bluuebunny/crossref_metadata_2025 huggingface dataset:
86 CREATION OF PORCINE TRANSGENIC NUCLEAR-TRANSFERRED EMBRYOS RECONSTRUCTED WITH EGFP-EXPRESSING ADULT DERMAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS
Authors: Skrzyszowska M. , Samiec M. , Smorag Z. , Lipinski D. , Slomski R.
Published: December 2006

The purpose of our study was to determine the in vitro developmental competences of porcine nuclear transfer (NT) embryos reconstructed with pWAPhGH-GFPBsd transgene-nucleofected gilt ear skin-descended fibroblast cells, which had been diagnosed on apoptosis through the live-plasma membrane fluorescent tagging. Frozen–thawed fibroblast cells, which had been in vitro-cultured up to a total confluency after 2–8 passages, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, single nuclear donor cell suspension was labeled with the conjugate of Annexin V and eGFP protein. The source of recipient cells were in vitro-matured oocytes. Maternal chromosomes were eliminated by a chemically assisted microsurgical technique. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of adult dermal fibroblast cells, it was shown that a relatively high proportion (ranging from 20 to 30%) of donor cells exhibited ultrastructural late-apoptotic or necrotic changes. In contrast, from among the morphologically normal cells, an extremely low rate (ranging from 0 to 2%) of the cells emitted the Annexin V-eGFP-derived green fluorescence, but the other ones did not emit this biochemiluminescence. This suggests that the former subpopulation of the cells was early-apoptotic, and the latter was non-apoptotic. A total of 158 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells and intended to be in vitro-cultured. Out of 158 reconstructed oocytes, 106 (67.1%) NT embryos were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/158 (30.4%) and 21/158 (13.3%), respectively. In conclusion, the nucleofection efficiency of in vitro-cultured porcine dermal fibroblast cells as estimated by nuclear donor live-fluorescent evaluation based on the expression index of the eGFP reporter transgene was nearly 100%. Moreover, our results demonstrate that the morphological criteria commonly used for cell viability classification are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. It was also found that porcine nuclear-transferred morulae and blastocysts exhibited an approximately 100% index of xenogeneic eGFP gene transcriptional activity, which revealed the live diagnostics of emission intensity for green fluorescent protein-derived biochemiluminescence.
This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.
87 EVALUATION OF MITOCHONDRIAL FUNCTION IN SINGLE CLONED MINIATURE PIG EMBRYOS BY MEASURING OXYGEN CONSUMPTION
Authors: Sugimura S. , Yokoo M. , Yamanaka K.-I. , Wakai T. , Abe H. , Shoji T. , Sasada H. , Kobayashi J. , Abe H. , Sato E.
Published: December 2006

Mitochondria are organelles that produce energy for embryogensis. Their function [oxidative phosphorylation (OXPHOS) and electron transport] is regulated by intercommunication with the nucleus. In somatic cell nuclear transfer (SCNT) embryos, incomplete reprogramming may lead to dysfunction of the intercommunication before or after embryonic activation, or both, although it is unknown whether reprogramming for energy synthesis is required. In the previous report (Abe et al. 2004 J. Mamm. Ova Rec. 21, 22), we developed a noninvasive method using a scanning electrochemical microscopy (SECM) for measurement of oxygen consumption that provides more direct information about mitochondrial function (Trimarch et al. 2000 Biol. Reprod. 62, 1866–1874). In the present study to evaluate mitochondrial function in individual miniature pig SCNT embryos, we measured oxygen consumption by SECM. Oocytes in pig ovaries collected from the local slaughterhouse were matured for 44 h in NCSU23 and used as recipient. After SCNT with fetal miniature pig fibroblasts, reconstructed embryos were cultured in vitro in NCSU23 or PZM-3. Oxygen consumption in single 2- and 4-cell-stage embryos, morulae, and blastocysts were measured, and the values were compared with those derived from IVF. All data were analyzed by ANOVA. In IVF embryos, oxygen consumption was lowest at the 2- and 4-cell stages, and reached a peak at the blastocyst stage on Day 5. However, there were significant differences (P < 0.05) in blastocysts between NCSU23 and PZM-3: 0.61 � 0.14 vs. 0.83 � 0.18 at Day 5, 0.53 � 0.14 vs. 0.70 � 0.24 at Day 6, 0.47 � 0.11 vs. 0.73 � 0.20 � 10-14 mol s-1 at Day 7, respectively. In contrast, SCNT embryos showed no increase in oxygen consumption during pre-implantation stages in the 2 media, but there was a significant difference (P < 0.05) at the 2-cell stage between NCSU23 and PZM-3 (0.35 � 0.09 vs. 0.43 � 0.10, respectively). Comparison of the Day 5 IVF and SCNT blastocysts cultured in PZM-3 showed no difference in total cell numbers but significantly (P < 0.05) lower oxygen consumption in SCNT (0.83 � 0.18 vs. 0.40 � 0.13 � 10-14 mol s-1, respectively). After treatment with 1 �M CCCP (mitochondrial uncoupler) or 1 mM NaCN (mitochondrial electron transporter inhibitor), oxygen consumption in IVF and SCNT blastocysts at Day 5 increased (112 � 18 and 51 � 44%, respectively) or decreased (50 � 20 and 21 � 32%, respectively) compared with those of nontreated embryos. Sensitivity to these reagents differed significantly (P < 0.05) between IVF and SCNT, indicating that the SCNT blastocysts had a lower OXPHOS capacity than those from IVF. These results suggest that reprogramming for sustaining mitochondrial function during pre-implantation development may be required in miniature pig SCNT embryos.
89 ZYGOTICALLY ACTIVATED GENES ARE SUPPRESSED IN MOUSE NUCLEAR-TRANSFERRED EMBRYOS
Authors: Suzuki T. , Minami N. , Imai H.
Published: December 2006

Mammalian oocytes have the ability to confer totipotency to terminally differentiated somatic cell nuclei. Viable cloned animals have been produced by somatic cell nuclear transfer (NT) into oocytes in many mammalian species including mouse. However, the success rates of the production were quite low in all species. Many studies have measured differences in gene expression between NT and fertilized embryos in relatively advanced stages of development such as pre- and post-natal stages or the blastocyst stage. In the mouse, major zygotic gene activation (ZGA) occurs at the 2-cell stage after fertilization and leads to the transition of gene regulation from maternal control to embryonic control. Suppression of the ZGA by a transcription inhibitor was shown to decrease the viability of embryos, and causes developmental arrest at the 2-cell stage. An abnormal ZGA may therefore affect the viability of NT embryos and cause further abnormalities in later embryonic development. In the present study, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. The developmental rate of NT embryos to blastocysts (32.9%) was significantly lower than that of IVF (92.7%) or PA (92.8%). In addition, the cell numbers of NT embryos at the blastocyst stage (39.5 � 2.6; n = 19) were less than those of IVF (66.8 � 2.1; n = 30) or PA embryos (48.2 � 2.1; n = 30). Using these embryos, we first identified 4 genes that were differentially expressed between NT and IVF embryos at the 2-cell stage. Among the identified genes, Inpp5b and Chst12 were up-regulated, and MuERV-L and Dnaja2 were down-regulated in the NT embryos compared with IVF embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-γ, Dub-1, Spz1, DD2106, and DD2111 were not properly activated in NT embryos, suggesting that the cellular process involved in the control of the zygotic genome activation is not appropriately regulated. These results indicate that abnormal gene expression has already occurred at the early stage of pre-implantation development as a failure of nuclear reprogramming.
91 PRODUCTION OF A CLONED BOER GOAT (CAPRA HIRCUS) BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Tao Y. , Han W. , Zhang M. , Ding J. , Zhang X.
Published: December 2006

We reported the birth of a goat clone produced by somatic cell nuclear transfer. The fusion and activation protocols of reconstructed oocytes and embryo transfer procedure were optimized. The donors of somatic cells were fibroblasts derived from ear skin of a Boer goat while the recipient ooplasm was in vitro-matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were activated by ionomycin, 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB) singly or simultaneously (termed as Ionomycin, Ionomycin+6-DMAP, and Ionomycin+6-DMAP+CB). The result showed that the cleavage rate in single ionomycin was significantly lower than that in Ionomycin+6-DMAP and 6-DMAP+CB (34.38% vs. 69.85% and 72.02%; P < 0.05). However, the cleavage rates and blastocyst rates had no significant difference after in vitro culture (P > 0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development rate increased. The reconstructed embryos were equilibrated 1–3 h, 3–6 h, and 6–9 h after fusion, and then activation was undertaken by ionomycin+6-DMAP. We found that the cleavage rates had no significant difference during 1–3 h and 3–6 h (72.58% vs. 72.97%; P > 0.05), but both were significantly higher than during 6–9 h (64.40%) (P < 0.05). A total of 491 reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural estrus. One of those who were treated with hCG after transfer was pregnant and gave birth to a live kid on 153 days. The lamb died accidentally 8 h after birth. The cloned offspring was then dissected and proved well in all organs. Staining of paraffin tissue slices of the viscera suggested that the organs developed well. Microsatellite analysis indicated that the lamb was derived from the somatic cell donor doe genetically.
93 EFFECT OF SEASON ON QUALITY OF OOCYTES, RESULTS OF IN VITRO MATURATION, AND SOMATIC CELL NUCLEAR TRANSFER IN SWAMP BUFFALO
Authors: Uoc N. T. , de Rennis F. , Duc N. H. , Bui L. C. , Hanh N. V. , Linh N. V. , Thanh N. T. , Huu Q. X. , Long D. D. , Nguyen B. X.
Published: December 2006

Reproductive activity in swamp buffalo is characterized by a clearly demonstrated anestrus season. The aim of the present study was to evaluate season effect on the oocyte collection, in vitro maturation, and somatic cell nuclear transfer. The ovaries collected from a slaughterhouse were divided into 3 groups according to the collection period: (1) G1: from January to April; G2: from May to August, which is characterized by higher climate temperature and low reproductive activity; and G3: from September to December. Cumulus–oocyte complexes (COCs) were aspirated from follicles 2-6 mm in diameter using an 18-gauge needle, washed in HEPES-buffered TCM-199 (Sigma-Aldrich, St Louis, MO, USA), and classified following 3 different quality levels: A (with 4–6 layers of cumulus cells), B (with 2–3 layers of cumulus cells), and C (few or without cumulus cells). The oocytes of A and B categories were used for IVM in maturation media currently used in cattle (TCM-199 medium + 10% fetal bovine serum) with an increase of FSH concentration (30 �g mL-1) and estradiol-17β (3 �g mL-1). Maturation was carried out at 39�C in a water-saturated incubator, under 5% CO2 for 22 h. The oocytes were observed for the cumulus expanding and the presence of polar body (PB). The oocytes with PB were used for further enucleation and cell nuclear transfer using buffalo quiescent fibroblast cells and the technique described previously (Nguyen et al. 2000 Theriogenology 53, 235). The percentages of intact and fused oocytes as well as reconstructed embryos developed to blastocyst stage were compared for the oocytes from G1 and G2. The results indicated that the average number of good quality COCs collected per ovary for the G1, G2, and G3 period were 6.00 � 4.08 (n = 426), 2.93 � 2.55 (n = 346), and 4.78 � 1.05 (n = 445), respectively. The percentages of A and B oocytes were 62.4% (1.58 � 0.51 vs. 2.17 � 1.54), 63.2% (0.90 � 0.32 vs. 0.95 � 0.50), and 54.7% (1.12 � 0.25 vs. 1.49 � 0.53), respectively; the maturation rate was 55.08%, 56.28%, and 52.16%, respectively. There were no significant differences between G1 and G2 in the percentage of intact and fused oocytes (93.7% and 59% for G1; 100% and 60% for G2, respectively), but the rate of embryos developed to blastocyst stage was higher for oocytes from G1 (18.5% vs. 10.2%). In conclusion, in swamp buffalo, the hot season affected significantly the number of oocytes collected per animal and the subsequent results of somatic cell nuclear transfer. The optimal period for working with buffalo oocyte is from January to April.
This work was aupported by a grant from the Vietnam-Italy 3AB3 Project.
94 PRODUCTION OF AN α1,3-GALACTOSYLTRANSFERASE GENE KNOCKOUT CALF USING A SINGLE TARGETING VECTOR AND REJUVENATED CELL LINES
Authors: Urakawa M. , Sendai Y. , Sawada T. , Shinkai Y. , Ideta A. , Kubota K. , Hoshi H. , Aoyagi Y.
Published: December 2006

Gene targeting in large animals has the potential to be useful in agriculture. In this study, we examined whether an �1,3-galactosyltransferase-gene knockout (null) calf could be produced using a single targeting vector for disruption of both alleles and a rejuvenation of cell line by production of cloned fetuses. A promoter-less positive selection vector (pGT-22) containing an IRES (internal ribosome entry site)-antibiotic resistance gene(neo) cassette and loxP sequences was used to disrupt the bovine �1,3-galactosyltransferase (�1,3-GT) gene. In gene-targeting (GT), Japanese Black fetal fibroblasts were transfected with pGT-22, and were selected with 0.4 mg mL-1 G418. G418-resistant cells were monitored by PCR and Southern blot analysis. After PCR selection, a portion of the PCR-positive colonies were infected with the adenovirus AxCANCre, which transiently expresses Cre recombinase in the infected cells, to excise the antibiotic-resistance gene cassette (IRES/lacZ-neo) from the targeted allele. The targeted cells in which homologous recombination occurred were used for somatic cell nuclear transfer (SCNT). The cell cycle synchronization of donor cells in the early G1 phase and SCNT were performed according to the established procedure in our laboratory (Urakawa et al. 2004 Theriogenology 62, 714–728). As a result, in the first GT, one PCR-Southern blot-positive clone (0.26%, in 380 G418-resistant colony) was used for SCNT. Of 35 pulsed SCNT embryos, 4 (11.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT+/-) was established and was transfected with pGT-22 in the second GT. As a result, 8 PCR-Southern blot-positive clones (1.6%, in 508 G418-resistant colony) were recovered. The 2nd GT and the following SCNT were accomplished by the same methods used in the 1st GT and SCNT procedure. Of 36 pulsed SCNT embryos, 7 (19.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT-/-) was established, and was used for SCNT to obtain the �1,3-GT homozygous knockout offspring. Finally, 52 of 148 pulsed SCNT embryos (35.1%) developed to the blastocyst stage and 6 cloned embryos were transferred into 3 recipient heifers. A single knockout female offspring with normal morphology in all organs was born (Day 287) but died about one hour after birth. In this study, we established both heterozygous and homozygous �1,3-GT knockout cell lines from primary fetal fibroblasts and produced an �1,3-GT knockout offspring using a single targeting vector and rejuvenated cell lines.
Divergence of reproductive phenology under climate warming
Authors: Sherry Rebecca A. , Zhou Xuhui , Gu Shiliang , Arnone John A. , Schimel David S. , Verburg Paul S. , Wallace Linda L. , Luo Yiqi
Published: December 2006

Because the flowering and fruiting phenology of plants is sensitive to environmental cues such as temperature and moisture, climate change is likely to alter community-level patterns of reproductive phenology. Here we report a previously unreported phenomenon: experimental warming advanced flowering and fruiting phenology for species that began to flower before the peak of summer heat but delayed reproduction in species that started flowering after the peak temperature in a tallgrass prairie in North America. The warming-induced divergence of flowering and fruiting toward the two ends of the growing season resulted in a gap in the staggered progression of flowering and fruiting in the community during the middle of the season. A double precipitation treatment did not significantly affect flowering and fruiting phenology. Variation among species in the direction and magnitude of their response to warming caused compression and expansion of the reproductive periods of different species, changed the amount of overlap between the reproductive phases, and created possibilities for an altered selective environment to reshape communities in a future warmed world.
Natural selection for rash-forming genotypes of the varicella-zoster vaccine virus detected within immunized human hosts
Authors: Quinlivan Mark L. , Gershon Anne A. , Al Bassam Mahmoud M. , Steinberg Sharon P. , LaRussa Philip , Nichols Richard A. , Breuer Judith
Published: December 2006

The Oka vaccine strain is a live attenuated virus that is routinely administered to children in the United States and Europe to prevent chickenpox. It is effective and safe but occasionally produces a rash. The vaccine virus has accumulated mutations during its attenuation, but the rashes are not explained by their reversion, unlike complications reported for other viral vaccines. Indeed, most of the novel mutations distinguishing the Oka vaccine from the more virulent parental virus have not actually become fixed. Because the parental alleles are still present, the vaccine is polymorphic at >30 loci and therefore contains a mixture of related viruses. The inoculation of >40 million patients has consequently created a highly replicated evolutionary experiment that we have used to assess the competitive ability of these different viral genotypes in a human host. Using virus recovered from rash vesicles, we show that two vaccine mutations, causing amino acid substitutions in the major transactivating protein IE62, are outcompeted by the ancestral alleles. Standard interpretations of varicella disease severity concentrate on the undeniably important effects of host genotype and immune status, yet our results allow us to demonstrate that the viral genotype is associated with virulence and to identify the key sites. We propose that these loci have pleiotropic effects on the immunogenic properties of the virus, rash formation, and its epidemiological spread, which mould the evolution of its virulence. These findings are of practical importance for reducing the incidence of vaccine-associated rash and promoting public acceptance of the vaccine.
Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy
Authors: Brunelli Silvia , Sciorati Clara , D'Antona Giuseppe , Innocenzi Anna , Covarello Diego , Galvez Beatriz G. , Perrotta Cristiana , Monopoli Angela , Sanvito Francesca , Bottinelli Roberto , Ongini Ennio , Cossu Giulio , Clementi Emilio
Published: December 2006

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (α-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.
Evidence for a chemical-thermal structure at base of mantle from sharp lateral P-wave variations beneath Central America
Authors: Sun Xinlei , Song Xiaodong , Zheng Sihua , Helmberger Don V.
Published: December 2006

Compressional waves that sample the lowermost mantle west of Central America show a rapid change in travel times of up to 4 s over a sampling distance of 300 km and a change in waveforms. The differential travel times of the PKP waves (which traverse Earth's core) correlate remarkably well with predictions for S-wave tomography. Our modeling suggests a sharp transition in the lowermost mantle from a broad slow region to a broad fast region with a narrow zone of slowest anomaly next to the boundary beneath the Cocos Plate and the Caribbean Plate. The structure may be the result of ponding of ancient subducted Farallon slabs situated near the edge of a thermal and chemical upwelling.
Validation of the Revised Self‐Administered Alcohol Screening Test (SAAST‐R)
Authors: Patten Christi A. , Vickers Kristin S. , Offord Kenneth P. , Decker Paul A. , Colligan Robert C. , Bronars Carrie , Reese Michael M. , Seime Richard J. , Hall‐Flavin Daniel K. , Hurt Richard D.
Published: December 2006

This study examined the sensitivity and specificity of the revised Self‐Administered Alcohol Screening Test (SAAST‐R)1 among adult outpatients evaluated at community addictions and psychiatry and psychology clinics. A total of 417 adults (mean ± SD age of 41.4 ± 15.0 years, 52% female, 91% Caucasian) completed the 36‐item SAAST‐R prior to their evaluation. Based on the clinician's documentation of the patient's alcohol use, two criterion groups were established: one composed of subjects classified as having no alcohol problems (n = 214), and one with alcohol problems (n = 203). The SAAST‐R was found to have high internal consistency reliability (Cronbach's alpha −0.96), sensitivity (90%), and specificity (82%) for differentiating outpatients with and without alcohol problems, respectively, using a cut point of > 4 to indicate risk for alcohol‐related problems. The SAAST‐R was found to have good sensitivity and specificity among both males and females.
Cocaine Use and Syphilis Trends: Findings from the Arrestee Drug Abuse Monitoring (ADAM) Program and Syphilis Epidemiology in Houston
Authors: Ross Michael W. , Risser Jan , Peters Ronald J. , Johnson Regina J.
Published: December 2006

There has been speculation that trends in syphilis have been fueled by crack cocaine use. This study examined the data on syphilis notifications and arrestee drug abuse monitoring (ADAM) to ascertain the relationships between syphilis and cocaine use trends in three racial/ethnic groups. Syphilis notifications and data from the ADAM project were compared in Houston/Harris County, Texas, from 1991–1998 using a linear regression equation. Data indicated significant relationships between the data for cocaine use and syphilis in African Americans but not Hispanics or non‐Hispanic whites. For African Americans, 58% of the variance between cocaine use and syphilis was explained. When data limited to jail syphilis notifications and ADAM cocaine in African Americans were examined, the association was stronger for males than for females. For African Americans, cocaine (probably crack cocaine) use trends were significantly associated with syphilis trends in this population. These data suggest that control of crack cocaine may have an impact on syphilis rates and that there may be close relationships between some STDs and drug abuse.
Changing Provider Practices, Program Environment, and Improving Outcomes by Transporting Multidimensional Family Therapy to an Adolescent Drug Treatment Setting
Authors: Liddle Howard A. , Rowe Cynthia L. , Gonzalez Alina , Henderson Craig E. , Dakof Gayle A. , Greenbaum Paul E.
Published: December 2006

Effective interventions for drug abusing adolescents are underutilized. Using an interrupted time series design, this study tested a multicomponent, multi‐level technology transfer intervention developed to train clinical staff within an existing day treatment program to implement multidimensional family therapy (MDFT), an evidence‐based adolescent substance abuse treatment. The sample included 10 program staff and 104 clients. MDFT was incorporated into the program and changes were noted in the program environment, therapist behavior, and in most (e.g., drug abstinence, and out of home placements) but not all (e.g., drug use frequency) client outcomes. These changes remained after MDFT supervision was withdrawn.
Development and Validation of the GAIN Short Screener (GSS) for Internalizing, Externalizing and Substance Use Disorders and Crime/Violence Problems Among Adolescents and Adults
Authors: Dennis Michael L. , Chan Ya‐Fen , Funk Rodney R.
Published: December 2006

The Global Appraisal of Individual Needs (GAIN)1 is a 1–2 hour standardized biopsychosocial that integrates clinical and research assessment for people presenting to substance abuse treatment. The GAIN – Short Screener (GSS) is 3–5 minute screener to quickly identify those who would have a disorder based on the full 60–120 minute GAIN and triage the problem and kind of intervention they are likely to need along four dimensions (internalizing disorders, externalizing disorders, substance disorders, and crime/ violence). Data were collected from 6,177 adolescents and 1,805 adults as part of 77 studies in three dozen locations around the United States that used the GAIN. For both adolescents and adults the 20‐item total disorder screener (TDScr) and its four 5‐item sub‐screeners (internalizing disorders, externalizing disorders, substance disorders, and crime/violence) has good internal consistency (alpha of .96 on total screener), is highly correlated (r—.84 to .94) with the 123‐item longer scales in the full GAIN. The GSS also does well in terms of its receiver operator characteristics (90% or more under the curve in all analyses) and has clinical decision‐making cut points with excellent sensitivity (90% or more) for identifying people with a disorder and excellent specificity (92% or more) for correctly ruling out people who did not have a disorder. The GSS has good potential as an efficient screener for identifying people with co‐occurring disorders across multiple systems and routing them to the right services and more detailed assessments.
An Interactive Voice Response (IVR) System for Adolescents with Alcohol Use Disorders: A Pilot Study
Authors: Kaminer Yifrah , Litt Mark D. , Burke Rebecca H. , Burleson Joseph A.
Published: December 2006

In order to understand predictors of relapse among adolescents treated for alcohol use disorders (AUD), it is important to accurately assess the daily circumstances associated with use. This pilot study investigates the feasibility and acceptability of an interactive voice response (IVR) system in adolescents with AOSUD. Twenty‐six adolescents 14 to 19 years old, with a mean age of 16.8, who were enrolled into an adolescent treatment program for AUD consented to make phone calls for 14 successive evenings to an IVR system and answer 14 questions pertaining to daily use of alcohol and other drugs. The subjects were compensated for their participation. A satisfaction questionnaire was administered at the end of the study. Participants completed 72% of scheduled recordings, with an average of 10.1 calls per subject. Most participants reported that they answered the questions honestly and accurately and were very much satisfied with the IVR system. The preliminary data presented here suggests that the use of IVR for the purpose of generating daily reports in youth is feasible and acceptable. The utilization of IVR systems should be explored to improve efficacy and attainment of generalizability to heterogeneous adolescent populations and lifestyles including for other psychiatric disorders.
Suicidal Ideation among Adolescents with Alcohol Use Disorders during Treatment and Aftercare
Authors: Kaminer Yifrah , Burleson Joseph A. , Goldston David B. , Burke Rebecca H.
Published: December 2006

The objectives of this study are to assess the magnitude and course of suicidal ideation during outpatient treatment and aftercare for adolescents with alcohol use disorders (AUD). One hundred seventy‐seven adolescents meeting eligibility criteria, including no past 30‐day suicidal behavior, participated in 9 weeks of outpatient cognitive‐behavioral group therapy. Treatment completers were randomized into: (1) No‐Active, (2) In‐Person, or (3) Telephone aftercare conditions for a period of 12 weeks. No specific intervention for suicidal behavior was provided during the study. The Suicide Ideation Questionnaire (SIQ‐JR, Reynolds, 1988) was administered at baseline, end of treatment, and end of aftercare. The results are as follows, a higher baseline suicidal ideation was associated with higher retention at the end of treatment and through aftercare. The In‐Person Aftercare condition showed a significant decrease in suicidal ideation, relative to the No‐Active Aftercare condition. There was a trend for similarly reduced severity of suicidal ideation in the Telephone Aftercare condition. In conclusion, the type of aftercare and resulting decrease in AUD may play a role in the reduction in suicidal ideation. The mechanism of change by which suicidal ideation is reduced in adolescents in treatment for AUD needs to be further explored.
GCP6 Binds to Intermediate Filaments: A Novel Function of Keratins in the Organization of Microtubules in Epithelial Cells
Authors: Oriolo Andrea S. , Wald Flavia A. , Canessa Gisella , Salas Pedro J.I.
Published: December 2006

In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the γ-tubulin ring complex, γ-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in γ-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced γ-tubulin from the cytoskeleton and resulted in depolarization of γ-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia.
PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells
Authors: Wang Qian , Chen Xiao-Wei , Margolis Ben
Published: December 2006

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.
Cell Fate Determination Factor DACH1 Inhibits c-Jun–induced Contact-independent Growth
Authors: Wu Kongming , Liu Manran , Li Anping , Donninger Howard , Rao Mahadev , Jiao Xuanmao , Lisanti Michael P. , Cvekl Ales , Birrer Michael , Pestell Richard G.
Published: December 2006

The cell fate determination factor DACH1 plays a key role in cellular differentiation in metazoans. DACH1 is engaged in multiple context-dependent complexes that activate or repress transcription. DACH1 can be recruited to DNA via the Six1/Eya bipartite transcription (DNA binding/coactivator) complex. c-Jun is a critical component of the activator protein (AP)-1 transcription factor complex and can promote contact-independent growth. Herein, DACH1 inhibited c-Jun–induced DNA synthesis and cellular proliferation. Excision of c-Jun with Cre recombinase, in c-junf1/f13T3 cells, abrogated DACH1-mediated inhibition of DNA synthesis. c-Jun expression rescued DACH1-mediated inhibition of cellular proliferation. DACH1 inhibited induction of c-Jun by physiological stimuli and repressed c-jun target genes (cyclin A, β-PAK, and stathmin). DACH1 bound c-Jun and inhibited AP-1 transcriptional activity. c-jun and c-fos were transcriptionally repressed by DACH1, requiring the conserved N-terminal (dac and ski/sno [DS]) domain. c-fos transcriptional repression by DACH1 requires the SRF site of the c-fos promoter. DACH1 inhibited c-Jun transactivation through the δ domain of c-Jun. DACH1 coprecipitated the histone deacetylase proteins (HDAC1, HDAC2, and NCoR), providing a mechanism by which DACH1 represses c-Jun activity through the conserved δ domain. An oncogenic v-Jun deleted of the δ domain was resistant to DACH1 repression. Collectively, these studies demonstrate a novel mechanism by which DACH1 blocks c-Jun-mediated contact-independent growth through repressing the c-Jun δ domain.
Transferrin Receptor 2: Evidence for Ligand-induced Stabilization and Redirection to a Recycling Pathway
Authors: Johnson Martha B. , Chen Juxing , Murchison Nicholas , Green Frank A. , Enns Caroline A.
Published: December 2006

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe2Tf). TfR2 also binds Fe2Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe2Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe2Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe2Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe2Tf, increases the rate of receptor turnover and prevents stabilization by Fe2Tf, indicating a direct role of Fe2Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.
Exophilin4/Slp2-a Targets Glucagon Granules to the Plasma Membrane through Unique Ca2+-inhibitory Phospholipid-binding Activity of the C2A Domain
Authors: Yu Miao , Kasai Kazuo , Nagashima Kazuaki , Torii Seiji , Yokota-Hashimoto Hiromi , Okamoto Koichi , Takeuchi Toshiyuki , Gomi Hiroshi , Izumi Tetsuro
Published: December 2006

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic β cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic α cells, in contrast to another effector, granuphilin, in β cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca2+concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca2+-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca2+-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.
Enrichment or depletion of a GO category within a class of genes: which test?
Authors: Rivals Isabelle , Personnaz Léon , Taing Lieng , Potier Marie-Claude
Published: December 2006

Motivation: A number of available program packages determine the significant enrichments and/or depletions of GO categories among a class of genes of interest. Whereas a correct formulation of the problem leads to a single exact null distribution, these GO tools use a large variety of statistical tests whose denominations often do not clarify the underlying P-value computations.

Summary: We review the different formulations of the problem and the tests they lead to: the binomial, χ2, equality of two probabilities, Fisher's exact and hypergeometric tests. We clarify the relationships existing between these tests, in particular the equivalence between the hypergeometric test and Fisher's exact test. We recall that the other tests are valid only for large samples, the test of equality of two probabilities and the χ2-test being equivalent. We discuss the appropriateness of one- and two-sided P-values, as well as some discreteness and conservatism issues.

Contact:  isabelle.rivals@espci.fr

Supplementary information: Supplementary data are available at Bioinformatics online.
Robust prediction of consensus secondary structures using averaged base pairing probability matrices
Authors: Kiryu Hisanori , Kin Taishin , Asai Kiyoshi
Published: December 2006

Motivation: Recent transcriptomic studies have revealed the existence of a considerable number of non-protein-coding RNA transcripts in higher eukaryotic cells. To investigate the functional roles of these transcripts, it is of great interest to find conserved secondary structures from multiple alignments on a genomic scale. Since multiple alignments are often created using alignment programs that neglect the special conservation patterns of RNA secondary structures for computational efficiency, alignment failures can cause potential risks of overlooking conserved stem structures.

Results: We investigated the dependence of the accuracy of secondary structure prediction on the quality of alignments. We compared three algorithms that maximize the expected accuracy of secondary structures as well as other frequently used algorithms. We found that one of our algorithms, called McCaskill-MEA, was more robust against alignment failures than others. The McCaskill-MEA method first computes the base pairing probability matrices for all the sequences in the alignment and then obtains the base pairing probability matrix of the alignment by averaging over these matrices. The consensus secondary structure is predicted from this matrix such that the expected accuracy of the prediction is maximized. We show that the McCaskill-MEA method performs better than other methods, particularly when the alignment quality is low and when the alignment consists of many sequences. Our model has a parameter that controls the sensitivity and specificity of predictions. We discussed the uses of that parameter for multi-step screening procedures to search for conserved secondary structures and for assigning confidence values to the predicted base pairs.

Availability: The C++ source code that implements the McCaskill-MEA algorithm and the test dataset used in this paper are available at

Contact:  kiryu-h@aist.go.jp

Supplementary information: Supplementary data are available at Bioinformatics online.
Form
Authors: Praeger Ferdinand
Published: January 2007

It has been, and will doubtless again be urged, that the term “Form” is misapplied in reference to music, and that “plan” or “design” are the more correct expressions. But when men like Ouseley and Stainer in their standard text books, and all modern German writers of note, adopt the word “form,” I think I have sufficient grounds for continuing its use. Still, those who incline to the term “plan or design” will find no difficulty in following my argument, as it i precisely in that sense that I use the word “Form.” I shall be forced to traverse well-known roads before reaching my destination, but will endeavour to be as brief as the case will admit.
Bax and the Ballet
Authors: Foreman Lewis
Published: January 2007

On 27 July 1913 Colin Taylor, a music master at Eton and confidant of the young Philip Heseltine, wrote to his protégé:

By the way I was introduced to Bax at the Russian Ballet the other night – do you know him? I have always pictured him as a slow speaking dreamy man – dreamy he may be, but in his speech he is short, dry and quite unpoetical. He told me he has a splendid ‘Book’ for a ballet and he is longing to get to work on it. At present however he is finishing a work for (I think he said) the Norwich Triennial Festival. A ballet should suit him.
The Solo Motet in Venice (1625–1775)
Authors: Arnold Denis
Published: January 2007

The genre of motet for a single voice of the baroque and pre-classical eras has been little investigated. Bukofzer mentions only its origins in a brief paragraph; Abraham admittedly has half a page in his Concise Oxford History of Music, putting it quickly into context; but Fellerer's ample Geschichte der katholischen Kirchenmusik (Kassel, 1976) treats it only sketchily and even Finscher's article on the motet in Die Musik in Geschichte und Gegenwart mentions it only in passing, the more important discussion being devoted to the motet in the stile antico, perhaps not surprisingly, since that was what the term really implied in Germany during Bach's lifetime. But everyone knows at least one solo motet, Mozart's Exsultate jubilate (K. 158a); most people will know a piece in a similar tradition, Bach's Jauchzet Gott (BVW 51), significantly included among the cantatas; to which may be added a work which some of us feel is finer than either, Handel's Silete venti. In monographs on the various composers, these pieces usually get short shrift, being considered either a-typical or at least of minor importance. This paper will attempt to provide a background to these works.
Frescobaldi's Toccatas and their Stylistic Ancestry
Authors: Newcomb Anthony
Published: January 2007

Since there is no standard description of the style of a Frescobaldi toccata, this study must begin by setting out the stylistic characteristics of these pieces, especially as manifested in what one might call the normal toccata type – the first eleven of Book I and the first, second and seventh of Book II. In this initial stage, the reader is referred for more detailed development of my points to my paper Ascoltare e guardare le toccate, soon to be published as part of the acts of the International Frescobaldi Congress, held in Ferrara in September 1983 to celebrate the quadri-centenary of Frescobaldi's birth. That paper illustrates the distinctive features of Frescobaldi's toccata style through close analysis of a single toccata (the ninth of Book I). I shall briefly lay out these features, proceeding from the make-up of the individual section to the make-up of the entire piece, and stressing not only what kind of things one finds in an individual section or entire piece, but also what gives those things coherence and shape.
Tallis's First and Second Thoughts
Authors: Milsom John
Published: January 2007

Thomas Tallis, in common with virtually every other Tudor composer, left no legacy of autograph copies of his music. A glance at the footnotes and critical commentaries that gloss his works in the various collected editions is enough to show how frequently the sources from which his music can be recovered are in disagreement with one another, often in detail, sometimes in greater substance. Looking at these sources and their readings, one is left with the impression that these are the testaments of lesser authorities: the musicians and scribes, of varying competence or trustworthiness, who copied Tallis's music into their manuscripts, not only during the composer's lifetime but also for many decades (or in certain cases centuries) after his death.
Another ‘Imitation’ of Busnoys'sMissa L'Homme armé—and Some Observations onImitatioin Renaissance Music
Authors: Wegman Rob C.
Published: January 2007

Antoine Busnoys'sMissa L'Homme armémust have been one of the most highly esteemed polyphonic Mass cycles of its time. It survives in no fewer than seven sources, an exceptionally large number not equalled by any other cycle from the 1460s or 1470s – even those by Dufay and Ockeghem. In this respect Busnoys's Mass stands alongside a work such as the anonymous EnglishMissa Caput, whose widespread popularity in the fifteenth century is also well established. It is a measure of their extraordinary esteem that both Masses served as models for ‘imitations’ by later composers. The entire cantus firmus layout of theCaputMass was copied in Ockeghem's and Obrecht's cycles on the same tune, and possibly in a fourthCaputcycle of which only the Agnus dei survives. Busnoys'sMissa L'Homme armélikewise served as the model for a later work, Obrecht'sL'Homme arméMass. There exists however a second ‘imitation’ of Busnoys's cycle, an anonymousMissa de Sancto Johanne Baptista, which probably dates from the 1480s or 1490s. This Mass has a less conspicuous relationship with its model than the other ‘imitations’ mentioned here, but it raises as many important questions concerning Renaissance practices of borrowing and structural modelling.