Science.gov

Sample records for acid cas reg

  1. 21 CFR 184.1069 - Malic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Malic acid. 184.1069 Section 184.1069 Food and....1069 Malic acid. (a) Malic acid (C4H6O5, CAS Reg. No. of L-form 97-67-6, CAS Reg. No. of DL-form 617-48-1) is the common name for 1-hydroxy-1, 2-ethanedicarboxylic acid. L (+) malic acid, referred to as...

  2. 21 CFR 184.1069 - Malic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Malic acid. 184.1069 Section 184.1069 Food and... Substances Affirmed as GRAS § 184.1069 Malic acid. (a) Malic acid (C4H6O5, CAS Reg. No. of L-form 97-67-6, CAS Reg. No. of DL-form 617-48-1) is the common name for 1-hydroxy-1, 2-ethanedicarboxylic acid....

  3. 21 CFR 184.1069 - Malic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Malic acid. 184.1069 Section 184.1069 Food and... Substances Affirmed as GRAS § 184.1069 Malic acid. (a) Malic acid (C4H6O5, CAS Reg. No. of L-form 97-67-6, CAS Reg. No. of DL-form 617-48-1) is the common name for 1-hydroxy-1, 2-ethanedicarboxylic acid....

  4. 21 CFR 184.1069 - Malic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Malic acid. 184.1069 Section 184.1069 Food and... Substances Affirmed as GRAS § 184.1069 Malic acid. (a) Malic acid (C4H6O5, CAS Reg. No. of L-form 97-67-6, CAS Reg. No. of DL-form 617-48-1) is the common name for 1-hydroxy-1, 2-ethanedicarboxylic acid....

  5. 21 CFR 184.1069 - Malic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Malic acid. 184.1069 Section 184.1069 Food and... Substances Affirmed as GRAS § 184.1069 Malic acid. (a) Malic acid (C4H6O5, CAS Reg. No. of L-form 97-67-6, CAS Reg. No. of DL-form 617-48-1) is the common name for 1-hydroxy-1, 2-ethanedicarboxylic acid....

  6. Chemical mechanical planarization of Ge2Sb2Te5 using IC1010 and Politex reg pads in acidic slurry

    NASA Astrophysics Data System (ADS)

    He, Ao-Dong; Liu, Bo; Song, Zhi-Tang; Wang, Liang-Yong; Liu, Wei-Li; Feng, Gao-Ming; Feng, Song-Lin

    2014-08-01

    In the paper, chemical mechanical planarization (CMP) of Ge2Sb2Te5 (GST) is investigated using IC1010 and Politex reg pads in acidic slurry. For the CMP with blank wafer, it is found that the removal rate (RR) of GST increases with the increase of pressure for both pads, but the RR of GST polished using IC1010 is far more than that of Politex reg. To check the surface defects, GST film is observed with an optical microscope (OM) and scanning electron microscope (SEM). For the CMP with Politex reg, many spots are observed on the surface of the blank wafer with OM, but no obvious spots are observed with SEM. With regard to the patterned wafer, a few stains are observed on the GST cell, but many residues are found on other area with OM. However, from SEM results, a few residues are observed on the GST cell, more dielectric loss is revealed about the trench structure. For the CMP with IC1010, the surface of the polished blank wafer suffers serious scratches found with both OM and SEM, which may result from a low hardness of GST, compared with those of IC1010 and abrasives. With regard to the patterned wafer, it can achieve a clean surface and almost no scratches are observed with OM, which may result from the high-hardness SiO2 film on the surface, not from the soft GST film across the whole wafer. From the SEM results, a clean interface and no residues are observed on the GST surface, and less dielectric loss is revealed. Compared with Politex reg, the patterned wafer can achieve a good performance after CMP using IC1010.

  7. 21 CFR 173.45 - Polymaleic acid and its sodium salt.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the Division of... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Polymaleic acid and its sodium salt. 173.45... and its sodium salt. Polymaleic acid (CAS Reg. No. 26099-09-2) and its sodium salt (CAS Reg. No....

  8. 21 CFR 173.45 - Polymaleic acid and its sodium salt.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the Division of Product Policy... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Polymaleic acid and its sodium salt. 173.45 Section... sodium salt. Polymaleic acid (CAS Reg. No. 26099-09-2) and its sodium salt (CAS Reg. No. 70247-90-4)...

  9. 21 CFR 173.45 - Polymaleic acid and its sodium salt.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the Division of... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Polymaleic acid and its sodium salt. 173.45... and its sodium salt. Polymaleic acid (CAS Reg. No. 26099-09-2) and its sodium salt (CAS Reg. No....

  10. 21 CFR 184.1097 - Tannic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Tannic acid. 184.1097 Section 184.1097 Food and... Substances Affirmed as GRAS § 184.1097 Tannic acid. (a) Tannic acid (CAS Reg. No. 1401-55-4), or hydrolyzable gallotannin, is a complex polyphenolic organic structure that yields gallic acid and either glucose or...

  11. 21 CFR 172.345 - Folic acid (folacin).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Folic acid (folacin). 172.345 Section 172.345 Food... Special Dietary and Nutritional Additives § 172.345 Folic acid (folacin). Folic acid (CAS Reg. No. 59-30-3... following prescribed conditions: (a) Folic acid is the chemical N- amino]benzoyl]-L-glutamic acid. (b)...

  12. 21 CFR 172.345 - Folic acid (folacin).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... following prescribed conditions: (a) Folic acid is the chemical N- amino]benzoyl]-L-glutamic acid. (b) Folic... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Folic acid (folacin). 172.345 Section 172.345 Food... Special Dietary and Nutritional Additives § 172.345 Folic acid (folacin). Folic acid (CAS Reg. No....

  13. 21 CFR 172.345 - Folic acid (folacin).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... following prescribed conditions: (a) Folic acid is the chemical N- amino]benzoyl]-L-glutamic acid. (b) Folic... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Folic acid (folacin). 172.345 Section 172.345 Food... Special Dietary and Nutritional Additives § 172.345 Folic acid (folacin). Folic acid (CAS Reg. No....

  14. 21 CFR 184.1061 - Lactic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ....1061 Lactic acid. (a) Lactic acid (C3H6O3, CAS Reg. Nos.: dl mixture, 598-82-3; l-isomer, 79-33-4; d-isomer, 10326-41-7), the chemical 2-hydroxypropanoic acid, occurs naturally in several foods. It...

  15. 21 CFR 184.1081 - Propionic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Propionic acid. 184.1081 Section 184.1081 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT FOOD....1081 Propionic acid. (a) Propionic acid (C3H6O2, CAS Reg. No. 79-09-4) is an oily liquid having...

  16. 21 CFR 184.1095 - Sulfuric acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sulfuric acid. 184.1095 Section 184.1095 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1095 Sulfuric acid. (a) Sulfuric acid (H2SO4, CAS Reg. No. 7664-93-9),...

  17. 21 CFR 184.1095 - Sulfuric acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sulfuric acid. 184.1095 Section 184.1095 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1095 Sulfuric acid. (a) Sulfuric acid (H2SO4, CAS Reg. No. 7664-93-9),...

  18. 21 CFR 184.1095 - Sulfuric acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sulfuric acid. 184.1095 Section 184.1095 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1095 Sulfuric acid. (a) Sulfuric acid (H2SO4, CAS Reg. No. 7664-93-9),...

  19. 21 CFR 184.1095 - Sulfuric acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sulfuric acid. 184.1095 Section 184.1095 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1095 Sulfuric acid. (a) Sulfuric acid (H2SO4, CAS Reg. No. 7664-93-9),...

  20. 21 CFR 184.1095 - Sulfuric acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sulfuric acid. 184.1095 Section 184.1095 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT FOOD....1095 Sulfuric acid. (a) Sulfuric acid (H2SO4, CAS Reg. No. 7664-93-9), also known as oil of vitriol,...

  1. 21 CFR 173.395 - Trifluoromethane sulfonic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Trifluoromethane sulfonic acid. 173.395 Section... HUMAN CONSUMPTION Specific Usage Additives § 173.395 Trifluoromethane sulfonic acid. Trifluoromethane sulfonic acid has the empirical formula CF3SO3H (CAS Reg. No. 1493-13-6). The catalyst...

  2. 21 CFR 173.395 - Trifluoromethane sulfonic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Trifluoromethane sulfonic acid. 173.395 Section... HUMAN CONSUMPTION Specific Usage Additives § 173.395 Trifluoromethane sulfonic acid. Trifluoromethane sulfonic acid has the empirical formula CF3SO3H (CAS Reg. No. 1493-13-6). The catalyst...

  3. 21 CFR 173.395 - Trifluoromethane sulfonic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Trifluoromethane sulfonic acid. 173.395 Section... HUMAN CONSUMPTION Specific Usage Additives § 173.395 Trifluoromethane sulfonic acid. Trifluoromethane sulfonic acid has the empirical formula CF3SO3H (CAS Reg. No. 1493-13-6). The catalyst...

  4. 21 CFR 184.1009 - Adipic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Adipic acid. 184.1009 Section 184.1009 Food and... Substances Affirmed as GRAS § 184.1009 Adipic acid. (a) Adipic acid (C6H10O4, CAS Reg. No. 00124-04-9) is also known as 1,4-butanedicarboxylic acid or hexane-dioic acid. It is prepared by nitric acid...

  5. 21 CFR 184.1009 - Adipic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Adipic acid. 184.1009 Section 184.1009 Food and... Substances Affirmed as GRAS § 184.1009 Adipic acid. (a) Adipic acid (C6H10O4, CAS Reg. No. 00124-04-9) is also known as 1,4-butanedicarboxylic acid or hexane-dioic acid. It is prepared by nitric acid...

  6. 21 CFR 184.1091 - Succinic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Succinic acid. 184.1091 Section 184.1091 Food and... Substances Affirmed as GRAS § 184.1091 Succinic acid. (a) Succinic acid (C4H6O4, CAS Reg. No. 110-15-6), also referred to as amber acid and ethylenesuccinic acid, is the chemical 1,4-butanedioic acid. It...

  7. 21 CFR 184.1091 - Succinic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Succinic acid. 184.1091 Section 184.1091 Food and....1091 Succinic acid. (a) Succinic acid (C4H6O4, CAS Reg. No. 110-15-6), also referred to as amber acid and ethylenesuccinic acid, is the chemical 1,4-butanedioic acid. It is commercially prepared...

  8. 21 CFR 184.1009 - Adipic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Adipic acid. 184.1009 Section 184.1009 Food and... Substances Affirmed as GRAS § 184.1009 Adipic acid. (a) Adipic acid (C6H10O4, CAS Reg. No. 00124-04-9) is also known as 1,4-butanedicarboxylic acid or hexane-dioic acid. It is prepared by nitric acid...

  9. 21 CFR 184.1091 - Succinic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Succinic acid. 184.1091 Section 184.1091 Food and... Substances Affirmed as GRAS § 184.1091 Succinic acid. (a) Succinic acid (C4H6O4, CAS Reg. No. 110-15-6), also referred to as amber acid and ethylenesuccinic acid, is the chemical 1,4-butanedioic acid. It...

  10. 21 CFR 184.1009 - Adipic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Adipic acid. 184.1009 Section 184.1009 Food and... Substances Affirmed as GRAS § 184.1009 Adipic acid. (a) Adipic acid (C6H10O4, CAS Reg. No. 00124-04-9) is also known as 1,4-butanedicarboxylic acid or hexane-dioic acid. It is prepared by nitric acid...

  11. 21 CFR 184.1091 - Succinic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Succinic acid. 184.1091 Section 184.1091 Food and... Substances Affirmed as GRAS § 184.1091 Succinic acid. (a) Succinic acid (C4H6O4, CAS Reg. No. 110-15-6), also referred to as amber acid and ethylenesuccinic acid, is the chemical 1,4-butanedioic acid. It...

  12. 21 CFR 184.1091 - Succinic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Succinic acid. 184.1091 Section 184.1091 Food and... Substances Affirmed as GRAS § 184.1091 Succinic acid. (a) Succinic acid (C4H6O4, CAS Reg. No. 110-15-6), also referred to as amber acid and ethylenesuccinic acid, is the chemical 1,4-butanedioic acid. It...

  13. 21 CFR 184.1009 - Adipic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Adipic acid. 184.1009 Section 184.1009 Food and....1009 Adipic acid. (a) Adipic acid (C6H10O4, CAS Reg. No. 00124-04-9) is also known as 1,4-butanedicarboxylic acid or hexane-dioic acid. It is prepared by nitric acid oxidation of cyclohexanol...

  14. 21 CFR 186.1316 - Formic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Formic acid. 186.1316 Section 186.1316 Food and... Substances Affirmed as GRAS § 186.1316 Formic acid. (a) Formic acid (CH2O2, CAS Reg. No. 64-18-6) is also referred to as methanoic acid or hydrogen carboxylic acid. It occurs naturally in some insects and...

  15. 21 CFR 186.1316 - Formic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Formic acid. 186.1316 Section 186.1316 Food and... Substances Affirmed as GRAS § 186.1316 Formic acid. (a) Formic acid (CH2O2, CAS Reg. No. 64-18-6) is also referred to as methanoic acid or hydrogen carboxylic acid. It occurs naturally in some insects and...

  16. 21 CFR 186.1316 - Formic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Formic acid. 186.1316 Section 186.1316 Food and... Substances Affirmed as GRAS § 186.1316 Formic acid. (a) Formic acid (CH2O2, CAS Reg. No. 64-18-6) is also referred to as methanoic acid or hydrogen carboxylic acid. It occurs naturally in some insects and...

  17. 21 CFR 186.1316 - Formic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Formic acid. 186.1316 Section 186.1316 Food and....1316 Formic acid. (a) Formic acid (CH2O2, CAS Reg. No. 64-18-6) is also referred to as methanoic acid or hydrogen carboxylic acid. It occurs naturally in some insects and is contained in the free...

  18. 21 CFR 186.1316 - Formic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Formic acid. 186.1316 Section 186.1316 Food and... Substances Affirmed as GRAS § 186.1316 Formic acid. (a) Formic acid (CH2O2, CAS Reg. No. 64-18-6) is also referred to as methanoic acid or hydrogen carboxylic acid. It occurs naturally in some insects and...

  19. 21 CFR 184.1099 - Tartaric acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Tartaric acid. 184.1099 Section 184.1099 Food and... Substances Affirmed as GRAS § 184.1099 Tartaric acid. (a) Food grade tartaric acid (C4H6O6, CAS Reg. No. 87-69-4) has the l configuration. The l form of tartaric acid is dextrorotatory in solution and is...

  20. 21 CFR 184.1099 - Tartaric acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Tartaric acid. 184.1099 Section 184.1099 Food and... Substances Affirmed as GRAS § 184.1099 Tartaric acid. (a) Food grade tartaric acid (C4H6O6, CAS Reg. No. 87-69-4) has the l configuration. The l form of tartaric acid is dextrorotatory in solution and is...

  1. 21 CFR 184.1099 - Tartaric acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Tartaric acid. 184.1099 Section 184.1099 Food and....1099 Tartaric acid. (a) Food grade tartaric acid (C4H6O6, CAS Reg. No. 87-69-4) has the l configuration. The l form of tartaric acid is dextrorotatory in solution and is also known as l−(+)−tartaric...

  2. 21 CFR 186.1093 - Sulfamic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sulfamic acid. 186.1093 Section 186.1093 Food and....1093 Sulfamic acid. (a) Sulfamic acid (H3NO3S, CAS Reg. No. 5329-14-6) is a white crystalline solid manufactured from urea, sulfur trioxide, and sulfuric acid. It is soluble and highly ionized in water. (b)...

  3. 21 CFR 184.1099 - Tartaric acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Tartaric acid. 184.1099 Section 184.1099 Food and... Substances Affirmed as GRAS § 184.1099 Tartaric acid. (a) Food grade tartaric acid (C4H6O6, CAS Reg. No. 87-69-4) has the l configuration. The l form of tartaric acid is dextrorotatory in solution and is...

  4. 21 CFR 184.1061 - Lactic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Lactic acid. 184.1061 Section 184.1061 Food and... Substances Affirmed as GRAS § 184.1061 Lactic acid. (a) Lactic acid (C3H6O3, CAS Reg. Nos.: dl mixture, 598-82-3; l-isomer, 79-33-4; d-isomer, 10326-41-7), the chemical 2-hydroxypropanoic acid,...

  5. 21 CFR 184.1061 - Lactic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Lactic acid. 184.1061 Section 184.1061 Food and... Substances Affirmed as GRAS § 184.1061 Lactic acid. (a) Lactic acid (C3H6O3, CAS Reg. Nos.: dl mixture, 598-82-3; l-isomer, 79-33-4; d-isomer, 10326-41-7), the chemical 2-hydroxypropanoic acid,...

  6. 21 CFR 186.1093 - Sulfamic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sulfamic acid. 186.1093 Section 186.1093 Food and... Substances Affirmed as GRAS § 186.1093 Sulfamic acid. (a) Sulfamic acid (H3NO3S, CAS Reg. No. 5329-14-6) is a white crystalline solid manufactured from urea, sulfur trioxide, and sulfuric acid. It is soluble...

  7. 21 CFR 186.1093 - Sulfamic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sulfamic acid. 186.1093 Section 186.1093 Food and... Substances Affirmed as GRAS § 186.1093 Sulfamic acid. (a) Sulfamic acid (H3NO3S, CAS Reg. No. 5329-14-6) is a white crystalline solid manufactured from urea, sulfur trioxide, and sulfuric acid. It is soluble...

  8. 21 CFR 184.1099 - Tartaric acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Tartaric acid. 184.1099 Section 184.1099 Food and... Substances Affirmed as GRAS § 184.1099 Tartaric acid. (a) Food grade tartaric acid (C4H6O6, CAS Reg. No. 87-69-4) has the l configuration. The l form of tartaric acid is dextrorotatory in solution and is...

  9. 21 CFR 184.1061 - Lactic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Lactic acid. 184.1061 Section 184.1061 Food and... Substances Affirmed as GRAS § 184.1061 Lactic acid. (a) Lactic acid (C3H6O3, CAS Reg. Nos.: dl mixture, 598-82-3; l-isomer, 79-33-4; d-isomer, 10326-41-7), the chemical 2-hydroxypropanoic acid,...

  10. 21 CFR 184.1061 - Lactic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Lactic acid. 184.1061 Section 184.1061 Food and... Substances Affirmed as GRAS § 184.1061 Lactic acid. (a) Lactic acid (C3H6O3, CAS Reg. Nos.: dl mixture, 598-82-3; l-isomer, 79-33-4; d-isomer, 10326-41-7), the chemical 2-hydroxypropanoic acid,...

  11. 21 CFR 186.1093 - Sulfamic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sulfamic acid. 186.1093 Section 186.1093 Food and... Substances Affirmed as GRAS § 186.1093 Sulfamic acid. (a) Sulfamic acid (H3NO3S, CAS Reg. No. 5329-14-6) is a white crystalline solid manufactured from urea, sulfur trioxide, and sulfuric acid. It is soluble...

  12. 21 CFR 186.1093 - Sulfamic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sulfamic acid. 186.1093 Section 186.1093 Food and... Substances Affirmed as GRAS § 186.1093 Sulfamic acid. (a) Sulfamic acid (H3NO3S, CAS Reg. No. 5329-14-6) is a white crystalline solid manufactured from urea, sulfur trioxide, and sulfuric acid. It is soluble...

  13. 21 CFR 184.1077 - Potassium acid tartrate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium acid tartrate. 184.1077 Section 184.1077... Listing of Specific Substances Affirmed as GRAS § 184.1077 Potassium acid tartrate. (a) Potassium acid tartrate (C4H5KO6, CAS Reg. No. 868-14-4) is the potassium acid salt of l−(+)−tartaric acid and is...

  14. 21 CFR 184.1077 - Potassium acid tartrate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium acid tartrate. 184.1077 Section 184.1077... Listing of Specific Substances Affirmed as GRAS § 184.1077 Potassium acid tartrate. (a) Potassium acid tartrate (C4H5KO6, CAS Reg. No. 868-14-4) is the potassium acid salt of l−(+)−tartaric acid and is...

  15. 21 CFR 184.1077 - Potassium acid tartrate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium acid tartrate. 184.1077 Section 184.1077... GRAS § 184.1077 Potassium acid tartrate. (a) Potassium acid tartrate (C4H5KO6, CAS Reg. No. 868-14-4) is the potassium acid salt of l−(+)−tartaric acid and is also called potassium bitartrate or cream...

  16. 21 CFR 184.1077 - Potassium acid tartrate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium acid tartrate. 184.1077 Section 184.1077... Listing of Specific Substances Affirmed as GRAS § 184.1077 Potassium acid tartrate. (a) Potassium acid tartrate (C4H5KO6, CAS Reg. No. 868-14-4) is the potassium acid salt of l−(+)−tartaric acid and is...

  17. 21 CFR 184.1081 - Propionic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Propionic acid. 184.1081 Section 184.1081 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1081 Propionic acid. (a) Propionic acid (C3H6O2, CAS Reg. No. 79-09-4)...

  18. 21 CFR 184.1097 - Tannic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... fat in accordance with 9 CFR 318.7. (d) Prior sanctions for this ingredient different from the uses... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Tannic acid. 184.1097 Section 184.1097 Food and... Substances Affirmed as GRAS § 184.1097 Tannic acid. (a) Tannic acid (CAS Reg. No. 1401-55-4), or...

  19. 21 CFR 184.1081 - Propionic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Propionic acid. 184.1081 Section 184.1081 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1081 Propionic acid. (a) Propionic acid (C3H6O2, CAS Reg. No. 79-09-4)...

  20. 21 CFR 184.1081 - Propionic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Propionic acid. 184.1081 Section 184.1081 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1081 Propionic acid. (a) Propionic acid (C3H6O2, CAS Reg. No. 79-09-4)...

  1. 21 CFR 184.1081 - Propionic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Propionic acid. 184.1081 Section 184.1081 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1081 Propionic acid. (a) Propionic acid (C3H6O2, CAS Reg. No. 79-09-4)...

  2. 21 CFR 184.1097 - Tannic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... fat in accordance with 9 CFR 318.7. (d) Prior sanctions for this ingredient different from the uses... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Tannic acid. 184.1097 Section 184.1097 Food and... Substances Affirmed as GRAS § 184.1097 Tannic acid. (a) Tannic acid (CAS Reg. No. 1401-55-4), or...

  3. Cas9 Functionally Opens Chromatin.

    PubMed

    Barkal, Amira A; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K; Sherwood, Richard I

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  4. 21 CFR 172.345 - Folic acid (folacin).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Folic acid (folacin). 172.345 Section 172.345 Food... Special Dietary and Nutritional Additives § 172.345 Folic acid (folacin). Folic acid (CAS Reg. No....

  5. NUHOMS{reg_sign} update

    SciTech Connect

    Rich, N.

    1995-12-31

    NUHOMS{reg_sign} is the dry spent fuel storage and transportation technology selected to date by the majority of commercial nuclear utilities. The author first gives a system overview of the NUHOMS{reg_sign}. Next she discusses the project status and licensing status. She closes with an update of the multi-purpose canister.

  6. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

    PubMed

    Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

    2016-01-01

    Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. PMID:27387532

  7. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

    PubMed

    Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

    2016-08-15

    Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.

  8. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

    PubMed Central

    Nakagawa, Yoshiko; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

    2016-01-01

    ABSTRACT Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. PMID:27387532

  9. Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen--Cas s 1.

    PubMed

    Kos, T; Hoffmann-Sommergruber, K; Ferreira, F; Hirschwehr, R; Ahorn, H; Horak, F; Jager, S; Sperr, W; Kraft, D; Scheiner, O

    1993-11-15

    Pollens from trees of the order Fagales (e.g. birch, alder, hazel, and hornbeam) all contain one major allergen--the main cause for tree pollen allergy. So far the major allergens from birch (Bet v 1), alder (Aln g 1), hazel (Cor a 1), and hornbeam (Car b 1) have been characterized, showing high sequence similarity with each other (1-4). We present the molecular and immunologic characterization of Cas s 1, the major allergen from the European chestnut (Castanea sativa). From aqueous pollen extracts from European chestnut a protein was purified to homogeneity and was subjected to further investigation. The protein revealed a Mr of 22 kDa and was shown to represent the major allergen of the European chestnut (immunoblotting, histamine release) and designated Cas s 1. Despite a marked difference in Mr, Cas s 1 shows significant amino acid sequence similarity at the N-terminus and is antigenically closely related to the major birch pollen allergen Bet v 1 (17 kDa), as shown by binding to the anti-Bet v 1 monoclonal antibody BIP-1 and by IgE-inhibition tests using recombinant Bet v 1.

  10. 75 FR 31713 - 2-Propenoic acid polymer, with 1,3-butadiene and ethenylbenzene; Tolerance Exemption

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-04

    ...,3- butadiene and ethenylbenzene, minimum number average molecular weight (in AMU) 9400 (CAS Reg. No...) 305-5805. II. Background and Statutory Findings In the Federal Register of March 19, 2010 (75 FR 13277..., minimum number average molecular weight (in AMU) 9400; CAS Reg. No. 25085-39-6. That notice included...

  11. Flemion {reg_sign}S and PEFC

    SciTech Connect

    Yoshitake, M.; Yoshida, N.; Ishisaki, T.

    1996-12-31

    Asahi Glass Co., Ltd. (AGC) had commercialized chloro-alkali electrolysis process using perfluorinated ion exchange membranes (Flemion{reg_sign}). AGC participated in the PEFC project of NEDO (New Energy and Industrial Technology Development Organization) which started in the fiscal year 1992. In this program, AGC selected Flemion{reg_sign}S as one of the candidate membranes for PEFC. The properties of Flemion{reg_sign}S and Nafion{reg_sign}117 such as thermal stability, mechanical strength in dry and wet state, water content, AC specific resistance and gas permeability in dry and wet state, were evaluated and a new method of the preparation of membrane-electrode composites was developed.

  12. The CAS Classroom

    ERIC Educational Resources Information Center

    Garner, Sue

    2004-01-01

    The Victorian Curriculum and Assessment Authority (VCAA) Computer Algebra System (CAS)Pilot study (2001-2005) is monitoring the use of CAS in senior secondary mathematics. This article explores the author's experiences in the CAS classroom and delineates changes in teaching style, as a result of the introduction of CAS into the senior mathematics…

  13. Stage-specific hypermutability of the regA locus of Volvox, a gene regulating the germ-soma dichotomy

    SciTech Connect

    Kirk, D.L.; Baran, G.J.; Harper, J.F.; Huskey, R.J.; Huson, K.S.; Zagris, N.

    1987-01-16

    Mutation at the regA locus confers on somatic cells of Volvox (which otherwise undergo programmed death) ability to redifferentiate as reproductive cells. Stable mutations at the regA locus, but not at other loci, were induced at high frequency when embryos at one particular stage were exposed to either UV irradiation, novobiocin, nalidixic acid, bleomycin, 4-hydroxyaminoquinoline-1-oxide, 5-bromodeoxyuridine, or 5-fluorouracil. All treatments led to some mutations that were not expressed until the second generation after treatment. The sensitive period was after somatic and reproductive cells of the next generation had been set apart, but before they had undergone cytodifferentiation. Hypermutability occurs in presumptive reproductive cells (in which regA is normally not expressed) somewhat before regA normally acts in somatic cells. We postulate that hypermutability of regA in the reproductive cells at this time reflects a change of state that the locus undergoes as it is inactivated.

  14. Pilot test of Pickliq{reg_sign} process to determine energy and environmental benefits & economic feasibility

    SciTech Connect

    Olsen, D.R.

    1997-07-13

    Green Technology Group (GTG) was awarded Grant No. DE-FG01-96EE 15657 in the amount of $99,904 for a project to advance GTG`s Pickliq{reg_sign} Process in the Copper and Steel Industries. The use of the Pickliq{reg_sign} Process can significantly reduce the production of waste acids containing metal salts. The Pickliq{reg_sign} Process can save energy and eliminate hazardous waste in a typical copper rod or wire mill or a typical steel wire mill. The objective of this pilot project was to determine the magnitude of the economic, energy and environmental benefits of the Pickliq{reg_sign} Process in two applications within the metal processing industry. The effectiveness of the process has already been demonstrated at facilities cleaning iron and steel with sulfuric acid. 9207 companies are reported to use sulfuric and hydrochloric acid in the USA. The USEPA TRI statistics of acid not recycled in the US is 2.4 x 10{sup 9} lbs (net) for Hydrochloric Acid and 2.0 x 10{sup 9} lbs (net) for Sulfuric Acid. The energy cost of not reclaiming acid is 10.7 x 10{sup 6} BTU/ton for Hydrochloric Acid and 21.6 x 10{sup 6} BTU/Ton for Sulfuric Acid. This means that there is a very large market for the application of the Pickliq{reg_sign} Process and the widespread use of the process will bring significant world wide savings of energy to the environment.

  15. 12 CFR 742.4 - RegFlex relief.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 6 2011-01-01 2011-01-01 false RegFlex relief. 742.4 Section 742.4 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.4 RegFlex relief. (a) Exemptions. RegFlex credit unions are exempt from the...

  16. 12 CFR 742.4 - RegFlex relief.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 7 2012-01-01 2012-01-01 false RegFlex relief. 742.4 Section 742.4 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.4 RegFlex relief. (a) Exemptions. RegFlex credit unions are exempt from the...

  17. MAECTITE{reg_sign} chemical treatment technology: Selected case histories for lead and cadmium

    SciTech Connect

    Yost, K.W.; Elia, A. Jr.; Chisick, S.A.

    1994-12-31

    Chemical treatment for lead and other heavy metals, including cadmium, in soil and solid waste with the MAECTITE{reg_sign} process creates hard, insoluble mixed forms. Heavy metals are rendered non-hazardous CRA definition, therefore, treated material may be disposed as a special waste. The MAECTITE{reg_sign} chemical treatment process forms non-leachable minerals through isomorphic reaction-series induced nucleation. Unlike cementitious, silicic, and/or pozzolanic methods where contaminants are restrained in a mixture with binding agents or hydroxides subject to degradation from physical and chemical conditions, MAECTITE{reg_sign} utilizes true bonding and crystal nucleation from disassociated metal species and yields metal-substituted crystal compounds. Waste subjected to optimized MAECTITE{reg_sign} chemical treatment complies with RCRA limits as determined by USEPA`s EPTOX and TCLP test methods, and the Multiple Extraction Procedure (MEP) that simulates 1,000 year exposure to acidic environments. The MAECTITE{reg_sign} chemical treatment process often reduces waste volume by over 20% with limited or no mass increase partially due to increased particle density, eliminated interstitial space, dehydration of the waste matrix, and destruction of semi-stable carbonates.

  18. Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system.

    PubMed

    Wilkinson, Max E; Nakatani, Yoshio; Staals, Raymond H J; Kieper, Sebastian N; Opel-Reading, Helen K; McKenzie, Rebecca E; Fineran, Peter C; Krause, Kurt L

    2016-04-15

    CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity. PMID:26929403

  19. T(reg) stability: to be or not to be.

    PubMed

    Overacre, Abigail E; Vignali, Dario Aa

    2016-04-01

    Regulatory T cell (T(reg)) stability has been primarily determined by the maintained expression of the transcription factor Forkhead box P3 (Foxp3). However, T(regs) can exhibit instability while maintaining Foxp3 expression, requiring a re-examination of what defines T(reg) stability. Recent work suggests that the establishment and stability of T(regs) is mediated by a number of mechanisms besides Foxp3 expression, such as epigenetic modifications, Foxo1/3a localization, expression of Eos and signaling via Neuropilin-1. Additional studies may help to define approaches that can undermine T(reg) stability in cancer or enhance T(reg) stability in transplantation, autoimmune or inflammatory diseases and therefore have substantial therapeutic utility. In this review, we will discuss how T(reg) stability is defined and the mechanisms utilized to maintain stability.

  20. Identification of the major regenerative III protein (RegIII) in the porcine intestinal mucosa as RegIIIγ, not RegIIIα.

    PubMed

    Soler, L; Miller, I; Nöbauer, K; Carpentier, S; Niewold, T

    2015-09-15

    During the last years, an antimicrobial protein from the RegIII family has been consistently identified as one of the main up-regulated mRNA transcripts in the pig small intestinal mucosa during different infections such as enterotoxigenic Escherichia coli (ETEC). This transcript has been mainly referred to in the literature as pancreatitis-associated protein (PAP/RegIIIα). However, the identity of this transcript has not been confirmed, and no evidence of its expression at the protein level is available in the literature, because the absence of a specific antibody. In this study, we first unequivocally identified the PAP/RegIII family protein mainly expressed in ETEC infected pig intestine as RegIIIγ by 2D-DIGE and MALDI-TOF/TOF. This shows that the pig differs from species like human and mice in that RegIIIγ (and not RegIIIα) might be the major RegIII isotype during intestinal infection. Immunoblotting analysis with a specifically generated polyclonal rabbit antibody revealed that pig RegIIIγ is expressed throughout the intestinal tract, but most abundantly in the ileum. Although a higher abundance of mRNA was paralleled by higher protein abundance, a lack of linear relationship was found between RegIIIγ mRNA and protein abundances in the jejunal mucosa, the latter most pronounced in the case of natural infection. This may be related to the secretory nature of RegIIIγ. This would mean that the antimicrobial protein RegIIIγ is a good candidate as a non-invasive faecal intestinal health biomarker in swine. PMID:26187439

  1. Orimulsion{reg_sign} an alternative source of energy

    SciTech Connect

    Marruffo, F.; Sarmiento, W.

    1997-07-01

    This paper gives a brief summary of the Orimulsion{reg_sign} capabilities, current applications and its potential as an alternative energy source. Orimulsion{reg_sign} is a fuel based on natural Bitumen dispersed in water. The characteristics of Orimulsion{reg_sign} permit its handling in a manner similar to that used for conventional liquid fuels. Conclusive proof that Orimulsion{reg_sign} maintains its stability during long periods of time has been provided by the favorable experience over the past years in the storage facilities used by Bitor and its clients. More than 4 million metric tons per year of Orimulsion{reg_sign} are being used worldwide for power generation with excellent results, not only in economic aspects but also in environmental matter. Furthermore, Orimulsion{reg_sign} has been tested successfully with most existing combustion technologies, as well as with sulfur abatement and recovery equipment. Orimulsion{reg_sign} represents an excellent cost-effective alternative for clean power generation. It has been demonstrated that Orimulsion{reg_sign} CO{sub 2} emission are 20 % lower than Coal, also Orimulsion{reg_sign} has an enormous potential to lower NO{sub x} emissions as a reburning fuel, due to its inherent pre-atomization nature, water content and lower flame temperature. The paper will describe the Orimulsion{reg_sign} performance in different combustion technologies, like: Conventional Boilers, Diesel Engines, Gasification Process and Cement Kilns.

  2. Crystal Structure of Streptococcus pyogenes Cas1 and Its Interaction with Csn2 in the Type II CRISPR-Cas System.

    PubMed

    Ka, Donghyun; Lee, Hasup; Jung, Yi-Deun; Kim, Kyunggon; Seok, Chaok; Suh, Nayoung; Bae, Euiyoung

    2016-01-01

    CRISPRs and Cas proteins constitute an RNA-guided microbial immune system against invading nucleic acids. Cas1 is a universal Cas protein found in all three types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated adaptive immunity. Here, we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and characterize its interaction with S. pyogenes Csn2 (SpCsn2). The SpCas1 structure reveals a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of potential metal-binding sites for catalysis. We demonstrate that SpCas1 directly interacts with SpCsn2, and identify the binding interface and key residues for Cas complex formation. These results provide structural information for a type II Cas1 protein, and lay a foundation for studying multiprotein Cas complexes functioning in type II CRISPR-Cas systems.

  3. Biomonitoring Equivalents (BE) dossier for 2,4-dichlorophenoxyacetic acid (2,4-D) (CAS No. 94-75-7).

    PubMed

    Aylward, Lesa L; Hays, Sean M

    2008-08-01

    Recent efforts by the US Centers for Disease Control and Prevention and other researchers have resulted in a growing database of measured concentrations of chemical substances in blood or urine samples taken from the general population. However, few tools exist to assist in the interpretation of the measured values in a health risk context. Biomonitoring Equivalents (BEs) are defined as the concentration or range of concentrations of a chemical or its metabolite in a biological medium (blood, urine, or other medium) that is consistent with an existing health-based exposure guideline. This document reviews available pharmacokinetic data and models for 2,4-dichlorophenoxyacetic acid (2,4-D) and applies these data and models to existing health-based exposure guidance values from the US Environmental Protection Agency to estimate corresponding BE values for 2,4-D in plasma and urine. These values can be used as screening tools for evaluation of biomonitoring data for 2,4-D in the context of the existing USEPA risk assessment and for prioritization of the potential need for additional risk assessment efforts for 2,4-D.

  4. [CAS General Standards 2012

    ERIC Educational Resources Information Center

    Council for the Advancement of Standards in Higher Education, 2011

    2011-01-01

    The mission of the Council for the Advancement of Standards in Higher Education (CAS) is to promote the improvement of programs and services to enhance the quality of student learning and development. CAS is a consortium of professional associations who work collaboratively to develop and promulgate standards and guidelines and to encourage…

  5. Expanding the catalog of cas genes with metagenomes.

    PubMed

    Zhang, Quan; Doak, Thomas G; Ye, Yuzhen

    2014-02-01

    The CRISPR (clusters of regularly interspaced short palindromic repeats)-Cas adaptive immune system is an important defense system in bacteria, providing targeted defense against invasions of foreign nucleic acids. CRISPR-Cas systems consist of CRISPR loci and cas (CRISPR-associated) genes: sequence segments of invaders are incorporated into host genomes at CRISPR loci to generate specificity, while adjacent cas genes encode proteins that mediate the defense process. We pursued an integrated approach to identifying putative cas genes from genomes and metagenomes, combining similarity searches with genomic neighborhood analysis. Application of our approach to bacterial genomes and human microbiome datasets allowed us to significantly expand the collection of cas genes: the sequence space of the Cas9 family, the key player in the recently engineered RNA-guided platforms for genome editing in eukaryotes, is expanded by at least two-fold with metagenomic datasets. We found genes in cas loci encoding other functions, for example, toxins and antitoxins, confirming the recently discovered potential of coupling between adaptive immunity and the dormancy/suicide systems. We further identified 24 novel Cas families; one novel family contains 20 proteins, all identified from the human microbiome datasets, illustrating the importance of metagenomics projects in expanding the diversity of cas genes.

  6. Protein engineering of Cas9 for enhanced function

    PubMed Central

    Oakes, Benjamin L.; Nadler, Dana C.; Savage, David F.

    2015-01-01

    CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complimentary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted directly into the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome modifying tools. PMID:25398355

  7. Diversity of CRISPR-Cas immune systems and molecular machines.

    PubMed

    Barrangou, Rodolphe

    2015-01-01

    Bacterial adaptive immunity hinges on CRISPR-Cas systems that provide DNA-encoded, RNA-mediated targeting of exogenous nucleic acids. A plethora of CRISPR molecular machines occur broadly in prokaryotic genomes, with a diversity of Cas nucleases that can be repurposed for various applications.

  8. xdamp: An IDL{reg_sign}-based Data Manipulation Program

    SciTech Connect

    Ballard, W.P.

    1995-04-01

    The original DAMP (DAta Manipulation Program) was written by Mark Hedemann of Sandia National Laboratories and used the CA-DISSPLA{trademark} (available from Computer Associates International, Inc., Garden City, NY) graphics package as its engine. It was used to plot, modify, and otherwise manipulate the one-dimensional data waveforms (data vs time) from a wide variety of accelerators. With the waning of CA-DISSPLA and the increasing popularity of UNIX{reg_sign}-based workstations, a replacement was needed. This package uses the IDL{reg_sign} software, available from Research Systems Incorporated in Boulder, Colorado, as the engine, and creates a set of widgets to manipulate the data in a manner similar to the original DAMP. IDL is currently supported on a wide variety of UNIX platforms such as IBM{reg_sign} workstations, Hewlett Packard workstations, SUN{reg_sign} workstations, Microsoft{reg_sign} Windows{trademark} computers, Macintosh{reg_sign} computers and Digital Equipment Corporation VMS{reg_sign} systems. Thus, this program should be portable across many platforms. We have verified operation, albeit with some IDL bugs, on IBM UNIX platforms, DEC Alpha systems, HP 9000/7OO series workstations, and Macintosh computers, both regular and PowerPC{trademark} versions.

  9. The role of Cas8 in type I CRISPR interference.

    PubMed

    Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

    2015-01-01

    CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. PMID:26182359

  10. The role of Cas8 in type I CRISPR interference

    PubMed Central

    Cass, Simon D.B.; Haas, Karina A.; Stoll, Britta; Alkhnbashi, Omer S.; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L.

    2015-01-01

    CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use ‘cascade’ [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5–Cas7–crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. PMID:26182359

  11. Cas9-mediated targeting of viral RNA in eukaryotic cells.

    PubMed

    Price, Aryn A; Sampson, Timothy R; Ratner, Hannah K; Grakoui, Arash; Weiss, David S

    2015-05-12

    Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense.

  12. Utilization of Orimulsion{reg_sign} in utility boilers

    SciTech Connect

    Woodworth, L.

    1994-08-01

    Orimulsion{reg_sign}, a bitumen-in-water emulsion with excellent combustion characteristics, abundantly available from Venezuela, is being increasingly considered a a reliable economical fuel for relatively low cost conversion of utility boilers. Given the increasing deregulation of power generation both in the United States and worldwide, the economics and related advantages of Orimulsion{reg_sign} applications may provide opportunities for electric utilities to compete in power markets and do so in an environmentally acceptable manner.

  13. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

    PubMed

    Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

    2015-10-01

    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing.

  14. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

    PubMed Central

    Jinek, Martin; Jiang, Fuguo; Taylor, David W.; Sternberg, Samuel H.; Kaya, Emine; Ma, Enbo; Anders, Carolin; Hauer, Michael; Zhou, Kaihong; Lin, Steven; Kaplan, Matias; Iavarone, Anthony T.; Charpentier, Emmanuelle; Nogales, Eva; Doudna, Jennifer A.

    2014-01-01

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation. PMID:24505130

  15. Genome sequence of the moderately thermophilic, amino-acid-degrading and sulfur-reducing bacterium Thermovirga lienii type strain (Cas60314T)

    SciTech Connect

    Goker, Markus; Saunders, Elizabeth H; Lapidus, Alla L.; Nolan, Matt; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Han, Cliff; Tapia, Roxanne; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Mavromatis, K; Pagani, Ioanna; Ivanova, N; Mikhailova, Natalia; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Chang, Yun-Juan; Jeffries, Cynthia; Brambilla, Evelyne-Marie; Rohde, Manfred; Spring, Stefan; Detter, J. Chris; Woyke, Tanja; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter

    2012-01-01

    Thermovirga lienii Dahle and Birkeland 2006 is a member to the genomically so far uncharacterized genus Thermovirga in the phylum 'Synergistetes'. Members of the only recently (2007) proposed phylum 'Synergistetes' are of interest because of their isolated phylogenetic position and their diverse habitats, e.g. from man to oil well. The genome of T. lienii Cas60314T is only the 5th genome sequence (3rd completed) from this phylum to be published. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,999,646 bp long genome (including one plasmid) with its 1,914 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  16. Cigarette advertising and children's smoking: why Reg was withdrawn.

    PubMed Central

    Hastings, G. B.; Ryan, H.; Teer, P.; MacKintosh, A. M.

    1994-01-01

    OBJECTIVE--To examine the appeal of the Embassy Regal "Reg" campaign to young people. DESIGN--Three quantitative surveys and one piece of qualitative research: (a) self completion questionnaire administered in classrooms, (b) questionnaire led interviews with children, (c) questionnaire led interviews with adults, and (d) group discussions with children and adults. SETTINGS--(a) Secondary and middle schools in England; (b) north of England, Scotland, and Wales; (c) north of England, Scotland, and Wales; and (d) Glasgow. SUBJECTS--(a) 5451 schoolchildren aged 11-15 recruited by stratified random sampling; (b) 437 children aged 5-10 recruited by quota sampling; (c) 814 adults aged 15-65 recruited by quota sampling; and (d) 12 groups of children aged 10-15, three groups of adults aged 18-24, and three groups of adults aged 35-55. RESULTS--Children were familiar with cigarette advertising and in particular the Reg campaign. Although younger children struggled to understand the creative content of the adverts, older and smoking children could understand and appreciate the humour. They considered Reg to be amusing and could relate to the type of joke used in the advert. In addition Reg's flippant attitude towards serious issues appealed to the children. While adults aged 18-24 understood the campaign they did not identify with it, and 35-55 year olds (the campaign's supposed target) were unappreciative of the campaign. CONCLUSIONS--The Reg campaign was getting through to children more effectively than it was to adults and held most appeal for teenagers, particularly 14-15 year old smokers. It clearly contravened the code governing tobacco advertising, which states that advertising must not appeal to children more than it does to adults, and it may have had a direct impact on teenage smoking. In view of these findings the Advertising Standards Authority's decision to withdraw the Reg campaign seems appropriate. Images p935-a PMID:7950668

  17. CV Protection in the EMPA-REG OUTCOME Trial: A "Thrifty Substrate" Hypothesis.

    PubMed

    Ferrannini, Ele; Mark, Michael; Mayoux, Eric

    2016-07-01

    The striking and unexpected relative risk reductions in cardiovascular (CV) mortality (38%), hospitalization for heart failure (35%), and death from any cause (32%) observed in the EMPA-REG OUTCOME trial using an inhibitor of sodium-glucose cotransporter 2 (SGLT2) in patients with type 2 diabetes and high CV risk have raised the possibility that mechanisms other than those observed in the trial-modest improvement in glycemic control, small decrease in body weight, and persistent reductions in blood pressure and uric acid level-may be at play. We hypothesize that under conditions of mild, persistent hyperketonemia, such as those that prevail during treatment with SGLT2 inhibitors, β-hydroxybutyrate is freely taken up by the heart (among other organs) and oxidized in preference to fatty acids. This fuel selection improves the transduction of oxygen consumption into work efficiency at the mitochondrial level. In addition, the hemoconcentration that typically follows SGLT2 inhibition enhances oxygen release to the tissues, thereby establishing a powerful synergy with the metabolic substrate shift. These mechanisms would cooperate with other SGLT2 inhibition-induced changes (chiefly, enhanced diuresis and reduced blood pressure) to achieve the degree of cardioprotection revealed in the EMPA-REG OUTCOME trial. This hypothesis opens up new lines of investigation into the pathogenesis and treatment of diabetic and nondiabetic heart disease. PMID:27289126

  18. Sediment toxicity in the Duluth-Superior Harbor: Use of Microtox{reg_sign} and Mutatox{reg_sign} as screening assays

    SciTech Connect

    Schubauer-Berigan, M.; Hubbard, C.; Schubauer-Berigan, J.; Tesser, G.

    1995-12-31

    Sediment toxicity tests were conducted in the Duluth-Superior Harbor at 40 sites as part of an integrated sediment assessment during the fall of 1993. Two rapid assays conducted with Photobacterium phosphoreum (Microtox{reg_sign} and Mutatox{reg_sign}) were compared with three standard US EPA sediment toxicity tests: Hyalella azteca (acute tests) and Chironomus tentans (acute and sub-lethal tests). The response in the two microbial assays was also evaluated for sensitivity to various contaminants analyzed simultaneously in the Duluth-Superior Harbor sediments. Microtox{reg_sign} and Mutatox{reg_sign} were found to be sensitive to approximately one-third and one-half the sediments, respectively; Chironomus tentans was sensitive to 15% of the sediments (either acutely or sub-lethally), while Hyalella azteca was not sensitive to any of the sediments. In almost all cases, Microtox{reg_sign} and Mutatox{reg_sign} correctly identified samples that were toxic to the chironomid, making it useful as a screening tool for toxicity, to reduce the number of sites to be tested with the benthic organisms. The subsequent application of Microtox{reg_sign} as a screen for sediment toxicity in an EMAP survey in the St. Louis River (MN) estuary will be discussed. Correlation of Microtox{reg_sign} and Mutatox{reg_sign} toxicity to environmental contaminants found in the sediments will be presented.

  19. Structure Principles of CRISPR-Cas Surveillance and Effector Complexes.

    PubMed

    Tsui, Tsz Kin Martin; Li, Hong

    2015-01-01

    The pathway of CRISPR-Cas immunity redefines the roles of RNA in the flow of genetic information and ignites excitement for next-generation gene therapy tools. CRISPR-Cas machineries offer a fascinating set of new enzyme assemblies from which one can learn principles of molecular interactions and chemical activities. The interference step of the CRISPR-Cas immunity pathway congregates proteins, RNA, and DNA into a single molecular entity that selectively destroys invading nucleic acids. Although much remains to be discovered, a picture of how the interference process takes place is emerging. This review focuses on the current structural data for the three known types of RNA-guided nucleic acid interference mechanisms. In it, we describe key features of individual complexes and we emphasize comparisons across types and along functional stages. We aim to provide readers with a set of core principles learned from the three types of interference complexes and a deep appreciation of the diversity among them.

  20. IMHEX{reg_sign} fuel cells progress toward commercialization

    SciTech Connect

    Laurens, R.M.; Petraglia, V.J.

    1995-08-01

    The overall goal of M-C Power is the development and subsequent commercialization of Molten Carbonate Fuel Cell (MCFC) stacks. More specifically, MCFC`s based upon the Internally Manifolded. Heat EXchange (IMHEX{reg_sign}) plate design created by the Institute of Technology. In order to achieve the aforementioned goal, M-C Power assembled a formidible team of industry leaders. This group, referred to as the IMHEX{reg_sign} Team, has developed a strategy to move decisively through the stages of Technology Development and Product Design and Improvement through commercialization. This paper is to review the status of the overall commercialization program and activities. It will also provide an overview of the market entry product. Furthermore, we will evaluate the opportunities and benefits this product brings to a competive power industry.

  1. FieldREG II: consciousness field effects: replications and explorations.

    PubMed

    Nelson, R D; Jahn, R G; Dunne, B J; Dobyns, Y H; Bradish, G J

    2007-01-01

    Based on formal analysis of 18 exploratory applications, 12 of which have been reported previously, a testable general hypothesis for FieldREG experiments has been postulated, namely that data taken in environments fostering relatively intense or profound subjective resonance will show larger deviations of the mean relative to chance expectation than those generated in more pragmatic assemblies. The 61 subsequent FieldREG applications reported here comprise 21 hypothesis-based formal replications, along with 40 further explorations designed to learn more about the circumstances that favor anomalous deviations. The results of the formal replications strongly confirm the general hypothesis, yielding a composite probability against chance for the resonant subset of 2.2 x 10(-6) compared to 0.91 for the mundane subset. The exploratory work suggests other venues in which anomalous effects of group consciousness can be expected, and also identifies a number of situations that do not appear to be conducive to such responses.

  2. The on-line removal of non-regenerable salts from amine solutions using the UCARSEP{reg_sign} Process

    SciTech Connect

    Burns, D.; Gregory, R.A.

    1995-11-01

    Amine unit contamination with non-regenerable salts, whether as a result of acid or inorganic salt incursion, or solvent degradation, is a common industry problem. In MEA systems this is usually addressed by the use of a reclaimer but this is not a practical solution for DEA, MDEA or formulated solvents. Similarly, the old approach of purging solvent is no longer economically or environmentally justifiable. Neutralization of amine salts with a strong base can significantly prolong the useful life of the amine solution but eventually some of the salt may have to be removed, especially if mechanical losses are low. Electrodialysis (ED) has recently been applied to this problem and has been found to overcome many of the disadvantages of vacuum distillation and ion exchange technologies, both of which have been used in recent years for solvent clean-up. Union Carbide adapted ED technology to the unique conditions encountered in an amine system and developed the UCARSEP{reg_sign} Process. A mobile UCARSEP{reg_sign} unit has been built to achieve on-line salt removal rates of 40 lbmol/day (about 3,300 lb/day). This has been successfully used to clean up UCARSOL{reg_sign} solvents as well as DEA. Case studies are presented and the relative merits of this and other clean-up options are discussed.

  3. REG4 promotes peritoneal metastasis of gastric cancer through GPR37

    PubMed Central

    Zang, Mingde; Zhang, Baogui; Duan, Yantao; Fan, Zhiyuan; Li, Jianfang; Su, Liping; Yan, Min; Zhu, Zhenggang; Liu, Bingya; Yang, Qiumeng

    2016-01-01

    Being the major reason of recurrence and death after surgery, peritoneal metastasis of gastric cancer dooms the prognosis of advanced gastric cancer patients. Regenerating islet-derived family, member 4 (REG4) is believed to promote peritoneal metastasis, however, its mechanism is still a moot point at present. In the present study, we show that high expression of REG4 correlates with advanced stage and poor survival prognosis for gastric cancer patients. REG4 overexpression significantly enhances peritoneal metastasis by increasing adhesion ability. Moreover, SP1 is proved to be a transcription factor of REG4 and induce REG4 expression upon TGF-alpha stimulation. Also, G protein-coupled receptor 37 (GPR37) is identified to be in the same complex of REG4, which mediates REG4′s signal transduction and promotes peritoneal metastasis of gastric cancer cell. Interestingly, we also discover a positive feedback loop triggered by REG4, amplifying itself through EGFR transactivation, consisting of GPR37, ADAM17, TGF-alpha, EGFR, SP1 and REG4. In conclusion, REG4 promotes peritoneal metastasis of gastric cancer through GPR37 and triggers a positive feedback loop. PMID:27036049

  4. Basic Use of SExtractor Catalogs With TweakReg - II

    NASA Astrophysics Data System (ADS)

    Lucas, Ray A.

    2015-05-01

    The TweakReg task in the DrizzlePac software package for aligning and drizzling images is an effective tool for the initial processing of HST images. However, the internal object-finding software which it uses to generate catalogs to be used for object matching and alignment, ImageFindPars, is based on the well-known stellar-oriented daofind routine, and though it is appropriate for most images, there are some for which the task is not well-suited, such as some extragalactic fields which have lots of galaxies but almost no stars, and in some cases, if taken over long time-baselines, even the few stars present may have moved due to proper motion, thereby making them unsuitable for use in image alignment. In that case, other object-detection programs such as SExtractor can be used to generate external catalogs of primarily galaxies which can then be fed to the TweakReg task. Even multiwavelength morphological differences may make some galaxies less suitable than others for use in image alignment and registration. The basic use of simple external SExtractor catalogs with the DrizzlePac TweakReg task is described here, using ACS/WFC data as an example. This includes updating geometric distortion files, initial drizzling to make cosmic ray-cleaned images, the set-up and use of required SExtractor files to generate SExtractor catalogs, basic manipulation of those catalogs, and their use with the TweakReg task in the DrizzlePac package. More options are explored for iteratively improving the astrometric solution than in Paper I (ACS ISR 2015-04) but this still represents an intermediate level of sophistication in methods. This paper introduces several methods but the ultimate best methods can vary with the nature of the objects and the data, etc. and are for the user to explore. Also, the use of crclean.fits images may be more suited to ACS/WFC and WFC3/UVIS than WFC3/IR.

  5. A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii*

    PubMed Central

    Brendel, Jutta; Stoll, Britta; Lange, Sita J.; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A.; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

    2014-01-01

    The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA. PMID:24459147

  6. A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii.

    PubMed

    Brendel, Jutta; Stoll, Britta; Lange, Sita J; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

    2014-03-01

    The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

  7. Fiber optic chemical sensor (FOCS{reg_sign}) technology for the detection of hydrocarbons in air and dissolved in water: PetroSense{reg_sign} Portable Hydrocarbon Analyzer (PHA 100) and the PetroSense{reg_sign} CMS 5000

    SciTech Connect

    Saini, D.P.; Himka, R.; Klainer, S.; Arrasate, M.; Leclerc, R.

    1995-12-31

    The PetroSense{reg_sign} Portable Hydrocarbon Analyzer (PHA 100) and the PetroSense{reg_sign} CMS 5000 for continuous monitoring, are instruments which can detect the presence of hydrocarbons dissolved in water as well as detect hydrocarbons in the air. They allow the user to obtain real time, in situ data on hydrocarbon levels present. They can also measure the progress of any remediation that is occurring. The technology used by these instruments is described in this paper.

  8. REG and GREAT, two networks to optimize Gaia scientific exploitation

    NASA Astrophysics Data System (ADS)

    Figueras, F.; Jordi, C.; Spanish Participants in Reg; Great

    2013-05-01

    The launch of Gaia satellite by the European Space Agency is a year ahead (last quarter of 2013), and Spanish and European community have already out in place two networks devoted to the preparation of the scientific exploitation of the data acquired by the satellite: GREAT (Gaia Research for European Astronomy Training), funded by the European Science Foundation and by Marie Curie Actions in its People 7th Programme, and REG (Spanish Network for the scientific exploitation of Gaia) funded by MINECO. These networks, which are open to the international community, have adopted the challenges of Gaia mission: to revolutionize our understanding of the Milky Way and its components, trace the distribution of dark matter in the local universe, validate and improve models of stellar structure and evolution, characterizing solar system objects, ... and many more. Both networks promote the close interaction among researchers of different institutes, by supporting short and long exchange visits, workshops, schools and large conferences. Currently, 128 Spanish people actively participate in the several working groups in GREAT and REG and 2 students are performing their PhD in the framework of the GREAT-ITN Spanish node. This paper provides detailed information about the structure of these networks, the Spanish participation, and present and future tasks that are foreseen.

  9. The revised National Electrical Code reg sign for new restaurants

    SciTech Connect

    Hart, G.K. ); Claar, C.N. )

    1991-08-01

    A recent change in the 1990 National Electrical Code{reg sign} now makes it possible for restaurant owners and other foodservice operators to save substantial amounts of money on the electrical systems for any new facilities they build. This report discusses the code change in detail, shows how to apply the new code change effectively, and provides detailed information on the code change process. This report is a user's guide which includes a user's manual, containing all technical details necessary for a full understanding of the new provision in Section 220-36 of the 1990 National Electrical Code{reg sign}. The manual can help utility representatives, food-service consultants, and others determine the size of the electric service entrance and the main electrical panel for a new restaurant. It can also be used to determine if additional electrical loads can be added to an existing restaurant's electric service. Only a calculator is needed to work with the manual. In addition, this report details a cost impact study undertaken by EPRI to estimate likely savings resulting from implementing the new code section. In this study, 60% of the cases averaged savings of $9300 per facility. The report also details steps leading to successful adoption of a code change and the supporting documentation presented in the code change process. 40 figs.

  10. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    PubMed

    Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2011-04-01

    Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA. PMID:21343909

  11. CAS-Induced Difficulties in Learning Mathematics?

    ERIC Educational Resources Information Center

    Jankvist, Uffe Thomas; Misfeldt, Morten

    2015-01-01

    In recent years computer algebra systems (CAS) have become an integrated part of the upper secondary school mathematics program. Despite the many positive possibilities of CAS, there also seems to be a flip side of the coin in relation to actual difficulties in learning mathematics, not least because a strong dependence on CAS for mathematical…

  12. CRISPR/Cas9 genome editing technique and its application in site-directed genome modification of animals.

    PubMed

    Jinwei, Zhou; Qipin, Xu; Jing, Yao; Shumin, Yu; Suizhong, Cao

    2015-10-01

    CRISPR/Cas system, which uses CRISPR RNAs (crRNAs) to guide Cas nuclease to silence invading nucleic acids, is self-defense system against exogenous virus or plasmid in bacteria and archaea. Through molecular modification, the typeⅡCRISPR/Cas system has become a highly efficient site-directed genome editing technique, which is simpler than zinc-finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs) and easier to be designed and applied. In this review, we summarize the evolutionary history of CRISPR/Cas9 system, the working principle and modification process of type Ⅱ CRISPR/Cas and its application in animal genome modification. We also analyze the existing problems and improvement program of the CRISPR/Cas9 system as well as its application prospect combined with successful cases, which may provide innovative perspectives on improving animal traits and establishing animal models of human diseases.

  13. Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)*

    PubMed Central

    Makkinje, Anthony; Vanden Borre, Pierre; Near, Richard I.; Patel, Prayag S.; Lerner, Adam

    2012-01-01

    The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. PMID:22711540

  14. Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion.

    PubMed

    Chopra, Martin; Biehl, Marlene; Steinfatt, Tim; Brandl, Andreas; Kums, Juliane; Amich, Jorge; Vaeth, Martin; Kuen, Janina; Holtappels, Rafaela; Podlech, Jürgen; Mottok, Anja; Kraus, Sabrina; Jordán-Garrote, Ana-Laura; Bäuerlein, Carina A; Brede, Christian; Ribechini, Eliana; Fick, Andrea; Seher, Axel; Polz, Johannes; Ottmüller, Katja J; Baker, Jeanette; Nishikii, Hidekazu; Ritz, Miriam; Mattenheimer, Katharina; Schwinn, Stefanie; Winter, Thorsten; Schäfer, Viktoria; Krappmann, Sven; Einsele, Hermann; Müller, Thomas D; Reddehase, Matthias J; Lutz, Manfred B; Männel, Daniela N; Berberich-Siebelt, Friederike; Wajant, Harald; Beilhack, Andreas

    2016-08-22

    Donor CD4(+)Foxp3(+) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo. PMID:27526711

  15. xdamp Version 3: An IDL{reg_sign}-based data and image manipulation program

    SciTech Connect

    Ballard, W.P.

    1998-05-01

    The original DAMP (DAta Manipulation Program) was written by Mark Hedemann of Sandia National Laboratories and used the CA-DISSPLA{trademark} (available from Computer Associates International, Inc., Garden City, NY) graphics package as its engine. It was used to plot, modify, and otherwise manipulate the one-dimensional data waveforms (data vs. time) from a wide variety of accelerators. With the waning of CA-DISSPLA and the increasing popularity of Unix{reg_sign}-based workstations, a replacement was needed. This package uses the IDL{reg_sign} software, available from Research Systems Incorporated in Boulder, Colorado, as the engine, and creates a set of widgets to manipulate the data in a manner similar to the original DAMP and earlier versions of xdamp. IDL is currently supported on a wide variety of Unix platforms such as IBM{reg_sign} workstations, Hewlett Packard workstations, SUN{reg_sign} workstations, Microsoft{reg_sign} Windows{trademark} computers, Macintosh{reg_sign} computers and Digital Equipment Corporation VMS{reg_sign} and Alpha{reg_sign} systems. Thus, xdamp is portable across many platforms. The author has verified operation, albeit with some minor IDL bugs, on personal computers using Windows 95 and Windows NT; IBM Unix platforms; and DEC alpha and VMS systems; HP 9000/700 series workstations; and Macintosh computers, both regular and PowerPC{trademark} versions. Version 3 adds the capability to manipulate images to the original xdamp capabilities.

  16. mReg2 inhibits nuclear entry of apoptosis-inducing factor in mouse insulinoma cells.

    PubMed

    Liu, Lu; Chowdhury, Subrata; Uppal, Sadaf; Fang, Xin; Liu, Jun-Li; Srikant, Coimbatore B

    2015-02-01

    We have reported earlier that murine-regenerating gene mReg2 protects MIN6 mouse insulinoma cells from ER stress and caspase-mediated apoptosis. In apoptotic cells, DNA damage is induced by the nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). Here we tested the hypothesis that mReg2 may regulate Scythe and/or hsp70 which influence the nuclear import of AIF. Treatment with thapsigargin (Tg) or doxorubicin induced an increase in nuclear AIF in MIN6 cells carrying the empty transfection vector (MIN6-VC) but not in cells overexpressing mReg2 (MIN6-mReg2). On one hand, nuclear Scythe was higher in the nucleus of MIN6-mReg2 compared with that in MIN6-VC cells. mReg2 did not alter the expression of AIF or Scythe. On the other hand, mReg2 induced the expression of hsp70 which is known to promote cytosolic retention of AIF. We conclude that mReg2 inhibits AIF-mediated apoptosis by promoting the nuclear presence of Scythe and inducing hsp70.

  17. 12 CFR 742.2 - Criteria to qualify for RegFlex designation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Criteria to qualify for RegFlex designation. 742.2 Section 742.2 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.2 Criteria to qualify for RegFlex designation....

  18. 12 CFR 742.2 - Criteria to qualify for RegFlex designation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 6 2011-01-01 2011-01-01 false Criteria to qualify for RegFlex designation. 742.2 Section 742.2 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.2 Criteria to qualify for RegFlex designation....

  19. 12 CFR 742.3 - Loss and revocation of RegFlex designation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Loss and revocation of RegFlex designation. 742.3 Section 742.3 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.3 Loss and revocation of RegFlex designation. (a) Loss...

  20. 12 CFR 742.3 - Loss and revocation of RegFlex designation.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 7 2012-01-01 2012-01-01 false Loss and revocation of RegFlex designation. 742.3 Section 742.3 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.3 Loss and revocation of RegFlex designation. (a) Loss...

  1. 12 CFR 742.2 - Criteria to qualify for RegFlex designation.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 7 2012-01-01 2012-01-01 false Criteria to qualify for RegFlex designation. 742.2 Section 742.2 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.2 Criteria to qualify for RegFlex designation....

  2. 12 CFR 742.3 - Loss and revocation of RegFlex designation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 6 2011-01-01 2011-01-01 false Loss and revocation of RegFlex designation. 742.3 Section 742.3 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS REGULATORY FLEXIBILITY PROGRAM § 742.3 Loss and revocation of RegFlex designation. (a) Loss...

  3. CoRegNet: reconstruction and integrated analysis of co-regulatory networks

    PubMed Central

    Nicolle, Rémy; Radvanyi, François; Elati, Mohamed

    2015-01-01

    CoRegNet is an R/Bioconductor package to analyze large-scale transcriptomic data by highlighting sets of co-regulators. Based on a transcriptomic dataset, CoRegNet can be used to: reconstruct a large-scale co-regulatory network, integrate regulation evidences such as transcription factor binding sites and ChIP data, estimate sample-specific regulator activity, identify cooperative transcription factors and analyze the sample-specific combinations of active regulators through an interactive visualization tool. In this study CoRegNet was used to identify driver regulators of bladder cancer. Availability: CoRegNet is available at http://bioconductor.org/packages/CoRegNet Contact: remy.nicolle@issb.genopole.fr or mohamed.elati@issb.genopole.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25979476

  4. HIsmelt{reg_sign} technology: the future of ironmaking

    SciTech Connect

    Leczo, T.

    2009-03-15

    The unique liquid ironmaking process of HIsmelt{reg_sign} technology produces LD-quality hot metal or pig iron using lower-quality iron ore and non-coking coal, and a variety of iron and carbon-bearing mill wastes, without blending or agglomeration. A HIsmelt facility can replace an obsolete blast furnace in a brownfield application or can be the iron-producing component of a greenfield state-of-the-art steelmaking plant. Two companies in China have signed license agreements to build HIsmelt facilities, but are waiting until the plant in Kwinana, Australia operates for 3 months without any shutdowns or delays before they start construction on their plants.

  5. FASTPLOT: An interface to Microsoft{reg_sign} FORTRAN graphics

    SciTech Connect

    Ward, R.C.

    1994-03-01

    Interface routines to the Microsoft{reg_sign} FORTRAN graphics library (GRAPHICS.LIB) are provided to facilitate development of graphics codes. These routines are collected into the FASTPLOT library (FASTPLOT.LIB). The FASTPLOT routines simplified the development of applications utilizing graphics and add capabilities not available in GRAPHICS.LIB such as plotting histograms, splines, symbols, and error bars. Specifically, these routines were utilized in the development of the mortality data viewing code, MORTVIEW, for the US Environmental Protection Agency. Routines for color imaging, developed for use with the X-ray Computer Tomography (XCT) imaging code, and examples are also provided in the FASTPLOT library. Many example uses of FASTPLOT.LIB are contained in this document to facilitate applications development. The FASTPLOT.LIB library, source, and applications programs are supplied on the accompanying FASTPLOT diskette.

  6. 21 CFR 177.2910 - Ultra-filtration membranes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... with zirconium oxide (CAS Reg. No. 1314-23-4) containing up to 12 percent yttrium oxide (CAS Reg. No... with zirconium oxide (CAS Reg. No. 1314-23-4) containing up to 5 percent yttrium oxide (CAS Reg....

  7. 75 FR 47893 - Proposed Collection; Comment Request for REG-111583-07, (T.D. 9405) (Final)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-09

    ... Internal Revenue Service Proposed Collection; Comment Request for REG-111583-07, (T.D. 9405) (Final) AGENCY... regulations (REG-111583-07) (T.D. 9405), Employment Tax Adjustments. DATES: Written comments should be...: 1545-2097. Form Number: REG-111583-07 (T.D. 9405) (final). Abstract: This document contains...

  8. Anti-cas spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR-Cas I-F systems.

    PubMed

    Almendros, Cristóbal; Guzmán, Noemí M; García-Martínez, Jesús; Mojica, Francisco J M

    2016-01-01

    Archaea and bacteria harbour clustered regularly interspaced short palindromic repeats (CRISPR) loci. These arrays encode RNA molecules (crRNA), each containing a sequence of a single repeat-intervening spacer. The crRNAs guide CRISPR-associated (Cas) proteins to cleave nucleic acids complementary to the crRNA spacer, thus interfering with targeted foreign elements. Notably, pre-existing spacers may trigger the acquisition of new spacers from the target molecule by means of a primed adaptation mechanism. Here, we show that naturally occurring orphan CRISPR arrays that contain spacers matching sequences of the cognate (absent) cas genes are able to elicit both primed adaptation and direct interference against genetic elements carrying those genes. Our findings show the existence of an anti-cas mechanism that prevents the transfer of a fully equipped CRISPR-Cas system. Hence, they suggest that CRISPR immunity may be undesired by particular prokaryotes, potentially because they could limit possibilities for gaining favourable sequences by lateral transfer. PMID:27573106

  9. Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

    PubMed

    Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

    2015-09-01

    Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

  10. Spectroscopic Observations of UU Cas

    NASA Astrophysics Data System (ADS)

    Markov, H.; Vince, I.; Markova, N.; Djurasevic, G.

    2010-09-01

    High dispersion (R = 30000) spectra of the eclipsing binary system UU Cas are presented for the first time. Spectra covering 15 different phases were taken in two spectral regions - the vicinity of H_α and another centered on 5800Å. Seven spectral lines were identified in common. Four of them belonging to the brighter component were used to construct radial velocity curve. Some constrains on the spectral classification of the components are presented.

  11. Reg2 protects mouse insulinoma cells from streptozotocin-induced mitochondrial disruption and apoptosis.

    PubMed

    Liu, Lu; Liu, Jun-Li; Srikant, Coimbatore B

    2010-10-01

    We reported previously that pancreas-specific ablation of IGF-I in mice induced an increased expression of regenerating family proteins Reg2 and Reg3β in the pancreas and protected them from streptozotocin (Stz)-induced β-cell damage. We, therefore, assessed the effect of ectopically introduced Reg2 on Stz-induced apoptosis in MIN6 mouse insulinoma cells and report here that Reg2 protects MIN6 cells from Stz-induced apoptosis by attenuating its ability to disrupt mitochondrial membrane integrity, activate caspase-3 and promote poly-ADP ribose polymerase cleavage, and induce apoptosis. These changes correlated with suppression of c-jun N-terminal kinase (JNK) phosphorylation by Stz. Reg2 inhibited Stz-induced proapoptotic events as well as the inactivation of JNK. Inclusion of chemical inhibitor of JNK to Reg2 expressing cells rendered them sensitive to Stz. These data demonstrate that Reg2 protects insulin-producing cells against Stz-induced apoptosis by interfering with its cytotoxic signaling upstream of the intrinsic proapoptotic events by preventing its ability to inactivate JNK.

  12. RegR virulence regulon of rabbit-specific enteropathogenic Escherichia coli strain E22.

    PubMed

    Srikhanta, Yogitha N; Hocking, Dianna M; Praszkier, Judyta; Wakefield, Matthew J; Robins-Browne, Roy M; Yang, Ji; Tauschek, Marija

    2013-04-01

    AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli, enteroaggregative E. coli, and Citrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22. PMID:23340312

  13. xdamp Version 2: An IDL{reg_sign}-based data manipulation program

    SciTech Connect

    Ballard, W.P.

    1996-12-01

    The original DAMP (DAta Manipulation Program) was written by Mark Hedemann of Sandia National Laboratories and used the CA-DISSPLA{trademark} (available from Computer Associates International, Inc., Garden City, NY) graphics package as its engine. It was used to plot, modify, and otherwise manipulate the one-dimensional data waveforms (data vs. time) from a wide variety of accelerators. With the waning of CA-DISSPLA and the increasing popularity of UNIX{reg_sign}-based workstations, a replacement was needed. This package uses the IDL{reg_sign} software, available from Research Systems Incorporated in Boulder, Colorado, as the engine, and creates a set of widgets to manipulate the data in a manner similar to the original DAMP.IDL is currently supported on a wide variety of UNIX platforms such as IBM{reg_sign} workstations, Hewlett Packard workstations, SUN{reg_sign} workstations, Microsoft{reg_sign} Windows{trademark} computers, Macintosh{reg_sign} computers and Digital Equipment Corporation VMS{reg_sign} systems. Thus, xdamp is portable across many platforms. The authors have verified operation, albeit with some minor IDL bugs, on IBM PC computers using Windows, Windows 95 and Windows NT; IBM UNIX platforms; DEC Alpha and VMS systems; HP 9000/700 series workstations; and Macintosh computers, both regular and PowerPC{trademark} versions. Version 2 updates xdamp to require IDL version 4.0.1, adds many enhancements, and fixes a number of bugs.

  14. Enhancing the fuel Orimulsion{reg{underscore}sign}-400, the next generation: Environmental fate and effects

    SciTech Connect

    Johnson, I.C.; Tavel, N.G.; Marrufo, F.

    1999-07-01

    Orimulsion First Generation commonly known as Orimulsion{reg{underscore}sign}-100, is a commercial boiler fuel presently used at power plants worldwide (e.g., Canada, Denmark, Japan, Italy, Lithuania, and China). This fuel is manufactured by PDVSA-BITOR from the vast bitumen reserves of the Orinoco Belt, Venezuela. Orimulsion{reg{underscore}sign} is a liquid fossil fuel made up of 70% bitumen, and 30% water. Orimulsion{reg{underscore}sign}-400 is part of the New Generation of Orimulsion-type fuels being developed by PDVSA-BITOR; this formulation includes modifications which enhance the fuels's combustion and transport, while continuing to improve its environmental and economic benefits. The characteristics of Orimulsion{reg{underscore}sign} fuels are conducive to this fuel in any kind of boiler designed to burn Fuel Oil No.6 or coal. The introduction of this fuel alternative for utility repowering will have a significant impact on power generation in North America. The environmental fate and effects of Orimulsion{reg{underscore}sign} in aquatic systems have been studied over the past 10 years by PDVSA-INTEVEP and PDVSA-BITOR; research in the US, Canada, and Europe has been carried out for the past 8 years. This presentation is a summary of recent Orimulsion{reg{underscore}sign} environmental fate and effects studies, including: chemical and physical properties; behavior of bitumen particles in freshwater and seawater; environmental toxicity studies using Orimulsion{reg{underscore}sign} and its surfactant package; and spill prevention and cleanup methods.

  15. 21 CFR 74.706 - FD&C Yellow No. 6.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... compound is coupled with 6-hydroxy-2-naphthalene-sulfonic acid. The dye is isolated as the sodium salt and...&C Yellow No. 6 is principally the disodium salt of 6-hydroxy-5- -2-naphthalenesulfonic acid (CAS Reg. No. 2783-94-0). The trisodium salt of 3-hydroxy-4- -2,7-naphthalenedisulfonic acid (CAS Reg....

  16. 21 CFR 74.706 - FD&C Yellow No. 6.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... compound is coupled with 6-hydroxy-2-naphthalene-sulfonic acid. The dye is isolated as the sodium salt and...&C Yellow No. 6 is principally the disodium salt of 6-hydroxy-5- -2-naphthalenesulfonic acid (CAS Reg. No. 2783-94-0). The trisodium salt of 3-hydroxy-4- -2,7-naphthalenedisulfonic acid (CAS Reg....

  17. 21 CFR 74.706 - FD&C Yellow No. 6.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... compound is coupled with 6-hydroxy-2-naphthalene-sulfonic acid. The dye is isolated as the sodium salt and...&C Yellow No. 6 is principally the disodium salt of 6-hydroxy-5- -2-naphthalenesulfonic acid (CAS Reg. No. 2783-94-0). The trisodium salt of 3-hydroxy-4- -2,7-naphthalenedisulfonic acid (CAS Reg....

  18. 21 CFR 74.706 - FD&C Yellow No. 6.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...&C Yellow No. 6 is principally the disodium salt of 6-hydroxy-5- -2-naphthalenesulfonic acid (CAS Reg. No. 2783-94-0). The trisodium salt of 3-hydroxy-4- -2,7-naphthalenedisulfonic acid (CAS Reg. No... compound is coupled with 6-hydroxy-2-naphthalene-sulfonic acid. The dye is isolated as the sodium salt...

  19. 21 CFR 74.706 - FD&C Yellow No. 6.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...&C Yellow No. 6 is principally the disodium salt of 6-hydroxy-5- -2-naphthalenesulfonic acid (CAS Reg. No. 2783-94-0). The trisodium salt of 3-hydroxy-4- -2,7-naphthalenedisulfonic acid (CAS Reg. No... compound is coupled with 6-hydroxy-2-naphthalene-sulfonic acid. The dye is isolated as the sodium salt...

  20. Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

    PubMed Central

    Millen, Anne M.; Horvath, Philippe; Boyaval, Patrick; Romero, Dennis A.

    2012-01-01

    Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes. PMID:23240053

  1. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

    PubMed Central

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  2. Genome modification by CRISPR/Cas9.

    PubMed

    Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

    2014-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9-mediated genome modification enables us to edit the genomes of a variety of organisms rapidly and efficiently. The advantages of the CRISPR-Cas9 system have made it an increasingly popular genetic engineering tool for biological and therapeutic applications. Moreover, CRISPR-Cas9 has been employed to recruit functional domains that repress/activate gene expression or label specific genomic loci in living cells or organisms, in order to explore developmental mechanisms, gene expression regulation, and animal behavior. One major concern about this system is its specificity; although CRISPR-Cas9-mediated off-target mutation has been broadly studied, more efforts are required to further improve the specificity of CRISPR-Cas9. We will also discuss the potential applications of CRISPR-Cas9.

  3. Diphonix{reg_sign} resin: A review of its properties and applications

    SciTech Connect

    Chiarizia, R.; Horwitz, E.P.; Alexandratos, S.D.; Gula, M.J.

    1997-01-01

    The recently developed Diphonix{reg_sign} resin is a new multifunctional chelating ion exchange resin containing seminally substituted diphosphonic acid ligands chemically bonded to a styrene-based polymeric matrix. Diphonix can be regarded as a dual mechanism polymer, with a sulfonic acid cation exchange group allowing for rapid access, mostly non-specific, of ions into the polymeric network, and the diphosphonic acid group responsible for specificity (recognition) for a number of metal cations. The Diphonix resin exhibits an extraordinarily strong affinity for actinides, especially in the tetra- and hexavalent oxidation states. Therefore the resin has potential for applications in TRU and mixed waste treatment and characterization, and in the development of new procedures for rapid actinide preconcentration and separation from environmental samples. Metal uptake studies have been extended to alkaline earth cations, to transition and post-transition metal species, and to metal sorption from neutral or near neutral solutions. Also the kinetic behavior of the resin has been investigated in detail. In view of the above applications the influence of the most commonly occurring matrix constituents (Na, Ca, Al, Fe, hydrofluoric, sulfuric, oxalic and phosphoric acids) on the uptake of actinide ions has been measured. This review paper summarizes the most important results obtained in the studies on the properties of the Diphonix resin and gives an overview of the applications already in existence or under development in the fields of mixed waste treatment, actinide separation procedures, treatment of radwaste from nuclear power and fuel processing plants, and removal of iron from copper electrowinning solutions.

  4. Immune response of apoptosis-related cysteine peptidases from the red abalone Haliotis rufescens (HrCas8 and HrCas3): molecular characterization and transcription expression.

    PubMed

    Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

    2014-07-01

    Caspases play an important role in the different stages of programmed cell death, or apoptosis, which has been related to the immune response in multicellular organisms. The present study characterized an initiator caspase (HrCas8) and an effector caspase (HrCas3) from the red abalone Haliotis rufescens using the RACE method and qPCR analysis. HrCas8 showed a complete sequence of 2529 base pairs (bp) with an open-reading frame (ORF) of 1911 bp, a 5'UTR of 201 bp, and a 3'UTR of 417 bp. The estimated molecular mass for the 636 amino acids from HrCas8 was 71.5 kDa with an isoelectric point of 6.2. The HrCas8 sequence had two death-effector domains (DEDs) and the subunits p20 and p10, in addition to an active site characteristic of cysteine proteins. Meanwhile, the effector caspase HrCas3 showed a complete sequence of 1404 bp, a 5'UTR of 82 bp, and a 3'UTR of 574 bp. The ORF of this caspase had 747 bp that coded for 248 residues. Moreover, the predicted molecular mass of HrCas3 was 29.4 kDa; the theoretical isoelectric point was 5.70, and the sequence evidenced a conserved caspase recruitment domain (CARD). The distribution of the caspases in distinct tissues revealed that HrCas8 was principally expressed in the hemolymph, while HrCas3 had a higher expression in the gills. A basal level of expression was found for both caspases in muscle tissue. The immune response of caspases in H. rufescens was evaluated through an injection of Vibrio anguillarum. The results showed an increase in the transcription of HrCas8 post-challenge, as well as an activation of HrCas3, which together suggest the initiation of apoptosis as a response to bacterial infection in H. rufescens. PMID:24821426

  5. Comparative Study of a Real-Time PCR Assay Targeting senX3-regX3 versus Other Molecular Strategies Commonly Used in the Diagnosis of Tuberculosis

    PubMed Central

    Sanjuan-Jimenez, Rocio; Toro-Peinado, Inmaculada; Bermudez, Pilar; Colmenero, Juan D.; Morata, Pilar

    2015-01-01

    Background Nucleic acid amplification tests are increasingly used for the rapid diagnosis of tuberculosis. We undertook a comparative study of the efficiency and diagnostic yield of a real-time PCR senX3-regX3 based assay versus the classical IS6110 target and the new commercial methods. Methods This single-blind prospective comparative study included 145 consecutive samples: 76 from patients with culture-confirmed tuberculosis (86.8% pulmonary and 13.2% extrapulmonary tuberculosis: 48.7% smear-positive and 51.3% smear-negative) and 69 control samples (24 from patients diagnosed with non-tuberculous mycobacteria infections and 45 from patients with suspected tuberculosis which was eventually ruled out). All samples were tested by two CE-marked assays (Xpert®MTB/RIF and AnyplexTM plus MTB/NTM) and two in-house assays targeting senX3-regX3 and the IS6110 gene. Results The detection limit ranged from 1.00E+01 fg for Anyplex, senX3-regX3 and IS6110 to 1.00E+04 fg for Xpert. All three Xpert, senX3-regX3 and IS6110 assays detected all 37 smear-positive cases. Conversely, Anyplex was positive in 34 (91.9%) smear-positive cases. In patients with smear-negative tuberculosis, differences were observed between the assays; Xpert detected 22 (56.41%) of the 39 smear-negative samples, Anyplex 24 (61.53%), senX3-regX3 28 (71.79%) and IS6110 35 (89.74%). Xpert and senX3-regX3 were negative in all control samples; however, the false positive rate was 8.7% and 13% for Anyplex and IS6110, respectively. The overall sensitivity was 77.6%, 85.7%, 77.3% and 94.7% and the specificity was 100%, 100%, 90.8% and 87.0% for the Xpert, senX3-regX3, Anyplex and IS6110 assays, respectively. Conclusion Real-time PCR assays targeting IS6110 lack the desired specificity. The Xpert MTB/RIF and in-house senX3-regX3 assays are both sensitive and specific for the detection of MTBC in both pulmonary and extrapulmonary samples. Therefore, the real time PCR senX3-regX3 based assay could be a useful and

  6. Topical safety analysis report for the transportation of the NUHOMS{reg_sign} dry shielded canister. Volume 2

    SciTech Connect

    1993-08-01

    A thermal analyses for the 10CFR71 normal conditions of . transport and hypothetical accident conditions is presented for the conceptual NUHOMS{reg_sign} Transportation Cask and NUHOMS{reg_sign}-24P DSC system. The purpose of the thermal analyses presented herein is to demonstrate that the conceptual NUHOMS{reg_sign} Transportation Cask with the NUHOMS{reg_sign}-24P DSC provides suitable heat dissipation to maintain the heat removal capacity of the loaded NUHOMS{reg_sign} Transportation Cask. The thermal analyses results show that the maximum temperatures and pressures of the NUHOMS{reg_sign} Transportation Cask and the NUHOMS{reg_sign}-24P DSC are within their allowable material temperature and pressure limits.

  7. [EMPA-REG OUTCOME: Empagliflozin reduces mortality in patients with type 2 diabetes at high cardiovascular risk].

    PubMed

    Scheen, A J

    2015-11-01

    EMPA-REG OUTCOME is an international, prospective, placebo-controlled clinical trial investigating the cardiovascular outcomes of empagliflozin, an inhibitor of sodium-glucose cotransporters type 2 (SGLT2), in patients with type 2 diabetes mellitus and known cardiovascular disease. The trial succeeded in reaching the primary objective of non-inferiority and, in addition, showed, after a median follow up of 3.1 years, a superiority of empagliflozin (10 or 25 mg/day) versus placebo as regards the primary composite cardiovascular endpoint (hasard ratio or HR = 0.86; 95% CI 0.74-0.99; P = 0.04), hospitalisations for heart failure (-35%), cardiovascular mortality (-38%) and all-cause mortality (-32%, each p < 0.001). The reductionin mortality appeared early (< 6 months) and concerned all subgroups, without any obvious heterogeneity. This reduction in mortality does not seem to be fully explained by the concomitant slight reductions in HbA1c, body weight, waist circumference, blood pressure and serum uric acid levels in the empagliflozin groups versus the placebo group. Finally, the tolerance and safety profile of empagliflozin was good, with only a moderate increase in benign mycotic genital infections, a well-known adverse event with SGLT2 inhibitors. The remarkable effects of empagliflozin in the EMPA-REG OUTCOME trial, especially on mortality, should modify the management of patients with type 2 diabetes and a high cardiovascular risk in a near future.

  8. ROTO PEEN Scalar and VAC-PAC{reg_sign} system

    SciTech Connect

    1998-02-01

    The Pentek, Inc., milling technology, comprising the ROTO PEEN Scaler and the VAC-PAC{reg_sign} waste collection system, is a fully developed and commercialized technology used to remove hazardous coatings from concrete and steel floors, walls, ceilings, and structural components. This report describes a demonstration of the Pentek, Inc., milling system to remove the paint coating from 650 ft{sup 2} of concrete flooring on the service floor of the Chicago Pile-5 (CP-5) Research Reactor. CP-5 is a heavy-water moderated and cooled, highly enriched, uranium-fueled thermal reactor designed to supply neutrons for research. The reactor had a thermal-power rating of 5 megawatts and was operated continuously for 25 years until its final shutdown in 1979. These 25 years of operation produced activation and contamination characteristics representative of other nuclear facilities within the Department of Energy (DOE) complex and the commercial nuclear sector. CP-5 contains many of the essential features of other DOE and commercial nuclear facilities and can be used safely as a demonstration facility for the evaluation of innovative technologies for the future D and D of much larger, more highly contaminated facilities.

  9. Reg proteins and their roles in inflammation and cancer of the human digestive system.

    PubMed

    Zhao, Jie; Wang, Jingyu; Wang, Hao; Lai, Maode

    2013-01-01

    The regenerating gene (Reg) family is a group of small molecules that includes four members found in various species, although only three are found in human tissues. Their expression is stimulated by certain growth factors or cytokines. The Reg family plays different roles in proliferation, migration, and anti-apoptosis through activating different signaling pathways. Their dysexpression is closely associated with a number of human conditions and diseases such as inflammation and cancer, especially in the human digestive system. Clinically, upregulation of Reg proteins is usually demonstrated in histological sections and sera from cancer patients. Therefore, Reg proteins can predict the progression and prognosis of cancers, especially those of the digestive tract, and can also act as diagnostic markers and therapeutic targets.

  10. The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas.

    PubMed

    Du, Qingyou; Schilde, Christina; Birgersson, Elin; Chen, Zhi-hui; McElroy, Stuart; Schaap, Pauline

    2014-02-01

    Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.

  11. Recombinant Reg3β protein protects against streptozotocin-induced β-cell damage and diabetes

    PubMed Central

    Luo, Chen; Yu, Lu-Ting; Yang, Meng-Qi; Li, Xiang; Zhang, Zhi-Yuan; Alfred, Martin O; Liu, Jun-Li; Wang, Min

    2016-01-01

    Regenerating genes (Reg) have been found during the search for factors involved in pancreatic islet regeneration. Our recent study discovered that pancreatic β-cell-specific overexpression of Reg3β protects against streptozotocin (Stz) -induced diabetes in mice. To investigate its potential roles in the treatment of diabetes, we produced a recombinant Reg3β protein and provided evidence that it is active in promoting islet β-cell survival against Stz- triggered cell death. Though ineffective in alleviating preexisting diabetes, pretreatment of recombinant Reg3β was capable of minimizing the Stz-induced hyperglycemia and weight loss, by preserving serum and pancreatic insulin levels, and islet β-cell mass. No obvious changes were observed in the rate of cell proliferation and hypertrophy in α- or acinar-cells after treatment with recombinant Reg3β. The underlying mechanism of Reg3β-mediated protection seems to involve Akt activation which upregulates Bcl-2 and Bcl-xL levels and consequently promotes cell survival. PMID:27767186

  12. Translational control of regA, a key gene controlling cell differentiation in Volvox carteri.

    PubMed

    Babinger, Karin; Hallmann, Armin; Schmitt, Rüdiger

    2006-10-01

    The complete division of labour between the reproductive and somatic cells of the green alga Volvox carteri is controlled by three types of genes. One of these is the regA gene, which controls terminal differentiation of the somatic cells. Here, we examined translational control elements located in the 5' UTR of regA, particularly the eight upstream start codons (AUGs) that have to be bypassed by the translation machinery before regA can be translated. The results of our systematic mutational, structural and functional analysis of the 5' UTR led us to conclude that a ribosome-shunting mechanism--rather than leaky scanning, ribosomal reinitiation, or internal ribosome entry site (IRES)-mediated initiation--controls the translation of regA mRNA. This mechanism, which involves dissociation of the 40S initiation complex from the message, followed by reattachment downstream, in order to bypass a secondary structure block in the mRNA, was validated by deleting the predicted ;landing site' (which prevented regA expression) and inserting a stable 64 nucleotide hairpin just upstream of this site (which did not prevent regA expression). We believe that this is the first report suggesting that translation of an mRNA in a green eukaryote is controlled by ribosome shunting. PMID:16971469

  13. Requirements and tasks of cohorts and registers, the German KoRegIT project.

    PubMed

    Michalik, Claudia; Dress, Jochen; Ngouongo, Sylvie; Stäubert, Sebastian; Weber, Ulrike; Brockmeyer, Norbert; Paulus, Ursula; Stausberg, Jürgen

    2014-01-01

    Epidemiological cohorts and registers (KoReg) are long lasting and complex research projects, which need systematic and extensive planning and steering. The aim of the KoRegIT project was to develop a generic catalogue of requirements to support the organisational- and IT-structure of KoReg. The catalogue of requirements comprises the top level (TL) tasks of the core processes. All TL were classified into the following project phases: 1. Development, 2. Operation, 3. Completion. According to the defined TL tasks, the appropriate use cases (UC) were identified. The catalogue currently specifies 45 TL tasks and 207 UC. The UC were elaborated by a short and standardized description of the task, the involved actors (human or external systems), the preconditions, which have to be fulfilled in order to realize this task, the normal flow of the task and the post conditions. The developed catalogue was reviewed by representatives of different KoReg in Germany. The draft catalogue of requirements was revised according to the reviewer's feedback and discussion. The revised and complete catalogue with all elaborated UC was reviewed again by further experts. The developed KoRegIT catalogue of requirements offers a supporting tool to set-up the organisational structures and processes of KoReg as well as the definition of the needed IT-infrastructure. In addition it can be used to optimize or to expand these structures. PMID:25160356

  14. CAS as Environments for Implementing Mathematical Microworlds.

    ERIC Educational Resources Information Center

    Alpers, Burkhard

    2002-01-01

    Investigates whether computer algebra systems (CAS) are suitable environments for implementing mathematical microworlds. Recalls what constitutes a microworld and explores how CAS can be used for implementation, stating potentials as well as limitations. Provides as an example the microworld "Formula 1", implemented in Maple Software. (Author/KHR)

  15. Clean power generation resulting from orimulsion{reg_sign} utilization

    SciTech Connect

    Quig, R.H.; Woodworth, L.

    1997-07-01

    In addition to the need to resolve and manage electric utility industry deregulation, mergers, hostile takeovers, divesture of generation assets, retail wheeling and accelerating competition, the industry must now prepare to also cope with oncoming proposed regulations by the E.P.A. to again revise standards for power plant emissions. Referred to as CAPI (Clean Air Power Initiative), the E.P.A. is focusing on new regulations to: (1) Further reduce NO{sub x} to emission caps ranging from 0.1 5-0.25 lb. per million BTU by 2005. (2) Reduce the present 1990 Clean Air Act SO{sub 2} emission cap by 50-60% by the year 2010. (3) Further reduce fine particulates and other hazardous pollutants, i.e.; air toxics. A variety of enforcement concepts and related formulas seem to be emerging from the E.P.A. covering multiple pollutants, caps and trades. Final details are not yet available, but it would appear that an integrated approach to organize the trading of multi-pollutant credits could emerge. Given the foregoing CAPI issue combined with constantly changing business and environment control climates, Bitor America Corporation (BAC) who is a subsidiary of Bitomenes Orinoco, S.A. (BITOR), in turn a subsidiary of Petroleos de Venezuela, S.A. (PDVSA) and the marketer and supplier of Orimulsion{reg_sign} in North America (known as the {open_quotes}fourth fossil fuel{close_quotes}) has undertaken-a series of major tests and power plant modification design studies. The objective was to determine the cleanliness and competitiveness of various Orimulsion power system applications. Hence the purpose of this paper is to share with the reader, BAC`s work to date.

  16. Using Pro/ENGINEER`s{reg_sign} interface module

    SciTech Connect

    Schulze, J.

    1994-06-01

    When the ACCORD Process introduced Pro/ENGINEER to Sandians several years ago, a new process for design/definition was implemented. Prior to ACCORD, engineers and draftsmen worked in the 2-D mode with a program caned ANVIL{reg_sign}, which had limited capabilities. Although the transition from 2-D modeling to 3-D modeling met with some resistance, most engineers have embraced this new concept with enthusiasm They are now able to work in the 3-D mode and at increased levels of productivity with appropriate time savings never achieved before. One area that Pro/ENGINEER is noted for that this report will concentrate on, is the powerful interface module with its wide selection of transfer file configurations. This allows the engineer to create parts or assemblies and transfer them to many different second party software packages whose vendors can provide the capability for stress analysis, rapid prototypes, virtual reality environments, or many other forms of advanced manufacturing modes of communication. The ACCORD Program has at its core, the Pro/ENGINEER program from Parametric Technology Inc. Included in the ACCORD program, are several supporting programs from other vendors to make this cooperation between software packages a reality. It is possible to create parts in Pro/ENG transfer those parts to another package that has the capability to analyze the parts for deficiencies, then optimize those parts, and allow for changes to be made. Also included in this report, are other packages closely tied to Pro/ENGINEER, but not necessarily supported under the ACCORD program. Some of these packages allow you to create very impressive video productions, or allow you to meander through a virtual reality scenario. All of these new software packages will give you a new perspective on performance. This report will show how some of these interfaces work, and how you can improve your productivity if you utilize the ACCORD program as it is implemented here at Sandia.

  17. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference

    PubMed Central

    Hochstrasser, Megan L.; Taylor, David W.; Bhat, Prashant; Guegler, Chantal K.; Sternberg, Samuel H.; Nogales, Eva; Doudna, Jennifer A.

    2014-01-01

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)–associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA–E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage. PMID:24748111

  18. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

    PubMed

    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-01

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  19. Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins

    PubMed Central

    Ménoret, Séverine; De Cian, Anne; Tesson, Laurent; Remy, Séverine; Usal, Claire; Boulé, Jean-Baptiste; Boix, Charlotte; Fontanière, Sandra; Crénéguy, Alison; Nguyen, Tuan H.; Brusselle, Lucas; Thinard, Reynald; Gauguier, Dominique; Concordet, Jean-Paul; Cherifi, Yacine; Fraichard, Alexandre; Giovannangeli, Carine; Anegon, Ignacio

    2015-01-01

    The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals. PMID:26442875

  20. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation.

    PubMed

    Huo, Yanwu; Nam, Ki Hyun; Ding, Fang; Lee, Heejin; Wu, Lijie; Xiao, Yibei; Farchione, M Daniel; Zhou, Sharleen; Rajashankar, Kanagalaghatta; Kurinov, Igor; Zhang, Rongguang; Ke, Ailong

    2014-09-01

    CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.

  1. CAS

    SciTech Connect

    Martinez, B.; Pomeroy, G. )

    1989-12-02

    The Security Alarm System is a data acquisition and control system which collects data from intrusion sensors and displays the information in a real-time environment for operators. The Access Control System monitors and controls the movement of personnel with the use of card readers and biometrics hand readers.

  2. Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species.

    PubMed

    Pawluk, April; Staals, Raymond H J; Taylor, Corinda; Watson, Bridget N J; Saha, Senjuti; Fineran, Peter C; Maxwell, Karen L; Davidson, Alan R

    2016-01-01

    CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids(1,2). They are present in approximately half of all sequenced prokaryotes(3) and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function(4,5). We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems. PMID:27573108

  3. New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

    PubMed

    Laughery, Marian F; Hunter, Tierra; Brown, Alexander; Hoopes, James; Ostbye, Travis; Shumaker, Taven; Wyrick, John J

    2015-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double-strand breaks. Targeting of the DNA break is typically controlled by a single-guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR-Cas9 technology can be used for efficient, marker-free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time-consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9-sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter-selection using 5-fluoro-orotic acid (5-FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR-Cas9 technology in yeast genome editing.

  4. Study on analysis of waste edible oil with deterioration and removal of acid value, carbonyl value, and free fatty acid by a food additive (calcium silicate).

    PubMed

    Ogata, Fumihiko; Tanaka, Yuko; Tominaga, Hisato; Kangawa, Moe; Inoue, Kenji; Ueda, Ayaka; Iwata, Yuka; Kawasaki, Naohito

    2013-01-01

    This study investigated the regeneration of waste edible oil using a food additive (calcium silicate, CAS). Waste edible oil was prepared by combined heat and aeration treatment. Moreover, the deterioration of edible oil by combined heat and aeration treatment was greater than that by heat treatment alone. The acid value (AV) and carbonyl value (CV) increased with increasing deterioration; conversely, the tocopherol concentration decreased with increasing deterioration. The specific surface area, pore volume, and mean pore diameter of the 3 CAS formulations used (CAS30, CAS60, and CAS90) were evaluated, and scanning electron microscopic images were taken. The specific surface area increased in the order of CAS30 (115.54 m(2)/g) < CAS60 (163.93 m(2)/g) < CAS90 (187.47 m(2)/g). The mean pore diameter increased in the order of CAS90 (170.59 Å) < CAS60 (211.60 Å) < CAS30 (249.70 Å). The regeneration of waste edible oil was possible with CAS treatment. The AV reduced by 15.2%, 10.8%, and 23.1% by CAS30, CAS60, and CAS90 treatment, respectively, and the CV was reduced by 35.6%, 29.8%, and 31.3% by these 3 treatments, respectively. Moreover, the concentrations of tocopherol and free fatty acids did not change with CAS treatment. The characteristics of CAS were not related to the degree of change of AV and CV. However, the adsorption mechanism of polar and non-polar compounds generated in waste edible oil by CAS was related with the presence of silica gel molecules in CAS. The findings indicated that CAS was useful for the regeneration of waste edible oil.

  5. AND-34/BCAR3 Regulates Adhesion-Dependent p130Cas Serine Phosphorylation and Breast Cancer Cell Growth Pattern

    PubMed Central

    Makkinje, Anthony; Near, Richard I.; Infusini, Giuseppe; Borre, Pierre Vanden; Bloom, Alexander; Cai, Dongpo; Costello, Catherine E.; Lerner, Adam

    2009-01-01

    NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS PAGE while NSP-1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype. PMID:19454314

  6. Assisting Students' Cognitive Strategies with the Use of CAS

    ERIC Educational Resources Information Center

    Sarvari, Csaba; Lavicza, Zsolt; Klincsik, Mihaly

    2010-01-01

    This paper examines various cognitive strategies applied while CAS (Computer Algebra System) are used in undergraduate-level engineering mathematics teaching and learning. We posed some questions in relation to such CAS use: What kind of tools can CAS offer to enhance different cognitive strategies of students? How can the use of CAS widen the…

  7. Data Communications Requirements for CAS Applications

    NASA Technical Reports Server (NTRS)

    McCabe, James D.; Blaylock, Bruce T. (Technical Monitor)

    1994-01-01

    This paper discusses the long-haul data communications requirements needed to support visualization of Computational Aeroscience (CAS) simulations. A scientific computing model is presented, from which current communications requirements are derived. This model is then expanded to describe the CAS Collaborative Visualization Environment, in which scientists from multiple NASA centers will collaborate on multidisciplinary, time-accurate simulations. An analysis of this model, along with data from the TFCC Workload Model, shows that CAS data communications requirements will increase to 10-100 megabytes/second by 1996.

  8. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2015-12-01

    During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

  9. RegPredict: an integrated system for regulon inference in prokaryotes by comparative genomics approach

    SciTech Connect

    Novichkov, Pavel S.; Rodionov, Dmitry A.; Stavrovskaya, Elena D.; Novichkova, Elena S.; Kazakov, Alexey E.; Gelfand, Mikhail S.; Arkin, Adam P.; Mironov, Andrey A.; Dubchak, Inna

    2010-05-26

    RegPredict web server is designed to provide comparative genomics tools for reconstruction and analysis of microbial regulons using comparative genomics approach. The server allows the user to rapidly generate reference sets of regulons and regulatory motif profiles in a group of prokaryotic genomes. The new concept of a cluster of co-regulated orthologous operons allows the user to distribute the analysis of large regulons and to perform the comparative analysis of multiple clusters independently. Two major workflows currently implemented in RegPredict are: (i) regulon reconstruction for a known regulatory motif and (ii) ab initio inference of a novel regulon using several scenarios for the generation of starting gene sets. RegPredict provides a comprehensive collection of manually curated positional weight matrices of regulatory motifs. It is based on genomic sequences, ortholog and operon predictions from the MicrobesOnline. An interactive web interface of RegPredict integrates and presents diverse genomic and functional information about the candidate regulon members from several web resources. RegPredict is freely accessible at http://regpredict.lbl.gov.

  10. The new generation of Orimulsion{reg{underscore}sign}: Enhancing the fuel

    SciTech Connect

    Marruffo, F.; Sarmiento, W.; Tavel, N.G.

    1999-07-01

    After 10 years of commercial experience and proven efficacy, Orimulsion {reg{underscore}sign} has been enhanced to improve its performance as a power plant fuel. The new generation of Orimulsion{reg{underscore}sign} is called Orimulsion{reg{underscore}sign} 400, and its constituents remain 70% bitumen (extracted from the Orinoco belt in Venezuela) and 30% water with a stabilizer agent or surfactant. The main changes in this new generation of fuels are a new surfactant package (NSP) and the exclusion of magnesium nitrate from the formulation. The NSP was specially designed to meet new regulations regarding tense-active compounds established by the European Economic Community (EEC) for the next millennium. The fuel reengineering process was focused on power producers' needs, taking into consideration the new, higher standards required to compete in a deregulated market. Orimulsion{reg{underscore}sign} 400 has undergone extensive tests in different pilot-scale facilities. The tests have included handling, combustion, and performance characteristics. Results obtained in these tests show that the new formulation has improved handling and combustion characteristics compared to the original formulation. As summary, combustion results show: better heat transfer capacity in all boiler zones, less ash produced, less nitrogen oxides (NO{sub x}) generated, and improved atomization properties. Two commercial trials have been performed to test the new formulation. These tests were carried out during 1998 at power plants currently using Orimulsion{reg{underscore}sign}.

  11. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  12. Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

    PubMed

    Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

    2015-03-12

    Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

  13. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

    PubMed Central

    Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S.; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V.; Charpentier, Emmanuelle

    2014-01-01

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. PMID:24270795

  14. Assessment of cerebral blood flow autoregulation (CBF AR) with rheoencephalography (REG): studies in animals

    NASA Astrophysics Data System (ADS)

    Popovic, Djordje; Bodo, Michael; Pearce, Frederick; van Albert, Stephen; Garcia, Alison; Settle, Tim; Armonda, Rocco

    2013-04-01

    The ability of cerebral vasculature to regulate cerebral blood flow (CBF) in the face of changes in arterial blood pressure (SAP) or intracranial pressure (ICP) is an important guard against secondary ischemia in acute brain injuries, and official guidelines recommend that therapeutic decisions be guided by continuous monitoring of CBF autoregulation (AR). The common method for CBF AR monitoring, which rests on real-time derivation of the correlation coefficient (PRx) between slow oscillations in SAP and ICP is, however, rarely used in clinical practice because it requires invasive ICP measurements. This study investigated whether the correlation coefficient between SAP and the pulsatile component of the non-invasive transcranial bioimpedance signal (rheoencephalography, REG) could be used to assess the state and lower limit of CBF AR. The results from pigs and rhesus macaques affirm the utility of REG; however, additional animal and clinical studies are warranted to assess selectivity of automatic REG-based evaluation of CBF AR.

  15. Regulatory Interactions in ProKaryotes from RegTransBase

    DOE Data Explorer

    Dubchak, Inna; Gelfand, Mikhail

    RegTransBase, a manually curated database of regulatory interactions in prokaryotes, captures the knowledge in published scientific literature using a controlled vocabulary. RegTransBase describes a large number of regulatory interactions reported in many organisms and contains various types of experimental data, in particular: the activation or repression of transcription by an identified direct regulator determining the transcriptional regulatory function of a protein (or RNA) directly binding to DNA or RNA mapping or prediction of binding sites for a regulatory protein characterization of regulatory mutations. RegTransBase also contains manually created position weight matrices (PWM) that can be used to identify candidate regulatory sites in over 60 species. (Specialized Interface)

  16. Composite molding of SPECTRA{reg_sign} extended chain polyethylene fibers in a flexible rubber matrix

    SciTech Connect

    McKeehan, K.

    1997-08-01

    THETA Technologies, Inc. is a multidisciplinary research and development firm involved in the design and development of affordable, lightweight, high ballistic protection modular body armor ensemble for future military, law enforcement, and specialized commercial market applications. In the course of their research, THETA Technologies, Inc. identified that current state-of-the-art in ballistic protection and overall composite reinforcement is a high density extended chain polyethylene (HDECPE) fiber known as SPECTRA{reg_sign}, a product of AlliedSignal Fibers, Petersburg, VA. SPECTRA{reg_sign} is ten times stronger than steel of equal weight. As a non-aramid polyethylene, it offers highly desirable properties in areas of resistance to chemical degradation, virtual neutral buoyancy (0.97), and increased capacity for composite bonding over competing nylon-based fibers, such as KEVLAR{reg_sign}. SPECTRA Shield{trademark}, a woven ballistic-resistant fabric using the SPECTRA{reg_sign} fiber, is presently the most effective ballistic-resistant component for both flexible and hard plate composite armors. THETA Technologies, Inc. identified a market need for a boot sole design that would measurably increase protection to the wearer without significantly degrading performance in other areas, such as flexibility and overall weight. THETA Technologies, Inc. proposed a nitrile rubber and SPECTRA{reg_sign} fiber matrix to produce an optimal boot sole. The objective of this CRADA effort was to develop and test a process for combining the SPECTRA{reg_sign} fiber, in both chopped fiber and SPECTRA Shield{trademark} form, within a semiflexible nitrile composite having desirable and marketable properties in areas of resistance to penetration and mechanical stress.

  17. The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

    PubMed

    Ma, Xiuying; Wu, Deqing; Zhou, Shu; Wan, Feng; Liu, Hua; Xu, Xiaorong; Xu, Xuanfu; Zhao, Yan; Tang, Maochun

    2016-01-01

    In the periphery of pancreatic ductal adenocarcinoma (PDAC), high accumulation of tumor-associated macrophages (TAMs), which exhibit M2 phenotype, has been shown to be correlated with extra-pancreatic invasion, lymph vessel invasion, lymph node involvement and shortened survival time. However, mechanisms by which tumor cells educate and reprogram TAMs remain largely unclear. The phenotype of TAMs in PDAC tissues was confirmed by immunofluoresence and confocal microscopy. Human CD14+ monocytes were incubated with recombinant human REG4 (rREG4) before being stimulated with LPS and IL-10 and IL-6 were measured with ELISA. A panel of M1 and M2 genes were measured by quantitative real-time PCR. Panc1, AsPC1 and BxPC3 cells were cultured in the conditioned medium (CM) and treated with REG4. The macrophages were infected with CREB shRNA or cultured by the CM of Panc1 cells infected with REG4 shRNA. The expression of CD163, CD206 and REG4 and the phosphorylation levels of epidermal growth factor receptor (EGFR), AKT and cAMP response element-binding protein (CREB) in cells were assessed with western blotting. Cell proliferation and invasiveness were also assessed. The rREG4 or the conditioned medium of Panc1 cells which secreted REG4 induced the polarization macrophages to M2 phenotype. Treatment of human macrophages with REG4 resulted in phosphorylation of EGFR, AKT and CREB. The latter was responsible for REG4-mediated macrophage polarization to M2. The conditioned medium of macrophages treated with rREG4 promoted the proliferation and invasion of pancreatic cancer cell lines. REG4, overexpressed in PDAC and secreted by cancer cells, promoted macrophage polarization to M2, through at least in part, activation of ERK1/2 and CREB and changed the microenvironment to facilitate cancer growth and metastasis.

  18. CRISPR-Cas: biology, mechanisms and relevance

    PubMed Central

    Hille, Frank

    2016-01-01

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue ‘The new bacteriology’. PMID:27672148

  19. CRISPR-Cas: biology, mechanisms and relevance.

    PubMed

    Hille, Frank; Charpentier, Emmanuelle

    2016-11-01

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes-termed spacers-into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent.This article is part of the themed issue 'The new bacteriology'.

  20. CRISPR-Cas: biology, mechanisms and relevance.

    PubMed

    Hille, Frank; Charpentier, Emmanuelle

    2016-11-01

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes-termed spacers-into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent.This article is part of the themed issue 'The new bacteriology'. PMID:27672148

  1. Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

    PubMed Central

    Paradkar, A S; Jensen, S E

    1995-01-01

    A Streptomyces clavuligerus mutant disrupted in cas2, encoding the clavaminate synthase (CAS2) isoenzyme, was constructed by a gene replacement procedure. The resulting cas2 mutant showed no clavulanic acid production when grown in starch-asparagine medium. However, in soy medium, the cas2 mutant did produce clavulanic acid, although in amounts less than those produced by wild-type cultures. This medium-dependent leaky phenotype correlated well with the presence of the cas1 transcript, encoding the CAS1 isoenzyme, in cultures grown in soy medium and with its absence from those grown in starch-asparagine medium. This suggested that CAS1 and CAS2 both contribute to clavulanic acid production but that their production is regulated differently. Under nutritional conditions in which cas1 expression is blocked, cas2 becomes essential for clavulanic acid production. Northern (RNA) analysis revealed that while cas1 is transcribed as a 1.4-kb monocistronic transcript only, cas2 is transcribed both as a 1.2-kb monocistronic transcript and as part of a 5.3-kb polycistronic transcript. High-resolution S1 nuclease analysis located the transcription start point of the monocistronic cas2 transcript at a C residue 103 nucleotides upstream from the cas2 start codon. PMID:7868606

  2. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

    PubMed

    Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H

    2015-09-22

    Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

  3. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

    PubMed Central

    Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H.

    2015-01-01

    Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis. PMID:26324940

  4. Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3.

    PubMed

    Gong, Bei; Shin, Minsang; Sun, Jiali; Jung, Che-Hun; Bolt, Edward L; van der Oost, John; Kim, Jeong-Sun

    2014-11-18

    Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

  5. Orthologs and paralogs of regA, a master cell-type regulatory gene in Volvox carteri.

    PubMed

    Duncan, Leonard; Nishii, Ichiro; Howard, Alicia; Kirk, David; Miller, Stephen M

    2006-07-01

    The multicellular green alga Volvox carteri forma nagariensis has only two cell types: terminally differentiated somatic cells and reproductive cells. The regA gene maintains the terminally differentiated state of the somatic cells, apparently by repressing transcription of genes required for chloroplast biogenesis and thereby preventing cell growth. Because the RegA protein sequence bore no obvious motifs, we are attempting to identify regions of functional importance by searching for strongly conserved domains in RegA orthologs. Here we report the cloning and characterization of regA from the most closely related known taxon, V. carteri f. kawasakiensis. Given the closeness of the relationship between these two formas, their regA genes are surprisingly different: they differ in the number of introns and by several lengthy indels, and they encode proteins that are only 80% identical. We also serendipitously discovered a paralogous gene immediately upstream of each regA locus. The two regA genes, both upstream paralogs and several genes in Chlamydomonas (the closest unicellular relative of Volvox) encode a conserved region (the VARL domain) that contains what appears to be a DNA-binding SAND domain. This discovery has opened up a new avenue for exploring how regA and the terminally differentiated state that it controls evolved. PMID:16622701

  6. Eichrom`s Diphonix{reg_sign} resin: Production-scale applications in radioactive waste treatment and iron control in copper electrowinning

    SciTech Connect

    Gula, M.J.; Chang, F.; Dreisinger, D.B.; Horwitz, E.P.

    1997-12-31

    Eichrom`s Diphonix{reg_sign} resin has been phased through synthetic scale-up, pilot testing, and production installation in radioactive waste treatment and hydrometallurgical applications. The geminal diphosphonic acid groups of Diphonix resin allow selective retention by cation-exchange and/or chelation. The resin is effective at low pH where sulfonic and carboxylic acid resins are ineffective. Diphonix resin has been used in nuclear facilities to reduce actinide concentrations in radioactive waste effluents and to reduce waste volumes. The high retention of iron(III) by Diphonix resin in acidic sulfate media has led to an installation capable of removing one ton of iron per day from a copper electrowinning stream. This iron control process diminishes cobalt losses in the electrowinning circuit and significantly reduces operating costs. The authors will discuss the development of these Diphonix resin applications.

  7. 78 FR 48771 - Proposed Collection; Comment Request for REG 209446-82 (TD 8852)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-09

    ... corporation to its shareholders. ] DATES: Written comments should be received on or before October 8, 2013 to... Items of an S Corporation to its Shareholders. OMB Number: 1545-1613. Regulation Project Number: REG-209446-82 (TD 8852) Abstract: Section 1366 requires shareholders of an S corporation to take into...

  8. NTP technical report on the toxicity studies of Formic Acid (CAS No. 64-18-6) Administered by Inhalation to F344/N Rats and B6C3F1 Mice.

    PubMed

    Thompson, Morrow

    1992-07-01

    Formic acid occurs in a variety of plants and fruits, mammalian tissues, and insect venoms. It is used industrially in preparing a variety of drugs, dyes, and chemicals; as a decalcifier; and in leather tanning. Formic acid also is an environmental contaminant of air and water and has been identified as the toxic intermediate (formate) in methanol poisoning. Two- and 13-week toxicity studies of formic acid were conducted in male and female F344/N rats and B6C3F1 mice by whole body inhalation exposure to formic acid vapors. In addition, in vitro genetic toxicity studies were performed with Salmonella typhimurium, with or without metabolic activation. Formic acid was not mutagenic in this assay. In 2-week studies, groups of 5 F344/N rats and 5 B6C3F1 mice of each sex were exposed to formic acid for 6 hours a day, 5 days a week, at concentrations of 0, 31, 62.5, 125, 250, or 500 ppm. Deaths occurred in animals exposed to 500 ppm (rats and mice) and 250 ppm (1 female mouse). Microscopic lesions in the respiratory and olfactory epithelia occurred in rats and mice exposed to 62.5 ppm and higher concentrations, with the severity related to the exposure concentration. The lesions consisted of squamous metaplasia, necrosis, and inflammation. Exposures had minimal or no effects on coagulation times, blood pH and electrolytes, or on concentrations and activities of urine analytes in rats during the 2-week studies. In 13-week studies, groups of 10 animals of each species and sex were exposed to formic acid at concentrations of 0, 8, 16, 32, 64, and 128 ppm for 6 hours a day, 5 days a week. Two mice, 1 male and 1 female, died in the 128 ppm groups. Body weight gains were significantly decreased in mice exposed to 64 and 128 ppm formic acid. Microscopic changes in rats and mice ranged from minimal to mild in severity and generally were limited to animals in the 128 ppm groups. Lesions related to exposure to formic acid consisted of squamous metaplasia and degeneration of the

  9. Variations in temperature and precipitation during Indian summer monsoon simulated by RegCM3

    NASA Astrophysics Data System (ADS)

    Dash, S. K.; Mamgain, A.; Pattnayak, K. C.; Giorgi, F.

    2012-04-01

    Variations in temperature and precipitation due to global changes have large societal impact in sectors such as agriculture and health. It is therefore very important to examine their temporal and spatial variations at the regional level in order to access the impact of climate change. In India, the most important quasi-periodic system to affect the weather and climate is the Indian summer monsoon. The local changes in the temperature and precipitation can be well examined by a regional model. RegCM3 is one such model best suited for the Indian region. This model has been integrated in the ensemble mode at 55km resolution over India for the summer monsoon season during the years 1982-2009. The model simulations are compared with observed values in detail. Comparison with observations shows that RegCM3 has slightly underestimated summer monsoon precipitation over the Central and Northeast India. Nevertheless, over these regions, RegCM3 simulated rainfall is closer to the observations when compared to other regions where rainfall is highly overestimated. The model simulated mid-tropospheric temperature shows a warm bias over the Himalayan and Tibetan regions that gives leads to the low pressure in the region. Thus the position of the monsoon trough as simulated by the model lies to the north of its original position. This is similar to the usual monsoon break condition leading to less rainfall over Central India. RegCM3 simulated surface maximum temperature shows large negative bias over the country while the surface minimum temperature is close to the observation. Nevertheless, there is a strong correlation between the all India weighted average surface temperature simulated by RegCM3 and IMD observations. At the regional level, in the Central India, both rainfall and temperature show the best correlation with the respective observed values. While examining the extreme condition in Central India, it is found that RegCM3 simulated frequencies of very wet and

  10. New Development of the Online Integrated Climate-Chemistry model framwork (RegCM-CHEM4)

    NASA Astrophysics Data System (ADS)

    Zakey, A. S.; Shalaby, A. K.; Solmon, F.; Giorgi, F.; Tawfik, A. B.; Steiner, A. L.; Baklanov, A.

    2012-04-01

    The RegCM-CHEM4 is a new online integrated climate-chemistry model based on the regional climate model (RegCM4). The RegCM4 developed at the Abdus Salam International Centre for Theoretical Physics (ICTP), is a hydrostatic, sigma coordinate model. Tropospheric gas-phase chemistry is integrated into the climate model using the condensed version of the Carbon Bond Mechanism CBM-Z with lumped species that represent broad categories of organics based on carbon bond structure. The computationally rapid radical balance method RBM is coupled as a chemical solver to the gas-phase mechanism. Photolysis rates are determined as a function of meteorological and chemical inputs and interpolated from an array of pre-determined values based on the Tropospheric Ultraviolet-Visible Model (TUV) with cloud cover corrections. Cloud optical depths and cloud altitudes from RegCM-CHEM4 are used in the photolysis calculations, thereby directly coupling the photolysis rates and chemical reactions to meteorological conditions at each model time step. In this study, we evaluate the model over Europe for two different time scales: (1) an event-based analysis of the ozone episode associated with the heat wave of August 2003 and (2) a climatological analysis of a six-year simulation (2000-2005). For the episode analysis, model simulations show a good agreement with the European Monitoring and Evaluation Program (EMEP) observations of hourly ozone over different regions in Europe and capture ozone concentrations during and after the summer 2003 heat wave event. Analysis of the full six years of simulation indicates that the coupled chemistry-climate model can reproduce the seasonal cycle of ozone, with an overestimation of ozone in the non-event years of 5-15 ppb depending on the geographic region. Overall, the ozone and ozone precursor evaluation shows the feasibility of using RegCM-CHEM4 for decadal-length simulations of chemistry-climate interactions.

  11. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  12. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  13. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  14. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

    PubMed

    Richter, Corinna; Dy, Ron L; McKenzie, Rebecca E; Watson, Bridget N J; Taylor, Corinda; Chang, James T; McNeil, Matthew B; Staals, Raymond H J; Fineran, Peter C

    2014-07-01

    Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

  15. Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection

    PubMed Central

    Spencer, John David; Jackson, Ashley R.; Li, Birong; Ching, Christina B.; Vonau, Martin; Easterling, Robert S.; Schwaderer, Andrew L.; McHugh, Kirk M.; Becknell, Brian

    2015-01-01

    Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain. PMID:26658437

  16. RegR, a Global LacI/GalR Family Regulator, Modulates Virulence and Competence in Streptococcus pneumoniae

    PubMed Central

    Chapuy-Regaud, Sabine; Ogunniyi, A. David; Diallo, Nicole; Huet, Yvette; Desnottes, Jean-François; Paton, James C.; Escaich, Sonia; Trombe, Marie-Claude

    2003-01-01

    The homolactic and catalase-deficient pathogen Streptococcus pneumoniae is not only tolerant to oxygen but requires the activity of its NADH oxidase, Nox, to develop optimal virulence and competence for genetic transformation. In this work, we show that the global regulator RegR is also involved in these traits. Genetic dissection revealed that RegR regulates competence and the expression of virulence factors, including hyaluronidase. In bacteria grown in vitro, RegR represses hyaluronidase. At neutral pH, it increases adherence to A549 epithelial cells, and at alkaline pH, it acts upstream of the CiaRH two-component signaling system to activate competence. These phenotypes are not associated with changes in antibiotic resistance, central metabolism, and carbohydrate utilization. Although the RegR0 (where 0 indicates the loss of the protein) mutation is sufficient to attenuate experimental virulence of strain 23477 in mice, the introduction of an additional hyl0 (where 0 indicates the loss of function) mutation in the RegR0 strain 23302 dramatically reduces its virulence. This indicates that residual virulence of the RegR0 Hyl+ derivative is due to hyaluronidase and supports the dual role of RegR in virulence. This LacI/GalR regulator, not essential for in vitro growth in rich media, is indeed involved in the adaptive response of the pneumococcus via its control of competence, adherence, and virulence. PMID:12704136

  17. Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells.

    PubMed

    Liu, Lu; Chowdhury, Subrata; Fang, Xin; Liu, Jun-Li; Srikant, Coimbatore B

    2014-05-29

    Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt-mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt-mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.

  18. Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection.

    PubMed

    Spencer, John David; Jackson, Ashley R; Li, Birong; Ching, Christina B; Vonau, Martin; Easterling, Robert S; Schwaderer, Andrew L; McHugh, Kirk M; Becknell, Brian

    2015-01-01

    Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain. PMID:26658437

  19. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis7. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  20. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    PubMed

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

  1. CRISPR-Cas immunity in prokaryotes.

    PubMed

    Marraffini, Luciano A

    2015-10-01

    Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny.

  2. CRISPR-Cas: Revolutionising genome engineering.

    PubMed

    Nicholson, Samantha Anne; Pepper, Michael Sean

    2016-09-01

    The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application. PMID:27601107

  3. CRISPR-Cas: Revolutionising genome engineering.

    PubMed

    Nicholson, Samantha Anne; Pepper, Michael Sean

    2016-08-01

    The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application.

  4. The Regional Earth System Model (RegESM) using RegCM4 coupled with the MITgcm ocean model: First assessments over the MED-CORDEX domain

    NASA Astrophysics Data System (ADS)

    Mariotti, Laura; Utku Turunçoǧlu, Ufuk; Farneti, Riccardo; Sannino, Gianmaria; Vittoria Struglia, Maria; Carillo, Adriana; Giorgi, Filippo

    2016-04-01

    In the framework of global climate studies, there is an increasingly growing concern about the vulnerability of the Mediterranean region, where high population density and intense exploitation activities pose severe questions on the sustainability of terrestrial water management, both for the present and the future. Ocean modeling studies suggest that the Mediterranean thermohaline circulation could be weakened in conditions of global greenhouse warming, an event which would undoubtedly affect regional climate, possibly triggering global feedback processes. Experiments with the atmosphere-ocean coupled system confirmed that a good comprehension of Mediterranean processes requires the explicit inclusion of the feedbacks between the atmospheric and the oceanic components, thus achieving a complete, fully coupled description of the Mediterranean hydrological cycle, at the same time gaining new insights in our current ability to reproduce the atmospheric hydrological processes and to close the hydrological balance. These issues are addressed by the upgraded PROTHEUS system which was jointly developed by ENEA and ICTP. Here we present a first evaluation of the performances of the new PROTHEUS system (called PROTHEUS 2.0) composed by the regional climate model RegCM4 (Giorgi et al. 2012) coupled with both the ocean model MITgcm (Marshall J. et al. 1997a,b) and the HD river model (Max-Planck's HD model; Hagemann and Dümenil, 1998) using RegESM (Regional Earth System Model) as a driver. The three-component (atmosphere, ocean and river routing) fully coupled model exchanges sea surface temperature (SST) from the ocean to the atmospheric model, surface wind stress, energy and freshwater fluxes from the atmosphere to the ocean model, surface and sub-surface runoff from the atmospheric component to the river routing model (Max-Planck's HD model; Hagemann and Dümenil, 1998). In order to have water conservation within the system, the river routing component sends the

  5. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false CAS applicability....

  6. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Types of CAS coverage....

  7. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false CAS applicability....

  8. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false Types of CAS coverage....

  9. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  10. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Types of CAS coverage....

  11. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  12. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Types of CAS coverage....

  13. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false CAS applicability....

  14. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  15. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false CAS applicability....

  16. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true CAS applicability. 9903.201... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  17. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Types of CAS coverage....

  18. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false CAS applicability....

  19. DNA targeting by the type I-G and type I-A CRISPR–Cas systems of Pyrococcus furiosus

    PubMed Central

    Elmore, Joshua; Deighan, Trace; Westpheling, Jan; Terns, Rebecca M.; Terns, Michael P.

    2015-01-01

    CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated. PMID:26519471

  20. Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR–Cas system

    PubMed Central

    Staals, Raymond H. J.; Jackson, Simon A.; Biswas, Ambarish; Brouns, Stan J. J.; Brown, Chris M.; Fineran, Peter C.

    2016-01-01

    CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition') is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome. PMID:27694798

  1. Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis

    PubMed Central

    Elmore, Joshua R.; Yokooji, Yuusuke; Sato, Takaaki; Olson, Sara; Glover, III, Claiborne V.C.; Graveley, Brenton R.; Atomi, Haruyuki; Terns, Rebecca M.; Terns, Michael P.

    2013-01-01

    CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5′ end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry. PMID:23535213

  2. Effects of Using a Computer Algebra System (CAS) on Junior College Students' Attitudes towards CAS and Achievement in Mathematics

    ERIC Educational Resources Information Center

    Leng, Ng Wee; Choo, Kwee Tiow; Soon, Lau Hock; Yi-Huak, Koh; Sun, Yap Yew

    2005-01-01

    This study examines the effects of using Texas Instruments' Voyage 200 calculator (V200), a graphing calculator with a built-in computer algebra system (CAS), on attitudes towards CAS and achievement in mathematics of junior college students (17 year olds). Students' attitudes towards CAS were examined using a 40-item Likert-type instrument…

  3. Toxicology and Carcinogenesis Studies of 4,4'-Diamino-2,2'-Stilbenedisulfonic Acid Disodium Salt (CAS No. 7336-20-1) in F344 Rats and B6C3F1 Mice (Feed Studies).

    PubMed

    1992-08-01

    4,4'-Diamino-2,2'-stilbenedisulfonic acid, disodium salt, is used in the synthesis of dyes and optical brighteners or fluorescent whitening agents. Toxicology and carcinogenesis studies were conducted by administering the chemical (approximately 14% water, 6% sodium chloride, 4% impurities, and 76% 4,4'-diamino-2,2'-stilbenedisulfonic acid) in feed to groups of F344/N rats and B6C3F1 mice of each sex for 14 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Chinese hamster ovary cells. 14-Day Studies: Groups of five rats and five mice of each sex were given 0, 6,250, 12,500, 25,000, 50,000, or 100,000 ppm 4,4'-diamino-2,2'-stilbenedisulfonic acid, disodium salt, in feed for 14 days. All rats and mice survived to the end of the studies. The mean body weight gain of male rats receiving 50,000 or 100,000 ppm and of female rats and male and female mice receiving 100,000 ppm was significantly lower than those of the respective controls. Clinical findings included diarrhea in the rats and mice receiving 100,000 ppm. There were no chemical-related changes in absolute or relative organ weights in rats or mice. There were no gross or microscopic lesions related to chemical administration in rats or mice. 13-Week Studies: Groups of 10 rats and 10 mice of each sex were given 0, 6,250, 12,500, 25,000, 50,000, or 100,000 ppm 4,4'-diamino-2,2'-stilbenedisulfonic acid, disodium salt, in feed for 13 weeks. One female rat, six male mice, and one female mouse in the 100,000 ppm dose groups died during the studies. Mean body weight gain was significantly decreased in male rats and female mice receiving 50,000 or 100,000 ppm, in male mice receiving 25,000, 50,000, or 100,000 ppm, and in female rats receiving 100,000 ppm. Clinical findings in rats that received 50,000 or 100,000 ppm and in mice that received 100,000 ppm included diarrhea, emaciation, and hyperemia of the perineum. There were no biologically significant changes in

  4. Historic light curve of V890 Cas

    NASA Astrophysics Data System (ADS)

    Nesci, R.

    2016-05-01

    The variability of V890 Cas is studied with 87 -band plates of the Asiago archive. The star shows variations of about 5 mag with an average magnitude =13 and a period of 486 days. An 5.0 color index is also derived near the maximum luminosity.

  5. Spectra of Cas A's Highest Velocity Ejecta

    NASA Astrophysics Data System (ADS)

    Fesen, Robert A.; Milisavljevic, Dan

    2010-08-01

    The young age and close distance of the Galactic supernova remnant Cassiopeia A (Cas A) make it perhaps our best case study and clearest look at the explosion dynamics of a core-collapse supernova (CCSN). Interestingly, Cas A exhibits two nearly opposing streams of high velocity ejecta or `jets' in its NE and SW regions racing outward at speeds more than twice that of the main shell. The nature of these jets, however, and their possible association with an aspherical supernova explosion mechanism is controversial. A handful of existing low-resolution spectra of outer knots in the NE jet display chemical abundances hinting at an origin from the S-Si-Ca- Ar rich layer deep inside the progenitor. If these abundances could be firmly established in both the NE and SW jets, it would be very strong evidence in support of a highly asymmetrical explosion engine for Cas A's progenitor and, in turn, for CCSNe in general. We request KPNO 4m telescope + MARS time to obtain high quality multi-object spectroscopy of Cas A's highest velocity ejecta to measure their nitrogen, sulfur, oxygen, calcium, and argon abundances. These spectra will be analyzed with the metal-rich shock models of J. Raymond and then compared to current sets of CCSN models paying particular attention to knot composition vs. ejection velocity and ejecta mixing.

  6. Using the CAS Standards in Assessment Projects

    ERIC Educational Resources Information Center

    Dean, Laura A.

    2013-01-01

    This chapter provides an overview of the use of professional standards of practice in assessment and of the Council for the Advancement of Standards in Higher Education (CAS). It outlines a model for conducting program self-studies and discusses the importance of implementing change based on assessment results.

  7. Chemical and radiation stability of SuperLig{reg_sign}644, resorcinol-formaldehyde, and CS-100 cesium ion exchange materials

    SciTech Connect

    Brown, G.N.; Adami, S.R.; Bray, L.A.

    1995-09-01

    At the request of the Initial Pretreatment Module Project within Westinghouse Hanford Company, Pacific Northwest Laboratory (PNL) conducted this study for the Efficient Separations and Processing Crosscutting Program (ESP) under the task ``Develop and Test Sorbents.`` The purpose of the study was to assess and compare the chemical and radiolytic stability of several cesium-selective ion exchange materials in simulated alkaline Hanford tank waste matrices. Pretreatment of nuclear process wastes to remove of cesium and other radionuclides by ion exchange was proposed previously as one method of minimizing the amount of high-level radioactive waste at Hanford. In this study, PNL evaluated three cesium-selective materials SuperLig{reg_sign}644, resorcinol-formaldehyde (R-F), and CS-100 for chemical and radiation stability in 1 M NaOH and a simulated neutralized current acid waste (NCAW). The objective of the study is to investigate the stability of the newly produced SuperLig{reg_sign}644 under a variety of conditions in an attempt to simulate and predict the degradation process. The following specific conclusions and recommendations resulted from the study.

  8. NTP Toxicology and Carcinogenesis Studies of Lauric Acid Diethanolamine Condensate (CAS NO. 120-40-1) in F344/N Rats and B6C3F1 Mice (Dermal Studies).

    PubMed

    1999-07-01

    Lauric acid diethanolamine condensate is widely used in cosmetics, shampoos, soaps, and related consumer products, to which there is extensive human exposure. Because of the lack of information about potential risks associated with long-term exposure, lauric acid diethanolamine condensate, coconut oil acid diethanolamine condensate, and oleic acid diethanolamine condensate were selected as representative of the class of diethanolamides for evaluation of prechronic toxicity and carcinogenic potential. Male and female F344/N rats and B6C3F1 mice were exposed to lauric acid diethanolamine condensate dermally for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were admin istered 0, 25, 50, 100, 200, or 400 mg lauric acid diethanolamine condensate/kg body weight in ethanol by dermal application for 14 weeks. All animals survived until study termination. Final mean body weights and body weight gains of males receiving 200 or 400 mg/kg were significantly less than those of the vehicle control group. Irritation of the skin at the site of application was observed in males receiving 100 mg/kg or greater and in females receiving 200 or 400 mg/kg. Kidney weights of females administered 200 or 400 mg/kg were significantly greater than those of the vehicle control group. There were dose-dependent increases in the incidences of nonneoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, chronic inflammation, parakeratosis, and ulcer. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice were admin istered 0, 50, 100, 200, 400, or 800 mg lauric acid diethanolamine condensate/kg body weight in ethanol by dermal application for 14 weeks. All animals survived until the end of the study, and final mean body weights and

  9. Gelcasting of CRYSTAR{reg_sign} silicon carbide ceramics. CRADA final report

    SciTech Connect

    Nunn, S.D.; Willkens, C.A.

    1998-12-31

    This Cooperative Research and Development Agreement (CRADA) was undertaken to assess the applicability the gelcasting process for forming ceramic green bodies using Saint-Gobain/Norton Industrial Ceramics Corporation`s proprietary CRYSTAR{reg_sign} silicon carbide powder. A gelcasting process, specifically tailored to Saint-Gobain/Norton`s powder composition, was developed and used successfully to form green bodies for property evaluation. This preliminary evaluation showed that the gelcast material had characteristics and properties comparable to Norton`s baseline material. Wafer carrier molds were received from Norton for gelcasting a complex-shaped configuration with CRYSTAR{reg_sign} silicon carbide. Gelcasting experiments showed that Norton`s standard plaster of paris molds were incompatible with the gelcasting process. Mold surface treatments and the use of alternative castable mold materials were investigated, however, a successful process was not identified. The highest quality parts were cast in either glass or aluminum molds.

  10. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    PubMed

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  11. Experience of Orimulsion{reg_sign} fired power plant in Japan

    SciTech Connect

    Kaneko, S.; Iwanaga, A.; Hishida, M.

    1996-12-31

    Orimulsion{reg_sign} is an emulsified fuel, a mixture of natural bitumen (Orinoco-bitumen), water (approx. 30% of Orimulsion), and a small quantity Orinoco-bitumen droplets are surrounded by water, and it can therefore be handled easily at atmospheric temperatures. The diversification of fuel resources has become an important matter for the power industry in recent years. Although Orimulsion is one possibility for diversification, it has several unique characteristics that should be taken into account.

  12. Conformational Dynamics of Response Regulator RegX3 from Mycobacterium tuberculosis

    PubMed Central

    Ahmad, Ashfaq; Cai, Yongfei; Chen, Xingqiang; Shuai, Jianwei; Han, Aidong

    2015-01-01

    Two-component signal transduction systems (TCS) are vital for adaptive responses to various environmental stresses in bacteria, fungi and even plants. A TCS typically comprises of a sensor histidine kinase (SK) with its cognate response regulator (RR), which often has two domains—N terminal receiver domain (RD) and C terminal effector domain (ED). The histidine kinase phosphorylates the RD to activate the ED by promoting dimerization. However, despite significant progress on structural studies, how RR transmits activation signal from RD to ED remains elusive. Here we analyzed active to inactive transition process of OmpR/PhoB family using an active conformation of RegX3 from Mycobacterium tuberculosis as a model system by computational approaches. An inactive state of RegX3 generated from 150 ns molecular dynamic simulation has rotameric conformations of Thr79 and Tyr98 that are generally conserved in inactive RRs. Arg81 in loop β4α4 acts synergistically with loop β1α1 to change its interaction partners during active to inactive transition, potentially leading to the N-terminal movement of RegX3 helix α1. Global conformational dynamics of RegX3 is mainly dependent on α4β5 region, in particular seven ‘hot-spot’ residues (Tyr98 to Ser104), adjacent to which several coevolved residues at dimeric interface, including Ile76-Asp96, Asp97-Arg111 and Glu24-Arg113 pairs, are critical for signal transduction. Taken together, our computational analyses suggest a molecular linkage between Asp phosphorylation, proximal loops and α4β5α5 dimeric interface during RR active to inactive state transition, which is not often evidently defined from static crystal structures. PMID:26201027

  13. NTP Carcinogenesis Bioassay of L-Ascorbic Acid (Vitamin C) (CAS No. 50-81-7) in F344/N Rats and B6C3F1 Mice (Feed Study).

    PubMed

    1983-03-01

    L-Ascorbic acid is essential for many physiologic functions in animals and humans, mostly biochemical reactions involving oxidation. L-Ascorbic acid is approved for use as a dietary supplement and chemical preservative by the U.S. Food and Drug Administration and is on the FDA's list of substances generally recognized as safe. L-Ascorbic acid may be used in soft drinks as an antioxidant for flavor ingredients, in meat and meat-containing products, for curing and pickling, in flour to improve baking quality, in beer as a stabilizer, in fats and oils as an antioxidant, and in a wide variety of foods for vitamin C enrichment. L-Ascorbic acid may also find use in stain removers, hair waving preparations; plastics manufacture, photography, and water treatment. A NTP Carcinogenesis bioassay of L-ascorbic acid (>97% pure) was conducted by administering diets containing 25,000 or 50,000 ppm L-ascorbic acid to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex for 103 weeks. Controls consisted of 50 untreated rats and untreated mice of each sex. Fifty-thousand ppm is the highest dose recommended for chronic studies. Survival of dosed and control female rats and of dosed and control female mice were comparable. Survival of high-dose male rats was slightly greater than that of the controls (P=0.087). Survival of high-dose male mice was significantly greater (P=0.009) than that of the controls. Throughout most of the study, mean body weights of dosed female rats and dosed female mice were lower than those of the controls. Final body weights were comparable among groups, except for the high-dose female rats (<13%); marginal differences (<8%) were observed for low-dose female rats and for dosed female mice (8%-11%). Food consumption was equivalent among groups. Most observational differences were confined to the female rat. The incidence of low-dose female rats with undifferentiated (mononuclear-cell) leukemias (control, 6/50, 12%; low-dose, 17/50, 34%; high-dose, 12/50, 24

  14. Controlling UCAVs by JTACs in CAS missions

    NASA Astrophysics Data System (ADS)

    Kumaş, A. E.

    2014-06-01

    By means of evolving technology, capabilities of UAVs (Unmanned Aerial Vehicle)s are increasing rapidly. This development provides UAVs to be used in many different areas. One of these areas is CAS (Close Air Support) mission. UAVs have several advantages compared to manned aircraft, however there are also some problematic areas. The remote controlling of these vehicles from thousands of nautical miles away via satellite may lead to various problems both ethical and tactical aspects. Therefore, CAS missions require a good level of ALI (Air-Land Integration), a high SA (situational awareness) and precision engagement. In fact, there is an aware friendly element in the target area in CAS missions, unlike the other UAV operations. This element is an Airman called JTAC (Joint Terminal Attack Controller). Unlike the JTAC, UAV operators are too far away from target area and use the limited FOV (Field of View) provided by camera and some other sensor data. In this study, target area situational awareness of a UAV operator and a JTAC, in a high-risk mission for friendly ground forces and civilians such as CAS, are compared. As a result of this comparison, answer to the question who should control the UCAV (Unmanned Combat Aerial Vehicle) in which circumstances is sought. A literature review is made in UAV and CAS fields and recent air operations are examined. The control of UCAV by the JTAC is assessed by SWOT analysis and as a result it is deduced that both control methods can be used in different situations within the framework of the ROE (Rules Of Engagement) is reached.

  15. Feasibility of integrating other federal information systems into the Global Network of Environment and Technology, GNET{reg_sign}

    SciTech Connect

    1998-05-01

    The Global Environment and Technology Enterprise (GETE) of the Global Environment and Technology Foundation (GETF) has been tasked by the US Department of Energy`s (DOE), Federal Energy Technology Center (FETC) to assist in reducing DOE`s cost for the Global Network of Environment and Technology (GNET{reg_sign}). As part of this task, GETE is seeking federal partners to invest in GNET{reg_sign}. The authors are also seeking FETC`s commitment to serve as GNET`s federal agency champion promoting the system to potential agency partners. This report assesses the benefits of partnering with GNET{reg_sign} and provides recommendations for identifying and integrating other federally funded (non-DOE) environmental information management systems into GNET{reg_sign}.

  16. Evaluation and analysis of RegCM3 simulated summer rainfall over the Huaihe River Basin of China

    NASA Astrophysics Data System (ADS)

    Zong, Peishu; Wang, Huijun

    2011-06-01

    This study evaluates the ability of the Abdus Salam International Center for Theoretical Physics (ICTP) version 3 Regional Climate Model (RegCM3) in simulating the summer rainfall amount and distribution and large-scale circulation over the Huaihe River basin of China. We conducted the simulation for the period of 1982-2001 and the wet year of 2003 to test the ensemble simulation capacity of RegCM3. First, by comparing the simulated rainfall amount and distribution against the observations, it is found that RegCM3 can reproduce the rainfall pattern and its annual variations. In addition, the simulated spatial patterns of 850-hPa wind and specific humidity fields are close to the observations, although the wind speed and humidity values are larger. Finally, the ensemble simulation of RegCM3 for summer 2003 failed to capture the spatial distribution and underestimated the magnitude of the precipitation anomalies, and the reasons are analyzed.

  17. Measurement of acute toxicity to Mysidopsis bahia using DaphniaQuant{reg_sign} instrument and protocol

    SciTech Connect

    Blankemeyer, J.T.; Nguyen, T.; Burks, S.L.

    1994-12-31

    DaphniaQuant{reg_sign} uses a fluorescent dye to permeate the cells of aquatic organisms. The technique has been used on frog embryos, fish embryos, and bovine erythrocytes. Two wavelengths of light are used to excite the fluorescent dye, Di-4-ANEPPS. The blue excitation wavelength measures the cell membrane potential while the yellow excitation wavelength measures the amount of dye loaded into the organisms. The authors applied the technique to the shrimp, Mysidopsis bahia, used in marine toxicity testing. The authors used from 1 to 10 shrimp, loaded into a 3 ml spectrofluorometry plastic cuvette. The fluorescent dye, Di-4-ANEPPS, was mixed with the 3 ml of ASW in the cuvette at a final Di-4ANEPPS concentration of 10{sub {minus}6} M. After a thirty minute incubation, the fluorescence of Di-4-ANEPPS was measured in the DaphniaQuant{reg_sign} instrument. A similar protocol was used to test various concentrations of standard assay chemicals and effluents. The test chemical was mixed with ASW and Di-4-ANEPPS and incubated with the shrimp for thirty minutes. After thirty minutes, the fluorescence was measured and compared to the fluorescence of the control shrimp. The authors found that the fluorescence from a single shrimp was detectable and gave similar toxicity data as did the replicates using 10 shrimp. They conclude that the DaphniaQuant{reg_sign} assay can be successfully adapted to marine organisms, particularly Mysidopsis bahia.

  18. Field evaluation of 69-kV outdoor Polysil reg sign insulators

    SciTech Connect

    Richenbacher, A.G. )

    1990-03-01

    This report, together with previous interim reports, documents and summarizes the field performance of 69 kV Polysil{reg sign} insulators during the field trial period from January, 1983 to December, 1988. These insulators were manufactured for the Electric Power Research Institute by Lindsey Industries in 1979. A description of the insulator development and manufacturing process is contained in the EPRI Final Report EL1281-1. Following their manufacture, the insulators were delivered, in the form of test racks of 17 Polysil{reg sign} insulators and one porcelain insulator, to twelve United States utilities and the Instituto de Investigaciones Electricas (IIE) in Mexico. These racks were subsequently installed and energized at twenty-five test sites during the latter half of 1979 and early 1980 by the project participants for the purpose of analyzing the outdoor field performance of these insulators and comparing the relative effect on performance of variations in composition, coating, electrical grading method, and shape represented by individual insulators within the test racks. This report documents the effects of the various Polysil{reg sign} insulator parameters on electrical performance in the field over a specific period of 6 years. However, the insulators had been energized for approximately 3 years prior to the initiation of this project and, although specific performance data is not available for that time period, the overall effects of field exposure for that additional time period (total of 9 years) are seen in the results of this report. 3 refs., 23 figs., 9 tabs.

  19. Intelligent HIP processing of a Spraycast-X{reg_sign} superalloy for aerospace applications

    SciTech Connect

    Zahrah, T.F.; Dalal, R.; Kissinger, R.

    1996-12-31

    An eddy current sensor system has been developed to monitor densification during hot isostatic pressing (HIP) of Spraycast-X{reg_sign} superalloy components for aerospace applications. The sensor system was designed, implemented and demonstrated by MATSYS personnel at the Howmet Corporation HIP facility. The eddy current sensor was used to monitor densification of Spraycast-X{reg_sign} Rene`41 ring segments from 95 to 98 percent relative density to full density. The sensor data were verified and validated by metallographic examinations of HIPed specimens. The grain size of the Spraycast-X{reg_sign} Rene`41 was not affected by HIP at both 1,066 C (1,950 F) and 1,121 C (2,050 F). Tensile strengths and 0.2% creep rupture properties were not sensitive to changes in HIP processing conditions. However, tensile ductilities and low cycle properties showed a strong correlation to HIP time at 1,121 C/103 MPa (2,050 F/15 KSI). As hole time at maximum temperature and pressure was increased from 1 to 4 hours, tensile ductilities and low cycle fatigue lives increased. The sensor system can be integrated with an intelligent closed loop control system to monitor and control densification rate and shape distortion.

  20. The EMPA-REG outcome study: critical appraisal and potential clinical implications.

    PubMed

    Perseghin, Gianluca; Solini, Anna

    2016-01-01

    Diabetes health care professionals have to face a study with results of incomparable success in secondary and tertiary cardiovascular disease prevention. In the past, no studies in patients with type 2 diabetes resulted to be successful in inducing an improvement of cardiovascular prognosis, no matter whether they were focused on a target, on life-style or on pharmacological intervention. On a clinical perspective, should the diabetologist's way to think about the anti-diabetic therapy of patients on secondary cardiovascular prevention change based on the results of Empa-Reg outcome? Due to the complexity of the clinical picture of patients with type 2 diabetes, a tailored therapy based on targets, complications, co-morbidity, familial and social environment, personal and cultural features must be conceived and applied in starting pharmacological therapy; however, the question whether should we consider empagliflozin as first choice therapy in individuals with type 2 diabetes exposed to high cardiovascular risk, the Empa-Reg outcome-like patient, awaits now for an answer. Waiting for data confirming the results of the Empa-Reg outcome study, this report goes through the good reasons in support of this way of thinking, but at the same time explores the many unanswered questions raising potential concerns about this clinical choice. PMID:27260022

  1. Effect of Orimulsion{reg_sign} physical properties and chemical composition on its behavior in water

    SciTech Connect

    Ojeda, M. de; Chirinos, M.L.

    1995-12-31

    Orimulsion{reg_sign} is the trade name given to a bitumen in water, 70:30, emulsion stabilized by a nonionic surfactant and used in industrial burners and power plants as a fuel. This paper summarizes the physical properties and chemical composition of Orimulsion{reg_sign} and their effect on the self dispersing properties of the product and its behavior in water. Also, the solubility and evaporation of the light hydrocarbon fraction from the bitumen, and the toxicity and biodegradation of the fuel are discussed. Salinity of the water affects the HLB of the surfactant and the stability of the bitumen droplets making them susceptible to coalesce. BTEX content of the bitumen was determined to be relatively low, an advantage in terms of its contribution to toxicity, when compared to other similar fuels (i.e., Fuel Oil No. 6). Because of the nature of the hydrocarbons components in the bitumen, biodegradation is estimated to be around 10% of the total bitumen fraction of the formulation. Detailed chemical characterization of each one of the constituents of the Orimulsion{reg_sign} formulation will be presented.

  2. Evaluation of the tool "Reg Refine" for user-guided deformable image registration.

    PubMed

    Johnson, Perry B; Padgett, Kyle R; Chen, Kuan L; Dogan, Nesrin

    2016-01-01

    "Reg Refine" is a tool available in the MIM Maestro v6.4.5 platform (www.mim-software.com) that allows the user to actively participate in the deformable image registration process. The purpose of this work was to evaluate the efficacy of this tool and investigate strategies for how to apply it effectively. This was done by performing DIR on two publicly available ground-truth models, the Pixel-based Breathing Thorax Model (POPI) for lung, and the Deformable Image Registration Evaluation Project (DIREP) for head and neck. Image noise matched in both magnitude and texture to clinical CBCT scans was also added to each model to simulate the use case of CBCT-CT alignment. For lung, the results showed Reg Refine effective at improving registration accuracy when controlled by an expert user within the context of large lung deformation. CBCT noise was also shown to have no effect on DIR performance while using the MIM algorithm for this site. For head and neck, the results showed CBCT noise to have a large effect on the accuracy of registration, specifically for low-contrast structures such as the brain-stem and parotid glands. In these cases, the Reg Refine tool was able to improve the registration accuracy when controlled by an expert user. Several strategies for how to achieve these results have been outlined to assist other users and provide feedback for developers of similar tools. PMID:27167273

  3. Rosebud syncoal partnership SynCoal{sup {reg_sign}} demonstration technology development update

    SciTech Connect

    Sheldon, R.W.; Heintz, S.J.

    1995-12-01

    Rosebud SynCoal{reg_sign} Partnership`s Advanced Coal Conversion Process (ACCP) is an advanced thermal coal upgrading process coupled with physical cleaning techniques to upgrade high moisture, low-rank coals to produce a high-quality, low-sulfur fuel. The coal is processed through two vibrating fluidized bed reactors where oxygen functional groups are destroyed removing chemically bound water, carboxyl and carbonyl groups, and volatile sulfur compounds. After thermal upgrading, the SynCoal{reg_sign} is cleaned using a deep-bed stratifier process to effectively separate the pyrite rich ash. The SynCoal{reg_sign} process enhances low-rank western coals with moisture contents ranging from 2555%, sulfur contents between 0.5 and 1.5 %, and heating values between 5,500 and 9,000 Btu/lb. The upgraded stable coal product has moisture contents as low as 1 %, sulfur contents as low as 0.3%, and heating values up to 12,000 Btu/lb.

  4. A novel putative lipoprotein receptor (CasLpR) in the hemocytes of the blue crab, Callinectes sapidus: cloning and up-regulated expression after the injection of LPS and LTA.

    PubMed

    Tsutsui, Naoaki; Chung, J Sook

    2012-03-01

    The full-length cDNA encoding a putative lipoprotein receptor (CasLpR) was isolated from the hemocytes of Callinectes sapidus using 5' and 3' RACEs. The open reading frame for CasLpR contains a precursor of putative CasLpR consisting of 1710 amino acid residues including 22 amino acid residues of the signal peptide (22 amino acids). Mature CasLpR (1688 amino acids with 5.6% of phosphorylation sites) has multiple, putative functional domains: five low-density lipoprotein receptor domains in the N-terminus, and a G-protein-coupled receptor proteolysis site domain and a 7 transmembrane receptor (secretin family) domain in the C-terminus. To date, there are no proteins with a similar domain structure in the GenBank. The expression pattern of CasLpR was exclusive in hemocytes among all tested tissues obtained from a juvenile female at intermolt stage: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis; and Y-organ (endocrine organ). There was no CasLpR expression in the ovary of an adult female. A putative function of CasLpR was examined after challenges of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in vivo using qRT-PCR assays. Animals at 24 h after injection of LPS or LTA up-regulated the expression of CasLpR in hemocytes by ∼3.5 and 1.4 folds, respectively, compared to the controls that received saline injection. LPS challenge also caused the greatest increment (∼55 folds) of heat shock protein 90 (Hsp90) expression in these samples. These data indicate that putative CasLpR and CasHsp90 may be involved in the defense system or the stress response of C. sapidus.

  5. Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations

    PubMed Central

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the “closure” of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and—more importantly—call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications. PMID:27800559

  6. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

    PubMed

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-04-28

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

  7. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

    PubMed Central

    2011-01-01

    Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III). Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to

  8. Reg3α Overexpression Protects Pancreatic β Cells from Cytokine-Induced Damage and Improves Islet Transplant Outcome

    PubMed Central

    Ding, Ying; Xu, Yuemei; Shuai, Xuanyu; Shi, Xuhui; Chen, Xiang; Huang, Wenbin; Liu, Yun; Liang, Xiubin; Zhang, Zhihong; Su, Dongming

    2014-01-01

    The process of islet transplantation for treating type 1 diabetes has been limited by the high level of graft failure. This may be overcome by locally delivering trophic factors to enhance engraftment. Regenerating islet-derived protein 3α (Reg3α) is a pancreatic secretory protein which functions as an antimicrobial peptide in control of inflammation and cell proliferation. In this study, to investigate whether Reg3α could improve islet engraftment, a marginal mass of syngeneic islets pretransduced with adenoviruses expressing Reg3α or control EGFP were transplanted under the renal capsule of streptozotocin-induced diabetic mice. Mice receiving islets with elevated Reg3α production exhibited significantly lower blood glucose levels (9.057 ± 0.59 mmol/L versus 13.48 ± 0.35 mmol/L, P < 0.05) and improved glucose-stimulated insulin secretion (1.80 ± 0.17 ng/mL versus 1.16 ± 0.16 ng/mL, P < 0.05) compared with the control group. The decline of apoptotic events (0.57% ± 0.15% versus 1.06% ± 0.07%, P < 0.05) and increased β-cell proliferation (0.70% ± 0.10% versus 0.36% ± 0.14%, P < 0.05) were confirmed in islet grafts overexpressing Reg3α by morphometric analysis. Further experiments showed that Reg3α production dramatically protected cultured islets and pancreatic β cells from cytokine-induced apoptosis and the impairment of glucose-stimulated insulin secretion. Moreover, exposure to cytokines led to the activation of MAPKs in pancreatic β cells, which was reversed by Reg3α overexpression in contrast to control group. These results strongly suggest that Reg3α could enhance islet engraftments through its cytoprotective effect and advance the therapeutic efficacy of islet transplantation. PMID:25826674

  9. The Reg3α (HIP/PAP) Lectin Suppresses Extracellular Oxidative Stress in a Murine Model of Acute Liver Failure

    PubMed Central

    Moniaux, Nicolas; Darnaud, Marion; Garbin, Kévin; Dos Santos, Alexandre; Guettier, Catherine; Samuel, Didier; Amouyal, Gilles; Amouyal, Paul; Bréchot, Christian; Faivre, Jamila

    2015-01-01

    Background and Aims Acute liver failure (ALF) is a rapidly progressive heterogeneous illness with high mortality rate and no widely accessible cure. A promising drug candidate according to previous preclinical studies is the Reg3α (or HIP/PAP) lectin, which alleviates ALF through its free-radical scavenging activity. Here we study the therapeutic targets of Reg3α in order to gain information on the nature of the oxidative stress associated with ALF. Methods Primary hepatocytes stressed with the reactive oxygen species (ROS) inducers TNFα and H2O2 were incubated with a recombinant Reg3α protein. ALF was induced in C57BL/6J mice by an anti-CD95 antibody. Livers and primary hepatocytes were harvested for deoxycholate separation of cellular and extracellular fractions, immunostaining, immunoprecipitation and malondialdehyde assays. Fibrin deposition was studied by immunofluorescence in frozen liver explants from patients with ALF. Results Fibrin deposition occurs during experimental and clinical acute liver injuries. Reg3α bound the resulting transient fibrin network, accumulated in the inflammatory extracellular matrix (ECM), greatly reduced extracellular ROS levels, and improved cell viability. Hepatocyte treatment with ligands of death receptors, e.g. TNFα and Fas, resulted in a twofold increase of malondialdehyde (MDA) level in the deoxycholate-insoluble fractions. Reg3α treatment maintained MDA at a level similar to control cells and thereby increased hepatocyte survival by 35%. No antioxidant effect of Reg3α was noted in the deoxycholate-soluble fractions. Preventing fibrin network formation with heparin suppressed the prosurvival effect of Reg3α. Conclusions Reg3α is an ECM-targeted ROS scavenger that binds the fibrin scaffold resulting from hepatocyte death during ALF. ECM alteration is an important pathogenic factor of ALF and a relevant target for pharmacotherapy. PMID:25938566

  10. Comparative analysis of Cas6b processing and CRISPR RNA stability

    PubMed Central

    Richter, Hagen; Lange, Sita J.; Backofen, Rolf; Randau, Lennart

    2013-01-01

    The prokaryotic antiviral defense systems CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) employs short crRNAs (CRISPR RNAs) to target invading viral nucleic acids. A short spacer sequence of these crRNAs can be derived from a viral genome and recognizes a reoccurring attack of a virus via base complementarity. We analyzed the effect of spacer sequences on the maturation of crRNAs of the subtype I-B Methanococcus maripaludis C5 CRISPR cluster. The responsible endonuclease, termed Cas6b, bound non-hydrolyzable repeat RNA as a dimer and mature crRNA as a monomer. Comparative analysis of Cas6b processing of individual spacer-repeat-spacer RNA substrates and crRNA stability revealed the potential influence of spacer sequence and length on these parameters. Correlation of these observations with the variable abundance of crRNAs visualized by deep-sequencing analyses is discussed. Finally, insertion of spacer and repeat sequences with archaeal poly-T termination signals is suggested to be prevented in archaeal CRISPR/Cas systems. PMID:23392318

  11. Programmable RNA Tracking in Live Cells with CRISPR/Cas9.

    PubMed

    Nelles, David A; Fang, Mark Y; O'Connell, Mitchell R; Xu, Jia L; Markmiller, Sebastian J; Doudna, Jennifer A; Yeo, Gene W

    2016-04-01

    RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags. PMID:26997482

  12. The role of CRISPR-Cas systems in virulence of pathogenic bacteria.

    PubMed

    Louwen, Rogier; Staals, Raymond H J; Endtz, Hubert P; van Baarlen, Peter; van der Oost, John

    2014-03-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.

  13. The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

    PubMed Central

    Staals, Raymond H. J.; Endtz, Hubert P.; van Baarlen, Peter; van der Oost, John

    2014-01-01

    SUMMARY Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular. PMID:24600041

  14. Programmable RNA Tracking in Live Cells with CRISPR/Cas9.

    PubMed

    Nelles, David A; Fang, Mark Y; O'Connell, Mitchell R; Xu, Jia L; Markmiller, Sebastian J; Doudna, Jennifer A; Yeo, Gene W

    2016-04-01

    RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.

  15. NTP Toxicology and Carcinogenesis Studies of Oleic Acid Diethanolamine Condensate (CAS No. 93-83-4) in F344/N Rats and B6C3F1 Mice (Dermal Studies).

    PubMed

    1999-07-01

    Oleic acid diethanolamine condensate is widely used as an emollient, thickener, and foam stabilizer present in cosmetic formulations of bath additives, shampoos, conditioners, lipsticks, and hair dyes. Male and female F344/N rats and B6C3F1 mice received dermal applications of diethanolamine in 95% ethanol for 13 weeks or 2 years. Genetic toxicology studies were performed in Salmonella typhimurium and L5178Y mouse lymphoma cells. 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were admin istered 0, 25, 50, 100, 200, or 400 mg oleic acid diethanolamine condensate/kg body weight in ethanol dermally for 13 weeks. All male and female rats survived until the end of the study. The final mean body weights and body weight gains of 200 and 400 mg/kg males and the mean body weight gain of 400 mg/kg females were significantly less than those of the vehicle controls. The only chemical-related clinical finding was irritation of the skin at the site of application in most males administered 100 mg/kg or greater and in all females administered 50 mg/kg or greater. Segmented neutrophil counts were increased relative to the vehicle controls in the 400 mg/kg male group on days 5 and 19, in the 200 mg/kg female group on day 19 and at week 13, and in the 400 mg/kg female group on days 5 and 19 and at week 13. Alkaline phosphatase concentrations were significantly increased in the 200 mg/kg male group on day 19, the 200 mg/kg female group at week 13, and in the 400 mg/kg groups of males and females at week 13. Kidney weights of 200 and 400 mg/kg females were significantly greater than those of the vehicle controls. Lesions of the skin at the site of application included epidermal hyperplasia, parakeratosis, chronic active dermal inflammation, suppurative epidermal inflammation, and sebaceous gland hypertrophy in dosed rats. The severities of these lesions generally increased with increasing dose. 13-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice were admin istered

  16. Annotation and Classification of CRISPR-Cas Systems.

    PubMed

    Makarova, Kira S; Koonin, Eugene V

    2015-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.

  17. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  18. Influence of maternal ingestion of Aroclor 1254[reg sign] (PCB) or FireMaster BP-6[reg sign] (PBB) on unstimulated and stimulated corticosterone levels in young rats

    SciTech Connect

    Meserve, L.A.; Murray, B.A.; Landis, J.A. )

    1992-05-01

    The organohalides polychlorinated biphenyl (PCB) and polybrominated biphenyl (PBB) remain troublesome environmental pollutants. For example, the percentage of the population in which PCB is detectable in adipose tissue remains high. These compounds are of particular interest to residents of the North Central United States, especially in regions surrounding the Great Lakes where contaminated fish may be a regular component of the diet. Additionally, PBB was mistakenly fed to cattle and chickens in Michigan during the early 1970s, products of which were ingested by humans. Among the physiological effects of ingestion of PCB or PBB is the depression of thyroid status, which has been reported in adult humans, in adult experimental animals, and in the offspring of these animals. In adult rats, circulating levels of thyroid hormones are inversely proportional to dose of PCB or PBB in the diet. On the other hand, reports of effects of these organohalides on adrenocortical function remain equivocal, describing both PCB- and PBB-induced depression, and absence of effect in rats and monkeys. Despite the possible consequences of maternal ingestion of PCB or PBB on future generations, little work has been done previously to determine whether consumption of these materials by pregnant and lactating animals confers hypothyroidism on their offspring, and/or influences other mechanisms of endocrine control in the young. Since early studies showed that hypothyroidism induced by feeding pregnant rats the goitrogen thiouracil altered the functional capabilities in their young of the hypothalamus-pituitary-adrenal (HPA) axis, as revealed by circulating corticosterone levels, the present study was done to determine whether ingestion of either PCB (Aroclor 1254[reg sign]) or PBB (FireMaster BP-6[reg sign]) by pregnant and lactating rats resulted in depressed thyroid status and/or modified HPA axis function in their 15 day old young. 19 refs., 1 fig., 1 tab.

  19. Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes.

    PubMed

    Oliveros, Juan C; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio

    2016-07-01

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.

  20. Alanine scan of α-conotoxin RegIIA reveals a selective α3β4 nicotinic acetylcholine receptor antagonist.

    PubMed

    Kompella, Shiva N; Hung, Andrew; Clark, Richard J; Marí, Frank; Adams, David J

    2015-01-01

    Activation of the α3β4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective α3β4 nAChR antagonists, α-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new α4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by α3β4, α3β2, and α7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the α3β4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the α3β4 than the α3β2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective α3β4 nAChR antagonist (IC50 of 370 nM) that could potentially be used in the treatment of lung cancer and nicotine addiction. Molecular dynamics simulations of RegIIA and [N11A,N12A]RegIIA bound to α3β4 and α3β2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of α3β2 (α3-Tyr(92), Ser(149), Tyr(189), Cys(192), and Tyr(196); β2-Trp(57), Arg(81), and Phe(119)) may form the molecular basis for the selectivity shift.

  1. Alanine scan of α-conotoxin RegIIA reveals a selective α3β4 nicotinic acetylcholine receptor antagonist.

    PubMed

    Kompella, Shiva N; Hung, Andrew; Clark, Richard J; Marí, Frank; Adams, David J

    2015-01-01

    Activation of the α3β4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective α3β4 nAChR antagonists, α-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new α4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by α3β4, α3β2, and α7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the α3β4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the α3β4 than the α3β2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective α3β4 nAChR antagonist (IC50 of 370 nM) that could potentially be used in the treatment of lung cancer and nicotine addiction. Molecular dynamics simulations of RegIIA and [N11A,N12A]RegIIA bound to α3β4 and α3β2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of α3β2 (α3-Tyr(92), Ser(149), Tyr(189), Cys(192), and Tyr(196); β2-Trp(57), Arg(81), and Phe(119)) may form the molecular basis for the selectivity shift. PMID:25411242

  2. Intestinal adaptation and Reg gene expression induced by antidiabetic duodenal-jejunal bypass surgery in Zucker fatty rats.

    PubMed

    Li, Bing; Lu, Yarong; Srikant, Coimbatore B; Gao, Zu-Hua; Liu, Jun-Li

    2013-04-01

    The antidiabetic mechanism of bariatric surgery includes specific changes in the secretion of incretins. To identify additional players originating from the gut, we evaluated the effects of duodenal-jejunal bypass (DJB) in morbidly obese Zucker fatty rats. A fast relief of hyperglycemia and hyperinsulinemia was achieved even before a significant weight loss occurred. Fourteen days after DJB, we characterized the changes in intestinal histochemistry in the bypassed duodenum and shortcut jejunum that was reanastomosed directly to the starting point of the duodenum and compared with the corresponding regions of sham-operated rats. The bypassed duodenum exhibited mucosal atrophy and apoptosis and decreased proliferative renewal. In shortcut jejunum, DJB resulted in 40% significantly enlarged intestinal circumference and increased epithelial proliferation, especially in putative transit-amplifying (TA) cells and the crypt. Because Reg family proteins promote cell growth and survival, we explored their expression in the intestine. With the use of immunohistochemistry, Reg1, -3α, and -3β were normally expressed in intestinal mucosa. After DJB, the level of Reg1 protein was reduced, whereas Reg3α and -3β were not changed in bypassed duodenum. Downstream in shortcut jejunum, the levels of Reg1 and -3β were greatly induced and especially concentrated in the putative TA cells. Our results revealed significant changes in the integrity and proliferation of the intestinal mucosa as a consequence of DJB, and in cell- and isoform-specific expression of Reg proteins within the replicating mucosal epithelium, and provide evidence indicating that the activation of Reg proteins may contribute to intestinal compensation against increased load and/or to improving insulin sensitivity.

  3. High resolution climate change simulation of the 21st century over East Asia by RegCM3

    NASA Astrophysics Data System (ADS)

    Gao, Xuejie

    2010-05-01

    To meet the increasing demands from the climate change impact assessment studies, a high resolution climate change simulation over East Asia region has being performed in the National Climate Center of the China Meteorological Administration. The model employed in the study is the Abdus Salam International Centre for Theoretical Physics (ICTP) Regional Climate Model (RegCM3). A global model of the CCSR/NIES/FRCGC MIROC3.2_hires is selected to drive RegCM3 because of its high resolution (T106) and its good performances in simulating the present day climate over the region. The simulation is conducted at 25-km grid spacing for the period of 1951-2100. Observed CO2 concentration are used for the present day simulation of 1951-2000 and the emission scenario of IPCC SRES A1B is used as the GHG (greenhouse gases) forcing. Simulations of present day climate over China by RegCM3 and MIROC3.2_hires are compared against observation to valid the model performances. Results show that both models reproduced the general pattern of surface air temperature and precipitation well over the region. Compared to the driving MIROC3.2_hires, RegCM3 provides with more spatial details of the surface fields. Differed from previous GCM-RegCM3 simulations, the RegCM3 did not improves the general pattern of the precipitation due to the good performances of MIROC3.2_hires. Preliminary analysis of the future changes simulated by the two models' show difference, in particular during June-July-August. For example while the MIROC3.2_hires projected a prevailing increase of precipitation in JJA over China, the RegCM3 projected extended areas of decreased precipitation. The data are available for those interested from the community of climate change impacts studies.

  4. Advances in therapeutic CRISPR/Cas9 genome editing.

    PubMed

    Savić, Nataša; Schwank, Gerald

    2016-02-01

    Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports.

  5. Chemical and radiation stability of a proprietary cesium ion exchange material manufactured from WWL membrane and SuperLig{reg_sign} 644

    SciTech Connect

    Brown, G.N.; Carson, K.J.; DesChane, J.R.; Elovich, R.J.; Berry, P.K.

    1996-09-01

    Pretreatment of nuclear process wastes for ion exchange removal of Cs and other radionuclides is one way to minimize amount of high-level radioactive waste at Hanford. This study evaluated Cs-selective SuperLig{reg_sign}644 (IBC Advanced Technologies, American Fork UT) entrapped in a proprietary WWL web membrane (3M) for chemical/radiation stability in simulated caustic neutralized current acid waste (NCAW), 0.5M HNO{sub 3}, water, and air. After exposure up to 2.0E+09 rad, the material was evaluated for Cs uptake in 5M sodium NCAW simulants with varying Cs contents. Radiolytic stability appears to be sufficient for ion exchange pretreatment of radioactive Cs: essentially no decrease in Cs selectivity or loading (Kd) was observed during {sup 60}Cs gamma irradiation in water or 0.5M HNO{sub 3} up to 1.0E+09 rad. Cs Kd decreased by a factor of 2 after 2.0E+09 rad exposure. Cs Kd did not change during irradiation in 5M NCAW or ambient air up to 1.0E+08 rad, but decreased by more than an order of magnitude between 1.0E+08 and 2.0E+09 rad (not typical of process conditions). Chemical stability under caustic conditions is lower than in air or under neutral/acidic conditions. Results indicate that this material is less stable in caustic solution irrespective of radiation exposure. Samples of the membrane retained their physical form throughout the entire experiment and were only slightly brittle after exposure to 2.0E+09 rad. (The material evaluated was a finely ground (400 mesh) particulate engineered to form a polymeric fiber (WWL), not the macroscopic form of SuperLig{reg_sign} 644 resin (20 to 50 mesh).)

  6. Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes.

    PubMed

    Copeland, Matthew F; Politz, Mark C; Pfleger, Brian F

    2014-10-01

    The last several years have witnessed an explosion in the understanding and use of novel, versatile trans-acting elements. TALEs, CRISPR/Cas, and sRNAs can be easily fashioned to bind any specific sequence of DNA (TALEs, CRISPR/Cas) or RNA (sRNAs) because of the simple rules governing their interactions with nucleic acids. This unique property enables these tools to repress the expression of genes at the transcriptional or post-transcriptional levels, respectively, without prior manipulation of cis-acting and/or chromosomal target DNA sequences. These tools are now being harnessed by synthetic biologists, particularly those in the eukaryotic community, for genome-wide regulation, editing, or epigenetic studies. Here we discuss the exciting opportunities for using TALEs, CRISPR/Cas, and sRNAs as synthetic trans-acting regulators in prokaryotes.

  7. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing.

    PubMed

    Sun, Wujin; Ji, Wenyan; Hall, Jordan M; Hu, Quanyin; Wang, Chao; Beisel, Chase L; Gu, Zhen

    2015-10-01

    CRISPR-Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn-like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA-binding proteins or functional nucleic acids.

  8. REG4 Is Highly Expressed in Mucinous Ovarian Cancer: A Potential Novel Serum Biomarker.

    PubMed

    Lehtinen, Laura; Vesterkvist, Pia; Roering, Pia; Korpela, Taina; Hattara, Liisa; Kaipio, Katja; Mpindi, John-Patrick; Hynninen, Johanna; Auranen, Annika; Davidson, Ben; Haglund, Caj; Iljin, Kristiina; Grenman, Seija; Siitari, Harri; Carpen, Olli

    2016-01-01

    Preoperative diagnostics of ovarian neoplasms rely on ultrasound imaging and the serum biomarkers CA125 and HE4. However, these markers may be elevated in non-neoplastic conditions and may fail to identify most non-serous epithelial cancer subtypes. The objective of this study was to identify histotype-specific serum biomarkers for mucinous ovarian cancer. The candidate genes with mucinous histotype specific expression profile were identified from publicly available gene-expression databases and further in silico data mining was performed utilizing the MediSapiens database. Candidate biomarker validation was done using qRT-PCR, western blotting and immunohistochemical staining of tumor tissue microarrays. The expression level of the candidate gene in serum was compared to the serum CA125 and HE4 levels in a patient cohort of prospectively collected advanced ovarian cancer. Database searches identified REG4 as a potential biomarker with specificity for the mucinous ovarian cancer subtype. The specific expression within epithelial ovarian tumors was further confirmed by mRNA analysis. Immunohistochemical staining of ovarian tumor tissue arrays showed distinctive cytoplasmic expression pattern only in mucinous carcinomas and suggested differential expression between benign and malignant mucinous neoplasms. Finally, an ELISA based serum biomarker assay demonstrated increased expression only in patients with mucinous ovarian cancer. This study identifies REG4 as a potential serum biomarker for histotype-specific detection of mucinous ovarian cancer and suggests serum REG4 measurement as a non-invasive diagnostic tool for postoperative follow-up of patients with mucinous ovarian cancer. PMID:26981633

  9. Rosebud SynCoal Partnership, SynCoal{reg_sign} demonstration technology update

    SciTech Connect

    Sheldon, R.W.

    1997-12-31

    An Advanced Coal Conversion Process (ACCP) technology being demonstrated in eastern Montana (USA) at the heart of one of the world`s largest coal deposits is providing evidence that the molecular structure of low-rank coals can be altered successfully to produce a unique product for a variety of utility and industrial applications. The product is called SynCoal{reg_sign} and the process has been developed by the Rosebud SynCoal Partnership (RSCP) through the US Department of Energy`s multi-million dollar Clean Coal Technology Program. The ACCP demonstration process uses low-pressure, superheated gases to process coal in vibrating fluidized beds. Two vibratory fluidized processing stages are used to heat and convert the coal. This is followed by a water spray quench and a vibratory fluidized stage to cool the coal. Pneumatic separators remove the solid impurities from the dried coal. There are three major steps to the SynCoal{reg_sign} process: (1) thermal treatment of the coal in an inert atmosphere, (2) inert gas cooling of the hot coal, and (3) removal of ash minerals. When operated continuously, the demonstration plant produces over 1,000 tons per day (up to 300,000 tons per year) of SynCoal{reg_sign} with a 2% moisture content, approximately 11,800b Btu/lb and less than 1.0 pound of SO{sub 2} per million Btu. This product is obtained from Rosebud Mine sub-bituminous coal which starts with 25% moisture, 8,600 Btu/lb and approximately 1.6 pounds of SO{sub 2} per million Btu.

  10. The Phase I CORDEX RegCM hyper-MAtrix (CREMA) experiment (Invited)

    NASA Astrophysics Data System (ADS)

    Giorgi, F.

    2013-12-01

    An ensemble of regional climate model (RCM) projections was produced with the RegCM4 modeling system as a first contribution to the CORDEX framework by the RegCM modeling community, the Phase I CORDEX RegCM hyper-MAtrix (CREMA) experiment. A total of 34 regional projections were completed covering the period 1970-2100 over five different CORDEX domains, Africa, Central America, South America, South Asia and the Mediterranean. The projections use different combinations of three driving GCMs (HadGEM, MPI and GFDL), two greenhouse gas concentration pathways (RCP4.5 and RCP8.5) and different land surface and convection schemes, which allows a first exploration of different sources of uncertainty. The paper will describe the CREMA phase I experiment and discuss some basic results from a first analysis of these runs, with emphasis on extreme events (including tropical storms), variability and regional circulations of relevance for the different domains (e.g. the monsoon). The CREMA Phase I experiment was completed as a collaboration between the Abdus Salam ICTP and the U. San Paolo, CICESE, and the Indian Institute of Technology, and the results from this first analysis are being published in a special issue of Climatic Change. The data from these projections is freely available following the CORDEX data policy for eventual further analysis and use in impact assessment studies. We plan to incrementally populate the CREMA ensemble with further simulations employing more driving GCMs and model configurations and to compare our resultswith other models participating to the CORDEX effort.

  11. Improvement of surface albedo parameterization within a regional climate model (RegCM3)

    NASA Astrophysics Data System (ADS)

    Bao, Y.; Lü, S.

    2009-03-01

    A parameterization for calculating surface albedo of Solar Zenith Angel (SZA) dependence with coefficient for each vegetation type determined on the Moderate Resolution Imaging Spectro-radiometer (MODIS) reformed by the Bidirectional Reflectance Distribution Function (BRDF) is incorporated within the latest Abdus Salam International Centre for Theoretical Physics (ICTP) Regional Climate Model (RegCM3), and evaluated with a high resolution one-way nesting simulation in China using the Climate Research Unit (CRU) data and the observations from the Field Experiment on Interaction between Land and Atmosphere in Arid Region of Northwest China (NWC-ALIEX). The performance of the SZA method modeling surface characteristic is investigated.Results indicate, RegCM with SZA method (RCM_SZA) considerably improve the cold bias of original RegCM (RCM_ORI) in air surface temperature in East Asia with 1.2 degree increased in summer due to the lower albedo produced by SZA method which makes more solar radiation absorbed by the surface and used for heating the atmosphere near to the surface. The simulated diurnal cycle of ground temperature conforms fairly well to the observation in the nesting simulation in Northwest China, especially during the noon time when the SZA has the lowest value. However, the modification can not obviously affect the East Asia summer monsoon precipitation simulation although RCM_SZA produce more evapo-transpiration in surface with more than 2 Wm-2 increases in simulated latent heat fluxes both in East Asia and in Northwest China compared to RCM_ORI.

  12. REG4 Is Highly Expressed in Mucinous Ovarian Cancer: A Potential Novel Serum Biomarker.

    PubMed

    Lehtinen, Laura; Vesterkvist, Pia; Roering, Pia; Korpela, Taina; Hattara, Liisa; Kaipio, Katja; Mpindi, John-Patrick; Hynninen, Johanna; Auranen, Annika; Davidson, Ben; Haglund, Caj; Iljin, Kristiina; Grenman, Seija; Siitari, Harri; Carpen, Olli

    2016-01-01

    Preoperative diagnostics of ovarian neoplasms rely on ultrasound imaging and the serum biomarkers CA125 and HE4. However, these markers may be elevated in non-neoplastic conditions and may fail to identify most non-serous epithelial cancer subtypes. The objective of this study was to identify histotype-specific serum biomarkers for mucinous ovarian cancer. The candidate genes with mucinous histotype specific expression profile were identified from publicly available gene-expression databases and further in silico data mining was performed utilizing the MediSapiens database. Candidate biomarker validation was done using qRT-PCR, western blotting and immunohistochemical staining of tumor tissue microarrays. The expression level of the candidate gene in serum was compared to the serum CA125 and HE4 levels in a patient cohort of prospectively collected advanced ovarian cancer. Database searches identified REG4 as a potential biomarker with specificity for the mucinous ovarian cancer subtype. The specific expression within epithelial ovarian tumors was further confirmed by mRNA analysis. Immunohistochemical staining of ovarian tumor tissue arrays showed distinctive cytoplasmic expression pattern only in mucinous carcinomas and suggested differential expression between benign and malignant mucinous neoplasms. Finally, an ELISA based serum biomarker assay demonstrated increased expression only in patients with mucinous ovarian cancer. This study identifies REG4 as a potential serum biomarker for histotype-specific detection of mucinous ovarian cancer and suggests serum REG4 measurement as a non-invasive diagnostic tool for postoperative follow-up of patients with mucinous ovarian cancer.

  13. REG4 Is Highly Expressed in Mucinous Ovarian Cancer: A Potential Novel Serum Biomarker

    PubMed Central

    Lehtinen, Laura; Vesterkvist, Pia; Roering, Pia; Korpela, Taina; Hattara, Liisa; Kaipio, Katja; Mpindi, John-Patrick; Hynninen, Johanna; Auranen, Annika; Davidson, Ben; Haglund, Caj; Iljin, Kristiina; Grenman, Seija; Siitari, Harri; Carpen, Olli

    2016-01-01

    Preoperative diagnostics of ovarian neoplasms rely on ultrasound imaging and the serum biomarkers CA125 and HE4. However, these markers may be elevated in non-neoplastic conditions and may fail to identify most non-serous epithelial cancer subtypes. The objective of this study was to identify histotype-specific serum biomarkers for mucinous ovarian cancer. The candidate genes with mucinous histotype specific expression profile were identified from publicly available gene-expression databases and further in silico data mining was performed utilizing the MediSapiens database. Candidate biomarker validation was done using qRT-PCR, western blotting and immunohistochemical staining of tumor tissue microarrays. The expression level of the candidate gene in serum was compared to the serum CA125 and HE4 levels in a patient cohort of prospectively collected advanced ovarian cancer. Database searches identified REG4 as a potential biomarker with specificity for the mucinous ovarian cancer subtype. The specific expression within epithelial ovarian tumors was further confirmed by mRNA analysis. Immunohistochemical staining of ovarian tumor tissue arrays showed distinctive cytoplasmic expression pattern only in mucinous carcinomas and suggested differential expression between benign and malignant mucinous neoplasms. Finally, an ELISA based serum biomarker assay demonstrated increased expression only in patients with mucinous ovarian cancer. This study identifies REG4 as a potential serum biomarker for histotype-specific detection of mucinous ovarian cancer and suggests serum REG4 measurement as a non-invasive diagnostic tool for postoperative follow-up of patients with mucinous ovarian cancer. PMID:26981633

  14. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process.

    PubMed

    Li, Ming; Wang, Rui; Zhao, Dahe; Xiang, Hua

    2014-02-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years.

  15. Evaluation of Boulder, CO,SmartRegs Ordinance and Better Buildings Program

    SciTech Connect

    Arena, L.; Vijayakumar, G.

    2012-04-01

    Under the SmartRegs ordinance in the city of Boulder, Colorado, all rental properties in the city must achieve an energy efficiency level comparable to a HERS Index of approximately 120 points or lower by the year 2019. The City of Boulder received a $12 million grant from the DOE’s Better Buildings initiative to create and incentivize their EnergySmart Program. In this report, the Consortium for Advanced Residential Buildings (CARB) describes its work with the program, including energy audits of rental properties, developing training programs for insulators and inspectors, and conducting interviews with property owners.

  16. Evaluation of Boulder, CO, SmartRegs Ordinance and Better Buildings Program

    SciTech Connect

    Arena, L.; Vijayakumar, G.

    2012-04-01

    Under the SmartRegs ordinance in the city of Boulder, Colorado, all rental properties in the city must achieve an energy efficiency level comparable to a HERS Index of approximately 120 points or lower by the year 2019. The City of Boulder received a $12 million grant from the DOE's Better Buildings initiative to create and incentivize their EnergySmart Program. In this report, the Consortium for Advanced Residential Buildings (CARB) describes its work with the program, including energy audits of rental properties, developing training programs for insulators and inspectors, and conducting interviews with property owners.

  17. Effects of Aroclor 1254 reg sign on hydrocortisone levels in adult Rhesus monkeys (Macaca mulatta)

    SciTech Connect

    Loo, J.C.K.; Tryphonas, H.; Jordan, N.; Brien, R.; Karpinski, K.R.; Arnold, D.L. )

    1989-11-01

    Researchers, using female Sprague Dawley rats, reported the effects of chronic (5-7 months) oral dosing with Aroclor 1254{reg sign} (Polychlorinated biphenyls-PCB) on the serum levels of corticosterone, the principle glucocorticoid in rats. Their findings indicated that corticosterone levels were significantly depressed at dose levels of 479 {mu}g/kg bw/day and above. The objective of the present study was to determine the effects of PCB on the hydrocortisone levels in Rhesus monkey (Macaca mulatta) serum. In the monkey the controlling hormone is hydrocortisone which is identical to that of humans.

  18. CRISPR-Cas9-guided Genome Engineering in C. elegans

    PubMed Central

    Kim, Hyun-Min; Colaiácovo, Monica P.

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms including the nematode C. elegans. Recent studies developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning and injection methods required for delivering Cas9, sgRNAs and repair template DNA into the C. elegans germline. PMID:27366893

  19. Foreign DNA capture during CRISPR–Cas adaptive immunity

    PubMed Central

    Nuñez, James K.; Harrington, Lucas B.; Kranzusch, Philip J.; Engelman, Alan N.; Doudna, Jennifer A.

    2015-01-01

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33 nucleotide (nt) protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′–OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2–4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci. PMID:26503043

  20. Foreign DNA capture during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Kranzusch, Philip J; Engelman, Alan N; Doudna, Jennifer A

    2015-11-26

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

  1. Sensitivity of the GCM driven summer monsoon simulations to cumulus parameterization schemes in nested RegCM3

    NASA Astrophysics Data System (ADS)

    Sinha, P.; Mohanty, U. C.; Kar, S. C.; Dash, S. K.; Kumari, S.

    2013-04-01

    The regional climate model (RegCM3) from the Abdus Salam International Centre for Theoretical Physics has been used to simulate the Indian summer monsoon for three different monsoon seasons such as deficit (1987), excess (1988) and normal (1989). Sensitivity to various cumulus parameterization and closure schemes of RegCM3 driven by the National Centre for Medium Range Weather Forecasting global spectral model products has been tested. The model integration of the nested RegCM3 is conducted using 90 and 30-km horizontal resolutions for outer and inner domains, respectively. The India Meteorological Department gridded rainfall (1° × 1°) and National Centre for Environment Prediction (NCEP)-Department of Energy (DOE) reanalysis-2 of 2.5° × 2.5° horizontal resolution data has been used for verification. The RegCM3 forced by NCEP-DOE reanalysis-2 data simulates monsoon seasons of 1987 and 1988 reasonably well, but the monsoon season of 1989 is not represented well in the model simulations. The RegCM3 runs driven by the global model are able to bring out seasonal mean rainfall and circulations well with the use of the Grell and Anthes-Kuo cumulus scheme at 90-km resolution. While the rainfall intensity and distribution is brought out well with the Anthes-Kuo scheme, upper air circulation features are brought out better by the Grell scheme. The simulated rainfall distribution is better with RegCM3 using the MIT-Emanuel cumulus scheme for 30-km resolution. Several statistical analyses, such as correlation coefficient, root mean square error, equitable threat score, confirm that the performance of MIT-Emanuel scheme at 30-km resolution is better in simulating all-India summer monsoon rainfall. The RegCM3 simulated rainfall amount is more and closer to observations than that from the global model. The RegCM3 has corrected its driven GCM in terms of rainfall distribution and magnitude over some parts of India during extreme years. This study brings out several

  2. RegTransBase - A Database Of Regulatory Sequences and Interactionsin a Wide Range of Prokaryotic Genomes

    SciTech Connect

    Kazakov, Alexei E.; Cipriano, Michael J.; Novichkov, Pavel S.; Minovitsky, Simon; Vinogradov, Dmitry V.; Arkin, Adam; Mironov, AndreyA.; Gelfand, Mikhail S.; Dubchak, Inna

    2006-07-01

    RegTransBase, a manually curated database of regulatoryinteractions in prokaryotes, captures the knowledge in publishedscientific literature using a controlled vocabulary. Although a number ofdatabases describing interactions between regulatory proteins and theirbinding sites are currently being maintained, they focus mostly on themodel organisms Escherichia coli and Bacillus subtilis, or are entirelycomputationally derived. RegTransBase describes a large number ofregulatory interactions reported in many organisms and contains varioustypes of experimental data, in particular: the activation or repressionof transcription by an identified direct regulator; determining thetranscriptional regulatory function of a protein (or RNA) directlybinding to DNA (RNA); mapping or prediction of binding site for aregulatory protein; characterization of regulatory mutations. Currently,the RegTransBase content is derived from about 3000 relevant articlesdescribing over 7000 experiments in relation to 128 microbes. It containsdata on the regulation of about 7500 genes and evidence for 6500interactions with 650 regulators. RegTransBase also contains manuallycreated position weight matrices (PWM) that can be used to identifycandidate regulatory sites in over 60 species. RegTransBase is availableat http://regtransbase.lbl.gov.

  3. Bicarbonate-mediated transcriptional activation of divergent operons by the virulence regulatory protein, RegA, from Citrobacter rodentium.

    PubMed

    Yang, Ji; Hart, Emily; Tauschek, Marija; Price, G Dean; Hartland, Elizabeth L; Strugnell, Richard A; Robins-Browne, Roy M

    2008-04-01

    Regulation of virulence gene expression plays a central role in the pathogenesis of enteric bacteria as they encounter diverse environmental conditions in the gastrointestinal tract of their hosts. In this study, we investigated environmental regulation of two putative virulence determinants adcA and kfc by RegA, an AraC/XylS-like regulator, from Citrobacter rodentium, and identified bicarbonate as the environmental signal which induced transcription of adcA and kfc through RegA. Primer extension experiments showed that adcA and kfc were divergently transcribed from sigma(70) promoters. In vivo and in vitro experiments demonstrated that bicarbonate facilitated and stabilized the binding of RegA to an operator located between the two promoters. The interaction of RegA with its DNA target resulted in the formation of a nucleosome-like structure, which evidently displaced the histone-like proteins, H-NS and StpA, from the adcA and kfc promoter regions, leading to transcriptional derepression. In addition, our results indicated that RegA also behaved as a Class I activator by directly stimulating transcription initiation by RNA polymerase. This is the first report to describe the molecular mechanism by which an environmental chemical stimulates transcription of virulence-associated genes of an enteric pathogen through an AraC/XlyS-like activator.

  4. Effects of modified soil water-heat physics on RegCM4 simulations of climate over the Tibetan Plateau

    NASA Astrophysics Data System (ADS)

    Wang, Xuejia; Pang, Guojin; Yang, Meixue; Wan, Guoning

    2016-06-01

    To optimize the description of land surface processes and improve climate simulations over the Tibetan Plateau (TP), a modified soil water-heat parameterization scheme (SWHPS) is implemented into the Community Land Model 3.5 (CLM3.5), which is coupled to the regional climate model 4 (RegCM4). This scheme includes Johansen's soil thermal conductivity scheme together with Niu's groundwater module. Two groups of climate simulations are then performed using the original RegCM4 and revised RegCM4 to analyze the effects of the revised SWHPS on regional climate simulations. The effect of the revised RegCM4 on simulated air temperature is relatively small (with mean biases changing by less than 0.1°C over the TP). There are overall improvements in the simulation of winter and summer air temperature but increased errors in the eastern TP. It has a significant effect on simulated precipitation. There is also a clear improvement in simulated annual and winter precipitation, particularly over the northern TP, including the Qilian Mountains and the source region of the Yellow River. There are, however, increased errors in precipitation simulation in parts of the southern TP. The precipitation difference between the two models is caused mainly by their convective precipitation difference, particularly in summer. Overall, the implementation of the new SWHPS into the RegCM4 has a significant effect not only on land surface variables but also on the overlying atmosphere through various physical interactions.

  5. RegScan: a GWAS tool for quick estimation of allele effects on continuous traits and their combinations

    PubMed Central

    Kals, Mart; Esko, Tõnu; Mägi, Reedik; Fischer, Krista

    2015-01-01

    Genome-wide association studies are becoming computationally more demanding with the growing amounts of data. Combinatorial traits can increase the data dimensions beyond the computational capabilities of the current tools. We addressed this issue by creating an application for quick association analysis that is ten to hundreds of times faster than the leading fast methods. Our tool (RegScan) is designed for performing basic linear regression analysis with continuous traits maximally fast on large data sets. RegScan specifically targets association analysis of combinatorial traits in metabolomics. It can both generate and analyze the combinatorial traits efficiently. RegScan is capable of analyzing any number of traits together without the need to specify each trait individually. The main goal of the article is to show that RegScan can be the preferred analytical tool when large amounts of data need to be analyzed quickly using the allele frequency test. Availability: Precompiled RegScan (all major platforms), source code, user guide and examples are freely available at www.biobank.ee/regscan. Requirements: Qt 4.4.3 or newer for dynamic compilations. PMID:24008273

  6. Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance.

    PubMed

    Esterházy, Daria; Loschko, Jakob; London, Mariya; Jove, Veronica; Oliveira, Thiago Y; Mucida, Daniel

    2016-05-01

    Oral tolerance prevents pathological inflammatory responses to innocuous foreign antigens by peripheral regulatory T cells (pT(reg) cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure for the 'instruction' of naive CD4(+) T cells to differentiate into pT(reg) cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs were dispensable, while classical dendritic cells (cDCs) were critical, for pT(reg) cell induction and oral tolerance. CD11b(-) cDCs from the gut-draining lymph nodes efficiently induced pT(reg) cells and, conversely, loss of transcription factor IRF8-dependent CD11b(-) cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in the induction of pT(reg) cells and their redundancy during the development of oral tolerance.

  7. Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.

    PubMed

    Inui, Masafumi; Miyado, Mami; Igarashi, Maki; Tamano, Moe; Kubo, Atsushi; Yamashita, Satoshi; Asahara, Hiroshi; Fukami, Maki; Takada, Shuji

    2014-06-23

    Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.

  8. Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system.

    PubMed

    Arslan, Zihni; Hermanns, Veronica; Wurm, Reinhild; Wagner, Rolf; Pul, Ümit

    2014-07-01

    The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.

  9. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  10. 21 CFR 573.210 - Benzoic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Benzoic acid. 573.210 Section 573.210 Food and... Listing § 573.210 Benzoic acid. The food additive, benzoic acid, may be safely used in the manufacture of... acid (CAS 65-85-0) by weight with the sum of 2-methylbiphenyl, 3-methylbiphenyl,...

  11. 21 CFR 184.1307a - Ferrous ascorbate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... GRAS § 184.1307a Ferrous ascorbate. (a) Ferrous ascorbate (CAS Reg. No. 24808-52-4) is a reaction product of ferrous hydroxide and ascorbic acid. It is a blue-violet product containing 16 percent iron....

  12. 21 CFR 184.1446 - Manganese chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... GRAS § 184.1446 Manganese chloride. (a) Manganese chloride (MnCl2, CAS Reg. No. 7773-01-5) is a pink... manganous oxide, pyrolusite ore (MnO2), or reduced manganese ore in hydrochloric acid. The...

  13. Recent Advances in Genome Editing Using CRISPR/Cas9

    PubMed Central

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

  14. Advanced Recyclable Media System{reg_sign}. Innovative technology summary report

    SciTech Connect

    1998-12-01

    The objective of the Large-Scale Demonstration Project (LSDP) is to select and demonstrate potentially beneficial technologies at the Argonne National Laboratory East`s (ANL) Chicago Pile-5 (CP-5) Research Reactor. The purpose of the LSDP is to demonstrate that using innovative and improved deactivation and decommissioning (D and D) technologies from various sources can result in significant benefits, such as decreased cost and increased health and safety, as compared with baseline D and D technologies. This report describes a demonstration of the Advanced Recyclable Media System{reg_sign} technology which was employed by Surface Technology Systems, Inc. to remove coatings from a concrete floor. This demonstration is part of the CP-5 LSDP sponsored by the US Department of Energy (DOE) Office of Science and Technology Deactivation and Decommissioning Focus Area (DDFA). The Advanced Recyclable Media System{reg_sign} (ARMS) technology is an open blast technology which uses a soft recyclable media. The patented ARMS Engineered Blast Media consists of a fiber-reinforced polymer matrix which can be manufactured in various grades of abrasiveness. The fiber media can be remade and/or reused up to 20 times and can clean almost any surface (e.g., metal, wood, concrete, lead) and geometry including corners and the inside of air ducts.

  15. High-temperature corrosion of Nicalon reg sign /SiC composites

    SciTech Connect

    Lowden, R.A.; James, R.D.

    1991-08-01

    The elevated-temperature stability of Nicalon{reg sign} (Nippon Carbon Company, Tokyo, Japan)/SiC composites with a graphitic carbon interface layer in oxidizing and simulated fossil fuel environments was investigated. Composite specimens with and without an external SiC surface coating were oxidizing in air and exposed to a variety of combustion environments at a temperature of 1273 K. A burner rig furnace was constructed for simulating corrosive fossil fuel environments containing water vapor, sulfur, and sodium. The mechanical properties of unprotected Nicalon{reg sign}/SiC specimens were degraded after short periods of exposure, due to the oxidation of the carbon interface coating. Longer exposures resulted in the oxidation of the fibers and matrix to form silica, which with time bonded the components together and produced brittle behavior. Combustion environments hastened the embrittlement of composites without an external SiC coating. Conversely, the specimens protected by a chemical vapor deposition (CVD) SiC surface coating exhibited only small decreases in strength after oxidation or corrosion in combustion environments. The SiC layer sealed off the surface of the composites, protecting the exposed fibers' ends, and thus prevented oxidation at the fiber-matrix interface. 26 refs., 11 figs., 1 tab.

  16. Leiomyoblastome gastrique: à propos de trois cas

    PubMed Central

    Moujahid, Mountassir; Ennafaa, Issam; EL Rhari, Ahmed; Serghini, Issam; Chekoura, Khalid; Tahiri, Moulay Hassan

    2015-01-01

    Le leiomyoblastome gastrique est une tumeur musculaire rare qui touche essentiellement l'adulte. Son développement est souvent exogastrique. Le diagnostic positif repose sur l'histologie et le traitement est basé sur la chirurgie. Nous rapportons trois cas de leiomyoblastome gastrique colligés dans le service de chirurgie générale au 5ème Hôpital Militaire. L’âge moyen des patients est de 47 ans; le motif de consultation était représenté par une hémorragie digestive et l'imagerie médicale a posé le diagnostic de masse tumorale dans tous les cas. Le traitement chirurgical consistait en une gastrectomie partielle et le compte rendu anatomopathologique a confirmé le leiomyoblastome gastrique dans les trois cas. Le siège de la tumeur a été posé par la fibroscopie oeso gastroduodénale, le traitement était chirurgical et les suites post opératoires étaient simples avec un contrôle par des fibroscopies répétitives sans aucun signe de récidive. Le leiomyoblastome gastrique est une tumeur rare. L’écho endoscopie joue un rôle primordial dans le diagnostic positif ainsi que dans l’évaluation de l'extension pariétale de ces tumeurs. Le traitement est essentiellement chirurgical. PMID:26090000

  17. Reg-2, A Downstream Signaling Protein in the Ciliary Neurotrophic Factor Survival Pathway, Alleviates Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Jiang, Hong; Tian, Ke-Wei; Zhang, Fan; Wang, Beibei; Han, Shu

    2016-01-01

    Ciliary neurotrophic factor (CNTF), originally described as a neurocytokine that could support the survival of neurons, has been recently found to alleviate demyelination, prevent axon loss, and improve functional recovery in a rat model of acute experimental autoimmune encephalomyelitis (EAE). However, poor penetration into the brain parenchyma and unfavorable side effects limit the utility of CNTF. Here, we evaluated the therapeutic potential of a protein downstream of CNTF, regeneration gene protein 2 (Reg-2). Using multiple morphological, molecular biology, and electrophysiological methods to assess neuroinflammation, axonal loss, demyelination, and functional impairment, we observed that Reg-2 and CNTF exert similar effects in the acute phase of EAE. Both treatments attenuated axonal loss and demyelination, improved neuronal survival, and produced functional improvement. With a smaller molecular weight and improved penetration into the brain parenchyma, Reg-2 may be a useful substitute for CNTF therapy in EAE and multiple sclerosis (MS). PMID:27242448

  18. Using CAS to Solve a Mathematics Task: A Deconstruction

    ERIC Educational Resources Information Center

    Berger, Margot

    2010-01-01

    I investigate how and whether a heterogeneous group of first-year university mathematics students in South Africa harness the potential power of a computer algebra system (CAS) when doing a specific mathematics task. In order to do this, I develop a framework for deconstructing a mathematics task requiring the use of CAS, into its primary…

  19. Interacting Parallel Constructions of Knowledge in a CAS Context

    ERIC Educational Resources Information Center

    Kidron, Ivy; Dreyfus, Tommy

    2010-01-01

    We consider the influence of a CAS context on a learner's process of constructing a justification for the bifurcations in a logistic dynamical process. We describe how instrumentation led to cognitive constructions and how the roles of the learner and the CAS intertwine, especially close to the branching and combining of constructing actions. The…

  20. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., OFFICE OF FEDERAL PROCUREMENT POLICY, OFFICE OF MANAGEMENT AND BUDGET PROCUREMENT PRACTICES AND COST... coverage. Full coverage requires that the business unit comply with all of the CAS specified in part 9904... later award of a CAS-covered contract. Full coverage applies to contractor business units that—...

  1. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  2. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  3. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  4. Transformation of OODT CAS to Perform Larger Tasks

    NASA Technical Reports Server (NTRS)

    Mattmann, Chris; Freeborn, Dana; Crichton, Daniel; Hughes, John; Ramirez, Paul; Hardman, Sean; Woollard, David; Kelly, Sean

    2008-01-01

    A computer program denoted OODT CAS has been transformed to enable performance of larger tasks that involve greatly increased data volumes and increasingly intensive processing of data on heterogeneous, geographically dispersed computers. Prior to the transformation, OODT CAS (also alternatively denoted, simply, 'CAS') [wherein 'OODT' signifies 'Object-Oriented Data Technology' and 'CAS' signifies 'Catalog and Archive Service'] was a proven software component used to manage scientific data from spaceflight missions. In the transformation, CAS was split into two separate components representing its canonical capabilities: file management and workflow management. In addition, CAS was augmented by addition of a resource-management component. This third component enables CAS to manage heterogeneous computing by use of diverse resources, including high-performance clusters of computers, commodity computing hardware, and grid computing infrastructures. CAS is now more easily maintainable, evolvable, and reusable. These components can be used separately or, taking advantage of synergies, can be used together. Other elements of the transformation included addition of a separate Web presentation layer that supports distribution of data products via Really Simple Syndication (RSS) feeds, and provision for full Resource Description Framework (RDF) exports of metadata.

  5. From Calculus to Dynamical Systems through DGS and CAS

    ERIC Educational Resources Information Center

    García, Jeanett López; Zamudio, Jorge Javier Jiménez

    2015-01-01

    Several factors have motivated the use of CAS or DGS in the teaching-learning process, such as: the development of new technologies, the availability of computers, and the widespread use of the Internet, among others. Even more, the trend to include CAS and DGS in the curricula of some undergraduate studies has resulted in the instruction of the…

  6. Cas Proteins in Normal and Pathological Cell Growth Control

    PubMed Central

    Tikhmyanova, Nadezhda; Little, Joy L.; Golemis, Erica A.

    2009-01-01

    Proteins of the CAS (Crk-Associated Substrate) family (BCAR1/p130Cas, NEDD9/HEF1/Cas-L, EFS/SIN and CASS4/HEPL) are integral players in normal and pathological cell biology. CAS proteins act as scaffolds to regulate protein complexes controlling migration and chemotaxis, apoptosis, cell cycle, and differentiation, and have more recently been linked to a role in progenitor cell function. Reflecting these complex functions, over-expression of CAS proteins has now been strongly linked to poor prognosis and increased metastasis in cancer, as well as resistance to first-line chemotherapeutics in multiple tumor types including breast and lung cancers, glioblastoma, and melanoma. Further, CAS proteins have also been linked to additional pathological conditions including inflammatory disorders, Alzheimer’s and Parkinson’s disease, as well as developmental defects. This review will explore the roles of the CAS proteins in normal and pathological states in the context of the many mechanistic insights into CAS protein function that have emerged in the past decade. PMID:19937461

  7. Optimization of genome editing through CRISPR-Cas9 engineering.

    PubMed

    Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F

    2016-04-01

    CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

  8. Recent Progress in CRISPR/Cas9 Technology.

    PubMed

    Mei, Yue; Wang, Yan; Chen, Huiqian; Sun, Zhong Sheng; Ju, Xing-Da

    2016-02-20

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, a simple and efficient tool for genome editing, has experienced rapid progress in its technology and applicability in the past two years. Here, we review the recent advances in CRISPR/Cas9 technology and the ways that have been adopted to expand our capacity for precise genome manipulation. First, we introduce the mechanism of CRISPR/Cas9, including its biochemical and structural implications. Second, we highlight the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Third, we review its current applications, in which the versatile CRISPR/Cas9 system was employed to edit the genome, epigenome, or RNA of various organisms. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, we should not ignore the significant ethical and biosafety concerns that it raises. Finally, we discuss the prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9.

  9. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    PubMed

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D

    2016-06-01

    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory.

  10. Teaching Undergraduate Mathematics Using CAS Technology: Issues and Prospects

    ERIC Educational Resources Information Center

    Tobin, Patrick C.; Weiss, Vida

    2016-01-01

    The use of handheld CAS technology in undergraduate mathematics courses in Australia is paradoxically shrinking under sustained disapproval or disdain from the professional mathematics community. Mathematics education specialists argue with their mathematics colleagues over a range of issues in course development and this use of CAS or even…

  11. Adaptive optics at the IOE, CAS

    NASA Astrophysics Data System (ADS)

    Jiang, Wenhan; Rao, Changhui; Zhang, Yudong; Ling, Ning; Guan, Chunlin

    2009-02-01

    R&D on Adaptive Optics in the Institute of Optics & Electronics (IOE), Chinese Academy of Sciences (CAS) began in 1979. In this paper, several recent achievements will be reported: 1. AO for astronomical telescopes. AO system for 1.2 m telescope at Yunnan Astronomical Observatory was built in 1998. It was updated in 2004, and high resolution images approaching diffraction limit were obtained. A new AO telescope with 1.8m aperture is being built, and a 4m AO telescope is being planned. 2. AO for ICF facility. A 19-element AO system with hill-climbing control algorithm for "Shenguang I" ICF facility was built in 1985, which was the first AO system used in ICF facility in the world. A set of 8 AO systems was installed in a bundle of ICF prototype for "Shenguang III" ICF facility. A new system is being built for further development of ICF facility. 3. AO for retinal imaging. In 1999, the first AO system with 19-element DM was built for retinal imaging. Several AO systems with 37-element deformable mirror (DM) were built and used for vision research and clinical inspection. It is being integrated with an OCT system for high resolution retinal imaging. All of the main subsystems, such as DMs, wavefront sensors, and high speed processors, were built in the Laboratory on Adaptive Optics of IOE, CAS.

  12. Implementing the CAS Standards: The Implementation of the CAS Standards in Student Affairs as a Comprehensive Assessment Approach

    ERIC Educational Resources Information Center

    Dorman, Jesse A.

    2012-01-01

    The increasing use of the CAS standards as a comprehensive assessment approach in divisions of student affairs necessitates a more in-depth understanding of how the CAS standards are being implemented in these settings. In response to increasing calls for improvement, accountability and professionalism in student affairs (Bresciani, 2006; Cooper…

  13. Overexpression of Reg3alpha increases cell growth and the levels of cyclin D1 and CDK4 in insulinoma cells.

    PubMed

    Cui, Wei; De Jesus, Kristine; Zhao, Hong; Takasawa, Shin; Shi, Bingyin; Srikant, Coimbatore B; Liu, Jun-Li

    2009-06-01

    Regenerating gene (Reg) family protein Reg3alpha is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet beta-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3alpha cDNA. Determined using real-time PCR and Western blots, the levels of Reg3alpha mRNA and protein in Reg3alpha-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3alpha-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3alpha-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that beta-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced beta-cell mass and diabetes. Our results suggest that Reg3alpha stimulates beta-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

  14. Simulating land use changes in the Upper Narew catchment using the RegCM model

    NASA Astrophysics Data System (ADS)

    Liszewska, Malgorzata; Osuch, Marzena; Romanowicz, Renata

    2010-05-01

    Catchment hydrology is influenced by climate forcing in the form of precipitation, temperature, evapotranspiration and human interactions such as land use and water management practices. The difficulty in separating different causes of change in a hydrological regime results from the complexity of interactions between those three factors and catchment responses and the uncertainty and scarcity of available observations. This paper describes an application of a regional climate model to simulate the variability in precipitation, temperature, evaporation and discharge under different land use parameterizations, using the Upper Narew catchment (north-east Poland) as a case study. We use RegCM3 model, developed at the International Centre for Theoretical Physics, Trieste, Italy. The model's dynamic core is based on the hydrostatic version of the NCAR/PSU Mesoscale Model version 5 (primitive equations, hydrostatic, compressible, sigma-vertical coordinate). The physical input includes radiation transfer, large-scale and convective precipitation, Planetary Boundary Layer, biosphere. The RegCM3 model has options to interface with a variety of re-analyses and GCM boundary conditions, and can thus be used for scenario assessments. The variability of hydrological conditions in response to regional climate model projections is modeled using an integrated Data Based Mechanistic (DBM) rainfall-flow/flow-routing model of the Upper River Narew catchment. The modelling tool developed is formulated in the MATLAB-SIMULINK language. The basic system structure includes rainfall-flow and flow routing modules, based on a Stochastic Transfer Function (STF) approach combined with a nonlinear transformation of rainfall into effective rainfall. We analyse the signal resulting from modified land use in a given region. 10 month-long runs have been performed from February to November for the period of 1991-2000 based on the NCEP re-analyses. The land use data have been taken from the GLCC

  15. Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

    PubMed

    Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

    2015-10-15

    Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.

  16. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    PubMed

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains.

  17. Function and regulation of clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated (Cas) systems.

    PubMed

    Richter, Corinna; Chang, James T; Fineran, Peter C

    2012-10-19

    Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous 'innate' mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific 'adaptive' immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.

  18. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    PubMed

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. PMID:27417121

  19. Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering

    PubMed Central

    Box, Allison M.; McGuffie, Matthew J.; O'Hara, Brendan J.

    2015-01-01

    ABSTRACT The classical and El Tor biotypes of Vibrio cholerae serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of V. cholerae, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in V. cholerae has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide in silico evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to V. cholerae El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3′ TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of V. cholerae lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting. IMPORTANCE Cholera, caused by the facultative pathogen Vibrio cholerae, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor

  20. EXpectation Propagation LOgistic REgRession (EXPLORER): Distributed Privacy-Preserving Online Model Learning

    PubMed Central

    Wang, Shuang; Jiang, Xiaoqian; Wu, Yuan; Cui, Lijuan; Cheng, Samuel; Ohno-Machado, Lucila

    2013-01-01

    We developed an EXpectation Propagation LOgistic REgRession (EXPLORER) model for distributed privacy-preserving online learning. The proposed framework provides a high level guarantee for protecting sensitive information, since the information exchanged between the server and the client is the encrypted posterior distribution of coefficients. Through experimental results, EXPLORER shows the same performance (e.g., discrimination, calibration, feature selection etc.) as the traditional frequentist Logistic Regression model, but provides more flexibility in model updating. That is, EXPLORER can be updated one point at a time rather than having to retrain the entire data set when new observations are recorded. The proposed EXPLORER supports asynchronized communication, which relieves the participants from coordinating with one another, and prevents service breakdown from the absence of participants or interrupted communications. PMID:23562651

  1. Assessment of TEES reg sign applications for Wet Industrial Wastes: Energy benefit and economic analysis report

    SciTech Connect

    Elliott, D.C.; Scheer, T.H.

    1992-02-01

    Fundamental work is catalyzed biomass pyrolysis/gasification led to the Thermochemical Environmental Energy System (TEES{reg sign}) concept, a means of converting moist biomass feedstocks to high-value fuel gases such as methane. A low-temperature (350{degrees}C), pressurized (3100 psig) reaction environment and a nickel catalyst are used to reduce volumes of very high-moisture wastes such as food processing byproducts while producing useful quantities of energy. A study was conducted to assess the economic viability of a range of potential applications of the process. Cases examined included feedstocks of cheese whey, grape pomace, spent grain, and an organic chemical waste stream. The analysis indicated that only the organic chemical waste process is economically attractive in the existing energy/economic environment. However, food processing cases will become attractive as alternative disposal practices are curtailed and energy prices rise.

  2. Spatial performance of RegEM climate field reconstruction techniques in a realistic pseudoproxy context

    NASA Astrophysics Data System (ADS)

    Wang, J.; Emile-Geay, J.; Guillot, D.

    2011-12-01

    Several methods of climate field reconstructions (CFRs) have been introduced in the past few years to estimate past climate variability from proxy data over the Common Era. The pseudoproxy framework has become a tool of choice for assessing the relative merits of such methods. Here we compare four variants of the RegEM algorithm [Schneider, 2001], using a pseudoproxy network mimicking the key spatio-temporal characteristics of the network of Mann et al., 2008 (hereinafter M08); the methods are (1) RegEM TTLS (2) RegEM iTTLS (3) GraphEM and (4) RegEM iRIDGE. To ensure continuity with previous work [Smerdon et al. 2011], pseudoproxy series are designed as a white-noise degraded version of the simulated temperature field [Amman et al. 2007] over 850-1980 C.E. colocated with 1138 M08 proxies. We use signal-to-noise ratios (SNRs) of: ∞ (no noise), 1.0, 0.5 and 0.25, to simulate differences in proxy quality. Two novelties in pseudoproxy design are introduced here: (1) the decrease in proxy availability over time follows that found in M08, (2) a realistic case where the SNR is empirically derived from correlations between each M08 proxy and the HadCRUT3v temperature field. It is found that this realistic SNR is clustered around 0.3, but ranges from 0.1 to 0.8. Verification statistics such as RE, CE, r2, bias, standard deviation ratio and RMSE are presented for each method at each SNR level. The results show that all methods perform relatively well at SNR levels higher than 0.5, but display drastically different performances at lower SNR levels. Compared with results using pseudoproxy network of Mann et al., 1998, (hereinafter MBH98), the reconstruction skill of the M08 network is relatively improved, in line with the findings of Smerdon et al., 2011. Overall, we find that GraphEM and iTTLS tend to produce more robust estimates of the temperature field at low SNR levels than other schemes, while preserving a higher amount of variance in the target field. Ammann, C. M., F

  3. Cure effects on microcracking in IM7 fiber/Matrimid 5292{reg_sign} BMI composites

    SciTech Connect

    Wilenski, M.S.; Shin, E.; Morgan, R.J.; Drzal, L.T.

    1995-12-31

    Initial results from a study of the microcracking behavior of the Matrimid 5292{reg_sign} BMI/IM7 carbon fiber system are presented. Studies were performed to determine the composite`s Stress Free Temperature (SFT) which is seen to control the presence and extent of microcracking. The SFT was determined using asymmetric laminates [0{sub 2}/90{sub 2}]. Varied post-cure cycles were utilized and it was found that until complete cure is obtained, the SFT is a function of the highest temperature experienced by the laminate, with little effect of previous thermal history. The reactions necessary to fully cure this system are not active at temperatures below 250 C, ruling out the possibility of obtaining a fully cured composite with a low SFT through extended post-cures at lower temperatures.

  4. LncReg: a reference resource for lncRNA-associated regulatory networks

    PubMed Central

    Zhou, Zhong; Shen, Yi; Khan, Muhammad Riaz; Li, Ao

    2015-01-01

    Long non-coding RNAs (lncRNAs) are critical in the regulation of various biological processes. In recent years, plethora of lncRNAs have been identified in mammalian genomes through different approaches, and the researchers are constantly reporting the regulatory roles of these lncRNAs, which leads to complexity of literature about particular lncRNAs. Therefore, for the convenience of the researchers, we collected regulatory relationships of the lncRNAs and built a database called ‘LncReg’. This database is developed by collecting 1081 validated lncRNA-associated regulatory entries, including 258 non-redundant lncRNAs and 571 non-redundant genes. With regulatory relationships information, LncReg can provide overall perspectives of regulatory networks of lncRNAs and comprehensive data for bioinformatics research, which is useful for understanding the functional roles of lncRNAs. Database URL: http://bioinformatics.ustc.edu.cn/lncreg/ PMID:26363021

  5. Bioremediation of TNT-contaminated soil by the TERRANOX{reg_sign} system

    SciTech Connect

    Stolpmann, H.; Warrelmann, J.; Heuermann, E.; Fruechtnicht, A.; Lenke, H.; Daun, G.; Knackmuss, H.J.

    1995-12-31

    A method for biological on-site treatment of 2,4,6-trinitrotoluene (TNT)-contaminated soil is presented. Laboratory and large-scale pilot experiments provide new data on how TNT and its bacterial metabolites (such as aminoaromatic chemicals) can be detoxified. The pilot project is performed in a controlled biological anaerobic/aerobic treatment process using a bioreactor called TERRANOX{reg_sign}. Experiences with TNT-contaminated soil from several former TNT production sites in Germany are based on extensive laboratory-scale experiments indicating that TNT disappears completely under strictly anaerobic conditions. A stable polymeric substances that is not able to leak out of the soil anymore is generated in the second step of the process from 2,4,6-triaminotoluene (TAT) by aerobic treatment. The careful analysis of this immobilization process, long-term behavior of the product, and ecotoxicological data are currently being investigated.

  6. Pilomatricome: étude de 22 cas

    PubMed Central

    Nasreddine, Fatima Zahra; Hali, Fouzia; Chiheb, Soumiya

    2016-01-01

    Le pilomatricome est une tumeur cutanée fréquente et bénigne du follicule pileux chez l'enfant. C'est une tumeur annexielle souvent méconnue et confondue avec d'autres lésions cutanées. Les localisations habituelles sont la tête et le cou. Le but de ce travail est de rapporter une série de 22 cas comportant des formes inhabituelles colligées au service de dermatologie sur une période allant de Janvier 2006 jusqu'au Mai 2015. L’étude a concerné 16 femmes et 6 hommes. La moyenne d’âge était de 23,3 ans (4-80 ans). La localisation cervico faciale a été observée dans 12 cas, 2 patients avaient des localisations multiples, un garçon de 4ans avait une localisation au niveau fronto-temporal et une fillette de 14 ans avait une localisation au niveau du visage et de l'avant-bras, et un patient de 48 ans avait une localisation sous unguéale. L'aspect clinique était typique dans tous les cas avec des nodules sous cutanés de consistance pierreuse. Tous les patients ont bénéficié d'une exérèse des nodules sous anesthésie locale. L’étude histologique était en faveur d'un épithélioma momifié de Malherbe d'exérèse complète sans signes de malignité. Aucun patient n'a présenté de rechute. L'originalité de notre étude réside dans la présence de localisations exceptionnelles au niveau latéro-vertébral, des membres et sous-unguéale, l’âge de survenue inhabituel à 80 ans et la présence de localisations multiples signalées chez 2 enfants. PMID:27516819

  7. Relationship between tropospheric temperature and Indian summer monsoon rainfall as simulated by RegCM3

    NASA Astrophysics Data System (ADS)

    Pattnayak, K. C.; Panda, S. K.; Saraswat, Vaishali; Dash, S. K.

    2016-05-01

    Relationship between rainfall and tropospheric temperature (TT) has been examined over the Indian subcontinent during four seasons of the year using Regional Climate Model Version 3.0 (RegCM3). The model has been integrated at 55 km horizontal resolution over India during the years 1980-2000 with prescribed lateral boundary forcing from the 40 years re-analysis (ERA40) of the European Centre for Medium-range Weather Forecasts. Results of this study show that RegCM3 in general is able to capture the spatial distributions of rainfall in all the seasons as compared to the corresponding IMD0.5 gridded rainfall. The model has simulated warmer TT over the Himalayan region in all the seasons as compared to ERA40. However, it is well captured over the peninsular India and the oceanic regions. In the model, larger warming by about 0.5 °C over the northwest and Central India in the summer monsoon months might have lead to lower surface pressure there. Also, the vertical extent of the monsoon trough is found to be up to 500 hPa in the model as compared to that in NCEP/NCAR reanalysis. As a consequence, the simulated monsoon circulation and rainfall are stronger than those observed. The two most important rainfall seasons, the summer monsoon and winter are reasonably well simulated with correlation coefficients (CC) of 0.60 and 0.59 respectively significant at 99 % confidence level with the corresponding observed values of IMD0.5. Further, Indian summer monsoon rainfall (ISMR) and TT during the contrasting monsoon years are also close to their respective observed values. Temporal CCs between the TT over Tibet, Pakistan and Central India during the summer monsoon season and gridded ISMR values reveals that the TT over Pakistan has been better correlated with the ISMR than those over Tibet and Central India. This relationship has been well supported by the model simulations.

  8. Automatic registration of serial sections of mouse lymph node by using Image-Reg.

    PubMed

    Ma, Bin; Lin, Zhuang; Winkelbach, Simon; Lindenmaier, Werner; Dittmar, Kurt E J

    2008-06-01

    The first step towards the three-dimensional (3D) reconstruction of histological structures from serial sectioned tissue blocks is the proper alignment of microscope image sequences. We have accomplished an automatic rigid registration program, named Image-Reg, to align serial sections from mouse lymph node and Peyer's patch. Our approach is based on the calculation of the pixel-correlation of objects in adjacent images. The registration process is mainly divided into two steps. Once the foreground images have been segmented from the original images, the first step (primary alignment) is performed on the binary images of segmented objects; this process includes rotation by using the moments and translation through the X, Y axes by using the centroid. In the second step, the matching error of two binary images is calculated and the registration results are refined through multi-scale iterations. In order to test the registration performance, Image-Reg has been applied to an image and its transformed (rotated) version and subsequently to an image sequence of three serial sections of mouse lymph node. In addition, to compare our algorithm with other registration methods, three other approaches, viz. manual registration with Reconstruct, semi-automatic landmark registration with Image-Pro Plus and the automatic phase-correlation method with Image-Pro Plus, have also been applied to these three sections. The performance of our program has been also tested on other two-image data sets. These include: (a) two light microscopic images acquired by the automatic microscope (stitched with other software); (b) two images fluorescent images acquired by confocal microscopy (tiled with other software). Our proposed approach provides a fast and accurate linear alignment of serial image sequences for the 3D reconstruction of tissues and organs. PMID:17512746

  9. Effects of GLC7 and REG1 deletion on maltose metabolism and leavening ability of baker's yeast in lean dough.

    PubMed

    Lin, Xue; Zhang, Cui-Ying; Bai, Xiao-Wen; Xiao, Dong-Guang

    2015-09-10

    Maltose metabolism and leavening ability of baker's yeast (Saccharomyces cerevisiae) in lean dough is negatively influenced by glucose repression. To improve maltose metabolism and leavening ability, it is necessary to alleviate glucose repression. In this study, we focus on the effects of regulators (GLC7 encoding the catalytic and REG1 encoding the regulatory subunits of protein phosphatase type 1) of glucose repression on maltose metabolism and leavening ability of baker's yeast in lean dough. To this end, GLC7 and/or REG1 deletions were constructed and characterized in terms of the growth characteristics, maltose metabolism, leavening ability, and enzyme activities. The results suggest that GLC7 and/or REG1 deletions increased maltose metabolism and leavening ability at different level with glucose derepression and increased enzymes (maltase and maltose permease) activities. In a medium containing glucose and maltose, at the point of glucose exhaustion the maltose metabolized and the leavening ability were increased 59.3% and 23.1%, respectively, in the case of a REG1 single gene deletion. PMID:26073997

  10. Effects of GLC7 and REG1 deletion on maltose metabolism and leavening ability of baker's yeast in lean dough.

    PubMed

    Lin, Xue; Zhang, Cui-Ying; Bai, Xiao-Wen; Xiao, Dong-Guang

    2015-09-10

    Maltose metabolism and leavening ability of baker's yeast (Saccharomyces cerevisiae) in lean dough is negatively influenced by glucose repression. To improve maltose metabolism and leavening ability, it is necessary to alleviate glucose repression. In this study, we focus on the effects of regulators (GLC7 encoding the catalytic and REG1 encoding the regulatory subunits of protein phosphatase type 1) of glucose repression on maltose metabolism and leavening ability of baker's yeast in lean dough. To this end, GLC7 and/or REG1 deletions were constructed and characterized in terms of the growth characteristics, maltose metabolism, leavening ability, and enzyme activities. The results suggest that GLC7 and/or REG1 deletions increased maltose metabolism and leavening ability at different level with glucose derepression and increased enzymes (maltase and maltose permease) activities. In a medium containing glucose and maltose, at the point of glucose exhaustion the maltose metabolized and the leavening ability were increased 59.3% and 23.1%, respectively, in the case of a REG1 single gene deletion.

  11. Sensitivity analysis of different parameterization schemes using RegCM4.3 for the Carpathian region

    NASA Astrophysics Data System (ADS)

    Pieczka, Ildikó; Pongrácz, Rita; Szabóné André, Karolina; Kelemen, Fanni Dóra; Bartholy, Judit

    2016-10-01

    In order to quantify the impact of the use of different parameterization schemes on regional climate model outputs, hindcast experiments have been completed applying the Regional Climate Model version 4.3 (RegCM4.3) for the Carpathian region and its surroundings at 10-km horizontal resolution with three different cumulus convection schemes. Besides, the sensitivity of outputs for subgrid-scale processes is also studied by activating the subgrid Biosphere-Atmosphere Transfer Scheme (BATS) model within other RegCM experiments. Among the analyzed factors, RegCM is most sensitive to the applied convection scheme. The impact of closure assumption related to the used convective parameterization is secondary, while the use of subgridding has less influence on the outputs. RegCM4.3 results show improved performance over our previous model simulations but still have larger amplitude for annual precipitation cycle than the measurement-based reference data. Our validation results for temperature and precipitation suggest that for the selected region, the overall best performance is achieved when using the mixed Grell-Emanuel scheme together with Fritsch and Chappell closure.

  12. Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon

    PubMed Central

    Sasaki, Nobuo; Sachs, Norman; Wiebrands, Kay; Ellenbroek, Saskia I. J.; Fumagalli, Arianna; Lyubimova, Anna; Begthel, Harry; van den Born, Maaike; van Es, Johan H.; Karthaus, Wouter R.; Li, Vivian S. W.; López-Iglesias, Carmen; Peters, Peter J.; van Rheenen, Jacco; van Oudenaarden, Alexander; Clevers, Hans

    2016-01-01

    Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5+) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5+ stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5+ stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4+ DCS cells promote organoid formation of single Lgr5+ colon stem cells. DCS cells can be massively produced from Lgr5+ colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4+ DCS cells serve as Paneth cell equivalents in the colon crypt niche. PMID:27573849

  13. Intestinal REG3 Lectins Protect against Alcoholic Steatohepatitis by Reducing Mucosa-Associated Microbiota and Preventing Bacterial Translocation.

    PubMed

    Wang, Lirui; Fouts, Derrick E; Stärkel, Peter; Hartmann, Phillipp; Chen, Peng; Llorente, Cristina; DePew, Jessica; Moncera, Kelvin; Ho, Samuel B; Brenner, David A; Hooper, Lora V; Schnabl, Bernd

    2016-02-10

    Approximately half of all deaths from liver cirrhosis, the tenth leading cause of mortality in the United States, are related to alcohol use. Chronic alcohol consumption is accompanied by intestinal dysbiosis and bacterial overgrowth, yet little is known about the factors that alter the microbial composition or their contribution to liver disease. We previously associated chronic alcohol consumption with lower intestinal levels of the antimicrobial-regenerating islet-derived (REG)-3 lectins. Here, we demonstrate that intestinal deficiency in REG3B or REG3G increases numbers of mucosa-associated bacteria and enhances bacterial translocation to the mesenteric lymph nodes and liver, promoting the progression of ethanol-induced fatty liver disease toward steatohepatitis. Overexpression of Reg3g in intestinal epithelial cells restricts bacterial colonization of mucosal surfaces, reduces bacterial translocation, and protects mice from alcohol-induced steatohepatitis. Thus, alcohol appears to impair control of the mucosa-associated microbiota, and subsequent breach of the mucosal barrier facilitates progression of alcoholic liver disease. PMID:26867181

  14. Evaluation of RegCM4 in simulating the interannual and interdecadal variations of Meiyu rainfall in China

    NASA Astrophysics Data System (ADS)

    Hu, Yijia; Zhong, Zhong; Lu, Wei; Zhang, Yaocun; Sun, Yuan

    2016-05-01

    In this paper, the performance of the regional climate model (RegCM4) in simulating the interannual and interdecadal variation of Meiyu rainfall over lower and middle reaches of Yangtze River (LMRYR) in China is evaluated. The results show that the bias of the simulated Meiyu rainfall exhibits some uncertainties and obvious interdecadal variation similar with that of Meiyu rainfall itself. The rainfall bias and its interdecadal variation have little impact on the simulation of the interannual variation of the Meiyu rainfall. Not only the amplitude but also the phase of the interannual variation of the Meiyu rainfall can be reproduced by the RegCM4. However, the interdecadal variation of the rainfall bias has large impacts on the model's performance in simulating the interdecadal component of the Meiyu rainfall, leading to lower skill of RegCM4 in simulating the interdecadal variation of the Meiyu rainfall than that in simulating the interannual variation. In addition, the interannual signals of the temperature, humidity, and vorticity in the lower troposphere in the large-scale NCEP lateral boundary force are stronger than the interdecadal signals, and the model can respond well to these interannual signals. Therefore, the RegCM4 has better performance in simulating the interannual variation of water vapor budget than the interdecadal variation, which further favors the simulation of the Meiyu rainfall at interannual time scale.

  15. Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon.

    PubMed

    Sasaki, Nobuo; Sachs, Norman; Wiebrands, Kay; Ellenbroek, Saskia I J; Fumagalli, Arianna; Lyubimova, Anna; Begthel, Harry; van den Born, Maaike; van Es, Johan H; Karthaus, Wouter R; Li, Vivian S W; López-Iglesias, Carmen; Peters, Peter J; van Rheenen, Jacco; van Oudenaarden, Alexander; Clevers, Hans

    2016-09-13

    Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4(+) DCS cells promote organoid formation of single Lgr5(+) colon stem cells. DCS cells can be massively produced from Lgr5(+) colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4(+) DCS cells serve as Paneth cell equivalents in the colon crypt niche.

  16. Light Curves and Analyses of the Eclipsing Binaries EG Cas and EP Cas

    NASA Astrophysics Data System (ADS)

    Bradstreet, David H.; Sanders, S. J.; McClain, T. R.

    2010-01-01

    New precision V & Rc light curves of the eclipsing binaries EG Cas and EP Cas have been obtained using the 41-cm telescope at the Eastern University Observatory equipped with an SBIG ST-10XME CCD. EG Cas (P = 0.6115 days, Vmax = 12.9) has no published light curves and only a few dozen (mostly visual) timings of minimum light. The system is being observed throughout the fall of 2009 and the current light curves distinctly show that the system is a totally eclipsing overcontact binary. The light curves are also significantly asymmetric (strong O'Connell effect) indicating the presence of large, cool starspots, most likely on both stars. Preliminary analysis indicates that the binary is an A-type (the larger, more massive star is the hotter component), has a mass ratio of 0.32, very large temperature difference between the stars greater than 1600 K, and a fillout of 0.26. The modern timings of minimum light combined with those in the literature indicate that the binary's period is decreasing. The large temperature difference coupled to a significant fillout factor seems contradictory, and further data acquisition and analysis will hopefully resolve this seeming enigma. EP Cas (P = 0.8134 days, Vmax = 11.2) is a partially eclipsing detached system with a relatively deep primary eclipse of 1.0 mag in Rc. No published light curves exist for this system although many timings of minimum light have been published. The O-C curve indicates that the period for the binary has remained relatively constant since observations were first published in 1936. Preliminary light curve models indicate a partially eclipsing system consisting of detached but tidally distorted stars. The complete light curve analyses as well as a period study of all published times of minimum light will be presented for both systems.

  17. Spatial and Temporal Variations in Indian Summer Monsoon Rainfall and Temperature: An Analysis Based on RegCM3 Simulations

    NASA Astrophysics Data System (ADS)

    Dash, S. K.; Mamgain, Ashu; Pattnayak, K. C.; Giorgi, F.

    2013-04-01

    Regional climate models are important tools to examine the spatial and temporal characteristics of rainfall and temperature at high resolutions. Such information has potential applications in sectors like agriculture and health. In this study, the Regional Climate Model Version 3 (RegCM3) has been integrated in the ensemble mode at 55 km resolution over India for the summer monsoon season during the years 1982-2009. Emphasis has been given on the validation of the model simulation at the regional level. In Central India, both rainfall and temperature show the best correlations with respective observed values. The model gives rise to large wet biases over Northwest and Peninsular India. RegCM3 slightly underestimates the summer monsoon precipitation over the Central and Northeast India. Nevertheless, over these regions, RegCM3 simulated rainfall is closer to the observations when compared to the other regions where rainfall is overestimated. The position of the monsoon trough simulated by the model lies to the north of its original observed position. This is similar to the usual monsoon break conditions leading to less rainfall over Central India. RegCM3 simulated surface maximum temperature shows a large negative bias over the country while the surface minimum temperature is close to the observation. Nevertheless, there is a strong correlation between the all India weighted average surface temperature simulated by RegCM3 and IMD observed values. While examining the extreme weather conditions in Central India, it is found that RegCM3 simulated frequencies of occurrence of very wet days, extremely wet days, warm days and warm nights more often as compared to those in IMD observed values. However, these are systematic biases. The model biases in the frequencies of distribution of rainfall extremes explain the wet and dry biases in different regions in the country. Overall, the inter-annual characteristics of both the rainfall and temperature extremes simulated by Reg

  18. Performance of the Cas9 nickase system in Drosophila melanogaster.

    PubMed

    Ren, Xingjie; Yang, Zhihao; Mao, Decai; Chang, Zai; Qiao, Huan-Huan; Wang, Xia; Sun, Jin; Hu, Qun; Cui, Yan; Liu, Lu-Ping; Ji, Jun-Yuan; Xu, Jiang; Ni, Jian-Quan

    2014-10-01

    Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9(D10A) nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs. PMID:25128437

  19. CRISPR/Cas9 in Genome Editing and Beyond.

    PubMed

    Wang, Haifeng; La Russa, Marie; Qi, Lei S

    2016-06-01

    The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

  20. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    PubMed Central

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-01-01

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

  1. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

    PubMed

    Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

    2016-07-15

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

  2. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  3. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  4. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  5. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  6. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  7. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  8. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  9. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  10. Adaptation in CRISPR-Cas Systems.

    PubMed

    Sternberg, Samuel H; Richter, Hagen; Charpentier, Emmanuelle; Qimron, Udi

    2016-03-17

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity.

  11. HPCCP/CAS Workshop Proceedings 1998

    NASA Technical Reports Server (NTRS)

    Schulbach, Catherine; Mata, Ellen (Editor); Schulbach, Catherine (Editor)

    1999-01-01

    This publication is a collection of extended abstracts of presentations given at the HPCCP/CAS (High Performance Computing and Communications Program/Computational Aerosciences Project) Workshop held on August 24-26, 1998, at NASA Ames Research Center, Moffett Field, California. The objective of the Workshop was to bring together the aerospace high performance computing community, consisting of airframe and propulsion companies, independent software vendors, university researchers, and government scientists and engineers. The Workshop was sponsored by the HPCCP Office at NASA Ames Research Center. The Workshop consisted of over 40 presentations, including an overview of NASA's High Performance Computing and Communications Program and the Computational Aerosciences Project; ten sessions of papers representative of the high performance computing research conducted within the Program by the aerospace industry, academia, NASA, and other government laboratories; two panel sessions; and a special presentation by Mr. James Bailey.

  12. RegNetwork: an integrated database of transcriptional and post-transcriptional regulatory networks in human and mouse.

    PubMed

    Liu, Zhi-Ping; Wu, Canglin; Miao, Hongyu; Wu, Hulin

    2015-01-01

    Transcriptional and post-transcriptional regulation of gene expression is of fundamental importance to numerous biological processes. Nowadays, an increasing amount of gene regulatory relationships have been documented in various databases and literature. However, to more efficiently exploit such knowledge for biomedical research and applications, it is necessary to construct a genome-wide regulatory network database to integrate the information on gene regulatory relationships that are widely scattered in many different places. Therefore, in this work, we build a knowledge-based database, named 'RegNetwork', of gene regulatory networks for human and mouse by collecting and integrating the documented regulatory interactions among transcription factors (TFs), microRNAs (miRNAs) and target genes from 25 selected databases. Moreover, we also inferred and incorporated potential regulatory relationships based on transcription factor binding site (TFBS) motifs into RegNetwork. As a result, RegNetwork contains a comprehensive set of experimentally observed or predicted transcriptional and post-transcriptional regulatory relationships, and the database framework is flexibly designed for potential extensions to include gene regulatory networks for other organisms in the future. Based on RegNetwork, we characterized the statistical and topological properties of genome-wide regulatory networks for human and mouse, we also extracted and interpreted simple yet important network motifs that involve the interplays between TF-miRNA and their targets. In summary, RegNetwork provides an integrated resource on the prior information for gene regulatory relationships, and it enables us to further investigate context-specific transcriptional and post-transcriptional regulatory interactions based on domain-specific experimental data. Database URL: http://www.regnetworkweb.org.

  13. RegPhos 2.0: an updated resource to explore protein kinase-substrate phosphorylation networks in mammals.

    PubMed

    Huang, Kai-Yao; Wu, Hsin-Yi; Chen, Yi-Ju; Lu, Cheng-Tsung; Su, Min-Gang; Hsieh, Yun-Chung; Tsai, Chih-Ming; Lin, Kuo-I; Huang, Hsien-Da; Lee, Tzong-Yi; Chen, Yu-Ju

    2014-01-01

    Protein phosphorylation catalyzed by kinases plays crucial roles in regulating a variety of intracellular processes. Owing to an increasing number of in vivo phosphorylation sites that have been identified by mass spectrometry (MS)-based proteomics, the RegPhos, available online at http://csb.cse.yzu.edu.tw/RegPhos2/, was developed to explore protein phosphorylation networks in human. In this update, we not only enhance the data content in human but also investigate kinase-substrate phosphorylation networks in mouse and rat. The experimentally validated phosphorylation sites as well as their catalytic kinases were extracted from public resources, and MS/MS phosphopeptides were manually curated from research articles. RegPhos 2.0 aims to provide a more comprehensive view of intracellular signaling networks by integrating the information of metabolic pathways and protein-protein interactions. A case study shows that analyzing the phosphoproteome profile of time-dependent cell activation obtained from Liquid chromatography-mass spectrometry (LC-MS/MS) analysis, the RegPhos deciphered not only the consistent scheme in B cell receptor (BCR) signaling pathway but also novel regulatory molecules that may involve in it. With an attempt to help users efficiently identify the candidate biomarkers in cancers, 30 microarray experiments, including 39 cancerous versus normal cells, were analyzed for detecting cancer-specific expressed genes coding for kinases and their substrates. Furthermore, this update features an improved web interface to facilitate convenient access to the exploration of phosphorylation networks for a group of genes/proteins. Database URL: http://csb.cse.yzu.edu.tw/RegPhos2/

  14. RegNetwork: an integrated database of transcriptional and post-transcriptional regulatory networks in human and mouse

    PubMed Central

    Liu, Zhi-Ping; Wu, Canglin; Miao, Hongyu; Wu, Hulin

    2015-01-01

    Transcriptional and post-transcriptional regulation of gene expression is of fundamental importance to numerous biological processes. Nowadays, an increasing amount of gene regulatory relationships have been documented in various databases and literature. However, to more efficiently exploit such knowledge for biomedical research and applications, it is necessary to construct a genome-wide regulatory network database to integrate the information on gene regulatory relationships that are widely scattered in many different places. Therefore, in this work, we build a knowledge-based database, named ‘RegNetwork’, of gene regulatory networks for human and mouse by collecting and integrating the documented regulatory interactions among transcription factors (TFs), microRNAs (miRNAs) and target genes from 25 selected databases. Moreover, we also inferred and incorporated potential regulatory relationships based on transcription factor binding site (TFBS) motifs into RegNetwork. As a result, RegNetwork contains a comprehensive set of experimentally observed or predicted transcriptional and post-transcriptional regulatory relationships, and the database framework is flexibly designed for potential extensions to include gene regulatory networks for other organisms in the future. Based on RegNetwork, we characterized the statistical and topological properties of genome-wide regulatory networks for human and mouse, we also extracted and interpreted simple yet important network motifs that involve the interplays between TF-miRNA and their targets. In summary, RegNetwork provides an integrated resource on the prior information for gene regulatory relationships, and it enables us to further investigate context-specific transcriptional and post-transcriptional regulatory interactions based on domain-specific experimental data. Database URL: http://www.regnetworkweb.org PMID:26424082

  15. Orbital period determination in an eclipsing dwarf nova HT Cas

    NASA Astrophysics Data System (ADS)

    Bąkowska, Karolina; Olech, Arkadiusz

    2014-09-01

    HT Cassiopeiae was discovered over seventy years ago (Hoffmeister 1943). Unfortunately, for 35 years this object did not receive any attention, until the eclipses of HT Cas were observed by Bond. After a first analysis, Patterson (1981) called HT Cas "a Rosetta stone among dwarf novae". Since then, the literature on this star is still growing, reaching several dozens of publications. We present an orbital period determination of HT Cas during the November 2010 super-outburst, but also during a longer time span, to check its stability.

  16. Study of Eclipsing Binary and Multiple Systems in OB Associations IV: Cas OB6 Member DN Cas

    NASA Astrophysics Data System (ADS)

    Bakış, V.; Bakış, H.; Bilir, S.; Eker, Z.

    2016-09-01

    An early-type, massive, short-period (Porb=2d.310951) eclipsing spectroscopic binary DN Cas has been re-visited with new spectral and photometric data. The masses and radii of the components have been obtained as M1=19.04± 0.07 M⊙, M2=13.73± 0.05 M⊙ and R1=7.22± 0.06 R⊙, R2=5.79± 0.06 R⊙, respectively. Both components present synchronous rotation (Vrot1=160 km s-1, Vrot2=130 km s-1) with their orbit. Orbital period analysis yielded a physically bound additional component in the system with a minimum mass of M3=0.88 M⊙ orbiting in an eccentric orbit (e = 0.37 ± 0.2) with an orbital period of P 12 = 42 ± 9 yr. High precision absolute parameters of the system allowed us to derive a distance to DN Cas as 1.7 ± 0.2 kpc which locates the system within the borders of the Cas OB6 association (d = 1.8 kpc). The space velocities and the age of DN Cas are in agreement with those of Cas OB6. The age of DN Cas (τ = 3-5 Myr) is found to be 1-2 Myr older than the embedded clusters (IC 1795, IC 1805, and IC 1848) in the Cas OB6 association, which implies a sequential star formation in the association.

  17. 21 CFR 74.102 - FD&C Blue No. 2.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Blue No. 2 is principally the disodium salt of 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid (CAS Reg. No. 860-22-0) with smaller amounts of the disodium salt... Reg. No. 54947-75-0) and the sodium salt of...

  18. 21 CFR 74.102 - FD&C Blue No. 2.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Blue No. 2 is principally the disodium salt of 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid (CAS Reg. No. 860-22-0) with smaller amounts of the disodium salt... Reg. No. 54947-75-0) and the sodium salt of...

  19. Crystal structure of the Csm3-Csm4 subcomplex in the type III-A CRISPR-Cas interference complex.

    PubMed

    Numata, Tomoyuki; Inanaga, Hideko; Sato, Chikara; Osawa, Takuo

    2015-01-30

    Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex.

  20. Genes Specific for the Biosynthesis of Clavam Metabolites Antipodal to Clavulanic Acid Are Clustered with the Gene for Clavaminate Synthase 1 in Streptomyces clavuligerus

    PubMed Central

    Mosher, Roy H.; Paradkar, Ashish S.; Anders, Cecilia; Barton, Barry; Jensen, Susan E.

    1999-01-01

    Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis. PMID:10223939

  1. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.

    PubMed

    Barrangou, Rodolphe; Marraffini, Luciano A

    2014-04-24

    Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control.

  2. CRISPR-Cas9-Guided Genome Engineering in C. elegans.

    PubMed

    Kim, Hyun-Min; Colaiácovo, Monica P

    2016-07-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans. Recent studies have developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc.

  3. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SUPPLEMENTARY REGULATIONS DOE MANAGEMENT AND OPERATING CONTRACTS Cost Accounting Standards Administration 970.3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix)...

  4. An updated evolutionary classification of CRISPR-Cas systems.

    PubMed

    Makarova, Kira S; Wolf, Yuri I; Alkhnbashi, Omer S; Costa, Fabrizio; Shah, Shiraz A; Saunders, Sita J; Barrangou, Rodolphe; Brouns, Stan J J; Charpentier, Emmanuelle; Haft, Daniel H; Horvath, Philippe; Moineau, Sylvain; Mojica, Francisco J M; Terns, Rebecca M; Terns, Michael P; White, Malcolm F; Yakunin, Alexander F; Garrett, Roger A; van der Oost, John; Backofen, Rolf; Koonin, Eugene V

    2015-11-01

    The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized. PMID:26411297

  5. CRISPR-Cas9-Guided Genome Engineering in C. elegans.

    PubMed

    Kim, Hyun-Min; Colaiácovo, Monica P

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans. Recent studies have developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc. PMID:27366893

  6. Editing plant genomes with CRISPR/Cas9.

    PubMed

    Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Patron, Nicola J; Nekrasov, Vladimir

    2015-04-01

    CRISPR/Cas9 is a rapidly developing genome editing technology that has been successfully applied in many organisms, including model and crop plants. Cas9, an RNA-guided DNA endonuclease, can be targeted to specific genomic sequences by engineering a separately encoded guide RNA with which it forms a complex. As only a short RNA sequence must be synthesized to confer recognition of a new target, CRISPR/Cas9 is a relatively cheap and easy to implement technology that has proven to be extremely versatile. Remarkably, in some plant species, homozygous knockout mutants can be produced in a single generation. Together with other sequence-specific nucleases, CRISPR/Cas9 is a game-changing technology that is poised to revolutionise basic research and plant breeding.

  7. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... to the same cost accounting practices. The term includes Government-owned contractor-operated (GOCO...), as applicable, shall be incorporated. (6) Continuity in fully CAS-covered contracts. Where...

  8. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... to the same cost accounting practices. The term includes Government-owned contractor-operated (GOCO...), as applicable, shall be incorporated. (6) Continuity in fully CAS-covered contracts. Where...

  9. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... to the same cost accounting practices. The term includes Government-owned contractor-operated (GOCO...), as applicable, shall be incorporated. (6) Continuity in fully CAS-covered contracts. Where...

  10. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SUPPLEMENTARY REGULATIONS DOE MANAGEMENT AND OPERATING CONTRACTS Cost Accounting Standards Administration 970.3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix)...

  11. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

    PubMed

    Tycko, Josh; Myer, Vic E; Hsu, Patrick D

    2016-08-01

    Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

  12. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity

    PubMed Central

    Barrangou, Rodolphe; Marraffini, Luciano A.

    2014-01-01

    Summary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing, and can be repurposed for numerous DNA targeting applications including transcriptional control. PMID:24766887

  13. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

    PubMed

    Tycko, Josh; Myer, Vic E; Hsu, Patrick D

    2016-08-01

    Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance.

  14. RegA, an AraC-like protein, is a global transcriptional regulator that controls virulence gene expression in Citrobacter rodentium.

    PubMed

    Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J; Frankel, Gad; Hartland, Elizabeth L; Robins-Browne, Roy M

    2008-11-01

    Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

  15. Functional Analysis of the Yeast Glc7-Binding Protein Reg1 Identifies a Protein Phosphatase Type 1-Binding Motif as Essential for Repression of ADH2 Expression

    PubMed Central

    Dombek, Kenneth M.; Voronkova, Valentina; Raney, Alexa; Young, Elton T.

    1999-01-01

    In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. Constitutive ADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2 expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressing ADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm. PMID:10454550

  16. Calibrated Ancillary System (CAS) user's guide, volume 6

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 6 describes ancillary products procedures, enhancement menu and processing task procedures for SDT/TDT (shuttle data tape/telemetry descriptor tape), database errors and network data driver (NDD) product menu procedures, and utility menu procedures.

  17. Potential pitfalls of CRISPR/Cas9-mediated genome editing.

    PubMed

    Peng, Rongxue; Lin, Guigao; Li, Jinming

    2016-04-01

    Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012.

  18. 21 CFR 184.1721 - Sodium acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and....1721 Sodium acetate. (a) Sodium acetate (C2H3O2Na, CAS Reg. No. 127-09-3 or C2H3O2Na·3H2O, CAS Reg. No. 6131-90-4) is the sodium salt of acetic acid and occurs naturally in plant and animal tissues....

  19. RegPrecise 3.0 – A resource for genome-scale exploration of transcriptional regulation in bacteria

    PubMed Central

    2013-01-01

    Background Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in prokaryotes is one of the critical tasks of modern genomics. Bacteria from different taxonomic groups, whose lifestyles and natural environments are substantially different, possess highly diverged transcriptional regulatory networks. The comparative genomics approaches are useful for in silico reconstruction of bacterial regulons and networks operated by both transcription factors (TFs) and RNA regulatory elements (riboswitches). Description RegPrecise (http://regprecise.lbl.gov) is a web resource for collection, visualization and analysis of transcriptional regulons reconstructed by comparative genomics. We significantly expanded a reference collection of manually curated regulons we introduced earlier. RegPrecise 3.0 provides access to inferred regulatory interactions organized by phylogenetic, structural and functional properties. Taxonomy-specific collections include 781 TF regulogs inferred in more than 160 genomes representing 14 taxonomic groups of Bacteria. TF-specific collections include regulogs for a selected subset of 40 TFs reconstructed across more than 30 taxonomic lineages. Novel collections of regulons operated by RNA regulatory elements (riboswitches) include near 400 regulogs inferred in 24 bacterial lineages. RegPrecise 3.0 provides four classifications of the reference regulons implemented as controlled vocabularies: 55 TF protein families; 43 RNA motif families; ~150 biological processes or metabolic pathways; and ~200 effectors or environmental signals. Genome-wide visualization of regulatory networks and metabolic pathways covered by the reference regulons are available for all studied genomes. A separate section of RegPrecise 3.0 contains draft regulatory networks in 640 genomes obtained by an conservative propagation of the reference regulons to closely related genomes. Conclusions RegPrecise 3.0 gives access to the

  20. Rational design of a split-Cas9 enzyme complex

    DOE PAGES

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interactmore » on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

  1. Rational design of a split-Cas9 enzyme complex

    SciTech Connect

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

  2. Internal guide RNA interactions interfere with Cas9-mediated cleavage.

    PubMed

    Thyme, Summer B; Akhmetova, Laila; Montague, Tessa G; Valen, Eivind; Schier, Alexander F

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation. PMID:27282953

  3. Internal guide RNA interactions interfere with Cas9-mediated cleavage

    PubMed Central

    Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.; Valen, Eivind; Schier, Alexander F.

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation. PMID:27282953

  4. Rational design of a split-Cas9 enzyme complex

    PubMed Central

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-01-01

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications. PMID:25713377

  5. CRISPR-Cas9 Based Engineering of Actinomycetal Genomes.

    PubMed

    Tong, Yaojun; Charusanti, Pep; Zhang, Lixin; Weber, Tilmann; Lee, Sang Yup

    2015-09-18

    Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes. We demonstrate our system by targeting two genes, actIORF1 (SCO5087) and actVB (SCO5092), from the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Our CRISPR-Cas9 system successfully inactivated the targeted genes. When no templates for homology-directed repair (HDR) were present, the site-specific DNA double-strand breaks (DSBs) introduced by Cas9 were repaired through the error-prone nonhomologous end joining (NHEJ) pathway, resulting in a library of deletions with variable sizes around the targeted sequence. If templates for HDR were provided at the same time, precise deletions of the targeted gene were observed with near 100% frequency. Moreover, we developed a system to efficiently and reversibly control expression of target genes, deemed CRISPRi, based on a catalytically dead variant of Cas9 (dCas9). The CRISPR-Cas9 based system described here comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes.

  6. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9

    PubMed Central

    Kaya, Hidetaka; Mikami, Masafumi; Endo, Akira; Endo, Masaki; Toki, Seiichi

    2016-01-01

    The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5′-NNGRRT-3′) differs from that of SpCas9 (5′-NGG-3′), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for ‘T’ at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5′-NNGRRV-3′) were much lower than those with a canonical PAM (5′-NNGRRT-3′). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop. PMID:27226350

  7. CCISS, Vascular and BP Reg: Canadian space life science research on ISS

    NASA Astrophysics Data System (ADS)

    Hughson, Richard L.; Shoemaker, J. Kevin; Arbeille, Philippe

    2014-11-01

    A comprehensive goal of the Canadian Space Agency studies (CCISS, Vascular and BP Reg) has been to investigate the efficacy of current exercise countermeasures to maintain cardiovascular and cerebrovascular health on return to Earth after up to 6-months in space. Results from the CCISS experiments revealed no significant change of in-flight heart rate during daily activities or sleep, and small, but variable between astronauts, post-flight elevation. The between astronaut differences were exaggerated during measurement of spontaneous baroreflex slope, which was reduced post-flight (P<0.05) during paced breathing with 3 astronauts having significant correlations between reduced baroreflex and reduced RR-interval (consistent with reduced fitness). Cerebrovascular autoregulation and CO2 response were mildly impaired after flight. Some loss of in-flight fitness of astronauts in Vascular was reflected by the increase in HR at a work rate of 161±46 W of 12.3±10.5 bpm, 10.4±5.9 bpm and 13.4±5.7 bpm for early-flight, late-flight and R+1, respectively. On return to gravity, changes in resting heart rate for supine (5.9±3.5 bpm), sit (8.1±3.3 bpm) and stand (10.3±10.0 bpm) were small but variable between individuals (from -5 bpm to +20 bpm in post-flight standing) and not related to the change in exercise heart rate. In Vascular astronauts, pulse wave transit time measured to the finger tended to be reduced post-flight and carotid artery distensibility was significantly reduced (P=0.03, and n=6). The heart rate and baroreflex data suggest that some astronauts return with cardiovascular deconditioning in spite of the exercise regimes. However, greater arterial stiffness is common among all astronauts studied to date. The new CSA project, BP Reg, will monitor inflight blood pressure in an attempt to identify astronauts in greater need for countermeasures. Future research should focus on whether Vascular changes in astronauts might make them an appropriate model to study

  8. Validation of newly designed regional earth system model (RegESM) for Mediterranean Basin

    NASA Astrophysics Data System (ADS)

    Turuncoglu, Ufuk Utku; Sannino, Gianmaria

    2016-06-01

    We present a validation analysis of a regional earth system model system (RegESM) for the Mediterranean Basin. The used configuration of the modeling system includes two active components: a regional climate model (RegCM4) and an ocean modeling system (ROMS). To assess the performance of the coupled modeling system in representing the climate of the basin, the results of the coupled simulation (C50E) are compared to the results obtained by a standalone atmospheric simulation (R50E) as well as several observation datasets. Although there is persistent cold bias in fall and winter, which is also seen in previous studies, the model reproduces the inter-annual variability and the seasonal cycles of sea surface temperature (SST) in a general good agreement with the available observations. The analysis of the near-surface wind distribution and the main circulation of the sea indicates that the coupled model can reproduce the main characteristics of the Mediterranean Sea surface and intermediate layer circulation as well as the seasonal variability of wind speed and direction when it is compared with the available observational datasets. The results also reveal that the simulated near-surface wind speed and direction have poor performance in the Gulf of Lion and surrounding regions that also affects the large positive SST bias in the region due to the insufficient horizontal resolution of the atmospheric component of the coupled modeling system. The simulated seasonal climatologies of the surface heat flux components are also consistent with the CORE.2 and NOCS datasets along with the overestimation in net long-wave radiation and latent heat flux (or evaporation, E), although a large observational uncertainty is found in these variables. Also, the coupled model tends to improve the latent heat flux by providing a better representation of the air-sea interaction as well as total heat flux budget over the sea. Both models are also able to reproduce the temporal evolution of

  9. Soft modes and superconductivity in the layered hexagonal carbides V2CAs, Nb2CAs, and Nb2CS

    NASA Astrophysics Data System (ADS)

    Halilov, S. V.; Singh, D. J.; Papaconstantopoulos, D. A.

    2002-05-01

    The electronic structure, selected phonon frequencies, and electron phonon coupling of the hexagonal layered compounds Nb2CS, Nb2CAs, and V2CAs are elucidated using density functional calculations. All materials are good three-dimensional metals with moderate values of the density of states, N(EF). The electronic structure of Nb2CS, the only superconductor within this family of materials, consists of well-hybridized Nb d-S p-C p derived bands characteristic around the Fermi energy. The S is more ionic in this compound than the As in the related arsenides Nb2CAs or V2CAs. This results in a relative softness for phonon modes involving S. Rigid muffin tin approximation calculations show moderate electron phonon couplings consistent with low-temperature superconductivity. The key difference from the carbo-arsenides is the presence of soft moderately coupled S modes.

  10. 40 CFR 721.10109 - Hexanoic acid, 2-ethyl-, mixed triesters with benzoic acid and trimethylolpropane.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... triesters with benzoic acid and trimethylolpropane. 721.10109 Section 721.10109 Protection of Environment... triesters with benzoic acid and trimethylolpropane. (a) Chemical substance and significant new uses subject... benzoic acid and trimethylolpropane (PMN P-04-508; CAS No. 610787-76-3) is subject to reporting under...

  11. 40 CFR 721.10109 - Hexanoic acid, 2-ethyl-, mixed triesters with benzoic acid and trimethylolpropane.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... triesters with benzoic acid and trimethylolpropane. 721.10109 Section 721.10109 Protection of Environment... triesters with benzoic acid and trimethylolpropane. (a) Chemical substance and significant new uses subject... benzoic acid and trimethylolpropane (PMN P-04-508; CAS No. 610787-76-3 ) is subject to reporting...

  12. 40 CFR 721.10109 - Hexanoic acid, 2-ethyl-, mixed triesters with benzoic acid and trimethylolpropane.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... triesters with benzoic acid and trimethylolpropane. 721.10109 Section 721.10109 Protection of Environment... triesters with benzoic acid and trimethylolpropane. (a) Chemical substance and significant new uses subject... benzoic acid and trimethylolpropane (PMN P-04-508; CAS No. 610787-76-3) is subject to reporting under...

  13. 40 CFR 721.10109 - Hexanoic acid, 2-ethyl-, mixed triesters with benzoic acid and trimethylolpropane.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... triesters with benzoic acid and trimethylolpropane. 721.10109 Section 721.10109 Protection of Environment... triesters with benzoic acid and trimethylolpropane. (a) Chemical substance and significant new uses subject... benzoic acid and trimethylolpropane (PMN P-04-508; CAS No. 610787-76-3 ) is subject to reporting...

  14. 40 CFR 721.10109 - Hexanoic acid, 2-ethyl-, mixed triesters with benzoic acid and trimethylolpropane.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... triesters with benzoic acid and trimethylolpropane. 721.10109 Section 721.10109 Protection of Environment... triesters with benzoic acid and trimethylolpropane. (a) Chemical substance and significant new uses subject... benzoic acid and trimethylolpropane (PMN P-04-508; CAS No. 610787-76-3) is subject to reporting under...

  15. A non-inheritable maternal Cas9-based multiple-gene editing system in mice

    PubMed Central

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection–based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create “Cas9 transgene-free” gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice. PMID:26817415

  16. Closing the knowledge gap on cardiovascular disease in type 2 diabetes: the EMPA-REG OUTCOME trial and beyond

    PubMed Central

    Oral, Elif A

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is associated with marked cardiovascular (CV) morbidity and mortality, including heart failure (HF). Until recently, an oral glucose-lowering agent that improved hyperglycemia as well as provided CV benefits in patients with T2DM and cardiovascular disease (CVD) was lacking. The newest class of glucose-lowering agents, sodium glucose cotransporter 2 (SGLT2) inhibitors, includes canagliflozin, dapagliflozin, and empagliflozin. Prior to the release of the LEADER trial results, the recent EMPA-REG OUTCOME study was the only dedicated CV trial to demonstrate a reduction in major adverse cardiac events, CV mortality, and all-cause mortality and a reduction in hospitalization for HF with empagliflozin, given on top of standard-of-care therapy in patients with T2DM and CVD. This paper summarizes the results from EMPA-REG OUTCOME and discusses their significance and clinical implications.

  17. Innovative remote monitoring of plant health for environmental applications: A joint effort between EPCOT{reg_sign} and the DOE

    SciTech Connect

    Robitaille, H.; Capelle, G.; Di Benedetto, J.

    1996-12-31

    In September of 1994, the US Department of Energy (DOE), Environmental Management, Office of Science and Technology for (OST) and Epcot{reg_sign} in the WALT DISNEY WORLD{reg_sign} Resort (Epcot) signed an agreement to cooperate on the research, development, and public communication and display of environmental technologies. Although Epcot and OST have distinctive missions, certain areas of their respective research and development efforts are common, including the integration of remote sensors with robotics platforms, airborne surveys for environmental characterization and monitoring, and ground based measurements of vegetation stress. The first area of cooperative R&D pursued under the agreement is the evaluation of laser-induced fluorescence imaging (LIFI), a technology developed by OST and proven effective for uranium detection. This paper describes the efforts being conducted under the Epcot-OST agreement and presents initial results. An appendix describing LIFI technology is also included.

  18. Closing the knowledge gap on cardiovascular disease in type 2 diabetes: the EMPA-REG OUTCOME trial and beyond.

    PubMed

    Oral, Elif A

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is associated with marked cardiovascular (CV) morbidity and mortality, including heart failure (HF). Until recently, an oral glucose-lowering agent that improved hyperglycemia as well as provided CV benefits in patients with T2DM and cardiovascular disease (CVD) was lacking. The newest class of glucose-lowering agents, sodium glucose cotransporter 2 (SGLT2) inhibitors, includes canagliflozin, dapagliflozin, and empagliflozin. Prior to the release of the LEADER trial results, the recent EMPA-REG OUTCOME study was the only dedicated CV trial to demonstrate a reduction in major adverse cardiac events, CV mortality, and all-cause mortality and a reduction in hospitalization for HF with empagliflozin, given on top of standard-of-care therapy in patients with T2DM and CVD. This paper summarizes the results from EMPA-REG OUTCOME and discusses their significance and clinical implications. PMID:27648101

  19. [Correlation of the level of Reg3α protein in plasma with gastrointestinal acute graft-versus-host disease].

    PubMed

    Cai, Cheng-Sen; Chen, Guang-Hua; Sun, Ai-Ning; Qiao, Man; Liu, Hui-Wen; Chen, Feng; Wang, Ying; Qiu, Hui-Ying; Han, Yue; Ma, Xiao; Tang, Xiao-Wen; Jin, Zheng-Ming; Fu, Cheng-Cheng; Wu, De-Pei

    2014-06-01

    This study was purposed to explore the correlation of regenerating Islet-derived 3-alpha(Reg3α) protein level in plasma with the diagnosis and prognosis of the gastrointestinal acute graft-versus-host disease (GI-aGVHD) after all-HSCT, 103 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were observed in our hospital from December 2011 to December 2012. Peripheral blood samples were routinely collected at 9 d before allo-HSCT, 0 d, 14 d, 28 d after allo-HSCT as well as in aGVHD and at the 1 and 4 weeks after aGVHD therapy. The plasma concentrations of Reg3α were measured by using ELISA kit. The results indicated that among the 103 patients, 17 cases never developed aGVHD symptoms (no-aGVHD), 27 cases presented with non-aGVHD associated diarrhea, 10 cases presented with isolated skin aGVHD, 17 cases developed grades I-II GI-aGVHD, 32 cases with grades III-IV GI-aGVHD. The plasma concentrations of Reg3α in group of patients with GI-aGVHD and group of non-aGVHD diarrhea were 111.5 (54.7-180.2) and 23.9 (14.5-89.5) ng/ml respectively with significant difference (P < 0.001). The plasma concentrations of Reg3α in 17 patients of grades III-IV GI-aGVHD who experienced a complete or partial response and 7 patients who had no response to therapy at 4 weeks were 137.2(51.7-205.4) and 679.4(122.3-896.8) ng/ml respectively with the significant difference (P = 0.028). All of the patients who had no response to therapy died of aGVHD associated multiple organ failure. The area under the ROC curve was 0.902 when plasma concentration of Reg3α was set at 87.73 ng/ml. The sensitivity was 81.48% and the specificity was 82.86% when the critical value was used in diagnosis of grades III-IV GI-aGVHD. The probability of grades III-IV GI-aGVHD had statistical difference above and below 87.73 ng/ml after allo-HSCT (P < 0.001). It is concluded that the increase of plasma Reg3α level after transplantation suggests the incidence of grades III-IV GI

  20. Closing the knowledge gap on cardiovascular disease in type 2 diabetes: the EMPA-REG OUTCOME trial and beyond

    PubMed Central

    Oral, Elif A

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is associated with marked cardiovascular (CV) morbidity and mortality, including heart failure (HF). Until recently, an oral glucose-lowering agent that improved hyperglycemia as well as provided CV benefits in patients with T2DM and cardiovascular disease (CVD) was lacking. The newest class of glucose-lowering agents, sodium glucose cotransporter 2 (SGLT2) inhibitors, includes canagliflozin, dapagliflozin, and empagliflozin. Prior to the release of the LEADER trial results, the recent EMPA-REG OUTCOME study was the only dedicated CV trial to demonstrate a reduction in major adverse cardiac events, CV mortality, and all-cause mortality and a reduction in hospitalization for HF with empagliflozin, given on top of standard-of-care therapy in patients with T2DM and CVD. This paper summarizes the results from EMPA-REG OUTCOME and discusses their significance and clinical implications. PMID:27648101

  1. Predicting relative toxicity and interactions of divalent metal ions: Microtox{reg_sign} bioluminescence assay

    SciTech Connect

    Newman, M.C.; McCloskey, J.T.

    1996-03-01

    Both relative toxicity and interactions between paired metal ions were predicted with least-squares linear regression and various ion characteristics. Microtox{reg_sign} 15 min EC50s (expressed as free ion) for Ca(II), Cd(II), Cu(II), Hg(II), Mg(II), Mn(II), Ni(II), Pb(II), and Zn(II) were most effectively modeled with the constant for the first hydrolysis (K{sub H} for M{sup n+} + H{sub 2}O {yields} MOH{sup a{minus}1} + H{sup +}) although other ion characteristics were also significant in regression models. The {vert_bar}log K{sub H}{vert_bar} is correlated with metal ion affinity to intermediate ligands such as many biochemical functional groups with O donor atoms. Further, ordination of metals according to ion characteristics, e.g., {vert_bar}log K{sub H}{vert_bar} facilitated prediction of paired metal interactions. Pairing metals with strong tendencies to complex with intermediate or soft ligands such as those with O or S donor atoms resulted in strong interactions.

  2. Exposing the impact of opp(reg)ressive policies on teacher development and on student learning

    NASA Astrophysics Data System (ADS)

    Rodriguez, Alberto J.

    2010-12-01

    This case study draws attention to Pedro's story, a Grade 6 Latino teacher who, along with other grade 4-6 teachers, participated in a three-year professional development research project. By using data analyzed from multiple ethnographic interviews with teachers and students, and by drawing from the quantitative analyzes of concept map unit tests, we illustrate how Pedro's significant professional growth and his students' learning were truncated by top-down school district policies. These policies were implemented because of the punitive nature of the No Child Left Behind Act. Simply put, this case study exposes the impact of opp(reg)ressive policies on learning, that is, policies simultaneously oppressive and regressive. The critical perspective of the project, and its emphasis on assisting teachers to make their pedagogy and curriculum more culturally and socially relevant, was informed by sociotransformative constructivism (sTc). This is a theoretical framework that affords equal importance to cross-cultural education (learning about and acting on socially/culturally relevant issues) and social constructivism (learning to critically produce and consume knowledge). We hope that this case study will provide additional insights into the slow progress we continue to make in science teacher professional development and in closing the achievement gap.

  3. New inorganic/organic copolymers (ORMOCER{reg_sign}s) for dental applications

    SciTech Connect

    Wolter, H.; Storch, W.; Ott, H.

    1994-12-31

    Urethane and thioether (meth)acrylate alkoxysilanes have been used, as sol-gel precursors, for the preparation of a special family of inorganic/organic copolymers (ORMOCER{reg_sign}s). The basic silane type offers the following structural and synthetic possibilities for modifying the properties of the resulting copolymers: variation of the number of alkoxy groups, combination with other, different, sol-gel precursors, variation of the number of attached (meth)acrylate groups (1--5), and variation within the molecular segment (kind, structure and length) connecting the inorganic with the organic polymer structure. To achieve the additional organic polymer structure in the cured copolymer the use of different radically induced polymerization approaches (UV-, visible light-, thermal- and redox induced) is possible. Taking the incorporation of fillers into account, the Young`s modulus of these copolymers is adjustable in a range of 1--17,000 MPa, and the thermal expansion coefficient in a range of 17--250{center_dot}10{sup {minus}6}{center_dot}K{sup {minus}1} (5--50 C). Other advantages are the low shrinkage (1--2,8 vol.-%), the high flexural strength (up to 160 MPa), and the high abrasion resistance. This new copolymer type seems to have significant potential for medical applications, especially as dental filling material to replace the currently used controversial amalgam fillings.

  4. The Super Separator Spectrometer S3 and the associated detection systems: SIRIUS & LEB-REGLIS3

    NASA Astrophysics Data System (ADS)

    Déchery, F.; Savajols, H.; Authier, M.; Drouart, A.; Nolen, J.; Ackermann, D.; Amthor, A. M.; Bastin, B.; Berryhill, A.; Boutin, D.; Caceres, L.; Coffey, M.; Delferrière, O.; Dorvaux, O.; Gall, B.; Hauschild, K.; Hue, A.; Jacquot, B.; Karkour, N.; Laune, B.; Le Blanc, F.; Lecesne, N.; Lopez-Martens, A.; Lutton, F.; Manikonda, S.; Meinke, R.; Olivier, G.; Payet, J.; Piot, J.; Pochon, O.; Prince, V.; Souli, M.; Stelzer, G.; Stodel, C.; Stodel, M.-H.; Sulignano, B.; Traykov, E.; Uriot, D.

    2016-06-01

    The Super Separator Spectrometer (S3) facility is developed in the framework of the SPIRAL2 project [1]. S3 has been designed to extend the capability of the facility to perform experiments with extremely low cross sections, taking advantage of the very high intensity stable beams of the superconducting linear accelerator of SPIRAL2. It will mainly use fusion-evaporation reactions to reach extreme regions of the nuclear chart: new opportunities will be opened for super-heavy element studies and spectroscopy at and beyond the driplines. In addition to our previous article (Déchery et al. [2]) introducing the optical layout of the spectrometer and the expected performances, this article will present the current status of the main elements of the facility: the target station, the superconducting multipole, and the magnetic and electric dipoles, with a special emphasis on the status of the detection system SIRIUS and on the low-energy branch which includes the REGLIS3 system. S3 will also be a source of low energy radioactive isotopes for delivery to the DESIR facility.

  5. Remediation of groundwater containing radionuclides and heavy metals using ion exchange and the AlgaSORB[reg sign] biosorbent system

    SciTech Connect

    Feiler, H.D. ); Darnall, D.W. )

    1991-11-07

    Bio-Recovery Systems, Inc. (BRS) studied the application of an immobilized algal biomass, termed AlgaSORB[reg sign], which has high affinity for heavy metal ions to DOE-contaminated groundwaters. The material can be packed into columns similar to commercial ion exchange resins. Dilute solutions containing heavy metals are passed through columns where metals are absorbed by the AlgaSORB[reg sign] resins. Once saturated, metal ions can be stripped from the resin biomass in a highly concentrated solution. Groundwaters contaminated with heavy metal ions from three different Department of Energy (DOE) sites: Savannah River, Hanford and the Oak Ridge Y-12 Plant were studied. The objective was to perform bench-scale treatability studies to establish treatment protocols and to optimize an AlgaSORB[reg sign]/ion exchange technology system to remove and recover toxic metal ions from these contaminated groundwaters. The specialty ion exchange/AlgaSORB[reg sign] resins tested in these studies show promise for selectively removing chromium, mercury and uranium from contaminated groundwater at DOE sites. The data show that effluents which satisfy the allowable metal ion limits are possible and most likely achievable. The use of these highly selective resins also offer advantages in terms of cost/benefit, risk and scheduling. Their high selectivity allows for high capacity and opportunities for recovery of removed constituents due to high pollutant concentration possible (3 to 4 orders of magnitude). Ion exchange is a proven technology which is easily automated and can be cost-effective, depending on the application.

  6. Removal of contaminants from equipment and debris and waste minimization using TechXtract{reg_sign} technology

    SciTech Connect

    Bonem, M.W.

    1997-10-01

    Under this Program Research and Development Agreement (PRDA), EET, Inc., is extending its proprietary TechXtract{reg_sign} chemical decontamination technology into an effective, economical, integrated contaminant removal system. This integrated system will consist of a series of decontamination baths using the TechXtract{reg_sign} chemical formulas, followed by a waste treatment process that will remove the contaminants from the spent chemicals. Sufficient decontamination will result so that materials can be released without restriction after they have been treated, even those materials that have traditionally been considered to be {open_quotes}undecontaminable.{close_quotes} The secondary liquid waste will then be treated to separate any hazardous and radioactive contaminants, so that the spent chemicals and wastewater can be discharged through conventional, permitted outlets. The TechXtract{reg_sign} technology is a unique process that chemically extracts hazardous contaminants from the surface and substrate of concrete, steel, and other solid materials. This technology has been used successfully to remove contaminants as varied as PCBs, radionuclides, heavy metals, and hazardous organics. The process` advantage over other alternatives is its effectiveness in safe and consistent extraction of subsurface contamination. TechXtract{reg_sign} is a proprietary process developed, owned, and provided by EET, Inc. The objective of the PRDA is to demonstrate on a full-scale basis an economical system for decontaminating equipment and debris, with further treatment of secondary waste streams to minimize waste volumes. Contaminants will be removed from the contaminated items to levels where they can be released for unrestricted use. The entire system will be designed with maximum flexibility and automation in mind.

  7. Evaluation of the Brinecell{reg_sign} electrochemical oxidation unit for destruction of noncyanide strippers and other colored compounds in wastewaters

    SciTech Connect

    Wikoff, P.M.; Suciu, D.F.

    1994-08-01

    The noncyanide strippers used in the plating operations at Kelly Air Force Base, in San Antonio, Texas, develop an intense red color during use. Currently, the spent strippers cannot be treated in the Industrial Wastewater Treatment Plant because, even after biological treatment, the color remains too high for discharge. The Brinecell{reg_sign} process was evaluated for its application in treating the spent stripping solutions. The Brinecell{reg_sign} produces strong oxidizing agents that could treat the color causing compounds. The Brinecell{reg_sign} process was used to treat a 1 to 400 dilution of spent Clepo 204-T at 50 C and 60 g/L salt. After 8 hours of treatment, the color remaining was 8 units and the solution appeared colorless. Treatment of the Metalx B-9 reduced the color to 60 units. The effect of salt concentration, solution temperature, and solution pH was evaluated on the Brinecell{reg_sign} treatment process.

  8. Implementation and evaluation of online gas-phase chemistry within a regional climate model (RegCM-CHEM4)

    SciTech Connect

    Shalaby, A. K.; Zakey, A. S.; Tawfik, A. B.; Solmon, F.; Giorgi, Filippo; Stordal, F.; Sillman, S.; Zaveri, Rahul A.; Steiner, A. L.

    2012-05-22

    The RegCM-CHEM4 is a new online climate-chemistry model based on the International Centre for Theoretical Physics (ICTP) regional climate model (RegCM4). Tropospheric gas-phase chemistry is integrated into the climate model using the condensed version of the Carbon Bond Mechanism (CBM-Z; Zaveri and Peters, 1999) with a fast solver based on radical balances. We evaluate the model over Continental Europe for two different time scales: (1) an event-based analysis of the ozone episode associated with the heat wave of August 2003 and (2) a climatological analysis of a sixyear simulation (2000-2005). For the episode analysis, model simulations show good agreement with European Monitoring and Evaluation Program (EMEP) observations of hourly ozone over different regions in Europe and capture ozone concentrations during and after the August 2003 heat wave event. For long-term climate simulations, the model captures the seasonal cycle of ozone concentrations with some over prediction of ozone concentrations in non-heat wave summers. Overall, the ozone and ozone precursor evaluation shows the feasibility of using RegCM-CHEM4 for decadal-length simulations of chemistry-climate interactions.

  9. Use of RegCM gridded dataset for thunderstorm favorable conditions analysis over Poland—climatological approach

    NASA Astrophysics Data System (ADS)

    Walawender, Ewelina; Kielar, Rafał; Ustrnul, Zbigniew

    2015-09-01

    The paper analyzes equivalent data for a low density meteorological station network (spatially discontinuous data) and poor temporal homogeneity of thunderstorm observational data. Due to that, a Regional Climate Model (RegCM) dataset was tested. The Most Unstable Convective Available Potential Energy index value (MUCAPE) above the 200 J kg-1 threshold was selected as a predictor describing favorable conditions for the occurrence of thunderstorms. The quality of the dataset was examined through a comparison between model results and soundings from several aerological stations in Central Europe. Good, statistically significant (0.05 significance level) results were obtained through correlation analysis; the value of Pearson's correlation coefficient was above 0.8 in every single case. Then, using methods associated with gridded climatology, data series for 44 weather stations were derived and an analysis of correlation between RegCM modeled data and in situ thunderstorm observations was conducted with coefficients in the range of 0.75-0.90. The possibility of employing the dataset in thunderstorm climatology analysis was checked via a few examples by mapping monthly, seasonal, and annual means. Moreover, long-term variability and trend analysis along with modeled MUCAPE data were tested. As a result, the RegCM modeled MUCAPE gridded dataset was proposed as an easily available, suitable, and valuable predictor for thunderstorm climatology analysis and mapping. Finally, some limitations are discussed and recommendations for further improvements are given.

  10. Topical safety analysis report for the transportation of the NUHOMS{reg_sign} dry shielded canister. Volume 1

    SciTech Connect

    1993-08-01

    This Topical Safety Analysis Report (SAR) describes the design and the generic transportation licensing basis for utilizing the NUTECH HORIZONTAL MODULAR STORAGE (NUHOMS{reg_sign}) system dry shielded canister (DSC) containing twenty-four pressurized water reactor (PWR) spent fuel assemblies (SFA) in conjunction with a conceptually designed Transportation Cask. This SAR documents the design qualification of the NUHOMS{reg_sign} DSC as an integral part of a 10CFR71 Fissile Material Class III, Type B(M) Transportation Package. The package consists of the canister and a conceptual transportation cask (NUHOMS{reg_sign} Transportation Cask) with impact limiters. Engineering analysis is performed for the canister to confirm that the existing canister design complies with 10CFR71 transportation requirements. Evaluations and/or analyses is performed for criticality safety, shielding, structural, and thermal performance. Detailed engineering analysis for the transportation cask will be submitted in a future SAR requesting 10CFR71 certification of the complete waste package. Transportation operational considerations describe various operational aspects of the canister/transportation cask system. operational sequences are developed for canister transfer from storage to the transportation cask and interfaces with the cask auxiliary equipment for on- and off-site transport.

  11. Spectroscopic studies of three Cepheids with high positive pulsation period increments: SZ Cas, BY Cas, and RU Sct

    NASA Astrophysics Data System (ADS)

    Usenko, I. A.; Klochkova, V. G.

    2015-07-01

    Three high-resolution spectra have been taken at different times with the 6-m SAO RAS telescope (LYNX and PFES spectrographs) for three Cepheids exhibiting high positive period increments: the small-amplitude (DCEPS) SZ Cas and BY Cas and the classical (DCEP) RU Sct. SZ Cas and RU Sct are members of the Galactic open clusters χ and h Per and Trump 35, respectively. Analysis of the spectra has shown that the interstellar Na I D1 and D2 lines in all objects are considerably stronger than the atmospheric ones and are redshifted in SZ Cas and BY Cas and blushifted in RU Sct. The core of the H α absorption line in BY Cas has an asymmetric knifelike shape, while RU Sct exhibits an intense emission in the blue wing of this line. Such phenomena are observed in long-period Cepheids and bright hypergiants with an extended envelope. In this case, the strong Mg Ib 5183.62 Å and Ba II 5853.67, 6141.713, and 6496.90 Å lines with low χlow in SZ Cas and RU Sct also show characteristic knifelike profiles with an asymmetry in the red region, while the Ba II 4934.095 Å line shows similar profiles in the blue one. The absorption lines of neutral atoms and singly ionized metals with different lowerlevel excitation potentials exhibit different degrees of asymmetry: from a pronounced one with secondary components in BY Cas (similar to those in the small-amplitude Cepheid BG Cru pulsating in the first overtone and having an envelope) to its insignificance or virtual absence in SZ Cas and RU Sct. Analysis of the secular changes in mean T eff determined from photometric color indices and spectra over the last 55 years for these stars has revealed periodic fluctuations of 200 K for SZ Cas and BY Cas and 500 K for RU Sct. For SZ Cas and RU Sct, T eff determined in some years from some color indices show much lower values, which together with the temperature fluctuations can be associated with mass loss and dust formation. Based on these facts, we hypothesize the existence of

  12. Dynamic downscaling of South Asia summer monsoon precipitation over COREDEX South Asia using the regional climate model (RegCM4.3)

    NASA Astrophysics Data System (ADS)

    Hassan, Mujtaba; Pengfei, Du; Iqbal, Waheed; Ba, Wulong

    2015-04-01

    We used the latest version of Abdus Slam International Centre for Theoretical Physics (ICTP) regional climate model (RegCM4.3) in the present study to analyze the multiyear climate simulation over the CORDEX South Asia, which characterize by complex topographical features with extensive mountain ranges of Hindukush-Karakorum-Himalaya (HKH) regions. European Community-Hamburg atmospheric model (ECHAM5) and the European Centre for Medium-Range Weather Forecast (ECMWF) 40 years reanalysis data (ERA-40) are used to provide the initial and lateral boundary conditions. Two experiments hereafter referred as RegCM-ERA40 and RegCM-ECHAM5, are performed for present day climate from 1970-2000 with a horizontal resolution of 50 km. Seasonal mean climatology, Interannual variability and annual cycle are compared against the observational data. The main features of spatial distribution of South Asia Summer Monsoon (SASM) precipitation are reproduced well by RegCM4.3 than ECHAM5 GCM. Regional climate model show wet bias over south peninsular India and dry bias over the central India. However RegCM4.3 improves the simulation results for SASM precipitation as compared to both of the driving fields. Our results show that the performance of RegCM4.3 is sufficient for climate change simulation and impact assessment studies over the complex topographical domain of South Asia. Keywords: Regional climate modeling. South Asia. Summer monsoon precipitation.

  13. Pyo-pneumothorax tuberculeux: à propos de 18 cas

    PubMed Central

    Hicham, Souhi; Hanane, El Ouazzani; Hicham, Janah; Ismaïl, Rhorfi; Ahmed, Abid

    2016-01-01

    Le pyo-pneumothorax tuberculeux est une complication rare mais grave de la tuberculose pulmonaire évolutive. Nous rapportons une série de 18 cas de pyo-pneumothorax tuberculeux colligés au service de Pneumo-Phtisiologie de l'Hôpital Militaire d'Instruction Mohammed V de Rabat entre janvier 2005 et décembre 2009. Il s'agit de 15 hommes et 3 femmes d’âge moyen de 35 ans ±7 ans. 4 patients étaient diabétiques. Le tabagisme était retrouvé chez 9 cas. Le pyo-pneumothorax était du coté droit dans 13 cas. La radiographie thoracique avait montré des lésions cavitaires chez 15 patients et des lésions étendues et bilatérales chez 8 cas. La recherche de BK dans le liquide de tubage gastrique était positive chez 16 cas. Un drainage thoracique associé à un traitement antituberculeux selon le régime 2SRHZ/7RH et une kinésithérapie respiratoire ont été instaurés chez tous les cas. La durée moyenne de drainage pleural était de 4 semaines. Chez 3 cas on avait noté la persistance de la suppuration pleurale ayant nécessité une toilette pleurale sous thoracoscopie avec pleurectomie et exérèse pulmonaire limitée emportant la lésion parenchymateuse tuberculeuse et la persistance d'une volumineuse poche pleurale avec trouble ventilatoire restrictif ayant nécessité une décortication pleurale chirurgicale chez deux cas. L’évolution était favorable avec pachypleurite séquellaire minime chez le reste des cas. Le pyo-pneumothorax tuberculeux est une forme grave, qui est souvent en rapport avec une tuberculose cavitaire active. L’évolution est généralement trainante malgré le traitement antituberculeux et le drainage thoracique, d'où la nécessité d'un diagnostic et un traitement précoce de toute forme de tuberculose. PMID:27583090

  14. ENSO impacts in the South America springtime climate: present and future projections of RegCM4-CMIP5

    NASA Astrophysics Data System (ADS)

    da Rocha, Rosmeri; Dutra, Livia; Reboita, Michelle; Llopart, Marta; Coppola, Erika

    2013-04-01

    The climate variability over the South America (SA) is mainly controlled by the El Niño-Southern Oscillation (ENSO) phenomenon. ENSO positive phase (El Niño; EN) is typically associated with below (above) normal precipitation over northern and northeastern (southeastern) of SA. The opposite signal is normally observed during the ENSO negative phase (La Niña; LN). In the context of CORDEX, the objective of this study is to investigate the ENSO precipitation signal over SA in the present and future climates. For this, we used an ensemble of RegCM4 projections driven by three global models (MPI-ESM-MR, GFDL-ESM2M and HadGEM2-ES). Basically, we investigated two scenarios (rcp4.5 and rcp8.5) during austral spring (September-October-November; SON) for present (P: 1975-2005), near future (N: 2020-2050), and far future (F: 2070-2098) climates. EN and LN years are defined according to the Oceanic Niño Index (ONI) that needs to be higher (lower) than a positive (negative) threshold for least 5 consecutive over-lapping seasons. For present climate, the number of EL and LN years simulated by HadGEM2-ES has a good agreement with the observations, while MPI-ESM-MR and GFDL-ESM2M underestimate the number of EN years. All global models project an increase (decrease) of EN (LN) years in the rcp8.5 far future climate scenario - except for GFDL-ESM2M which shows a slight decrease of EN years; on the other hand, for the near future climate there is a large discrepancy in this signal. For present climate, the composites indicate that all RegCM4 simulations capture the positive anomalies of rainfall over Southeastern of SA (SESA) associated with EN years. Individual RegCM4 simulations show some differences in the spatial pattern and intensity of rainfall anomalies during EN years compared with observations. The RegCM4 simulations ensemble for present climate shows a good representation of the observed anomalies. For near and future climates, all RegCM4 projections indicate an

  15. Intra-field on-product overlay improvement by application of RegC and TWINSCAN corrections

    NASA Astrophysics Data System (ADS)

    Sharoni, Ofir; Dmitriev, Vladimir; Graitzer, Erez; Perets, Yuval; Gorhad, Kujan; van Haren, Richard; Cekli, Hakki E.; Mulkens, Jan

    2015-03-01

    The on product overlay specification and Advanced Process Control (APC) is getting extremely challenging particularly after the introduction of multi-patterning applications like Spacer Assisted Double Patterning (SADP) and multipatterning techniques like N-repetitive Litho-Etch steps (LEN, N >= 2). When the latter is considered, most of the intrafield overlay contributors drop out of the overlay budget. This is a direct consequence of the fact that the scanner settings (like dose, illumination settings, etc.) as well as the subsequent processing steps can be made very similar for two consecutive Litho-Etch layers. The major overlay contributor that may require additional attention is the Image Placement Error (IPE). When the inter-layer overlay is considered, controlling the intra-field overlay contribution gets more complicated. In addition to the IPE contribution, the TWINSCANTM lens fingerprint in combination with the exposure settings is going to play a role as well. Generally speaking, two subsequent functional layers have different exposure settings. This results in a (non-reticle) additional overlay contribution. In this paper, we have studied the wafer overlay correction capability by RegC® in addition to the TWINSCANTM intrafield corrections to improve the on product overlay performance. RegC® is a reticle intra-volume laser writing technique that causes a predictable deformation element (RegC® deformation element) inside the quartz (Qz) material of a reticle. This technique enables to post-process an existing reticle to correct for instance for IPE. Alternatively, a pre-determined intra-field fingerprint can be added to the reticle such that it results in a straight field after exposure. This second application might be very powerful to correct for instance for (cold) lens fingerprints that cannot be corrected by the scanner itself. Another possible application is the intra-field processing fingerprint. One should realize that a RegC® treatment of a

  16. Repurposing CRISPR/Cas9 for in situ functional assays.

    PubMed

    Malina, Abba; Mills, John R; Cencic, Regina; Yan, Yifei; Fraser, James; Schippers, Laura M; Paquet, Marilène; Dostie, Josée; Pelletier, Jerry

    2013-12-01

    RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.

  17. SD-CAS: Spin Dynamics by Computer Algebra System.

    PubMed

    Filip, Xenia; Filip, Claudiu

    2010-11-01

    A computer algebra tool for describing the Liouville-space quantum evolution of nuclear 1/2-spins is introduced and implemented within a computational framework named Spin Dynamics by Computer Algebra System (SD-CAS). A distinctive feature compared with numerical and previous computer algebra approaches to solving spin dynamics problems results from the fact that no matrix representation for spin operators is used in SD-CAS, which determines a full symbolic character to the performed computations. Spin correlations are stored in SD-CAS as four-entry nested lists of which size increases linearly with the number of spins into the system and are easily mapped into analytical expressions in terms of spin operator products. For the so defined SD-CAS spin correlations a set of specialized functions and procedures is introduced that are essential for implementing basic spin algebra operations, such as the spin operator products, commutators, and scalar products. They provide results in an abstract algebraic form: specific procedures to quantitatively evaluate such symbolic expressions with respect to the involved spin interaction parameters and experimental conditions are also discussed. Although the main focus in the present work is on laying the foundation for spin dynamics symbolic computation in NMR based on a non-matrix formalism, practical aspects are also considered throughout the theoretical development process. In particular, specific SD-CAS routines have been implemented using the YACAS computer algebra package (http://yacas.sourceforge.net), and their functionality was demonstrated on a few illustrative examples.

  18. SD-CAS: Spin Dynamics by Computer Algebra System

    NASA Astrophysics Data System (ADS)

    Filip, Xenia; Filip, Claudiu

    2010-11-01

    A computer algebra tool for describing the Liouville-space quantum evolution of nuclear 1/2-spins is introduced and implemented within a computational framework named Spin Dynamics by Computer Algebra System (SD-CAS). A distinctive feature compared with numerical and previous computer algebra approaches to solving spin dynamics problems results from the fact that no matrix representation for spin operators is used in SD-CAS, which determines a full symbolic character to the performed computations. Spin correlations are stored in SD-CAS as four-entry nested lists of which size increases linearly with the number of spins into the system and are easily mapped into analytical expressions in terms of spin operator products. For the so defined SD-CAS spin correlations a set of specialized functions and procedures is introduced that are essential for implementing basic spin algebra operations, such as the spin operator products, commutators, and scalar products. They provide results in an abstract algebraic form: specific procedures to quantitatively evaluate such symbolic expressions with respect to the involved spin interaction parameters and experimental conditions are also discussed. Although the main focus in the present work is on laying the foundation for spin dynamics symbolic computation in NMR based on a non-matrix formalism, practical aspects are also considered throughout the theoretical development process. In particular, specific SD-CAS routines have been implemented using the YACAS computer algebra package (http://yacas.sourceforge.net), and their functionality was demonstrated on a few illustrative examples.

  19. Measurement of Flux Density of Cas A at Low Frequencies

    NASA Astrophysics Data System (ADS)

    Patil, Ajinkya; Fisher, R.

    2012-01-01

    Cas A is used as a flux calibrator throughout the radio spectrum. Therefore it is important to know the spectral and secular variations in its flux density. Earlier observations by Scott et. al. (1969) and Baars et. al. (1972) suggested a secular decrease in flux density of Cas A at a rate of about 1% per year at all frequencies. However later observations by Erickson & Perley (1975) and Read (1977) indicated anomalously high flux from Cas A at 38 MHz. Also, these observations suggested that the original idea of faster decay of the flux density rate at low frequencies may be in error or that something more complex than simple decay is affecting the flux density at low frequencies. The source changes at 38 MHz still remains a mystery. We intend to present the results of follow up observations made from 1995 to 1998 with a three element interferometer in Green Bank operating in frequency range 30 to 120 MHz. We will discuss the problems at such low frequencies due to large beamwidth and unstable ionosphere. We will also discuss the strategies we have used so far to to find the flux density of Cas A by calculating the ratio of flux density of Cas A to that of Cyg A, assuming flux density of Cyg A to be constant. Above mentioned work was performed in summer student program sponsored by National Radio Astronomy Observatory.

  20. Rheoencephalographic (REG) Assessment of Head and Neck Cooling for use with Multiple Sclerosis Patients

    NASA Technical Reports Server (NTRS)

    Montogomery, Leslie D.; Ku, Yu-Tsuan E.; Webbon, Bruce W. (Technical Monitor)

    1995-01-01

    We have prepared a computer program (RHEOSYS:RHEOencephalographic impedance trace scanning SyStem) that can be used to automate the analysis of segmental impedance blood flow waveforms. This program was developed to assist in the post test analysis of recorded impedance traces from multiple segments of the body. It incorporates many of the blood flow, segmental volume, and vascular state indices reported in the world literature. As it is currently programmed, seven points are selected from each blood flow pulse and associated ECG waveforrn: 1. peak of the first ECG QRS complex, 2. start of systolic slope on the blood flow trace, 3. maximum amplitude of the impedance pulse, 4. position of the dicrotic notch, 5. maximum amplitude of the postdicrotic segment, 6. peak of the second ECG QRS complex, and 7. start of the next blood flow pulse. These points we used to calculate various geometric, area, and time-related values associated with the impedance pulse morphology. RHEOSYS then calculates a series of 34 impedance and cardiac cycle parameters which include pulse amplitudes; areas; pulse propagation times; cardiac cycle times; and various measures of arterial and various tone, contractility, and pulse volume. We used this program to calculate the scalp and intracranial blood flow responses to head and neck cooling as it may be applied to lower the body temperatures of multiple sclerosis patients. Twelve women and twelve men were tested using a commercially available head and neck cooling system operated at its maximum cooling capacity for a period of 30 minutes. Head and neck cooling produced a transient change in scalp blood flow and a significant, (P<0.05) decrease of approx. 30% in intracranial blood flow. Results of this experiment will illustrate how REG and RHEOSYS can be used in biomedical applications.

  1. Important considerations for radiochromic film dosimetry with flatbed CCD scanners and EBT GAFCHROMIC[reg] film

    SciTech Connect

    Lynch, Bart D.; Kozelka, Jakub; Ranade, Manisha K.; Li, Jonathan G.; Simon, William E.; Dempsey, James F.

    2006-12-15

    In this study, we present three significant artifacts that have the potential to negatively impact the accuracy and precision of film dosimetry measurements made using GAFCHROMIC[reg] EBT radiochromic film when read out with CCD flatbed scanners. Films were scanned using three commonly employed instruments: a Macbeth TD932 spot densitometer, an Epson Expression 1680 CCD array scanner, and a Microtek ScanMaker i900 CCD array scanner. For the two scanners we assessed the variation in optical density (OD) of GAFCHROMIC EBT film with scanning bed position, angular rotation of the film with respect to the scan line direction, and temperature inside the scanner due to repeated scanning. Scanning uniform radiochromic films demonstrated a distinct bowing effect in profiles in the direction of the CCD array with a nonuniformity of up to 17%. Profiles along a direction orthogonal to the CCD array demonstrated a 7% variation. A strong angular dependence was found in measurements made with the flatbed scanners; the effect could not be reproduced with the spot densitometer. An IMRT quality assurance film was scanned twice rotating the film 90 deg. between the scans. For films scanned on the Epson scanner, up to 12% variation was observed in unirradiated EBT films rotated between 0 deg. and 90 deg. , which decreased to approximately 8% for EBT films irradiated to 300 cGy. Variations of up to 80% were observed for films scanned with the Microtek scanner. The scanners were found to significantly increase the film temperature with repeated scanning. Film temperature between 18 and 33 deg. C caused OD changes of approximately 7%. Considering these effects, we recommend adherence to a strict scanning protocol that includes: maintaining the orientation of films scanned on flatbed scanners, limiting scanning to the central portion of the scanner bed, and limiting the number of consecutive scans to minimize changes in OD caused by film heating.

  2. Impact of land-use type on present climate over Romanian territory using RegCM

    NASA Astrophysics Data System (ADS)

    Bojariu, R.; Velea, L.; Birsan, M.; Dascalu, S. I.; Cica, R.

    2012-04-01

    Southern part of Romanian territory (Romanian Plain) is one of the most arid area in the country, while representing the main agricultural region with respect to cereal (wheat and maize) crops. Several local studies highlighted the increasing risk for enhanced aridity in three smaller areas in this region: Dobrogea (eastern side), South Moldova Plain (south-east) and Oltenia Plain (south-western part). A tendency for increased temperature and dry spells as well as prolonged periods with significant surface wind speed were identified in the Oltenia Plain. The main driving factors for the above-mentioned changes are both global signal of climate change and local factors. We have investigated these factors, trying to evaluate how present climate conditions would be affected if (a) the cultivated area in the Romanian Plain would be converted to natural vegetation; and (b) a more intensive agriculture would be practiced (i.e. irrigated crops). In this study, we perform a 10 years (2000-2010) simulation using regional climate model RegCM, at a horizontal resolution of 40 km, on a domain covering the entire Romanian territory, using as initial and lateral boundary conditions data from ERA-INTERIM at 0.75x0.75 deg. This larger-scale simulation is further used to provide boundary conditions for two nested experiments at 10 km, centered on a smaller area in the Romanian Plain. The simulations on the smaller domain are characterized in terms of land-use by (a) tall grass (considered as natural vegetation in the absence of crop/cultivated land) and (b) irrigated crop. The effects on the climatic conditions are evaluated with respect to precipitation, air temperature, surface wind speed (changes in spatial distribution, extremes, frequency of threshold values). Further work will assess these effects in the context of climate change scenarios, possibly providing useful information for agriculture planning.

  3. Development of internal manifold heat exchanger (IMHEX reg sign ) molten carbonate fuel cell stacks

    SciTech Connect

    Marianowski, L.G.; Ong, E.T.; Petri, R.J.; Remick, R.J.

    1991-01-01

    The Institute of Gas Technology (IGT) has been in the forefront of molten carbonate fuel cell (MCFC) development for over 25 years. Numerous cell designs have been tested and extensive tests have been performed on a variety of gas manifolding alternatives for cells and stacks. Based upon the results of these performance tests, IGT's development efforts started focusing on an internal gas manifolding concept. This work, initiated in 1988, is known today as the IMHEX{reg sign} concept. MCP has developed a comprehensive commercialization program loading to the sale of commercial units in 1996. MCP's role is in the manufacture of stack components, stack assembly, MCFC subsystem testing, and the design, marketing and construction of MCFC power plants. Numerous subscale (1 ft{sup 2}) stacks have been operated containing between 3 and 70 cells. These tests verified and demonstrated the viability of internal manifolding from technical (no carbonate pumping), engineering (relaxed part dimensional tolerance requirements), and operational (good gas sealing) aspects. Simplified fabrication, ease of assembly, the elimination of external manifolds and all associated clamping requirements has significantly lowered anticipated stack costs. Ongoing 1 ft{sup 2} stack testing is generating performance and endurance characteristics as a function of system specified operating conditions. Commercial-sized, full-area stacks (10 ft{sup 2}) are in the process of being assembled and will be tested in November. This paper will review the recent developments the MCFC scale-up and manufacture work of MCP, and the research and development efforts of IGT which support those efforts. 17 figs.

  4. Initial Husky BPU H-OIL{reg_sign} unit operations and performance

    SciTech Connect

    Faupel, T.H.; Tasker, K.G.; Bannayan, M.A.

    1995-12-31

    The Husky Bi-Provincial Upgrader (BPU) complex was successfully started up in the fall of 1992. The heart of this heavy oil upgrading plant is the H-Oil{reg_sign} unit, which utilizes ebullated-bed technology to hydrocrack and upgrade heavy atmospheric residue (400{degrees}C{sup +}) to synthetic crude oil. The H-Oil process, jointly licensed by Hydrocarbon Research, Inc. (HRI) and Texaco Development Corp. (TDC), employs daily catalyst additions and withdrawals, thereby maintaining constant catalytic activity. Since its successful start-up, the H-Oil unit, designed to process 5,088 m{sup 3}/sd (32,000 BPSD), has processed in excess of 5,850 m{sup 3}/sd (36,800 BPSD) of atmospheric residue at conversion levels in the 55 to 65 vol% range. The on-stream factor for the first year of operation has been a very respectable 81% for a new unit, which is significantly better than prior residue hydrocracking unit start-ups. The onstream factor for the second full year of operation was over 95% based on unit availability. The process performance and yields from the H-Oil unit have been in close agreement with the design values. The H-Oil unit was accepted by the BPU without a process guarantee test run which was waived. This paper summarizes the initial operations and performance of the H-Oil unit and provides a comparison of process performance and product yields between ebullated-bed laboratory units and the commercial unit.

  5. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.

    PubMed

    Zheng, Jun; Jia, Honglin; Zheng, Yonghui

    2015-02-01

    Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia and Trypanosoma. Previously, we characterised Toxoplasma gondii leucine aminopeptidase and demonstrated its role in regulating the levels of free amino acids. In this study, we evaluated the potential of T. gondii leucine aminopeptidase as a drug target in T. gondii by a knockout method. Existing knockout methods for T. gondii have many drawbacks; therefore, we developed a new technique that takes advantage of the CRISPR/Cas9 system. We first chose a Cas9 target site in the gene encoding T. gondii leucine aminopeptidase and then constructed a knockout vector containing Cas9 and the single guide RNA. After transfection, single tachyzoites were cloned in 96-well plates by limiting dilution. Two transfected strains derived from a single clone were cultured in Vero cells, and then subjected to expression analysis by western blotting. The phenotypic analysis revealed that knockout of T. gondii leucine aminopeptidase resulted in inhibition of attachment/invasion and replication; both the growth and attachment/invasion capacity of knockout parasites were restored by complementation with a synonymously substituted allele of T. gondii leucine aminopeptidase. Mouse experiments demonstrated that T. gondii leucine aminopeptidase knockout somewhat reduced the pathogenicity of T. gondii. An enzymatic activity assay showed that T. gondii leucine aminopeptidase knockout reduced the processing of a leucine aminopeptidase-specific substrate in T. gondii. The absence of leucine aminopeptidase activity could be slightly compensated for in T. gondii. Overall, T. gondii leucine aminopeptidase knockout influenced the growth of T. gondii, but did not completely block parasite development, virulence or enzymatic activity. Therefore, we conclude that leucine aminopeptidase would be useful only as an adjunctive drug target in T. gondii.

  6. CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

    PubMed

    Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

    2014-11-01

    Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. PMID:25193494

  7. CRISPR/Cas9 Genome Editing in Embryonic Stem Cells.

    PubMed

    Andrey, Guillaume; Spielmann, Malte

    2017-01-01

    Targeted mutagenesis is required to evaluate the function of DNA segments across the genome. In recent years the CRISPR/Cas9 technology has been widely used for functional genome studies and is partially replacing classical homologous recombination methods in different aspects. CRISPR/Cas9-derived tools indeed allow the production of a wide-range of engineered mutations: from point mutations to large chromosomal rearrangements such as deletions, duplications and inversions. Here we present a protocol to engineer Embryonic Stem Cells (ESC) with desired mutations using transfection of custom-made CRISPR/Cas9 vectors. These methods allow the in vivo modeling of congenital mutations and the functional interrogation of DNA sequences. PMID:27662879

  8. Genome engineering using CRISPR-Cas9 system.

    PubMed

    Cong, Le; Zhang, Feng

    2015-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

  9. CRISPR-Cas adaptation: insights into the mechanism of action.

    PubMed

    Amitai, Gil; Sorek, Rotem

    2016-02-01

    Since the first demonstration that CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR-Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR-Cas components that are involved in memory formation, and consider current models for their evolutionary origin.

  10. Harnessing CRISPR-Cas systems for bacterial genome editing.

    PubMed

    Selle, Kurt; Barrangou, Rodolphe

    2015-04-01

    Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair.

  11. CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling.

    PubMed

    Torres-Ruiz, Raul; Rodriguez-Perales, Sandra

    2015-09-14

    The cancer-modelling field is now experiencing a conversion with the recent emergence of the RNA-programmable CRISPR-Cas9 system, a flexible methodology to produce essentially any desired modification in the genome. Cancer is a multistep process that involves many genetic mutations and other genome rearrangements. Despite their importance, it is difficult to recapitulate the degree of genetic complexity found in patient tumors. The CRISPR-Cas9 system for genome editing has been proven as a robust technology that makes it possible to generate cellular and animal models that recapitulate those cooperative alterations rapidly and at low cost. In this review, we will discuss the innovative applications of the CRISPR-Cas9 system to generate new models, providing a new way to interrogate the development and progression of cancers.

  12. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... responsible for— (a) Establishing agency-specific Flight Program Standards, as defined in §§ 102-33.140... your aircraft hired as CAS; and (d) Reporting the cost and usage data for your CAS hires (for...

  13. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... responsible for— (a) Establishing agency-specific Flight Program Standards, as defined in §§ 102-33.140... your aircraft hired as CAS; and (d) Reporting the cost and usage data for your CAS hires (for...

  14. On the ability of RegCM4 regional climate model to simulate surface solar radiation patterns over Europe: an assessment using satellite-based observations

    NASA Astrophysics Data System (ADS)

    Alexandri, G.; Georgoulias, A. K.; Zanis, P.; Katragkou, E.; Tsikerdekis, A.; Kourtidis, K.; Meleti, C.

    2015-11-01

    In this work, we assess the ability of RegCM4 regional climate model to simulate surface solar radiation (SSR) patterns over Europe. A decadal RegCM4 run (2000-2009) was implemented and evaluated against satellite-based observations from the Satellite Application Facility on Climate Monitoring (CM SAF), showing that the model simulates adequately the SSR patterns over the region. The SSR bias between RegCM4 and CM SAF is +1.5 % for MFG (Meteosat First Generation) and +3.3 % for MSG (Meteosat Second Generation) observations. The relative contribution of parameters that determine the transmission of solar radiation within the atmosphere to the deviation appearing between RegCM4 and CM SAF SSR is also examined. Cloud macrophysical and microphysical properties such as cloud fractional cover (CFC), cloud optical thickness (COT) and cloud effective radius (Re) from RegCM4 are evaluated against data from CM SAF. Generally, RegCM4 underestimates CFC by 24.3 % and Re for liquid/ice clouds by 36.1 %/28.3 % and overestimates COT by 4.3 %. The same procedure is repeated for aerosol optical properties such as aerosol optical depth (AOD), asymmetry factor (ASY) and single-scattering albedo (SSA), as well as other parameters, including surface broadband albedo (ALB) and water vapor amount (WV), using data from MACv1 aerosol climatology, from CERES satellite sensors and from ERA-Interim reanalysis. It is shown here that the good agreement between RegCM4 and satellite-based SSR observations can be partially attributed to counteracting effects among the above mentioned parameters. The potential contribution of each parameter to the RegCM4-CM SAF SSR deviations is estimated with the combined use of the aforementioned data and a~radiative transfer model (SBDART). CFC, COT and AOD are the major determinants of these deviations on a monthly basis; however, the other parameters also play an important role for specific regions and seasons. Overall, for the European domain, CFC, COT and

  15. Cas9 gRNA engineering for genome editing, activation and repression

    PubMed Central

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-01-01

    We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  16. Cas9 gRNA engineering for genome editing, activation and repression.

    PubMed

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-11-01

    We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  17. Les sarcomes des tissus mous: à propos de 33 cas

    PubMed Central

    Abdou, Jiddou; Elkabous, Mustapha; M'rabti, Hind; Errihani, Hassan

    2015-01-01

    L'objectif de cette étude est de rapporter les particularités épidémiologiques, cliniques, histologiques, thérapeutiques et évolutives des sarcomes des tissus mous à l'Institut National d'Oncologie et de définir les facteurs influençant la survie des patients. C'est une étude rétrospective de 33 cas de sarcome des tissus mous, colligés entre janvier 2008 et décembre 2010. Les critères d’éligibilité étaient un âge supérieur à 16 ans, une épreuve histologique d'un sarcome des tissus mous à l'exclusion des tumeurs stromales gastro-intestinales (GIST). Les items recueillis étaient: épidémiologiques, cliniques, histologiques, Radiologiques, et thérapeutiques. Des analyses univariées puis multivariées ont été réalisées à la recherche de facteurs influençant la survie à 2 ans. Il s'agit de 33 cas, 17 Hommes et 16 Femmes, l’âge moyen était de 43,21 ans (Extrêmes= 18-76 ans). La tumeur était localisée aux extrémités dans 24 cas (72,72%). Le type histologique prédominant était le Liposarcome dans 9 cas (27,27%). Le stade tumoral était localisé dans 25 cas (75,8%), métastatique dans 8 cas (24,2%). Vingt-cinq tumeurs ont été traitées chirurgicalement dont 21 cas (84%) de chirurgie conservatrice et 4 cas (16%) de chirurgie radicale. La radiothérapie a été réalisée chez 10 patients (30,3%). La chimiothérapie a été faite chez 20 patients. En analyse univariée les facteurs pronostiques étaient l’âge (p=0,03) et le stade tumoral (p=0,09). L’âge et le stade tumoral sont des facteurs pronostiques influençant la survie des sarcomes des tissus mous. PMID:27022434

  18. Expanding the Biologist's Toolkit with CRISPR-Cas9.

    PubMed

    Sternberg, Samuel H; Doudna, Jennifer A

    2015-05-21

    Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control.

  19. Applications of CRISPR-Cas systems in neuroscience.

    PubMed

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

    Genome-editing tools, and in particular those based on CRISPR-Cas (clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research.

  20. Mouse Genome Editing using CRISPR/Cas System

    PubMed Central

    Harms, Donald W; Quadros, Rolen M; Seruggia, Davide; Ohtsuka, Masato; Takahashi, Gou

    2015-01-01

    The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much faster than the previously used techniques and more importantly multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one cell mouse embryos to create knockout or knock-in mouse models. PMID:25271839

  1. Applications of CRISPR-Cas9 mediated genome engineering.

    PubMed

    Yang, Xiao

    2015-01-01

    Targeted mutagenesis based on homologous recombination has been a powerful tool for understanding the mechanisms underlying development, normal physiology, and disease. A recent breakthrough in genome engineering technology based on the class of RNA-guided endonucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9, is further revolutionizing biology and medical studies. The simplicity of the CRISPR-Cas9 system has enabled its widespread applications in generating germline animal models, somatic genome engineering, and functional genomic screening and in treating genetic and infectious diseases. This technology will likely be used in all fields of biomedicine, ranging from basic research to human gene therapy.

  2. CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.

    PubMed

    Schiml, Simon; Fauser, Friedrich; Puchta, Holger

    2016-01-01

    The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species. To cover the concern for off-target activity, we also describe the generation of stable mutants using paired Cas9 nickases. PMID:27557689

  3. How Do Traditional Examination Questions Fare in the Presence of a Computer Algebra System (CAS)?

    ERIC Educational Resources Information Center

    Malabar, Ian; Pountney, Dave

    2001-01-01

    Describes the outcomes and discusses possible implications for the development of assessment with a Computer Algebra System (CAS) when a group of undergraduate mathematics students, familiar with using a CAS in examinations, tackled an assortment of traditional (i.e., non-CAS type) questions. (Author/MM)

  4. 48 CFR 9904.412-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CAS Pension Harmonization Rule. 9904.412-64.1 Section 9904.412-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. Contractors or subcontractors that become subject... through 7 Notes (Note 1) Actuarial Accrued Liability $2,470,500 $14,225,000 2 CAS Actuarial Value...

  5. 48 CFR 9904.412-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CAS Pension Harmonization Rule. 9904.412-64.1 Section 9904.412-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. Contractors or subcontractors that become subject... through 7 Notes (Note 1) Actuarial Accrued Liability $2,470,500 $14,225,000 2 CAS Actuarial Value...

  6. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  7. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  8. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  9. 76 FR 40817 - Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-12

    ... to the CAS Applicability Threshold for the Inflation Adjustment to the Truth in Negotiations Act... revising the threshold for the application of CAS from ``$650,000'' to ``the Truth in Negotiations Act... are open for comment. Simply type ``CAS-TINA Threshold'' (without quotation marks) in the Comment...

  10. On the Integration of Computer Algebra Systems (CAS) by Canadian Mathematicians: Results of a National Survey

    ERIC Educational Resources Information Center

    Buteau, Chantal; Jarvis, Daniel H.; Lavicza, Zsolt

    2014-01-01

    In this article, we outline the findings of a Canadian survey study (N = 302) that focused on the extent of computer algebra systems (CAS)-based technology use in postsecondary mathematics instruction. Results suggest that a considerable number of Canadian mathematicians use CAS in research and teaching. CAS use in research was found to be the…

  11. Mismatch Negativity Responses in Children with a Diagnosis of Childhood Apraxia of Speech (CAS)

    ERIC Educational Resources Information Center

    Froud, Karen; Khamis-Dakwar, Reem

    2012-01-01

    Purpose: To evaluate whether a hypothesis suggesting that apraxia of speech results from phonological overspecification could be relevant for childhood apraxia of speech (CAS). Method: High-density EEG was recorded from 5 children with CAS and 5 matched controls, ages 5-8 years, with and without CAS, as they listened to randomized sequences of CV…

  12. The Impact on Student Achievement of When CAS Technology Is Introduced

    ERIC Educational Resources Information Center

    Driver, David

    2012-01-01

    When a Computer Algebra System (CAS) is used as a pedagogical and functional tool in class and as a functional tool in exams, its effect on student achievement can be quite profound. The timing of when students are first introduced to a CAS has an impact on gains in student achievement. In this action research project, the CAS calculator was…

  13. RegCM4-HadGEM2-ES simulated cyclone climatology (1979-2005) over the Southwestern South Atlantic Ocean

    NASA Astrophysics Data System (ADS)

    Porfírio da Rocha, Rosmeri; Simões Reboita, Michelle

    2015-04-01

    Cyclones over the Southwestern South Atlantic Ocean (SAO) are a subject of great interest once they modify the weather and control the climate near east coast of South America (SA). In this study we compare the cyclones climatology in the period 1979-2005 simulated by Regional Climate Model version 4 (RegCM4) with that from ERA-Interim reanalysis (ECMWF). RegCM4 was nested in HadGEM2-ES output and the simulation used the SA domain of CORDEX project, with a horizontal grid of 50 km and 18 sigma-pressure levels in the vertical. The RegCM4 simulation used the land surface Biosphere-Atmosphere Transfer Scheme (BATS) and the mixed convection Emanuel-Grell scheme configurations. This simulation is part of the CREMA (CORDEX REgCM4 hyper-MAtrix) experiment. The cyclones were identified using an automated tracking scheme based on minima (cyclonic in Southern Hemisphere) of relative vorticity from the wind at 925 hPa. The threshold of -1.5 x 10-5s-1 was used in the algorithm. All cyclones in RegCM4 and ERA-Interim with relative vorticity lower than this threshold and with lifetime higher or equal 24 hours were included in the climatology. ERA-Interim shows three main cyclogenetic regions near east coast of SA. In general, RegCM4 simulated these same regions but with an underestimation of the number of cyclones. In each of these regions, there is a different season of higher cyclones frequency. Over extreme south of southern Brazil and Uruguay the higher frequency of cyclones occurs in winter, while southeastern Brazil and southeastern Argentina cyclones are most frequent during summer. RegCM4 is able to simulate this observed seasonality.

  14. Pancreatic islet-specific overexpression of Reg3β protein induced the expression of pro-islet genes and protected the mice against streptozotocin-induced diabetes mellitus.

    PubMed

    Xiong, Xiaoquan; Wang, Xiao; Li, Bing; Chowdhury, Subrata; Lu, Yarong; Srikant, Coimbatore B; Ning, Guang; Liu, Jun-Li

    2011-04-01

    Reg family proteins have been implicated in islet β-cell proliferation, survival, and regeneration. The expression of Reg3β (pancreatitis-associated protein) is highly induced in experimental diabetes and acute pancreatitis, but its precise role has not been established. Through knockout studies, this protein was shown to be mitogenic, antiapoptotic, and anti-inflammatory in the liver and pancreatic acinars. To test whether it can promote islet cell growth or survival against experimental damage, we developed β-cell-specific overexpression using rat insulin I promoter, evaluated the changes in normal islet function, gene expression profile, and the response to streptozotocin-induced diabetes. Significant and specific overexpression of Reg3β was achieved in the pancreatic islets of RIP-I/Reg3β mice, which exhibited normal islet histology, β-cell mass, and in vivo and in vitro insulin secretion in response to high glucose yet were slightly hyperglycemic and low in islet GLUT2 level. Upon streptozotocin treatment, in contrast to wild-type littermates that became hyperglycemic in 3 days and lost 15% of their weight, RIP-I/Reg3β mice were significantly protected from hyperglycemia and weight loss. To identify specific targets affected by Reg3β overexpression, a whole genome DNA microarray on islet RNA isolated from the transgenic mice revealed more than 45 genes significantly either up- or downregulated. Among them, islet-protective osteopontin/SPP1 and acute responsive nuclear protein p8/NUPR1 were significantly induced, a result further confirmed by real-time PCR, Western blots, and immunohistochemistry. Our results suggest that Reg3β is unlikely an islet growth factor but a putative protector that prevents streptozotocin-induced damage by inducing the expression of specific genes.

  15. On the ability of RegCM4 regional climate model to simulate surface solar radiation patterns over Europe: an assessment using satellite-based observations

    NASA Astrophysics Data System (ADS)

    Alexandri, G.; Georgoulias, A. K.; Zanis, P.; Katragkou, E.; Tsikerdekis, A.; Kourtidis, K.; Meleti, C.

    2015-07-01

    In this work, we assess the ability of RegCM4 regional climate model to simulate surface solar radiation (SSR) patterns over Europe. A decadal RegCM4 run (2000-2009) was implemented and evaluated against satellite-based observations from the Satellite Application Facility on Climate Monitoring (CM SAF) showing that the model simulates adequately the SSR patterns over the region. The bias between RegCM4 and CM SAF is +1.54 % for MFG (Meteosat First Generation) and +3.34 % for MSG (Meteosat Second Generation) observations. The relative contribution of parameters that determine the transmission of solar radiation within the atmosphere to the deviation appearing between RegCM4 and CM SAF SSR is also examined. Cloud macrophysical and microphysical properties such as cloud fractional cover (CFC), cloud optical thickness (COT) and cloud effective radius (Re) from RegCM4 are evaluated against data from CM SAF. The same procedure is repeated for aerosol optical properties such as aerosol optical depth (AOD), asymmetry factor (ASY) and single scattering albedo (SSA), as well as other parameters including surface broadband albedo (ALB) and water vapor amount (WV) using data from MACv1 aerosol climatology, from CERES satellite sensors and from ERA-Interim reanalysis. It is shown here that the good agreement between RegCM4 and satellite-based SSR observations can be partially attributed to counteracting effects among the above mentioned parameters. The contribution of each parameter to the RegCM4-CM SAF SSR deviations is estimated with the combined use of the aforementioned data and a radiative transfer model (SBDART). CFC, COT and AOD are the major determinants of these deviations; however, the other parameters also play an important role for specific regions and seasons.

  16. RNA Targeting by the Type III-A CRISPR-Cas Csm Complex of Thermus thermophilus

    PubMed Central

    Staals, Raymond H. J.; Zhu, Yifan; Taylor, David W.; Kornfeld, Jack E.; Sharma, Kundan; Barendregt, Arjan; Koehorst, Jasper J.; Vlot, Marnix; Neupane, Nirajan; Varossieau, Koen; Sakamoto, Keiko; Suzuki, Takehiro; Dohmae, Naoshi; Yokoyama, Shigeyuki; Schaap, Peter J.; Urlaub, Henning; Heck, Albert J. R.; Nogales, Eva; Doudna, Jennifer A.; Shinkai, Akeo; van der Oost, John

    2015-01-01

    SUMMARY CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1–Csm5) with an uneven stoichiometry and a single crRNA of variable size (35–53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence. PMID:25457165

  17. Apport de l'imagerie dans le diagnostic des sacroiliites infectieuses : à propos de 19 cas

    PubMed Central

    Abid, Hanen; Chaabouni, Salim; Frikha, Faten; Toumi, Nozha; Souissi, Basma; Lahiani, Dorra; Bahloul, Zouhir; Ben Mahfoudh, Khaireddine

    2014-01-01

    Les sacro-iliites infectieuses méritent d’être mieux connues. Leur diagnostic est souvent retardé en raison d'une symptomatologie trompeuse et des diffcultés d'exploration de l'articulation sacro-iliaque. Notre travail est basé sur une étude rétrospective portant sur les cas de SII, recueillis sur une période comprise entre 1997 et 2008 dans notre centre universitaire Sfax-Tunisie. Le diagnostic de sacro-iliite était retenu en présence d'arguments cliniques et radiologiques d'atteinte sacroiliaque. Nous rapportons dix neuf cas de sacroiliites infectieuses (10 hommes et 9 femmes), avec un âge moyen de 32 ans. L'atteinte était unilatérale dans tous les cas. Les radiographies standard faites dans tous les cas ont été suggestives dans 14 cas et normales dans les autres cas. La TDM faite dans 13 cas a montré, un abcès des parties molles dans 8 cas et un séquestre osseux dans 2 cas. L'IRM réalisée dans 8 cas, a objectivé une infiltration des parties molles dans tous les cas et un abcès dans 3 cas. Le germe a été identifié dans 9 cas (3 cas de tuberculose, 3 cas de brucellose, 2 sacro-iliites à pyogène et un cas de candidose). Cette identification était faite par biopsie dans 3 cas, hémocultures dans 2 cas, prélèvement au niveau de la porte d'entrée dans 1 cas et sérodiagnostic dans 3 cas. Pour les autres cas, l'origine pyogène a été retenue sur des arguments cliniques et biologiques. L'imagerie joue un rôle primordial dans le diagnostic précoce et l'orientation étiologique d'une sacroiliite infectieuse. PMID:25120884

  18. 40 CFR 721.650 - 11-Aminoundecanoic acid.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false 11-Aminoundecanoic acid. 721.650... Substances § 721.650 11-Aminoundecanoic acid. (a) Chemical substance and significant new use subject to reporting. (1) The chemical substance 11-ami-no-un-de-ca-noic acid, CAS Number 2432-99-7, is subject...

  19. 40 CFR 721.10066 - 1-Hexanesulfonic acid, (dimethylphenyl)-.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false 1-Hexanesulfonic acid, (dimethylphenyl... Specific Chemical Substances § 721.10066 1-Hexanesulfonic acid, (dimethylphenyl)-. (a) Chemical substance...-hexanesulfonic acid, (dimethylphenyl)- (PMN P-04-962; CAS No. 676143-36-5) is subject to reporting under...

  20. 40 CFR 721.650 - 11-Aminoundecanoic acid.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false 11-Aminoundecanoic acid. 721.650... Substances § 721.650 11-Aminoundecanoic acid. (a) Chemical substance and significant new use subject to reporting. (1) The chemical substance 11-ami-no-un-de-ca-noic acid, CAS Number 2432-99-7, is subject...

  1. 40 CFR 721.650 - 11-Aminoundecanoic acid.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false 11-Aminoundecanoic acid. 721.650... Substances § 721.650 11-Aminoundecanoic acid. (a) Chemical substance and significant new use subject to reporting. (1) The chemical substance 11-ami-no-un-de-ca-noic acid, CAS Number 2432-99-7, is subject...

  2. 40 CFR 721.650 - 11-Aminoundecanoic acid.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false 11-Aminoundecanoic acid. 721.650... Substances § 721.650 11-Aminoundecanoic acid. (a) Chemical substance and significant new use subject to reporting. (1) The chemical substance 11-ami-no-un-de-ca-noic acid, CAS Number 2432-99-7, is subject...

  3. 40 CFR 721.10065 - 1-Tetradecanesulfonic acid, (dimethylphenyl)-.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false 1-Tetradecanesulfonic acid... Specific Chemical Substances § 721.10065 1-Tetradecanesulfonic acid, (dimethylphenyl)-. (a) Chemical...-tetradecanesulfonic acid, (dimethylphenyl)- (PMN P-04-961; CAS No. 671756-61-9) is subject to reporting under...

  4. 40 CFR 721.2584 - Dodecanoic acid, 12-amino-.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Dodecanoic acid, 12-amino-. 721.2584... Substances § 721.2584 Dodecanoic acid, 12-amino-. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as dodecanoic acid, 12-amino- (PMN P-98-0823; CAS No....

  5. 40 CFR 721.2584 - Dodecanoic acid, 12-amino-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Dodecanoic acid, 12-amino-. 721.2584... Substances § 721.2584 Dodecanoic acid, 12-amino-. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as dodecanoic acid, 12-amino- (PMN P-98-0823; CAS No....

  6. 40 CFR 721.2584 - Dodecanoic acid, 12-amino-.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Dodecanoic acid, 12-amino-. 721.2584... Substances § 721.2584 Dodecanoic acid, 12-amino-. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as dodecanoic acid, 12-amino- (PMN P-98-0823; CAS No....

  7. 40 CFR 721.2584 - Dodecanoic acid, 12-amino-.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Dodecanoic acid, 12-amino-. 721.2584... Substances § 721.2584 Dodecanoic acid, 12-amino-. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as dodecanoic acid, 12-amino- (PMN P-98-0823; CAS No....

  8. 40 CFR 721.2584 - Dodecanoic acid, 12-amino-.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Dodecanoic acid, 12-amino-. 721.2584... Substances § 721.2584 Dodecanoic acid, 12-amino-. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as dodecanoic acid, 12-amino- (PMN P-98-0823; CAS No....

  9. Evaluation of freshwater sediment quality values including apparent effects thresholds of Hyalella azteca and Microtox{reg_sign}

    SciTech Connect

    Cubbage, J.; Batts, D.

    1995-12-31

    Data from 36 studies in Washington State were merged into a single database to derive Apparent Effects Thresholds (AETS) for freshwater sediments and to evaluate other sediment quality values. Sediments from 235 sites were analyzed for several contaminants, Bioassays tested at 30 or more of these sites included Hyalella azteca, Daphnia magna and Chironomus tentans mortality, Ceriodaphnia dubia reproduction and growth, and Microtox{reg_sign} luminescence reduction. Adequate sample size (n > 50) and wide range of response were available for Hyalella (n = 235) and Microtox{reg_sign} (n = 61) to determine AETs for organic carbon normalized PAHs, pesticides, and PCBs, and dry weight normalized metals, phenols and chlorinated phenols. The ability of these AETs to predict response in any bioassay at the sites in the database was compared with Ontario Ministry Environment and Energy (OMEE) Sediment Quality Guidelines and the Environment Canada (EC) Interim Sediment Quality Values. Measures of sensitivity (number correctly predicted/number impacted) and efficiency (number correctly predicted/number predicted) were compared. The EC Threshold Effects Level (TEL) was the most sensitive (fewest Type 2 errors) at 92%, but the least efficient (most Type 1 errors) at 35%. The lowest AET (LAET) of Hyalella and Microtox{reg_sign}, derived from the current study, was the next most sensitive (77%) and more efficient (44%). The OMEE Severe Effects Level was the least sensitive (59%) and the most efficient (53%). The EC Probable Effects Level (PEL) was 50% efficient and 72% sensitive. Of the four sets of sediment quality values, the OMEE Severe Effects Level had the highest overall reliability (number of correct predictions of effects and non-effects/number of sites examined) in predicting bioassay effects in the Washington State freshwater sediment database.

  10. Evaluation of the capability of RegCM4.0 in simulating East African climate

    NASA Astrophysics Data System (ADS)

    Ogwang, Bob Alex; Chen, Haishan; Li, Xing; Gao, Chujie

    2016-04-01

    The International Centre for Theoretical Physics (ICTP) regional climate model RegCM4.0 is used in this study to examine its ability to reproduce the climate of East Africa (EA) in regard to the annual cycle and June-to-August (JJA) seasonal climatology. Two domain sizes [large domain (LD) and small domain (SD)] and two cumulus convection schemes [Grell convection scheme with Fritsch-Chappell closure assumption (GRE scheme) and MIT scheme (EMA scheme)] are used. Simulations were done for the period 1989-2008 at a resolution of 50 km. The experiments were performed with the initial and lateral boundary conditions obtained from ERA-Interim-gridded reanalysis data at a 1.5° resolution. The variables investigated are precipitation, temperature, humidity, diurnal temperature range, and 850-hPa winds. Results show that the model realistically reproduces the East African climate, with a few discrepancies due to the different cumulus convection schemes and the domain sizes used. Grell with Fritsch-Chappell (Grell-FC) scheme captures well the observed climate in regard to the annual cycle and June-to-August seasonal climatology, with a tendency to underestimate rainfall over the JJA rainfall maximum region (RMR). This scheme performs better in LD than in SD. EMA scheme similarly captures well the observed climatology. It tends to overestimate rainfall over RMR. It however performs better in SD than in LD. The ensemble mean of simulations with GRE and EMA schemes (ENSM) tends to offer an improved representation of the observed climate, with a few discrepancies owing to the individual schemes used. In general, therefore, considering the performance of the model in both domains, the East African climate based on this study is better simulated by the Grell-FC scheme over LD. The observed biases in this study signify that the ability of the model in simulating climate over East Africa is still a significant challenge. Thus, future work must focus on improving the performance of

  11. RegCM4 Dynamical Downscaling of Seasonal Climate Predictions over the Southeast of Brazil

    NASA Astrophysics Data System (ADS)

    Reboita, Michelle S.; Dutra, Lívia M. M.; da Rocha, Rosmeri P.

    2013-04-01

    In this study the Regional Climate Model version 4 (RegCM4) was nested in the General Circulation Model from the Brazilian Center for Weather Forecasts and Climate Studies (CPTEC) to produce three month (seasonal) predictions to the southeast of Brazil (SB). The predictions for MAM (March-April-May), AMJ (April-May-June) and SON (September-October-November) of 2012 used six different parameterizations of convection: 1) Grell with Arakawa-Schubert (GAS) closure, 2) Grell with Fritsch-Chappell (GFC) closure, 3) Kuo, 4) Emanuel (EM), 5) Mixed-1 with GFC and Emanuel schemes over the land and ocean, respectively and 6) Mixed-2 with Emanuel and GFC over the land and ocean, respectively. The simulations started 48 days before the seasons of interest to permit a spin-up period. The predicted precipitation was compared with observation from Climate Prediction Center (CPC). For MAM/2012, the experiment with Kuo scheme presented the seasonal precipitation similar to CPC, while GAS, GFC and Mixed-1 experiments underestimated the precipitation (~1-2 mm/day) over the center of SB and EM and Mixed-2 schemes overestimated it (~4 mm/day) over most part of SB. For AMJ/2012 all experiments underestimated the precipitation (~2-3 mm/day) in the central-south part of SB, but they simulated the precipitation intensity close to the observation over the center-north SB (except the EM which shows overestimation in this area). For this season, Mixed-1 presented the smaller bias compared to the other convective schemes. For AMJ/2012, the experiments underestimated the precipitation (~2-4 mm/day) over the center-north of SB and overestimated it (~2-4 mm/day) in the eastern sector. In this period, the EM and Mixed-1 predictions presented the smaller bias compared to CPC. Considering the three seasons, this study suggests that the best convective scheme to seasonal predictions for SB is Mixed-1, while GFC, GAS and Kuo also produce satisfactory seasonal precipitation.

  12. Estimates of common ragweed pollen emission and dispersion over Europe using RegCM-pollen model

    NASA Astrophysics Data System (ADS)

    Liu, L.; Solmon, F.; Vautard, R.; Hamaoui-Laguel, L.; Torma, Cs. Zs.; Giorgi, F.

    2015-11-01

    Common ragweed (Ambrosia artemisiifolia L.) is a highly allergenic and invasive plant in Europe. Its pollen can be transported over large distances and has been recognized as a significant cause of hayfever and asthma (D'Amato et al., 2007; Burbach et al., 2009). To simulate production and dispersion of common ragweed pollen, we implement a pollen emission and transport module in the Regional Climate Model (RegCM) version 4 using the framework of the Community Land Model (CLM) version 4.5. In the online model environment where climate is integrated with dispersion and vegetation production, pollen emissions are calculated based on the modelling of plant distribution, pollen production, species-specific phenology, flowering probability, and flux response to meteorological conditions. A pollen tracer model is used to describe pollen advective transport, turbulent mixing, dry and wet deposition. The model is then applied and evaluated on a European domain for the period 2000-2010. To reduce the large uncertainties notably due to ragweed density distribution on pollen emission, a calibration based on airborne pollen observations is used. Resulting simulations show that the model captures the gross features of the pollen concentrations found in Europe, and reproduce reasonably both the spatial and temporal patterns of flowering season and associated pollen concentrations measured over Europe. The model can explain 68.6, 39.2, and 34.3 % of the observed variance in starting, central, and ending dates of the pollen season with associated root mean square error (RMSE) equal to 4.7, 3.9, and 7.0 days, respectively. The correlation between simulated and observed daily concentrations time series reaches 0.69. Statistical scores show that the model performs better over the central Europe source region where pollen loads are larger. From these simulations health risks associated common ragweed pollen spread are then evaluated through calculation of exposure time above health

  13. Regional modeling of Saharan dust events using the RegCM model

    NASA Astrophysics Data System (ADS)

    Santese, M.; Perrone, M. R.; Zakey, A.

    2009-04-01

    As one of the major components of the atmospheric aerosol, mineral dust plays an important role in the Earth's climate system. Dust has been found to redistribute the radiative energy from the surface to the dust loaded atmospheric column by cooling the surface while heating the dust layer. The resulting stabilizing effect on the vertical structure of the atmosphere can affect cloud formation and the dust production itself. In addition, dust may change the size number of cloud condensation nuclei (CCN) and thus the optical and precipitation properties of clouds. All these impacts are difficult to quantify due to the highly variable spatio-temporal distribution of mineral dust and uncertainties determining its optical and physicochemical properties (IPCC 2001). The distribution of dust has been modeled in many studies using general circulation models (GCMs). However, because the aerosol effects are especially important at the regional scale, the recent development of high-resolution regional climate models (RCMs) offers useful tools to assess the regional impacts of aerosols. Compared to global climate models (GCMs), the relatively high-resolution and detailed physical parameterizations by RCMs are particularly suitable to describe the complexity of aerosol processes (Solmon et al., 2006). Furthermore, the results from regional models are well suited for comparisons with measurements of individual events. Dust radiative effects on climate are likely to be especially important at the regional scale, thus RCMs can be particularly useful tools to investigate the regional climate effects of dust outbreaks (Zakey et al., 2006). In this work, we will use the regional climate model RegCM (Version 3.1), developed at the Abdus Salam International Centre for Theoretical Physics (ICTP), Trieste, to investigate dust event impacts over Mediterranean sites. The Sahara desert is the largest dust source on Earth, providing at least half of the emitted dust (Washington et al., 2003

  14. 40 CFR 721.10111 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and diethylene glycol.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... diesters with benzoic acid and diethylene glycol. 721.10111 Section 721.10111 Protection of Environment... diesters with benzoic acid and diethylene glycol. (a) Chemical substance and significant new uses subject... benzoic acid and diethylene glycol (PMN P-04-510; CAS No. 610787-78-5) is subject to reporting under...

  15. 40 CFR 721.10110 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and neopentlyl glycol.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... diesters with benzoic acid and neopentlyl glycol. 721.10110 Section 721.10110 Protection of Environment... diesters with benzoic acid and neopentlyl glycol. (a) Chemical substance and significant new uses subject... benzoic acid and neopentlyl glycol (PMN P-04-509; CAS No. 610787-77-4) is subject to reporting under...

  16. 40 CFR 721.10111 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and diethylene glycol.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... diesters with benzoic acid and diethylene glycol. 721.10111 Section 721.10111 Protection of Environment... diesters with benzoic acid and diethylene glycol. (a) Chemical substance and significant new uses subject... benzoic acid and diethylene glycol (PMN P-04-510; CAS No. 610787-78-5) is subject to reporting under...

  17. 40 CFR 721.10110 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and neopentlyl glycol.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... diesters with benzoic acid and neopentlyl glycol. 721.10110 Section 721.10110 Protection of Environment... diesters with benzoic acid and neopentlyl glycol. (a) Chemical substance and significant new uses subject... benzoic acid and neopentlyl glycol (PMN P-04-509; CAS No. 610787-77-4) is subject to reporting under...

  18. 40 CFR 721.10111 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and diethylene glycol.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... diesters with benzoic acid and diethylene glycol. 721.10111 Section 721.10111 Protection of Environment... diesters with benzoic acid and diethylene glycol. (a) Chemical substance and significant new uses subject... benzoic acid and diethylene glycol (PMN P-04-510; CAS No. 610787-78-5) is subject to reporting under...

  19. 40 CFR 721.10111 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and diethylene glycol.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... diesters with benzoic acid and diethylene glycol. 721.10111 Section 721.10111 Protection of Environment... diesters with benzoic acid and diethylene glycol. (a) Chemical substance and significant new uses subject... benzoic acid and diethylene glycol (PMN P-04-510; CAS No. 610787-78-5) is subject to reporting under...

  20. 40 CFR 721.10110 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and neopentlyl glycol.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... diesters with benzoic acid and neopentlyl glycol. 721.10110 Section 721.10110 Protection of Environment... diesters with benzoic acid and neopentlyl glycol. (a) Chemical substance and significant new uses subject... benzoic acid and neopentlyl glycol (PMN P-04-509; CAS No. 610787-77-4) is subject to reporting under...

  1. 40 CFR 721.10110 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and neopentlyl glycol.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... diesters with benzoic acid and neopentlyl glycol. 721.10110 Section 721.10110 Protection of Environment... diesters with benzoic acid and neopentlyl glycol. (a) Chemical substance and significant new uses subject... benzoic acid and neopentlyl glycol (PMN P-04-509; CAS No. 610787-77-4) is subject to reporting under...

  2. 40 CFR 721.10110 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and neopentlyl glycol.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... diesters with benzoic acid and neopentlyl glycol. 721.10110 Section 721.10110 Protection of Environment... diesters with benzoic acid and neopentlyl glycol. (a) Chemical substance and significant new uses subject... benzoic acid and neopentlyl glycol (PMN P-04-509; CAS No. 610787-77-4) is subject to reporting under...

  3. 40 CFR 721.10111 - Hexanoic acid, 2-ethyl-, mixed diesters with benzoic acid and diethylene glycol.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... diesters with benzoic acid and diethylene glycol. 721.10111 Section 721.10111 Protection of Environment... diesters with benzoic acid and diethylene glycol. (a) Chemical substance and significant new uses subject... benzoic acid and diethylene glycol (PMN P-04-510; CAS No. 610787-78-5) is subject to reporting under...

  4. Quantitative orientational analysis of a polymeric material (Kevlar{sup {reg_sign}} fibers) with x-ray microspectroscopy

    SciTech Connect

    Smith, A.P.; Ade, H.

    1996-12-01

    It has previously been shown that x-ray linear dichroism microscopy can be utilized to image and determine orientation in a polymeric material at high spatial resolution. We have now expanded on this technique and extracted quantitative information about the orientation of specific functional groups in a polymeric system from submicron areas. This is accomplished by acquiring and analyzing spectral data sets rather than just images at specific energies. It has allowed us to compare the relative lateral orientation of various grades of Kevlar{sup {reg_sign}} fibers. {copyright} {ital 1996 American Institute of Physics.}

  5. Genome Editing with CRISPR-Cas9: Can It Get Any Better?

    PubMed

    Haeussler, Maximilian; Concordet, Jean-Paul

    2016-05-20

    The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.

  6. CRISPR-Cas9 Genome Editing in Drosophila.

    PubMed

    Gratz, Scott J; Rubinstein, C Dustin; Harrison, Melissa M; Wildonger, Jill; O'Connor-Giles, Kate M

    2015-01-01

    The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining (NHEJ) or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target-site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification, and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags. PMID:26131852

  7. Preparing Students to Take SOA/CAS Exam FM/2

    ERIC Educational Resources Information Center

    Marchand, Richard J.

    2014-01-01

    This paper provides suggestions for preparing students to take the actuarial examination on financial mathematics, SOA/CAS Exam FM/2. It is based on current practices employed at Slippery Rock University, a small public liberal arts university. Detailed descriptions of our Theory of Interest course and subsequent Exam FM/2 prep course are provided…

  8. New Insight in Mathematics by Live CAS Documents.

    ERIC Educational Resources Information Center

    Cnop, Ivan

    Traditional education in mathematics is mostly a matter of hindsight, and many mathematics texts offer little opportunity for students and learners to gain insight. This paper shows how experiments in CAS (computer algebra systems) can lead to new ways of handling problems, new conjectures, new visualizations, new proofs, new correspondences…

  9. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY SUPPLEMENTARY REGULATIONS DOE MANAGEMENT AND OPERATING CONTRACTS Cost Accounting Standards Administration...

  10. Cas9-based genome editing in Arabidopsis and tobacco.

    PubMed

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2014-01-01

    Targeted modification of plant genome is key to elucidating and manipulating gene functions in plant research and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome-editing method in diverse plants that traditionally lacked facile and versatile tools for targeted genetic engineering. This technology utilizes easily reprogrammable guide RNAs (sgRNAs) to direct Streptococcus pyogenes Cas9 endonuclease to generate DNA double-stranded breaks in targeted genome sequences, which facilitates efficient mutagenesis by error-prone nonhomologous end-joining (NHEJ) or sequence replacement by homology-directed repair (HDR). In this chapter, we describe the procedure to design and evaluate dual sgRNAs for plant codon-optimized Cas9-mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applications for generating targeted genome modifications and gene regulations in plants.

  11. Targeted mutagenesis in chicken using CRISPR/Cas9 system

    PubMed Central

    Oishi, Isao; Yoshii, Kyoko; Miyahara, Daichi; Kagami, Hiroshi; Tagami, Takahiro

    2016-01-01

    The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens. PMID:27050479

  12. Does CAS Use Disadvantage Girls in VCE Mathematics?

    ERIC Educational Resources Information Center

    Forgasz, Helen; Tan, Hazel

    2010-01-01

    In 2009, four mathematics subjects were offered at the year 12 level in the Victorian Certificate of Education (VCE). The two subjects at the intermediate level--Mathematical Methods and Mathematical Methods CAS--run in parallel, that is, a student can be enrolled in only one or the other, the choice being made at the school level. The curricular…

  13. Prospective Mathematics Teachers' Interactions with CAS-Based Textbook Elements

    ERIC Educational Resources Information Center

    Davis, Jon D.

    2015-01-01

    This study investigated how a group of 10 prospective secondary mathematics teachers (PST) read, evaluated, and adapted a textbook lesson involving the symbolic manipulation capabilities of computer algebra systems (CASS). PST read the entire lesson and tended to focus on the organizing question at the beginning of the student lesson and the CAS-S…

  14. Toward Whole-Transcriptome Editing with CRISPR-Cas9.

    PubMed

    Heckl, Dirk; Charpentier, Emmanuelle

    2015-05-21

    Targeted regulation of gene expression holds huge promise for biomedical research. In a series of recent publications (Gilbert et al., 2014; Konermann et al., 2015; Zalatan et al., 2015), sophisticated, multiplex-compatible transcriptional activator systems based on the CRISPR-Cas9 technology and genome-scale libraries advance the field toward whole-transcriptome control. PMID:26000839

  15. CRISPR/Cas9 advances engineering of microbial cell factories.

    PubMed

    Jakočiūnas, Tadas; Jensen, Michael K; Keasling, Jay D

    2016-03-01

    One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering.

  16. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY SUPPLEMENTARY REGULATIONS DOE MANAGEMENT AND OPERATING CONTRACTS Cost Accounting Standards Administration...

  17. Equivalent Expressions Using CAS and Paper-and-Pencil Techniques

    ERIC Educational Resources Information Center

    Fonger, Nicole L.

    2014-01-01

    How can the key concept of equivalent expressions be addressed so that students strengthen their representational fluency with symbols, graphs, and numbers? How can research inform the synergistic use of both paper-and-pencil analysis and computer algebra systems (CAS) in a classroom learning environment? These and other related questions have…

  18. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    PubMed

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

  19. Medical marijuana: CAS releases report, government cuts research funding.

    PubMed

    Betteridge, Glenn

    2006-12-01

    In June 2006, the Canadian AIDS Society (CAS) released a comprehensive report with recommendations to overcome barriers to the use of cannabis for medical purposes faced by people living with HIV/AIDS in Canada. On 25 September 2006, as part of package of spending cuts, the federal government announced plans to eliminate its marijuana medical research program. PMID:17373064

  20. Problem Solving in Calculus with Symbolic Geometry and CAS

    ERIC Educational Resources Information Center

    Todd, Philip; Wiechmann, James

    2008-01-01

    Computer algebra systems (CAS) have been around for a number of years, as has dynamic geometry. Symbolic geometry software is new. It bears a superficial similarity to dynamic geometry software, but differs in that problems may be set up involving symbolic variables and constants, and measurements are given as symbolic expressions. Mathematical…