Connecting the Libraries and Athletics through Instruction and Outreach.

This column describes the approaches taken by librarians and staff at James Madison University (JMU) Libraries & Educational Technologies (LET) to extend library support to university athletics. The model resembles that used for outreach to academic programs and was first adapted to the semi-clinical, nonacademic Strength & Conditioning Department, then to JMU Athletics as a whole. Librarians offered targeted instructional sessions, orientations, and asynchronous learning modules embedded in the learning management system. This new relationship has provided an opportunity for broader collaboration, increasing LET's presence across campus.

An evaluation of pharmacogenomic information provided by five common drug information resources.

INTRODUCTION: This study evaluated whether pharmacogenomic information contained in the Food and Drug Administration (FDA)-approved package inserts of sixty-five drugs was present in five drug information resources. METHODS: The study searched for biomarkers from the FDA package inserts in 5 drug information sources: American Hospital Formulary Service Drug Information (AHFS), Facts & Comparisons 4.0 (Facts), ePocrates Online Free (ePocrates Free), Lexicomp Online (Lexicomp), and Micromedex 2.0. Each resource had the opportunity to present biomarker information for 65 drugs, a total of 325 opportunities. A binary system was used to indicate presence or absence of the biomarker information. A sub-analysis was performed on the 13 most frequently prescribed drugs in the United States. RESULTS: Package insert biomarker information was available, on average, for 81.5% of the 65 FDA-listed drugs in 2011. Percent availability for the individual resources was: Lexicomp, 95.3%; Micromedex 2.0, 92.3%; Facts, 76.9%; AHFS, 75.3%; and ePocrates Free, 67.7%. The sub-analysis of the 13 top drugs showed Lexicomp and Micromedex 2.0 had the most mentions, 92.3%; ePocrates Free had the least, 53.8%. CONCLUSION: The strongest resource for pharmacogenomic information was Lexicomp. The gap between Lexicomp and ePocrates Free is concerning. Clinicians would miss pharmacogenomic information 6.6 times more often in ePocrates Free than in Lexicomp. IMPLICATIONS: Health sciences librarians should be aware of the variation in biomarker availability when recommending drug resources for licensing and use. Librarians can also use this study to encourage publishers to include pharmacogenomics information from the package insert as a minimum standard.

Development of the research lifecycle model for library services.

QUESTION: Can the niche services of individual librarians across multiple libraries be developed into a suite of standard services available to all scientists that support the entire research lifecycle? SETTING: Services at a large, research-intensive state university campus are described. METHOD: Initial data were collected via concept mapping by librarians. Additional data were collected at conferences and meetings through interactive poster presentations. MAIN RESULTS: Services of interest to scientists for each of the stages in the research lifecycle were developed by the team to reflect the wide range of strengths of team members in aggregate. CONCLUSION: Input from researchers was the most effective tool for developing the model. A flexible research lifecycle model can be developed to match the needs of different service groups and the skills of different librarians.

A preliminary analysis of library holdings as compared to the basic resources for pharmacy education list.

The catalogs of 11 university libraries were analyzed against the Basic Resources for Pharmaceutical Education (BRPE) to measure the percent coverage of the core total list as well as the core sublist. There is no clear trend in this data to link school age, size, or rank with percentage of coverage of the total list or the "First Purchase" core list when treated as independent variables. Approximately half of the schools have significantly higher percentages of core titles than statistically expected. Based on this data, it is difficult to predict what percentage of titles on the BRPE a library will contain.

Development of targeted online modules for recurring reference questions.

When students are given assignments with specific information needs, they may turn to the library for help. The UNC Health Sciences Library developed three short online modules to teach first-year pharmacy students how to find early/animal studies, mechanism of action information, and specific study types in an effort to lessen demand on the reference desk. The modules filled two goals: to free up time that had been spent on three common low-level questions and to provide a pedagogically sound online tool to teach students how to find answers to these three questions. The modules were created using Adobe Captivate. Developing and promoting the modules took three hours of the pharmacy librarian's time compared with nearly 23 hours spent answering individual questions via e-mail, in consultations, and at the reference desk before the modules were introduced. After introducing the modules, only one student asked for help from the library compared to more than 60 who viewed the online modules at least once.

"Hidden treasures": librarian office hours for three health sciences schools.

The changing needs of students and faculty have prompted UNC Chapel Hill's Health Sciences Library to reconsider the delivery of library services. Several years of outreach and office hours have yielded an array of "hidden treasures," or secondary outcomes, of both online and in-person office hours. The online office hours are tailored for the Schools of Medicine, Pharmacy, and Public Health. This article examines the benefits that go beyond simple consultation statistics and encompass more qualitative aspects of success resulting from increased outreach, goodwill, and stronger library-departmental partnerships.

Vignettes: Diverse library staff offering diverse bioinformatics services.

OBJECTIVES: The paper gives examples of the bioinformatics services provided in a variety of different libraries by librarians with a broad range of educational background and training. METHODS: Two investigators sent an email inquiry to attendees of the "National Center for Biotechnology Information's (NCBI) Introduction to Molecular Biology Information Resources" or "NCBI Advanced Workshop for Bioinformatics Information Specialists (NAWBIS)" courses. The thirty-five-item questionnaire addressed areas such as educational background, library setting, types and numbers of users served, and bioinformatics training and support services provided. Answers were compiled into program vignettes. DISCUSSION: The bioinformatics support services addressed in the paper are based in libraries with academic and clinical settings. Services have been established through different means: in collaboration with biology faculty as part of formal courses, through teaching workshops in the library, through one-on-one consultations, and by other methods. Librarians with backgrounds from art history to doctoral degrees in genetics have worked to establish these programs. CONCLUSION: Successful bioinformatics support programs can be established in libraries in a variety of different settings and by staff with a variety of different backgrounds and approaches.

Developing an interdisciplinary collaboration center in an academic Health Sciences Library.

This article describes the evolution of the Health Sciences Library's plans for an interdisciplinary, technology-enhanced collaboration center, from a technology-driven space to one with a vision of support for peer-to-peer learning and research. The center offers an exciting opportunity to be an essential partner in collaborative and interdisciplinary programs such as the new Carolina Center for Exploratory Genetic Analysis. The Library is a centrally located and neutral place, which helps minimize geographical and territorial obstacles to effective collaboration. The collaboration center raises the Library's visibility and allows staff to demonstrate the value of knowledge resources, services, technology expertise, infrastructure, and facilities for group study and collaboration.

Building a bioinformatics community of practice through library education programs.

This paper addresses the following questions:What makes the community of practice concept an intriguing framework for developing library services for bioinformatics? What is the campus context and setting? What has been the Health Sciences Library's role in bioinformatics at the University of North Carolina (UNC) Chapel Hill? What are the Health Sciences Library's goals? What services are currently offered? How will these services be evaluated and developed? How can libraries demonstrate their value? Providing library services for an emerging community such as bioinformatics and computational biology presents special challenges for libraries including understanding needs, defining and communicating the library's role, building relationships within the community, preparing staff, and securing funding. Like many academic health sciences libraries, the University of North Carolina (UNC) at Chapel Hill Health Sciences Library is addressing these challenges in the context of its overall mission and goals.

Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome.

EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.

Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin.

Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued). To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains. We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150(Glued) in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.

Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors.

To exploit zebrafish as a transgenic model, tissue-specific promoters must be identified. We isolated a 20-kilobase (kbp) zebrafish rod opsin genomic clone, which consists of 18 kbp of 5'-flanking region, the entire coding region, and 0.5 kbp of 3'-flanking sequence. Polymerase chain reaction, Southern blotting, and DNA sequencing revealed the rod opsin gene lacks introns. The transcription start site was localized 94 nucleotides upstream of the translation initiation site. Sequence alignment with orthologous promoters revealed conserved cis-elements including glass, NRE, OTX/Bat-1, Ret-1/PCE-1, Ret-4, and TATA box. A 1.2-kbp promoter fragment was cloned upstream of the enhanced green fluorescent protein (EGFP) cDNA and microinjected into 1- to 2-cell stage zebrafish embryos. EGFP expression was detected in the ventral-nasal eye at 3 days postfertilization and spread throughout the eye. Progeny of the positive founder fish, which were identified by polymerase chain reaction amplification of fin genomic DNA, exhibited EGFP expression in the retina, confirming the germline transmission of the transgene. Frozen eye sections demonstrated the EGFP expression was rod-specific and exhibited a similar developmental expression profile as the rod opsin protein. This stable transgenic line provides a novel tool for identification of genes regulating development and maintenance of rod photoreceptors.

A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function.

Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.

The herpes simplex virus 1 U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane.

To express the function encoded in its genome, the herpes simplex virus 1 capsid-tegument structure released by deenvelopment during entry into cells must be transported retrograde to the nuclear pore where viral DNA is released into the nucleus. This path is essential in the case of virus entering axons of dorsal root ganglia. The objective of the study was to identify the viral proteins that may be involved in the transport. We report the following findings. (i) The neuronal isoform of the intermediate chain (IC-1a) of the dynein complex pulled down, from lysates of [(35)S]methionine-labeled infected cells, two viral proteins identified as the products of U(L)34 and U(L)31 open reading frames, respectively. U(L)34 protein is a virion protein associated with cellular membranes and phosphorylated by the viral kinase U(S)3. U(L)31 protein is a largely insoluble, evenly dispersed nuclear phosphoprotein required for optimal processing and packaging of viral DNA into preformed capsids. Reciprocal pulldown experiments verified the interaction of IC-1a and U(L)34 protein. In similar experiments, U(L)34 protein was found to interact with U(L)31 protein and the major capsid protein ICP5. (ii) To determine whether U(L)34 protein is transported to the nuclear membrane, a requirement if it is involved in transport, the U(L)34 protein was inserted into a baculovirus vector under the cytomegalovirus major early promoter. Cells infected with the recombinant baculovirus expressed U(L)34 protein in a dose-dependent manner, and the U(L)34 protein localized primarily in the nuclear membrane. An unexpected finding was that U(L)34-expressing cells showed a dissociation of the inner and outer nuclear membranes reminiscent of the morphologic changes seen in cells productively infected with herpes simplex virus 1. U(L)34, like many other viral proteins, may have multiple functions expressed both early and late in infection.

An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers.

Poor sperm motility characterized by a distinct aberration in flagellar waveform known as "curlicue" is a hallmark of t haplotype (t) homozygous male sterility. Previous studies have localized "curlicue" and a flagellar developmental defect, "whipless", to the Hybrid Sterility 6 locus (Hst6), between the markers Pim1 and Crya1. More recent heterospecific breeding experiments between Mus spretus (Spretus) and Mus musculus domesticus (Domesticus) have mapped the primary source(s) of both "curlicue" and "whipless" to a small sub-locus of Hst6, Curlicue a (Ccua). Here we report the complete physical isolation of the Ccua locus and the identification of a candidate gene for expression of both "whipless" and "curlicue" at its proximal end, an axonemal dynein heavy chain gene, Dnahc8, formerly mapped by interspecific backcross analysis near Pim1. Dnahc8 mRNA expression commences in the Domesticus wild-type testis just prior to flagellar assembly and is testis-specific in the adult male. However, expression of Dnahc8 is not readily evident in the testis of either Spretus or "whipless" animals (Domesticus males homozygous for the Spretus allele of Dnahc8). Our results argue that Dnahc8 is fundamental to flagellar organization and function in Domesticus, but not Spretus, and suggest that Dnahc8 is integral to both Hst6- and t-specific male infertility.

Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends.

Cytoplasmic dynein is a minus end-directed microtubule motor responsible for centripetal organelle movement and several aspects of chromosome segregation. Our search for cytoplasmic dynein-interacting proteins has implicated the dynactin complex as the cytoplasmic dynein 'receptor' on organelles and kinetochores. Immunofluorescence microscopy using a total of six antibodies generated against the p150Glued, Arp1 and dynamitin subunits of dynactin revealed a novel fraction of dynactin-positive structures aligned in linear arrays along the distal segments of interphase microtubules. Dynactin staining revealed that these structures colocalized extensively with CLIP-170. Cytoplasmic dynein staining was undetectable, but extensive colocalization with dynactin became evident upon transfer to a lower temperature. Overexpression of the dynamitin subunit of dynactin removed Arp1 from microtubules but did not affect microtubule-associated p150Glued or CLIP-170 staining. Brief acetate treatment, which has been shown to affect lysosomal and endosomal traffic, also dispersed the Golgi apparatus and eliminated the microtubule-associated staining pattern. The effect on dynactin was rapidly reversible and, following acetate washout, punctate dynactin was detected at microtubule ends within 3 minutes. Together, these findings identify a region along the distal segments of microtubules where dynactin and CLIP-170 colocalize. Because CLIP-170 has been reported to mark growing microtubule ends, our results indicate a similar relationship for dynactin. The functional interaction between dynactin and cytoplasmic dynein further suggests that this these regions represent accumulations of cytoplasmic dynein cargo-loading sites involved in the early stages of minus end-directed organelle transport.

The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes.

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

Multiple mouse chromosomal loci for dynein-based motility.

Dyneins are multisubunit mechanochemical enzymes capable of interacting with microtubules to generate force. Axonemal dyneins produce the motive force for ciliary and flagellar beating by inducing sliding between adjacent microtubules within the axoneme. Cytoplasmic dyneins translocate membranous organelles and chromosomes toward the minus ends of cytoplasmic microtubules. Dynactin is an accessory complex implicated in tethering cytoplasmic dynein to membranous organelles and mitotic kinetochores. In the studies described here, we have identified a number of new dynein genes and determined their mouse chromosomal locations by interspecific backcross analysis. We have also mapped several dynein and dynactin genes cloned previously. Our studies provide the first comprehensive attempt to map dynein and dynactin genes in mammals and provide a basis for the further analysis of dynein function in development and disease.

Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis.

Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and analyzed the effects of its overexpression on mitosis in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both dynactin and dynein in chromosome alignment and spindle organization.

Differential expression and phosphorylation of the 74-kDa intermediate chains of cytoplasmic dynein in cultured neurons and glia.

The 74-kDa intermediate chains (IC74) of the cytoplasmic dynein complex are believed to be involved in the association of dynein with membranous organelles. While each dynein molecule is thought to have two or three IC74 subunits, at least six different IC74 protein isoforms were found in dynein from brain. Therefore we investigated the relationships of the brain cytoplasmic dynein IC74 isoforms and their association in the dynein complex at the cellular level. We found that cultured cortical neurons and glia express distinct IC74 isoforms. The IC74 isoform pattern observed in dynein from cortical neurons was generally similar to that found in dynein from adult brain, indicating that there are different populations of cytoplasmic dynein in neurons. Two IC74 isoforms were observed on two-dimensional gels of dynein from glia, while a single glial IC74 mRNA was detected. Metabolic labeling of glial dynein with 32P followed by treatment of the isolated dynein with phosphatase in vitro demonstrated that one of the glial IC74 isoforms is the product of the single glial IC74 mRNA and that the other is its phosphoisoform. A single mRNA product and its phosphoisoform are therefore sufficient for constitutive dynein function and regulation in glial cells.