No abstract

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.synthetic biology | natural product | flow cytometry | single-cell resolution | modular regulatory elements S treptomycetes are well known as the most abundant source of bioactive secondary metabolites (1), including medically important antimicrobial agents [e.g., chloramphenicol from Streptomyces venezuelae (2)], agricultural chemicals [e.g., avermectin from Streptomyces avermitilis (3)], and anticancer agents and immunosuppressants [e.g., rapamycin from Streptomyces hygroscopicus (4)]. However, the increasing difficulty of discovering novel drugs via traditional high-throughput screening and the "one strain many compounds" approach is frustrating pharmaceutical productivity (5, 6). Deciphering the genome sequences of Streptomyces surprisingly established the presence of a plethora of gene clusters encoding for yet-unobserved molecules, even in intensively investigated Streptomyces coelicolor A3 (2), revealing a much higher potential of novel bioactive agent production than originally anticipated (7,8). Therefore, the enormous number of natural products that have been obtained likely represent only a tiny portion of the repertoire of bioactive compounds that can possibly be produced. This has brought about extensive research into applied genomics aimed at investigating these new gene clusters, generally referred to as "cryptic," "silent," or "orphan" (9-11). With data on more than 12,000 in-house draft bacterial genomes, the potential for the discovery of a number of novel chemicals encrypted in silent biosynthetic gene clusters has been detected by genome mining.Many new strategies have been documented for "awakening" poorly expressed and/or silent gene clusters in Strept...

The activities of transcription factors (TFs) require interactions with specific DNA sequences and other regulatory proteins. To detect such interactions in Arabidopsis, we developed a high-throughput screening system with a Gateway-compatible Gal4-AD-TF library of 1589 Arabidopsis TFs, which can be easily screened by mating-based yeast-one-hybrid (Y1H) and yeast-two-hybrid (Y2H) methods. The efficiency of the system was validated by examining two well-characterized TF-DNA and TF-protein interactions: the CHE-CCA1 promoter interaction by Y1H and NPR1-TGAs interactions by Y2H. We used this system to identify eight TFs that interact with a Mediator subunit, Med25, a key regulator in JA signaling. We identified five TFs that interacted with the GCC-box cis-element in the promoter of PDF1.2, a downstream gene of Med25. We found that three of these TFs, all from the AP2-EREBP family, interact directly both with Med25 and the GCC-box of PDF1.2, suggesting that Med25 regulates PDF1.2 expression through these three TFs. These results demonstrate that this high-throughput Y1H/Y2H screening system is an efficient tool for studying transcriptional regulation networks in Arabidopsis. This system will be available for other Arabidopsis researchers, and thus it provides a vital resource for the Arabidopsis community.

Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here, we report the functions of two proteins containing RNA recognition motifs, RZ-1B and RZ-1C, in Arabidopsis thaliana. RZ-1B and RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C termini. RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA binding activity of RZ-1C with SR proteins through overexpression of the C terminus of RZ-1C conferred defective phenotypes similar to those observed in rz-1b rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of RZ-1B and RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that RZ-1C directly targeted FLOWERING LOCUS C (FLC), promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins.

Neurofibrillary tangles (NFTs) made up of hyperphosphorylated tau are a histopathological hallmark of Alzheimer’s disease (AD) and related tauopathies. Hyperphosphorylation of tau is responsible for its loss of normal physiological function, gain of toxicity and its aggregation to form NFTs. Injection of misfolded tau seeds into mouse brain induces tau aggregation, but the nature of tau phosphorylation in pathologic tau seeded pathology is unclear. In the present study, we injected hyperphosphorylated and oligomeric tau isolated from AD brain (AD P-tau) into hippocampus of human tau transgenic mice and found that in addition to tau aggregation/pathology, tau was hyperphosphorylated at Ser202/Thr205, Thr212, Ser214, Thr217, Ser262, and Ser422 in AD P-tau injected hippocampus and at Ser422 in the contralateral hippocampus and in the ipsilateral cortex. AD P-tau-induced AD-like high molecular weight aggregation of tau that was SDS- and reducing agent-resistant and site-specifically hyperphosphorylated in the ipsilateral hippocampus. There were no detectable alterations in levels of tau phosphatases or tau kinases in AD P-tau-injected brains. Furthermore, we found that hyperphosphorylated tau was easier to be captured by AD P-tau and that aggregated tau was more difficult to be dephosphorylated than the non-aggregated tau by protein phosphatase 2A (PP2A). Based on these findings, we speculate that AD P-tau seeds hyperphosphorylated tau to form aggregates, which resist to the dephosphorylation by PP2A, resulting in hyperphosphorylation and pathology of tau.

The phytohormone jasmonic acid (JA) is an important signaling molecular involved in many developmental and physiological processes, especially in the response of plants to wounding. In this study, we adopted a new strategy, taking into consideration the microarray data of the CHX treatment, to identify 15 COI1-dependent JA-inducible transcription factors (JCTFs) that have distinct expression patterns in response to wounding. After the analysis on the JCTFs over-expressor plants, we identified four JCTFs, i.e., WRKY18, At1g74930 and At3g53600 in addition to AtMYC2, as the positive regulators in the JA-mediated signaling pathway in response to Arabidopsis wounding.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) is an important enzyme involved in the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway which provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the role of the MEP pathway in plant development and metabolism, we carried out detailed analyses on a dxr mutant (GK_215C01) and two DXR transgenic co-suppression lines, OX-DXR-L2 and OX-DXR-L7. We found that the dxr mutant was albino and dwarf. It never bolted, had significantly reduced number of trichomes and most of the stomata could not close normally in the leaves. The two co-suppression lines produced more yellow inflorescences and albino sepals with no trichomes. The transcription levels of genes involved in trichome initiation were found to be strongly affected, including GLABRA1, TRANSPARENT TESTA GLABROUS 1, TRIPTYCHON and SPINDLY, expression of which is regulated by gibberellic acids (GAs). Exogenous application of GA 3 could partially rescue the dwarf phenotype and the trichome initiation of dxr, whereas exogenous application of abscisic acid (ABA) could rescue the stomata closure defect, suggesting that lower levels of both GA and ABA contribute to the phenotype in the dxr mutants. We further found that genes involved in the biosynthetic pathways of GA and ABA were coordinately regulated. These results indicate that disruption of the plastidial MEP pathway leads to biosynthetic deficiency of photosynthetic pigments, GAs and ABA, and thus the developmental abnormalities, and that the flux from the cytoplasmic mevalonate pathway is not sufficient to rescue the deficiency caused by the blockage of the plastidial MEP pathway. These results reveal a critical role for the MEP biosynthetic pathway in controlling the biosynthesis of isoprenoids.

Passive-active immunoprophylaxis with hepatitis B immunoglobulin (HBIG) and hepatitis B vaccine provides a high level of protection against vertical transmission of hepatitis B virus (HBV). Nevertheless, 1 in 10 children of HBV carriers have chronic hepatitis B early in their lives despite immunoprophylaxis because they were infected in utero. This study of 469 pregnant women testing positive for hepatitis B surface antigen (HbsAg) was conducted to learn whether giving HBIG to HBV carriers in the third trimester can prevent vertical transmission of the virus. Of the women entered into the study, all of whom were asymptomatic, 126 (group 1) tested positive for hepatitis B e antigen (HbeAg), while 343 (group 2) did not. HBIG was given intramuscularly in a dose of 200 IU at 28, 32, and 36 weeks of pregnancy. All newborn infants received 100 IU of HBIG within 12 hours of birth as well as the first of 3 doses of hepatitis B vaccine (the subsequent doses were given at ages 1 and 6 months).In group 1, 16% of infants whose mothers received HBIG in the third trimester tested positive for HbsAg at birth and 7% at age 6 months. The respective figures for infants whose mothers were not treated were significantly higher at 39% and 23%. No such treatment-related difference was observed in group 2 infants. At age 6 months, rates of protective levels of anti-Hbs antibody were 32% in group 1 infants whose mothers were HbeAg-positive and 56% in group 2 infants when the mothers had not received HBIG while pregnant. The respective figures for infants whose mothers were treated in the third trimester were 76% and 89%. Neither mothers nor their infants experienced adverse effects related to the administration of HBIG or hepatitis B vaccine.The investigators conclude that, if all HbsAg-positive pregnant women, regardless of HbeAg status, were to receive HBIG in the last weeks of pregnancy, their infants would be better protected against chronic HBV infection. ABSTRACTFetal karyotyping of cells obtained by amniocentesis may take as long as 3 weeks, and many pregnant women experience anxiety during the interval. This randomized, controlled study evaluated 2 tactics for lessening anxiety: issuing karyotyping results on a prespecified date rather than when they become available and presenting early results Preconception and Prenatal Care 495 496 Obstetrical and Gynecological Survey ABSTRACTAbout 1 in 4 primary cesarean deliveries take place during the second stage of labor. Not only is second-stage surgery technically more difficult, but the fetus is at risk of hypoxia-related morbidity. This prospective observational study of primary cesarean deliveries was carried out at 13 university centers that make up the National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Of 11,981 primary cesarean deliveries, 9265 were done in the first and 2716 in the second stage of labor. Women in the latter group were likely to be older, nulliparous, and white, and they had a smaller body mass index at the time of del...