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Enlightened from our previous work of structural simplification of quinine and innovative application of natural products against phytopathogenic fungi, lead structure 2,8-bis(trifluoromethyl)-4-quinolinol (3) was selected to be a candidate and its diversified design, synthesis, and antifungal evaluation were carried out. All of the synthesized compounds Aa1-Db1 were evaluated for their antifungal activity against four agriculturally important fungi, Botrytis cinerea, Fusarium graminearum, Rhizoctonia solani, and Sclerotinia sclerotiorum. Results showed that compounds Ac3, Ac4, Ac7, Ac9, Ac12, Bb1, Bb10, Bb11, Bb13, Cb1. and Cb3 exhibited a good antifungal effect, especially Ac12 had the most potent activity with EC50 values of 0.52 and 0.50 μg/mL against S. sclerotiorum and B. cinerea, respectively, which were more potent than those of the lead compound 3 (1.72 and 1.89 μg/mL) and commercial fungicides azoxystrobin (both >30 μg/mL) and 8-hydroxyquinoline (2.12 and 5.28 μg/mL). Moreover, compound Ac12 displayed excellent in vivo antifungal activity, which was comparable in activity to the commercial fungicide boscalid. The preliminary mechanism revealed that compound Ac12 might cause an abnormal morphology of cell membranes, an increase in membrane permeability, and release of cellular contents. These results indicated that compound Ac12 displayed superior in vitro and in vivo fungicidal activities and could be a potential fungicidal candidate against plant fungal diseases.

Plant pathogenic fungi seriously affect agricultural production and are difficult to control. The discovery of new leads based on natural products is an important way to innovate fungicides. In this study, 30 natural-product-based magnolol derivatives were synthesized and characterized on the basis of NMR and mass spectroscopy. Bioactivity tests on phytopathogenic fungi (Rhizoctonia solani, Fusarium graminearum, Botrytis cinerea, and Sclerotinia sclerotiorum) in vitro of these compounds were performed systematically. The results showed that 11 compounds were active against four kinds of phytopathogenic fungi with EC50 values in the range of 1.40-20.00 μg/mL, especially compound L5 that exhibited excellent antifungal properties against B. cinerea with an EC50 value of 2.86 μg/mL, approximately 2.8-fold more potent than magnolol (EC50 = 8.13 μg/mL). Moreover, compound L6 showed the highest antifungal activity against F. graminearum and Rhophitulus solani with EC50 values of 4.39 and 1.40 μg/mL, respectively, and compound L7 showed good antifungal activity against S. sclerotiorum. Then, an in vivo experiment of compound L5 against B. cinerea was further investigated in vivo using infected tomatoes (curative effect, 50/200 and 36%/100 μg/mL). The physiological and biochemical studies illustrated that the primary action mechanism of compound L5 on B. cinerea might change the mycelium morphology, increase cell membrane permeability, and destroy the function of mitochondria. Furthermore, structure-activity relationship (SAR) studies revealed that hydroxyl groups play a key role in antifungal activity. To sum up, this study provides a reference for understanding the application of magnolol-based antifungal agents in crop protection.

Rhizoctonia solani causes serious plant diseases. Neocryptolepine presented the significant antifungal activity against R. solani, however the mode of action is unclear. In this paper, we investigated the potential mode of action of neocryptolepine against R. solani integrated the proteomics and transcriptomics. Results showed that after treatment with neocryptolepine, 1012 differentially expressed proteins and 10 920 differentially expressed genes of R. solani were found, most of them were enriched in mitochondrial respiratory chain. It affected oxidative phosphorylation led to the enrichment of ROS and the decrease of MMP, and inhibited complex III activity with the inhibition rate of 63.51% at 10 μg/mL. The mitochondrial structural and function were damaged. Cytochrome b-c1 complex subunit Rieske (UQCRFS1) with the high binding score to neocryptolepine was found as a potential target. In addition, it inhibited the sclerotia formation and presented antifungal efficacy by decreasing the diameter of a wound in potato in a concentration-dependent manner. Above results indicated that neocryptolepine inhibited the complex III activity by binding UQCRFS1 and blocked the ion transfer to cause the death of R. solani mycelia. This study laid the foundation for the future development of neocryptolepine as an alternative biofungicide.

The small brown planthopper (SBPH), Laodelphax striatellus (Fallen), is an economically important pest that affects rice. In this study, we analyzed the sublethal effects of triflumezopyrim on the biological and demographic parameters of SBPHs in the laboratory. Treatment of third-instar SBPHs with the LC30 of triflumezopyrim prolonged the lives of male adults. The F0 individuals exhibited a significant decrease in fecundity (25.8%) after exposure to triflumezopyrim. The fecundity and egg hatchability of the progeny (F1 individuals) were not significantly affected by treatment with the LC30 of triflumezopyrim, but the mean generation time increased significantly. In addition, the intrinsic rate of increase, finite rate of increase, net reproductive rate, and doubling time did not exhibit any significant changes. However, long-term exposure to the LC75 of triflumezopyrim resulted in a significant decrease in the number of males in the SBPH population. The results of this study indicate that triflumezopyrim can be utilized in pest control programs for SBPHs.

Humulus lupulus Linn. is a traditional medicinal and edible plant with several biological properties. The aims of this work were: (1) to evaluate the in vitro antifungal activity of H. lupulus ethanolic extract, (2) to study the in vitro and in vivo antifungal activity of isoxanthohumol, an isoprene flavonoid from H. lupulus, against Botrytis cinerea, and (3) to explore the antifungal mechanism of isoxanthohumol on B. cinerea. The present data revealed that the ethanolic extract of H. lupulus exhibited moderate antifungal activity against the five tested phytopathogenic fungi in vitro, and isoxanthohumol showed highly significant antifungal activity against B. cinerea, with an EC50 value of 4.32 µg/mL. Meanwhile, it exhibited moderate to excellent protective and curative efficacies in vivo. The results of morphologic observation, RNA-seq, and physiological indicators revealed that the antifungal mechanism of isoxanthohumol is mainly related to metabolism, it affected the carbohydrate metabolic process, destroyed the tricarboxylic acid (TCA) cycle, and hindered the generation of ATP by inhibiting respiration. Further studies indicated that isoxanthohumol caused membrane lipid peroxidation, thus accelerating the death of B. cinerea. This study demonstrates that isoxanthohumol can be used as a potential botanical fungicide for the management of phytopathogenic fungi.

Summary Taxane-based reagents, such as Taxol, Taxotere, and Abraxane, are popular anti-cancer drugs that can differ in their clinical efficacy. This difference is generally attributed to their active pharmaceutical ingredients. Here, we report a serendipitous discovery that Taxol induces metabolic dysregulation and unfolded protein response. Surprisingly, these effects of Taxol are entirely dependent on its excipient, Cremophor EL (CrEL). We show that CrEL promotes aerobic glycolysis and in turn results in drastic upregulation of angiopoietin like 4 (ANGPTL4), a major regulator of human blood lipid profile. Notably, premedication with dexamethasone further enhances the expression of ANGPTL4. Consistently, we find that the amplitude and frequency of increase in triglycerides is more prominent in Taxol-treated patients with breast cancer. In addition, we find that CrEL activates the unfolded protein response pathway to trigger proinflammatory gene expression and caspase/gasdermin E-dependent pyroptosis. Finally, we discuss the implications of these results in anti-cancer therapies., Graphical abstract, Highlights • Cremophor EL, the excipient of chemotherapy drug Taxol, is biologically active • Cremophor EL promotes aerobic glycolysis in cancer and primary human immune cells • Dexamethasone and Cremophor EL may cause dyslipidemia via ANGPTL4 upregulation • Cremophor EL promotes the unfolded protein response, Biological sciences; Molecular biology; Cell biology; Cancer

Despite advances in the characterization of colorectal cancer (CRC), it still faces a poor prognosis. There is growing evidence that gut microbiota and their metabolites potentially contribute to the development of CRC. Thus, microbial dysbiosis and their metabolites associated with CRC, based on stool samples, may be used to advantage to provide an excellent opportunity to find possible biomarkers for the screening, early detection, prevention, and treatment of CRC. Using 16S rRNA amplicon sequencing coupled with statistical analysis, this study analyzed the cause–effect shift of the microbial taxa and their metabolites that was associated with the fecal gut microbiota of 17 healthy controls, 21 polyps patients, and 21 cancer patients. The microbial taxonomic shift analysis revealed striking differences among the healthy control, polyps and cancer groups. At the phylum level, Synergistetes was reduced significantly in the polyps group compared to the healthy control and cancer group. Additionally, at the genus level and in association with the cancer group, a total of 12 genera were highly enriched in abundance. In contrast, only Oscillosprira was significantly higher in abundance in the healthy control group. Comparisons of the polyps and cancer groups showed a total of 18 significantly enriched genera. Among them, 78% of the genera associated with the cancer group were in higher abundance, whereas the remaining genera showed a higher abundance in the polyps group. Additionally, the comparison of healthy control and polyp groups showed six significantly abundant genera. More than 66% of these genera showed a reduced abundance in the polyps group than in healthy controls, whereas the remaining genera were highly abundant in the polyps group. Based on tumor presence and absence, the abundance of Olsenella and Lactobacillus at the genus level was significantly reduced in the patient group compared to healthy controls. The significant microbial function prediction revealed an increase in the abundance of metabolites in the polyps and cancer groups compared to healthy controls. A correlation analysis revealed a higher contribution of Dorea in the predicted functions. This study showed dysbiosis of gut microbiota at the taxonomic level and their metabolic functions among healthy subjects and in two stages of colorectal cancer, including adenoma and adenocarcinoma, which might serve as potential biomarkers for the early diagnosis and treatment of CRC.

The stem cell is the foundation of regenerative medicine and tissue engineering. Regulating specific stem cell fate, such as cell attachment, proliferation, differentiation, and even death, undergoes continuous development. Cold atmospheric plasma (CAP), the core technology of plasma medicine, is attracting tremendous attention due to its ability and versatility to manipulate various types of cells, including stem cells. Specifically, the direct and indirect applications of CAP in controlling cell fate are best exemplified by upfront irradiation of the stem cells and modification of the stem cell niche, respectively. This review will describe the recent advances in various CAP strategies, both direct and indirect, and their influence on the fate of healthy and cancer stem cells. Particular emphasis will be placed on the mechanism of connecting the physical and chemical cues carried by the plasma and biological changes presented by the cells, especially at the transcriptomic level. The ultimate goal is to exploit CAP’s potential in regenerative medicine.

Magnolia officinalis, as a well-known herb worldwide, has been widely used to treat multiple diseases for a long time. In this study, the petroleum ether extract from M. officinalis showed effective antifungal activity against seven plant pathogens (particularly against R. solani with an inhibition rate of 100.00% at 250 μg/mL). Honokiol and magnolol, isolated by the bioassay-guided method, exhibited greater antifungal activity than tebuconazole (EC50 = 3.07 μg/mL, p ≤ 0.001) against R. solani, which EC50 values were 2.18 μg/mL and 3.48 μg/mL, respectively. We used transcriptomics to explore the mechanism of action of honokiol against R. solani. Results indicated that honokiol may exert antifungal effects by blocking the oxidative phosphorylation metabolic pathway. Further studies indicated that honokiol induced ROS overproduction, disrupted the mitochondrial function, affected respiration, and blocked the TCA cycle, which eventually inhibited ATP production. Besides, honokiol also damaged cell membranes and caused morphological changes. This study demonstrated that the lignans isolated from M. officinalis possess the potential to be developed as botanical fungicides.

Nine antialgal active compounds, (i.e. trehalose (1), twenty-two methyl carbonate (2), (-)-dihydromenisdaurilide (3), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (4), isophytol (5), 8-hexadecenol (6), 17-hydroxyheptadecanoic acid (7), trans-asarone (8) and 2-amino-3-mercaptopropanoic acid (9)) were isolated from Ulva pertusa for the first time by sephadex LH-20 column chromatography, silica gel column chromatography and repeated preparative TLC. Except for compound 4, all compounds represented novel isolated molecules from marine macroalgae. Further, antialgal activities of these compounds against Amphidinium carterae, Heterosigma akashiwo, Karenia mikimitoi, Phaeocystis globosa, Prorocentrum donghaiense and Skeletonema costatum were investigated for the first time. Results showed these nine compounds have selectivity antialgal effects on all test red tide microalgae, and antialgal activities against red tide microalgae obviously enhanced with the increase of concentration of antialgal compounds. Based on this, EC50–96 h values of these nine compounds for six red tide microalgae were obtained for the first time. By analyzing and comparing EC50–96 h values, it has been determined that seven compounds (1, 3, 4, 6, 7, 8 and 9) showed the superior application potential than potassium dichromate or gossonorol and other six compounds as a characteristic antialgal agent against Heterosigma akashiwo, Karenia mikimitoi and Prorocentrum donghaiense. Overall this study has suggested that green algae Ulva pertusa is a new source of bioactive compounds with antialgal activity.

Among the 17 known species of the genus Cincticostella, only five of them have been reported as both nymphs and imagoes, which does not include the Chinese species C. fusca Kang et Yang (1995). Here the nymphs are redescribed and reared imagoes are reported for the first time, based on material collected from southeastern China. Morphologically, the nymph is unique because of its large body size, expanded genae, pale median stripe on the anterior of the head, and dark tarsi. The male imago can be differentiated from congeners by its large body size, pigmented forewings, and the mesothoracic scutellum having a pair of membranous lamellae. More importantly, its penes are distinct, being fused for most of their length and having complex ventral projections. Adults emerge in early spring (March to April).

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSC) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate hPSC-PCs that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs, and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hESC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile, mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides the first direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.Significance StatementTo compare global gene expression features of differentiating human pluripotent stem cell-derived Purkinje cells (hPSC-PC) and developing mouse Purkinje cells (PC) we derived hPSC-PCs and compared gene expression datasets from human and mouse PCs. We optimized a differentiation protocol that generated hPSC-PCs most similar in gene expression to mouse P21 PCs. Metagene projection analysis of mouse PC gene expression over postnatal development identified both classical PC marker genes as well as novel mitochondrial and autophagy gene pathways. These key gene expression patterns were conserved in differentiating hPSC-PCs. We further identified differences in timing and expression of key gene sets between mouse and hPSC-PCs and confirmed expression of a novel human PC marker, CD40LG, in human cerebellar tissue.

Inspired by quinine and its analogues, we designed, synthesized, and evaluated two series of quinoline small molecular compounds (a and 2a) and six series of quinoline derivatives (3a-f) for their antifungal activities. The results showed that compounds 3e and 3f series exhibited significant fungicidal activities. Significantly, compounds 3f-4 (EC50 = 0.41 μg/mL) and 3f-28 (EC50 = 0.55 μg/mL) displayed the superior in vitro fungicidal activity and the potent in vivo curative effect against Sclerotinia sclerotiorum. Preliminary mechanism studies showed that compounds 3f-4 and 3f-28 could cause changes in the cell membrane permeability, accumulation of reactive oxygen species, loss of mitochondrial membrane potential, and effective inhibition of germination and formation of S. sclerotiorum sclerotia. These results indicate that compounds 3f-4 and 3f-28 are novel potential fungicidal candidates against S. sclerotiorum derived from natural products.

It is currently not understood whether cigarette smoke exposure facilitates sensitisation to self-antigens and whether ensuing auto-reactive T cells drive chronic obstructive pulmonary disease (COPD)-associated pathologies.To address this question, mice were exposed to cigarette smoke for 2 weeks. Following a 2-week period of rest, mice were challenged intratracheally with elastin for 3 days or 1 month. Rag1−/−, Mmp12−/−, and Il17a−/− mice and neutralising antibodies against active elastin fragments were used for mechanistic investigations. Human GVAPGVGVAPGV/HLA-A*02:01 tetramer was synthesised to assess the presence of elastin-specific T cells in patients with COPD.We observed that 2 weeks of cigarette smoke exposure induced an elastin-specific T cell response that led to neutrophilic airway inflammation and mucus hyperproduction following elastin recall challenge. Repeated elastin challenge for 1 month resulted in airway remodelling, lung function decline and airspace enlargement. Elastin-specific T cell recall responses were dose dependent and memory lasted for over 6 months. Adoptive T cell transfer and studies in T cells deficient Rag1−/−mice conclusively implicated T cells in these processes. Mechanistically, cigarette smoke exposure-induced elastin-specific T cell responses were matrix metalloproteinase (MMP)12-dependent, while the ensuing immune inflammatory processes were interleukin 17A-driven. Anti-elastin antibodies and T cells specific for elastin peptides were increased in patients with COPD.These data demonstrate that MMP12-generated elastin fragments serve as a self-antigen and drive the cigarette smoke-induced autoimmune processes in mice that result in a bronchitis-like phenotype and airspace enlargement. The study provides proof of concept of cigarette smoke-induced autoimmune processes and may serve as a novel mouse model of COPD.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the world. It has been the most prevalent malignancy in Taiwan for consecutive thirteen years. Despite the diversity of its etiologic and pathophysiologic factors, a biological process named as epithelial-mesenchymal transition (EMT) is indispensable in the progression of epithelial cancer. Our aim is to investigate the correlation between the expression of 8 EMT-related proteins (E-cadherin, β-catenin, claudin-1, CD44, N-cadherin, fibronectin, vimentin, S100A4) and the clinicopathologic features of CRC in Taiwan, along with the DNA CpG epigenetic status of CD44 gene. In immunohistochemical assessment, decreased expression of E-cadherin is statistically associated with the progression of cancer stage, while decreased expression of claudin-1 as well as increased β-catenin nuclear translocation and N-cadherin expression is statistically associated with the progression of histopathologic grade. E-cadherin, nuclear β-catenin and claudin-1 are also associated with other important prognostic factors, including nodal metastasis, tumor deposits, and elevated serum CA 19–9 levels. In addition, the left-sided colon and rectal cancers show increased nuclear translocation of β-catenin compared to the right-sided colon cancers, while the rectal cancers show increased fibronectin expression compared to the right-sided and left-sided colon cancers. Moreover, vimentin is aberrantly expressed in one case of signet-ring cell carcinoma. The DNA methylation levels of CD44 gene promoter between the tumoral and non-tumorous tissues by NGS comparison showed statistical difference on six CpG sites. However, such difference may not be sufficient because these DNA methylation proportions are too low to inactivate CD44 gene. Our results demonstrate the expression of E-cadherin, claudin-1, and nuclear β-catenin is closely related to the clinicopathologic prognostic determinants of CRC in Taiwan. The DNA methylation level of CD44 gene and its protein expression, however, show no correlation with the clinicopathologic features in CRC.

Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ)-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications., The mechanisms controlling irreversible cell cycle exit in cerebellar granule progenitors (GCPs) have not been fully elucidated. Here, the authors performed RNA-sequencing of GCPs exiting the cell cycle to identify downregulation of Brd4 activity as an early event during cell cycle exit which subsequently regulates Shh activity and is needed for proper cerebellar development

Anemone flaccida Fr. Schmidt is a perennial medicinal herb that contains pentacyclic triterpenoid saponins as the major bioactive constituents. In China, the rhizomes are used as treatments for a variety of ailments including arthritis. However, yields of the saponins are low, and little is known about the plant's genetic background or phytohormonal responsiveness. Using one-quarter of the 454 pyrosequencing information from the Roche GS FLX Titanium platform, we performed a transcriptomic analysis to identify 157 genes putatively encoding 26 enzymes involved in the synthesis of the bioactive compounds. It was revealed that there are two biosynthetic pathways of triterpene saponins in A. flaccida. One pathway depends on β-amyrin synthase and is similar to that found in other plants. The second, subsidiary ("backburner") pathway is catalyzed by camelliol C synthase and yields β-amyrin as minor byproduct. Both pathways used cytochrome P450-dependent monooxygenases (CYPs) and family 1 uridine diphosphate glycosyltransferases (UGTs) to modify the triterpenoid backbone. The expression of CYPs and UGTs were quite different in roots treated with the phytohormones methyl jasmonate, salicylic acid and indole-3-acetic acid. This study provides the first large-scale transcriptional dataset for the biosynthetic pathways of triterpene saponins and their phytohormonal responsiveness in the genus Anemone.

Embryonic stem cells (ESCs) are self-renewing pluripotent cells, capable of differentiating into all somatic cell types. The molecular control of self-renewal is relatively well-characterized, whereas how ESCs exit pluripotent state to differentiate is poorly understood. Here we identify two genes are required for differentiation and dozens of intergenic regions that potentially regulate ESC differentiation. We used PiggyBac (PB) transposon-based approach to randomly mutate the genome of ESCs, and generated hundreds of clones that resisted differentiation signals. Each clone was sequenced to determine genomic regions mutated by PB insertion. Intriguingly, many mutations were localized among intergenic regions and we identified two genes are required for differentiation. This study should facilitate further exploration of novel molecular determinants of embryonic stem cell differentiation.

Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target. We thank Dr. David Rowitch (University of California, San Francisco) for providing the Atoh1-Cre deleter line and Dr. Marc Kirschner for helpful discussions and critical reading of the manuscript. We also thank all members of the Center for Therapeutic Innovation (University of Miami) and the Department of Cancer Biology (Scripps Florida) for helpful suggestions. We thank Dr. Angela MacArthur and the Department of Scientific Editing at St. Jude Children's Research Hospital for helpful suggestions. This work was supported by R21 NS056991 (N.G.A.), R01NS067289 (N.G.A. and M.E.H.), NIH grant NCI CA-096832 (M.F.R.), NIH Molecular Library Screening Center Network grant U54MH074404 (W.R.R., Hugh Rosen, PI), Core Grant CA-021765 (M.F.R., D.K., and D.F.), the Anderson Fellowship (D.K.), and the American Lebanese-Syrian Associated Charities (ALSAC) of St. Jude Children's Research Hospital (M.F.R., D.K., and D.F.) as well as the Spanish Ministerio de Economia y Competitividad (MINECO, SAF2012-38215), Fundacion Ramon Areces, Comunidad de Madrid (S2010/BMD-2470), and the European Union Seventh Framework Programme (MitoSys project; HEALTH-F5-2010-241548) (M.M.). Sí

Embryonic Six2-positive nephron progenitor cells adjacent to ureteric bud tips ultimately give rise to nephron structures, including proximal and distal tubules, podocytes, Bowman's capsules, and the glomeruli. This process requires an internal balance between self-renew and differentiation of the nephron progenitor cells, which is mediated by numerous molecules. Recent studies have shown that the neurofibromin (Nf1) null mutant mouse embryos have an 18- to 24-h developmental delay in metanephros manifesting retardation in its cephalad repositioning and reduction number of glomeruli. However, the underlying inter-/intracellular signaling mechanisms responsible for reducing number of glomeruli during nephrogenesis remain to be fully elucidated. Here, we originally detected the Nf1 expression in developing kidney and metanephric mesenchyme cells. Surprisingly, Nf1 knockdown by small interfering RNAs in the metanephric mesenchyme cells (mK3) resulted in a decreased expression of Six2, the key marker of renal progenitor cells, while the ratio of apoptotic cells was significantly increased. Furthermore, overexpression of Six2 in mk3 cells partially rescued apoptosis phenotype. Collectively, these results implied that knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro probably through down-regulation of Six2 expression. Collectively, we demonstrated that down-regulated Six2 by knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro. These results implied that inhibition of Nf1 may delay metanephros development via down-regulation of Six2.

Background.Ex vivoculture of intact embryonic kidney has become a powerful system for studying renal development. However, few methods have been available for gene manipulation and have impeded the identification and investigation of genes in this developmental process.Results. Here we systemically compared eight different serotypes of pseudotyped self-complementary adenoassociated viruses (scAAVs) transduction in cultured embryonic kidney with a modified culture procedure. We demonstrated that scAAV was highly effective in delivering genes into and expressing in compacted tissues. scAAV serotypes 2 and 8 exhibited higher efficiency of transduction compared to others. Expression kinetics assay revealed that scAAV can be used for gene manipulation at the study of UB branching and nephrogenesis. Repressing WT1 in cultured kidney using shRNA impairs tubule formation. We for the first time employed and validated scAAV as a gene delivery tool in cultured kidney.Conclusions. These findings are expected to expedite the use of theex vivoembryonic kidney cultures for kidney development research. For otherex vivocultured organ models, scAAV could also be a promising tool for organogenesis study.

Purpose Apolipoprotein E knockout (apoE−/−) mouse is one of the most popular models for cardiovascular research, especially in the study of atherosclerosis. Naturally, large amount of studies try to uncover the role of apoE in atherosclerosis, and indeed apoE plays an important role in this pathogenesis. Kidney is an organ that contains lots of capillaries and also largely expresses apoE. Moreover, a protective role of apoE in kidney as an autocrine regulator has been demonstrated previously, however, the underlying mechanism is largely unknown. Experimental design In this study, comparative proteomics is for the first time used to identify the differential proteins in kidneys of apoE−/− and wild type mice, respectively, and we try to reveal the signaling network of apoE in mice kidney using bioinformatics analysis. Results Our findings show that approximately 80 proteins are significantly differentially expressed in kidneys of apoE−/− and wild type mice, and the signaling network correlated to apoE is successfully established by employing bioinformatics assay. Conclusions and clinical relevance Taken together, we originally identify the proteins with differential expression and propose an apoE correlated molecular network in mice kidney. These findings further provide evidence of the role of apoE in mice kidney and a brand new perspective in the protection and treatment of kidney disease.

Previous studies showed that methanol extracts from Porphyra yezoensis significantly inhibited Karenia mikimitoi and Skeletonema costatum. Five sesquiterpenoids (1–5) were successfully isolated from this marine macroalga through a combination of silica gel column chromatography and repeated preparative thin-layer chromatography in this paper. Their structure was identified as gossonorol (1), 7,10-epoxy-ar-bisabol-11-ol (2), cyclonerodiol (3), cadinol, (4) and 4-cadinen-1-ol (5) on the basis of spectroscopic data. These sesquiterpenoids were isolated from Porphyra yezoensis for the first time, and cyclonerodiol (3) and cadinol (4) isolated from marine macroalgae for the first time. Further, a quantitative relationship between the inhibition of algal growth and the concentration of each antialgal sesquiterpenoid (gossonorol, 7,10-epoxy-ar-bisabol-11-ol and cyclonerodiol) was determined and important parameters, e.g., EC50-96h for future practical HAB control are to be obtained. Results showed that three sesquiterpenoids (1–3) had selective antialgal activity against the growth of red tide microalgae (Amphidinium carterae, Heterosigma akashiwo, Karenia mikimitoi, Phaeocystis globosa, Prorocentrum donghaiense, and Skeletonema costatum). More than two test red tide microalgae were significantly inhibited by these three sesquiterpenoids (1–3). Their antialgal activity against red tide microalgae has not been previously reported. Furthermore, EC50-96h of gossonorol (1) and 7,10-epoxy-ar-bisabol-11-ol (2) for specific test red microalgae were not only significantly less than 10 μg/mL, but also were smaller than/or very close to those of potassium dichromate. Gossonorol (1) and 7,10-epoxy-ar-bisabol-11-ol (2) possessed good application potential than potassium dichromate as a characteristic antialgal agent against the specific harmful red tide microalgae (Heterosigma akashiwo, Phaeocystis globosa, and Prorocentrum donghaiense) (or Heterosigma akashiwo and Karenia mikimitoi).

Proteomics method, based on NanoLC-LTQ-Orbitrap technology, was applied to explore the biological basis of intervention effect of "Qi enriching" herbs on "Qi deficiency" rats. The "Qi deficiency" rat model was established with caloric restriction combined with excessive swimming. Muscle proteins of vastus lateralis from the blank group, the model group and the ginseng group were detected by NanoLC-LTQ-Orbitrap system. The data were imported into Protein Discovery software to identify the proteins and all the raw datum were analyzed by SIEVE software. Compared with model group, 26 significant difference proteins were found in ginseng group, which the variation trend was consistent with the blank group. Through the biological function analysis, the found proteins could be classified into proteins involved in energy metabolism, proteins involved in glucose metabolism, electrolyte balance and material transfer related proteins, inflammation related protein and cytoskeleton protein. The above target proteins and their regulation pathways may be the biological basis which ginseng played a role of tonifying "Qi" of "Qi deficiency" symptom.

Introduction We have developed new urethane acrylate–based root canal sealers using polycarbonate (PC) as polyol and 2,2-azobis(2-methyl)butyronitrile (AMBN) as a thermal initiator. The purpose of this study was to compare the properties among a group of seven sealers: (1) polybutyleneadipate (PBA) with 2,2-azobis-isobutyronitrile (AIBN), (2) PBA-AMBN, (3) PC-AIBN, (4) PC-AMBN, (5) AH Plus (Dentsply DeTrey GmbH, Konstanz, Germany), (6) Epiphany (Pentron Clinical Technologies, Wallingford, CT), and (7) EndoREZ (Ultradent Products, Inc, South Jordan, UT). Methods Attenuated total reflectance-Fourier transforming infrared spectroscopy was used to analyze the degree of conversion. Cytotoxicity was evaluated using MTT assay. In addition, push-out bond strength, solubility, and dimensional stability were investigated. Results Urethane acrylate–based root canal sealers behaved better than Epiphany and EndoREZ for the properties evaluated in this study. The PC-AMBN sealer exhibited the highest degree of conversion even in the apical third, and its cytotoxicity for nonsetting and aged specimens at 24 hours was comparable to AH Plus sealer. In addition, the PC-AMBN sealer with zinc oxide/thermoplastic polyurethane cones had a significantly higher bond strength than AH Plus with gutta percha. Solubility and dimensional changes of the PC-AMBN sealer conformed to the American National Standards Institute/American Dental Association (ANSI/ADA) standard 57. Conclusions The PC-AMBN sealer has great potential for use in endodontic therapy.

Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α 2 β 2 -heterotetramer of the Archaeoglobus fulgidus , the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the α-subunit from another αβ-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the α-subunit and Asp159 in the C terminus of the β-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the β-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the β-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the β-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the β-subunit.

Rotaviruses cause acute diarrhea worldwide. Previous studies of rotavirus diarrhea in China found that rotavirus infection is the most common cause of severe diarrhea in young children. In the present study, surveillance of rotavirus diarrhea was conducted involving 9549 children aged

An efficient deuterium labelling of theaflavin, aiming at radiolabelling of theaflavin, was accomplished for the first time by using 4-deuterium-labelled epi-catechin as the precursor and the resulting D-labelled theaflavin was fully characterized by 1H NMR, 13C NMR, IR spectroscopy and ESI-MS. The synthesis features a novel efficient polyphenol oxidase (PPO) catalyzed benzotropolone formation. PPO was bound onto a selected polymer by a novel efficient method developed in this lab. Copyright © 2009 John Wiley & Sons, Ltd.

Noroviruses are an important cause of epidemic acute gastroenteritis and the viruses recognize human histo-blood group antigens (HBGAs) as receptors. The protruding (P) domain of noroviral capsid, the receptor-binding domain, forms subviral particles in vitro that retain the receptor-binding function. In this study we characterized the structure and HBGA-binding function of the P particle. Structure reconstruction using cryo-EM showed that the P particles are comprised of 12 P dimers that are organized in octahedral symmetry. The dimeric packing of the proteins in the P particles is similar to that in the norovirus capsid, in which the P2 subdomain with the receptor-binding interface is located at the outermost surface of the P particle. The P particles are immunogenic and reveal similar antigenic and HBGA-binding profiles with their parental virus-like particle, further confirming the shared surface structures between the two types of particles. The P particles are easily produced in E. coli and yeast and are stable, which are potentially useful for a broad application including vaccine development against noroviruses.

Background. Human bocavirus (HBoV) was recently discovered in children with respiratory tract disease and gastroenteritis. The causative role of HBoV in human gastroenteritis remains uncertain, and, to our knowledge, no previous case-control study has studied the relationship between HBoV and gastroenteritis. Methods. We conducted a case-control study that examined stool samples from 397 children with diarrhea and from 115 asymptomatic control subjects. HBoV was detected using polymerase chain reaction. Real-time polymerase chain reaction was used to quantify the HBoV loads in case and control groups. Common enteric viruses were examined using enzyme-linked immunosorbent assays, polymerase chain reaction, and reverse-transcription polymerase chain reaction. Results. At least 1 viral agent was discovered in 60.2% of cases. HBoV was detected in 14 samples, and 9 were coinfected with either rotavirus (7 of 14 samples) or human calicivirus (2 of 14). Many (8 [57.1%] of 14) of the HBoV infections occurred during September-December 2006. Most (12 [85.7%]) of the HBoV-infected children were 7–18 months of age. The percentage of children with HBoV infection did not differ significantly between case patients and control subjects (3.5% vs. 3.5%), and the statistical analysis did not support a correlation between HBoV infection and more-severe clinical symptoms. The viral load differences between the 2 groups were not statistically significant (P = .09, by log-normal Student's t test). In addition, the VP1/VP2 partial gene of HBoV from case patients and control subjects showed minimal sequence variation. Conclusions. A single genetic lineage of HBoV was revealed in persons in China. Despite its high prevalence in stool samples, our study does not support a causative role of HBoV in gastroenteritis.

Background We found an unusual human rotavirus, LL36755 of G5P[6] genotype, in a stool sample collected in Lulong County, Hebei Province, China. This is the first detection of rotavirus serotype G5 in Asia. Objectives To identify and characterize G5 rotaviruses in 988 stool samples collected from children under 5 years old with acute gastroenteritis. Study design We analyzed 459 rotavirus-positive samples with RT-PCR using G5 genotype-specific primers. The G5 strains were sequenced. Results Two additional G5-positive samples (LL3354 and LL4260) were identified. VP7, VP4, VP6 and NSP4 genes of LL3354, LL4260 and LL36755 strains were sequenced. The VP4 sequences formed a group with porcine P[6] strains. The VP6 sequences of strains LL3354 and LL36755 were phylogenetically close to the major clusters of SGI and SGII rotaviruses, respectively. The deduced VP6 protein of strain LL4260 had characteristics of both SGI and SGII strains, but best fit with a cluster of atypical SGI viruses. In addition, based on NSP4 sequences, the three G5 strains belonged to genogroup B and were closest to human strain Wa. Conclusion These results indicate a dynamic interaction of human and porcine rotaviruses and suggest that reassortment could result in the stable introduction and successful spread of porcine gene alleles into human rotaviruses.

gamma-Aminobutyric acid (GABA) transporters play a key role in the regulation of GABA neurotransmission. We reported previously that overexpression of the GABA transporter subtype 1 (GAT1), the major form of the GABA transporter in the CNS, led to hyperalgesia in mice. In the present study, nociceptive responses of GAT1-knockout mice (GAT1(-/-)) were compared with those of heterozygous (GAT(+/-)) and wild-type (GAT(+/+)) mice by four conventional pain models (tail-immersion test, hot-plate test, acetic acid-induced abdominal constriction test, and formalin test). In addition, the analgesic effects of two GAT1-selective inhibitors, NO-711 and tiagabine, were examined in all three genotypes using the same four models. Our data demonstrated that GAT1 deficiency because of genetic knockout or acute blockade by selective inhibitors leads to hypoalgesia in mice. These results confirmed the crucial role of GAT1 in the regulation of nociceptive threshold and suggested that GAT1 inhibitors have the potential for clinical use in pain therapy.

Environmental ultrafine particulate matter (PM) is capable of inducing airway injury, while the detailed molecular mechanisms remain largely unclear. Here, we demonstrate pivotal roles of autophagy in regulation of inflammation and mucus hyperproduction induced by PM containing environmentally persistent free radicals in human bronchial epithelial (HBE) cells and in mouse airways. PM was endocytosed by HBE cells and simultaneously triggered autophagosomes, which then engulfed the invading particles to form amphisomes and subsequent autolysosomes. Genetic blockage of autophagy markedly reduced PM-induced expression of inflammatory cytokines, e.g. IL8 and IL6, and MUC5AC in HBE cells. Mice with impaired autophagy due to knockdown of autophagy-related gene Becn1 or Lc3b displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo. Interference of the autophagic flux by lysosomal inhibition resulted in accumulated autophagosomes/amphisomes, and intriguingly, this process significantly aggravated the IL8 production through NFKB1, and markedly attenuated MUC5AC expression via activator protein 1. These data indicate that autophagy is required for PM-induced airway epithelial injury, and that inhibition of autophagy exerts therapeutic benefits for PM-induced airway inflammation and mucus hyperproduction, although they are differentially orchestrated by the autophagic flux.

During a rotavirus surveillance conducted in Lulong County, Hebei Province, China, a total of 331 stool specimens collected in 2003 from children under 5 years old with diarrhea were screened. We identified a novel group A human rotavirus of genotype G5P[6]. Phylogenetic analysis confirmed that the VP7 protein of this newly identified strain, LL36755, was closely related to those of the G5 strains. As such, it has 95.4% homology with its counterparts in the porcine G5 strains C134 and CC117 at the amino acid sequence level. On the other hand, the VP4 protein of the LL36755 strain was 94.5% homologous to those of the porcine P[6] strains 134/04-10, 134/04-11, 221/04-7, and 221/04-13. Our findings indicate a dynamic interaction between human and porcine rotaviruses.

Seven antialgal compounds (1–7) were successfully isolated from the red alga Gracilaria lemaneiformis through a combination of silica gel column chromatography and repeated preparative thin-layer chromatography. On the basis of the spectral data, the compounds were identified as gossonorol (1), 7,10-epoxy-ar-bisabol-11-ol (2), glycerol monopalmitate (3), stigmasterol (4), 15-hydroxymethyl-2, 6, 10, 18, 22, 26, 30-heptamethyl-14-methylene-17-hentriacontene (5), 4-hydroxyphenethyl alcohol (6), and margaric acid (7). These seven compounds were isolated from G. lemaneiformis for the first time, while the compounds 4, 6, and 7 were isolated from marine macroalgae for the first time. Furthermore, a quantitative relationship between the inhibition of algal growth and the concentration of each antialgal compound was determined and important parameters for future practical HAB control, e.g., EC50-96h, were also obtained. The results indicated that isolated compounds 1–7 possess selective antialgal activity against the growth of several red tide microalgae (including Amphidinium carterae, Heterosigma akashiwo, Karenia mikimitoi, Phaeocystis globsa, Prorocentrum donghaiense, and Skeletonema costatum). Their antialgal activity against test red tide microalgae has not been previously reported. Furthermore, the EC50-96h of one or more of the compounds towards the tested red microalgae was not only significantly less than 10 μg/mL but also was smaller than that of the characteristic antialgal agent potassium dichromate. The study demonstrates that compounds 1–7 possess significant application potential as antialgal agents against several harmful red tide microalgae.

Ten compounds (1~10) were successfully isolated from green algae Ulva prolifera through the combination of silica gel column chromatography, Sephadex LH-20 column chromatography and repeated preparative thin-layer chromatography. These ten compounds showed antialgal activity against red tide microalgae. Among them, compounds 3, 6, and 7 showed stronger antialgal activity against red tide microalgae. Furthermore, their structure was identified on the basis of spectroscopic data. There are three glycoglycerolipids: 1-O-octadecanoic acid-3-O-β-D-galactopyranosyl glycerol (2), 1-O-palmitoyl-3-O-β-D-galactopyranosyl glycerol (4), and 1-O-palmitoyl-2-O-oleoyl-3-O-β-D-galactopyranosyl glycerol (5); two monoglycerides: glycerol monopalmitate (1), 9-hexadecenoic acid, 2,3-dihydroxypropyl ester (3); two terpenoids: loliolide (6) and lsololiolide (7); one lipid-soluble pigments: zeaxanthin (8); one sterol: cholest-5-en-3-ol (9); and one alkaloid: pyrrolopiperazine-2,5-dione (10). These compounds were isolated from U. prolifera for the first time, and compounds 2, 3, 5, and 8 were isolated from marine macroalgae for the first time.

China has the second largest birth cohort in the world and the second highest number of deaths due to rotavirus infection. It is also the only country with a licensed rotavirus vaccine. Chinese policy makers now need credible estimates of the burden of rotavirus disease, to decide about vaccine use. From August 2001 through July 2003, prospective hospital-based surveillance for rotavirus diarrhea among children

We studied the sensitivity of several human cancer cell strains (HeLa, HT1080, SPC-A1, and ACHN) and normal cell strains (MRC-5 and Wish) to mumps virus (MuV) S79, a live attenuated vaccine strain. These cells exhibited a differential sensitivity to infection with MuV, and the susceptible sequences were ACHN > HeLa > HT1080 > SPC-A1 > Wish > MRC-5. In experiments in vivo, nude mice with HT1080 fibrosarcoma xenografts were randomly divided into three groups for intratumoral treatment with MuVS79, UV-inactivated MuVS79, and PBS. At 107 PFU, live MuVS79 injection caused in 7 of 9 mice complete regression by day 15 while rapid tumor growth occurred in all 9 mice treated with PBS. Rapid tumor progression also occurred in all 8 mice treated with UV-inactivated virus; however, tumor growth was delayed in the logarithmic phase relative to the PBS-treated tumors.

A new immunomodulatory protein (APP) was purified from the fruiting body of an edible Jew's Ear mushroom, Auricularia polytricha, by extraction using 5% cold acetic acid in the presence of 0.1% 2-mercaptoethanol, followed by ammonium sulfate fractionation, DE-52 and MonoQ anion-exchange chromatography. As determined by electrophoresis techniques, the molecular mass of APP is around 13.4 kDa and its pI is approximately 5.1. APP is a simple protein without carbohydrate, and can agglutinate mouse red blood cells. APP alone activates murine splenocytes, markedly increasing their proliferation and gamma-interferon (IFN-γ) secretion, and presents no cytotoxicity in vitro. Although murine splenocytes are stimulated by the mitogen concanavalin A (ConA), APP suppresses their proliferation in a dose-dependent manner. APP (5–20 μg/ml) also enhances the production of both nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) by LPS-induced RAW 264.7 macrophages. These findings suggest that APP is an immune stimulant and can strengthen the immune response of its host.

Despite abundant evidence that different nervous system control strategies may exist for human concentric and eccentric muscle contractions, no data are available to indicate that the brain signal differs for eccentric versus concentric muscle actions. The purpose of this study was to evaluate electroencephalography (EEG)-derived movement-related cortical potential (MRCP) and to determine whether the level of MRCP-measured cortical activation differs between the two types of muscle activities. Eight healthy subjects performed 50 voluntary eccentric and 50 voluntary concentric elbow flexor contractions against a load equal to 10% body weight. Surface EEG signals from four scalp locations overlying sensorimotor-related cortical areas in the frontal and parietal lobes were measured along with kinetic and kinematic information from the muscle and joint. MRCP was derived from the EEG signals of the eccentric and concentric muscle contractions. Although the elbow flexor muscle activation (EMG) was lower during eccentric than concentric actions, the amplitude of two major MRCP components—one related to movement planning and execution and the other associated with feedback signals from the peripheral systems—was significantly greater for eccentric than for concentric actions. The MRCP onset time for the eccentric task occurred earlier than that for the concentric task. The greater cortical signal for eccentric muscle actions suggests that the brain probably plans and programs eccentric movements differently from concentric muscle tasks.

In the developing cerebellar cortex, granule neuron precursors (GNPs) proliferate and commence differentiation in a superficial zone, the external granule layer (EGL). The molecular basis of the transition from proliferating precursors to immature differentiating neurons remains unknown. Notch signaling is an evolutionarily conserved pathway regulating the differentiation of precursor cells of many lineages. Notch2 is specifically expressed in proliferating GNPs in the EGL. Treatment of GNPs with soluble Notch ligand Jagged1, or overexpression of activated Notch2 or its downstream target HES1, maintains precursor proliferation. The addition of GNP mitogens Jagged1 or Sonic Hedgehog (Shh) upregulates the expression of HES1, suggesting a role for HES1 in maintaining precursor proliferation.

Eight serotypes of human astroviruses (HAstV-1 to HAstV-8) have been described. To date, the entire genomes of HAstV-1 and HAstV-2 as well as the ORF2 sequences of HAstV-1-6 and 8 have been reported. In this study, the ORF2 sequences of seventeen strains of HAstVs originating from different countries were determined, as well as the sequence ORF2 of one porcine astrovirus (PAstV) strain. Afterwards, comparison of the capsid protein precursors encoded by ORF2 of 46 strains of HAstVs, PAstV, and feline astrovirus (FAstV) was carried out. A phylogenetic tree showed eight genogroups of HAstVs that corresponded exactly to the serotypes. HAstV-3 and 7 were the most closely related, whereas HAstVs, FAstV, and PAstV segregated from each other. Compared to a PAstV, a FAstV is closer to HAstVs. Furthermore, the capsid protein precursors were divided into four regions (after amino acid residues 424, 688, and 776, respectively) based on sequence identity. Region I was the most conserved, and FAstV was very close in identity to HAstVs. Two amino acid motifs in region I were predicted to contain the common antigenic epitopes. Region II was relatively variable. Deletions and insertions were characteristic of region III, and region IV was relatively conserved. To our knowledge, this is the first comparative sequence analysis of the capsid protein precursors of eight serotypes of HAstVs as well as two animal astroviruses (FAstV and PAstV).

Sequence analyses of the VP4 and NSP4 genes were performed on twenty human isolates of serotype G3 rotavirus obtained from China and Japan. One isolate from China, CHW17, possessed P[4] genotype VP4 and KUN group NSP4 genes which are associated with G2. One isolate (02/92) from Japan, which was shown to have a wider spacing between RNA segments 10 and 11 by RNA polyacrylamide gel electrophoretic analysis like AU-1, possessed P[9] genotype VP4 and AU-1 group NSP4 genes. The other isolates had P[8] genotype VP4 and Wa group NSP4 genes. While the nucleotide sequence conservation among the G3 VP7 genes was more than 79% (Wen et al, Arch. Virol., 1997, 142: 1481-1489), the conservation of VP4 and NSP4 genes in the same genotypes or groups was more than 85%.

High mobility group (HMG)-box proteins, a large and functionally diverse superfamily of architectural protein, are involved in the regulation of DNA-dependent processes such as transcription, replication and DNA repair via the HMG-box domain. Bobby sox homolog (BBX), a newly identified HMG-Box protein, may function as a sequence-specific transcription factor. However, its expression pattern and biological functions are largely unknown. In this work, phylogenetic analysis showed that BBX is highly conserved and belongs to a novel subfamily of HMG-Box superfamily together with CIC (capicua homolog). Real time RT-PCR and whole-mount in situ hybridization in zebrafish embryo revealed that bbx, cica, and cicb were maternally highly expressed from 4 cell to 1K cell stage, and the zygote expression was primarily distributed in the central nervous system (CNS) from 24 to 60 h post-fertilization (hpf). Immunohistochemistry analysis in mouse brain revealed that BBX was weakly expressed in the cerebellum and highly expressed in the cortex and hippocampus. These findings indicate that as a novel HMG-box protein, BBX maybe associated with CNS development and provides useful clues to further study of its biological functions.

Three polysaccharides, IPSI-A, IPSI-B and IPSII, were successfully isolated from the marine microalgae Isochrysis galbana through a combination of anion-exchange column chromatography and repeated gel chromatography. These three polysaccharides were demonstrated to have moderate scavenging activities against superoxide and hydroxyl radicals and moderate reductive power in a concentration-dependent manner. The IPSII demonstrated more effective antioxidant activities than IPSI-A and IPSI-B. IPSII had a molecular weight of 15.934 kDa belonging to a β-type heteropolysaccharide with a pyran group and primarily contained mannose with variable amounts of glucose, galactose and rhamnose based on an analysis of infrared spectroscopy (IR), electrospray ionization-mass spectrometry (ESI-MS) and (1)H nuclear magnetic resonance ((1)H NMR).

MicroRNAs (miRNAs) are small non-coding RNAs that down-regulate gene expression by binding to target mRNA for cleavage or translational repression, and play important regulatory roles in renal development. Despite increasing genes have been predicted to be miRNA targets by bioinformatic analysis during kidney development, few of them have been verified by experiment. The objective of our study is to identify the miRNAs targeting Six2, a critical transcription factor that maintains the mesenchymal progenitor pool via self-renewal (proliferation) during renal development. We initially analyzed the 3′UTR of Six2 and found 37 binding sites targeted by 50 putative miRNAs in the 3′UTR of Six2. Among the 50 miRNAs, miR-181b is the miRNAs predicted by the three used websites. In our study, the results of luciferase reporter assay, realtime-PCR and Western blot demonstrated that miR-181b directly targeted on the 3′UTR of Six2 and down-regulate the expression of Six2 at mRNA and protein levels. Furthermore, EdU proliferation assay along with the Six2 rescue strategy showed that miR-181b suppresses the proliferation of metanephric mesenchymal by targeting Six2 in part. In our research, we concluded that by targeting the transcription factor gene Six2, miR-181b inhibits the proliferation of metanephric mesenchymal cells in vitro and might play an important role in the formation of nephrons.