vitamin E. The optimisation of N. oculata cultivation on an angled TL-PSBR system yielded promising results, and applications for commercial products need to be further explored.

Cytoglobin attenuates pancreatic cancer growth via scavenging reactive oxygen species.

Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.

Capacity of extracellular globins to reduce liver fibrosis via scavenging reactive oxygen species and promoting MMP-1 secretion.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are the primary cell type in liver fibrosis, a significant global health care burden. Cytoglobin (CYGB), a globin family member expressed in HSCs, inhibits HSC activation and reduces collagen production. We studied the antifibrotic properties of globin family members hemoglobin (HB), myoglobin (MB), and neuroglobin (NGB) in comparison with CYGB. APPROACH & RESULTS: We characterized the biological activities of globins in cultured human HSCs (HHSteCs) and their effects on carbon tetrachloride (CCl4)-induced cirrhosis in mice. All globins demonstrated greater antioxidant capacity than glutathione in cell-free systems. Cellular fractionation revealed endocytosis of extracellular MB, NGB, and CYGB, but not HB; endocytosed globins localized to intracellular membranous, cytoplasmic, and cytoskeletal fractions. MB, NGB, and CYGB, but not HB, scavenged reactive oxygen species generated spontaneously or stimulated by H2O2 or transforming growth factor ß1 in HHSteCs and reduced collagen 1A1 production via suppressing COL1A1 promoter activity. Disulfide bond-mutant NGB displayed decreased heme and superoxide scavenging activity and reduced collagen inhibitory capacity. RNA sequencing of MB- and NGB-treated HHSteCs revealed downregulation of extracellular matrix-encoding and fibrosis-related genes and HSC deactivation markers. Upregulation of matrix metalloproteinase (MMP)-1 was observed following MB and NGB treatment, and MMP-1 knockdown partially reversed globin-mediated effects on secreted collagen. Importantly, administration of MB, NGB, and CYGB suppressed CCl4-induced mouse liver fibrosis. CONCLUSIONS: These findings revealed unexpected roles for MB and NGB in deactivating HSCs and inhibiting liver fibrosis development, suggesting that globin therapy may represent a new strategy for combating fibrotic liver disease.

Cancer cells produce liver metastasis via gap formation in sinusoidal endothelial cells through proinflammatory paracrine mechanisms.

Intracellular gap (iGap) formation in liver sinusoidal endothelial cells (LSECs) is caused by the destruction of fenestrae and appears under pathological conditions; nevertheless, their role in metastasis of cancer cells to the liver remained unexplored. We elucidated that hepatotoxin-damaged and fibrotic livers gave rise to LSECs-iGap formation, which was positively correlated with increased numbers of metastatic liver foci after intrasplenic injection of Hepa1-6 cells. Hepa1-6 cells induced interleukin-23-dependent tumor necrosis factor-α (TNF-α) secretion by LSECs and triggered LSECs-iGap formation, toward which their processes protruded to transmigrate into the liver parenchyma. TNF-α triggered depolymerization of F-actin and induced matrix metalloproteinase 9 (MMP9), intracellular adhesion molecule 1, and CXCL expression in LSECs. Blocking MMP9 activity by doxycycline or an MMP2/9 inhibitor eliminated LSECs-iGap formation and attenuated liver metastasis of Hepa1-6 cells. Overall, this study revealed that cancer cells induced LSEC-iGap formation via proinflammatory paracrine mechanisms and proposed MMP9 as a favorable target for blocking cancer cell metastasis to the liver.

Traumatic cerebrospinal fluid oculorrhea managed with an external ventricular drain.

Cerebrospinal fluid (CSF) leaks following head injuries are rare complications with significant morbidity and mortality if left untreated. CSF oculorrhea secondary to a cranio-orbital fistula is a rare presentation of this complication. Standard treatment for a CSF leak involves management of intracranial pressure, CSF diversion and surgical repair of any dural defect. Lumbar drains have commonly been inserted to aid in diverting CSF. We describe a case of a 16-year-old male who presented with an open comminuted depressed skull fracture and CSF oculorrhea. Following a bifrontal decompressive craniectomy, he was successfully treated with CSF diversion following a conservative trial using an external ventricular drain. The use of an external ventricular drain for this purpose has not been described in the literature to date. We report this case as a method of demonstrating the use of an external ventricular drain to adequately divert CSF.

Extensive gene flow of white-backed planthopper in the Greater Mekong Subregion as revealed by microsatellite markers.

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is a destructive pest of rice in the Greater Mekong Subregion (GMS) countries including Cambodia, Laos, Myanmar, Thailand, Vietnam, and China's Yunnan Province. Our previous study not only confirmed the immigration sources of the WBPH in China's Yunnan Province were from Myanmar, Vietnam, and Laos, but also indicated that Cambodia was likely an additional migration source. To further clarify the migration sources and patterns of the WBPH in the GMS, we investigated the genetic structure of 42 WBPH populations using microsatellite loci markers. The analysis of genetic diversity, heterozygosity deficit, and heterozygosity excess based on the nuclear markers suggest that there is extensive gene flow between the 42 sampled populations from the GMS. The genetic structure confirmed the immigration sources of WBPH as revealed by mitochondrial markers and trajectory analyses methods in previous studies. These findings will aid in the sustainable regional management of this insect pest in the GMS.

Asymmetric construction of quaternary carbon stereocenters: high stereoselection in Mukaiyama aldol reactions of 2-siloxyindoles with chiral aldehydes.

[reaction: see text] A new synthesis of enantiopure 3,3-disubstituted oxindoles by stereoselective Mukaiyama aldol reaction of 3-substituted 2-siloxyindoles and chiral, enantiopure aldehydes having nitrogen or oxygen substituents at the alpha carbon is described. When the C3 substituent of the prochiral nucleophile is aryl or heteroaryl, stereoselectivity is high (10-80:1).

Sulfur mustard primes phagocytosis and degranulation in human polymorphonuclear leukocytes.

Sulfur mustard (2,2'-bis-chloroethyl-sulfide; SM) is a chemical warfare vesicant that causes debilitating skin lesions. Although a great deal of work has focused on the direct effects of SM exposure on the epithelium, it is unclear how much the inflammatory response, induced by exposure, contributes to lesion pathogenesis. Keratinocytes exposed to SM express a number of inflammatory mediators and elicit a cellular infiltrate consisting largely of polymorphonuclear leukocytes (PMN). PMN infiltration into SM lesions occurs as early as 30 min and peaks after several hours postexposure, and, despite the relatively short half-life of SM, PMN infiltrating a lesion could be exposed to micromolar concentrations of the agent. Previously, we have shown that exposure to low doses of sulfur mustard prime oxidative function in human PMN. The current study was undertaken to evaluate the effects of low-dose SM exposure on PMN phagocytosis, degranualtion and chemotaxis. PMN exposed to low doses of SM (50-200 microM) showed a dose-dependent enhancement of phagocytic function. Exocytosis of PMN azurophilic and specific granules [determined by analysis of granule-specific intravesicular receptors, Interleukin 10 receptor (IL-10R) and CD63] was also enhanced by SM exposure. Finally, we examined the effect of SM as a chemoattractant for PMN and show that SM is not itself a chemotaxin. These results suggest that SM injury may, in part, be caused by normal inflammatory function, and that therapeutic strategies aimed at down-regulating PMN activation could lessen the severity of SM injury and the time required for its resolution.

Low-dose sulfur mustard primes oxidative function and induces apoptosis in human polymorphonuclear leukocytes.

Although considerable work has focused on understanding the processes of direct tissue injury mediated by the chemical warfare vesicant, sulfur mustard (2,2'-bis-chloroethyl sulfide; SM), relatively little is known regarding the mechanisms of secondary injury caused potentially by the acute inflammatory response that follows SM exposure. Polymorphonuclear leukocytes (PMNs) play a central role in the initiation and propagation of inflammatory responses that, in some cases, result in autoimmune tissue damage. The potential for PMN-derived tissue damage following SM exposure may, in part, account for the protracted progression of the injury before it resolves. The current study was undertaken to evaluate the priming, oxidative function, and viability of PMN following exposure to low doses of SM such as those that might remain in tissues as a result of topical exposure. Our results demonstrate that doses of SM ranging from 25 to 100 microM primed PMN for oxidative burst in response to activation by fMLP, and that doses of SM ranging from 50 to 100 microM induced PMN apoptosis. Understanding the mechanisms through which SM directly affects PMN activation and apoptosis will be of critical value in developing novel treatments for inflammatory tissue injury following SM exposure.