bluuebunny/crossref_metadata_2025_split huggingface dataset:
Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version A
Authors: Tiscornia Gustavo , Singer Oded , Verma Inder M. 
Published: August 2008

INTRODUCTIONThis protocol combines the specificity of small interfering RNA (siRNA)-mediated silencing cassettes with the versatility of lentiviral vectors to stably transduce a wide range of cell types. A short hairpin RNA (shRNA) designed against a given target is cloned into a plasmid containing the pol III promoter. The design uses a 5′ forward primer upstream of the pol III promoter and a 3′ reverse primer that includes the entire shRNA sequence (i.e., sense, loop, and antisense sequences followed by five Ts), followed by 22 bases complementary to the last 22 bp upstream of the +1 transcriptional start site of the pol III promoter. An NheI-compatible restriction site is included at the 5′ end of both forward and reverse primers. A single round of PCR is used to amplify this template. The resulting DNA fragment contains an shRNA expression cassette that can be cloned into a simple cloning vector, tested, and then transferred to the lentiviral vector, or cloned into the lentiviral vector directly. This procedure uses a unique restriction site in the 3′ long terminal repeat (LTR). During integration, the 5′ LTR of the provirus is copied from the 3′ LTR, cloning the H1-driven shRNA into the 3′ LTR, resulting in duplication of the silencing cassette. This strategy maximizes the silencing power of the lentiviral vector. The combination of the lentiviral and siRNA technologies provides a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo.
Phage-Display Methodology for the Study of Protein-Protein Interactions: Stage 2, Panning: Figure 1.
Authors: Goodyear Carl S. , Silverman Gregg J. 
Published: August 2008

INTRODUCTIONThis method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 2 of the method, we describe the use of streptavidin-coated magnetic beads for library selection (panning). After selection, the elution strategy used to recover desirable clones should be chosen on the basis of the type of panning strategy employed, and the properties of the ligand that has been targeted. Of the several alternatives, we have compared the common recovery strategies of low pH, trypsinization of bound libraries, and competition with soluble antigen. When considering which option to employ, it is worth knowing how this may affect the type of binder that will be recovered. Low pH will dissociate most or all binders, whether they are specific or nonspecific for the selection antigen, whereas trypsin targets the fusion area between the protein and gene III, and specifically cleaves it. As a result, for the latter, only phage that bind via a protein-protein interaction will be recovered. In contrast, soluble antigen will provide specific binders of relatively high affinity because the elution will work in a competitive manner. We recommend an empiric approach to determine which is best for specific projects.
Phage-Display Methodology for the Study of Protein-Protein Interactions: Stage 3, Binding Analysis
Authors: Goodyear Carl S. , Silverman Gregg J. 
Published: August 2008

INTRODUCTIONThis method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 3 of the method, described here, the rounds of selection are analyzed to evaluate the enrichment for binders. To do this, polyethylene glycol (PEG)-precipitated phage from each round of panning are tested in an assay, be it ELISA-based or flow cytometric. The assay should include all rounds of phage samples, from the initial unselected phage to the last round and helper phage (as a negative control). The method chosen to evaluate whole phage libraries can also be used as a system of evaluating single clones from the appropriate round. The appropriate round is the one that yields a library exhibiting the best binding characteristics (or best signal in the detection assay) associated with the protein-protein interaction. This protocol outlines a basic ELISA-based analysis strategy.
Analyzing DNA Replication I: Labeling Animals, Tissues, and Cells with Bromodeoxyuridine (BrdU)
Authors: Jackson Dean , Cook Peter R. 
Published: August 2008

INTRODUCTIONThe number of cells traversing the cell cycle and the rate of progression through it provide important indices of cell growth and tumorigenicity. S-phase cells can also be identified by their high content of DNA polymerase and proliferating cell nuclear antigen, a component of the leading-strand polymerase. Although both these markers can be detected rapidly and conveniently using the appropriate antibodies, neither are found exclusively in S-phase cells. Immunolabeling after incorporation of modified DNA precursors (e.g., 5-bromodeoxyuridine [BrdU, bromodeoxyuridine]) allows more rapid and precise detection of cells in S-phase of the cell cycle. BrdU is phosphorylated by cells to give BrdUTP, and this precursor is incorporated into DNA instead of deoxythimidine triphosphate. In living cells, BrdU is incorporated into replication sites that can then be detected using fluorochrome or enzyme-coupled antibodies. Alternatively, DNA synthesis sites can be labeled at high resolution by incubating cells with analogs of the natural precursors of DNA. The cells are then fixed and the incorporation sites are detected using fluorochrome- or enzyme-tagged antibodies. Cells labeled in this way either in vivo or in vitro display a few hundred discrete nuclear sites early in S-phase, with distinct patterns of DNA replication that are characteristic of different stages of S-phase. “Foci” labeled after very short incubations correspond with sites where many replicons are duplicated simultaneously within massive protein complexes. This protocol provides details for incorporating DNA precursors into tissues in whole animals in vivo, isolated tissue in vitro, and cultured cells.
Whole Mount RNA In Situ Hybridization on Zebrafish Embryos: Probe Synthesis
Authors: Moens Cecilia 
Published: August 2008

INTRODUCTIONThis protocol describes methods for synthesizing digoxigenin- and fluorescein-labeled RNA probes from linearized plasmids or PCR products for use in whole-mount RNA in situ hybridization on zebrafish embryos.
Whole Mount RNA In Situ Hybridization on Zebrafish Embryos: Hybridization
Authors: Moens Cecilia 
Published: August 2008

INTRODUCTIONThis protocol describes procedures for detecting gene expression in zebrafish embryos in situ using labeled RNA probes. The labels (digoxigenin or fluorescein) are detected using alkaline phosphatase-conjugated antibodies that catalyze a chromogenic reaction, producing visible blue or red products. Two-color in situ hybridization can be performed by labeling with a combination of digoxigenin- and fluorescein-labeled probes and detecting the digoxigenin and fluorescein with sequential alkaline phosphatase reactions using different chromogenic substrates. In the method described here, the fluorescein-labeled probe is detected with the red substrate, but either probe can be detected with either substrate.
Fast, transient and specific intracellular ROS changes in living root hair cells responding to Nod factors (NFs)
Authors: Cárdenas Luis , Martínez Adán , Sánchez Federico , Quinto Carmen 
Published: August 2008

SummaryThe role of reactive oxygen species (ROS) in root‐nodule development and metabolism has been extensively studied. However, there is limited evidence showing ROS changes during the earliest stages of the interaction between legumes and rhizobia. Herein, using ratio‐imaging analysis, increasing and transient ROS levels were detected at the tips of actively growing root hair cells within seconds after addition of Nod factors (NFs). This transient response (which lasted up to 3 min) was Nod‐factor‐specific, as chitin oligomers (pentamers) failed to induce a similar response. When chitosan, a fungal elicitor, or ATP was used instead, a sustained increasing signal was observed. As ROS levels are transiently elevated after the perception of NFs, we propose that this ROS response is characteristic of the symbiotic interaction. Furthermore, we discuss the remarkable spatial and temporal coincidences between ROS and transiently increased calcium levels observed in root hair cells immediately after the detection of NFs.
Transgressive segregation for phenological traits in barley explained by two major QTL alleles with additivity
Authors: Castro A. J. , Hayes P. , Viega L. , Vales I. 
Published: August 2008

AbstractQuantitative trait loci (QTL) analysis can contribute to a deeper understanding of crop phenology. The parents of a barley mapping population have similar growth and development profiles, but the progeny show transgressive segregation for phenological traits. These phenotypes were measured in eight field experiments, using different planting dates over 3 years. Five QTL, on four chromosomes, were detected for anthesis date. The four maturity QTL were on the same chromosomes as the anthesis QTL. Five QTL for grain filling were detected on all chromosomes. Three QTL, on chromosomes 1H and 2H, were detected for photoperiod sensitivity. Both parents contributed higher value alleles for all traits, except for photoperiod sensitivity. QTL epistasis was not significant. Two QTL explained most of the phenotypic variation for anthesis and physiological maturity. Non‐parental combinations of alleles at these loci account for the phenotypic transgressive segregation. Candidate genes for these QTL effects are eps2S (2H) and denso (3H). QTL for other traits had smaller effects and were coincident with genes and/or QTL for the same traits reported in other germplasm.
STIM, ORAI AND TRPC CHANNELS IN THE CONTROL OF CALCIUM ENTRY SIGNALS IN SMOOTH MUSCLE
Authors: Wang Youjun , Deng Xiaoxiang , Hewavitharana Thamara , Soboloff Jonathan , Gill Donald L 
Published: August 2008

SUMMARY

Ca2+ entry signals are crucial in the control of smooth muscle contraction. Smooth muscle cells are unusual in containing plasma membrane (PM) Ca2+ entry channels that respond to voltage changes, receptor activation and Ca2+ store depletion.
Activation of these channel subtypes is highly coordinated. The TRPC6 channel, widely expressed in most smooth muscle cell types, is largely non‐selective to cations and is activated by diacylglycerol arising from receptor‐induced phosholipase C activation.
Receptor activation results largely in Na+ ion movement through TRPC6 channels, depolarization and subsequent activation of voltage‐dependent L‐type Ca2+ channels. The TRPC6 channels also appear to be activated by mechanical stretch, resulting again in depolarization and L‐type Ca2+ channel activation. Such a coupling may be crucial in mediating the myogenic tone response in vascular smooth muscle.
The emptying of stores mediated by inositol 1,4,5‐trisphosphate receptors triggers the endoplasmic reticulum (ER) Ca2+ sensing protein stromal‐interacting molecule (STIM) 1 to translocate into defined ER–PM junctional areas in which coupling occurs to Orai proteins, which serve as highly Ca2+‐selective low‐conductance Ca2+ entry channels.
These ER‐PM junctional domains may serve as crucial sites of interaction and integration between the function of store‐operated, receptor‐operated and voltage‐operated Ca2+ channels. The STIM, Orai and TRPC channels represent highly promising new pharmacological targets through which such control may be induced.
Effect of initial shock wave voltage on shock wave lithotripsy‐induced lesion size during step‐wise voltage ramping
Authors: Connors Bret A. , Evan Andrew P. , Blomgren Philip M. , Handa Rajash K. , Willis Lynn R. , Gao Sujuan 
Published: August 2008

OBJECTIVETo determine if the starting voltage in a step‐wise ramping protocol for extracorporeal shock wave lithotripsy (SWL) alters the size of the renal lesion caused by the SWs.MATERIALS AND METHODSTo address this question, one kidney from 19 juvenile pigs (aged 7–8 weeks) was treated in an unmodified Dornier HM‐3 lithotripter (Dornier Medical Systems, Kennesaw, GA, USA) with either 2000 SWs at 24 kV (standard clinical treatment, 120 SWs/min), 100 SWs at 18 kV followed by 2000 SWs at 24 kV or 100 SWs at 24 kV followed by 2000 SWs at 24 kV. The latter protocols included a 3–4 min interval, between the 100 SWs and the 2000 SWs, used to check the targeting of the focal zone. The kidneys were removed at the end of the experiment so that lesion size could be determined by sectioning the entire kidney and quantifying the amount of haemorrhage in each slice. The average parenchymal lesion for each pig was then determined and a group mean was calculated.RESULTSKidneys that received the standard clinical treatment had a mean (sem) lesion size of 3.93 (1.29)% functional renal volume (FRV). The mean lesion size for the 18 kV ramping group was 0.09 (0.01)% FRV, while lesion size for the 24 kV ramping group was 0.51 (0.14)% FRV. The lesion size for both of these groups was significantly smaller than the lesion size in the standard clinical treatment group.CONCLUSIONSThe data suggest that initial voltage in a voltage‐ramping protocol does not correlate with renal damage. While voltage ramping does reduce injury when compared with SWL with no voltage ramping, starting at low or high voltage produces lesions of the same approximate size. Our findings also suggest that the interval between the initial shocks and the clinical dose of SWs, in our one‐step ramping protocol, is important for protecting the kidney against injury.
STRATEGIC INVESTMENT IN A NEW MIXED MARKET WITH LABOR‐MANAGED AND PROFIT‐MAXIMIZING FIRMS
Authors: Ohnishi Kazuhiro 
Published: August 2008

ABSTRACTThis paper examines a continuous‐time mixed model of the strategic investment decisions of a labor‐managed income‐per‐worker‐maximizing firm and a profit‐maximizing firm in a new mixed market and constructs a set of perfect equilibria of the continuous‐time mixed model. The paper shows that there exists a particular equilibrium in which neither firm invests to its steady‐state reaction curve. The paper also finds that the existence of the particular equilibrium depends on each firm's being able to respond quickly to its rival's investment and that the particular equilibrium is profitable for each firm.
Clinical supervision: a means of promoting reciprocity between practitioners and academics
Authors: Pettifer Annie , Clouder Lynn 
Published: August 2008

AbstractThis paper explores the potential of adopting an alternative approach to the facilitation of clinical supervision in a practice context by focusing on the experiences of academic staff of facilitating clinical supervision for clinical colleagues. The exploratory study, which forms the basis for ideas generated, was very small and hence it might be better conceptualized as an analysis of conversations about practice understandings rather than research per se, and certainly, we make no claims of any possibility of generalizability. Nonetheless, we anticipate that our findings will be of interest to a wide audience involved in clinical supervision given that the supervision by academic staff of colleagues in practice is an unusual and unconventional departure from the norm.The experiences of six academic staff from one higher education institution who were brought together to form a focus group provide the basis for discussion. Analysis of the structured discussion led to emergence of four major themes which are presented in an order that moves from the individuals’ experiences and perceptions of themselves in the process, to explore the relational nature of clinical supervision. The first theme highlights issues of professional identity or academics’ sense of themselves as health professionals and/or academics. This leads to the second theme that explores the tangible benefits identified by participants of providing clinical supervision as academics. The third theme about the perceived contribution that academics make to supervision, links, finally, to the perceived reciprocity that springs from clinical supervision relationships between academics providing supervision in practice, and clinicians. Our findings, based on this modest inquiry, suggest that this model of clinical supervision has potential to prove highly fruitful for both academic staff and their clinical colleagues.
Characterization of the Drosophila Gene‐Switch system in aging studies: a cautionary tale
Authors: Poirier Luc , Shane Amanda , Zheng Jie , Seroude Laurent 
Published: August 2008

SummaryGenetic studies have shown that in many model organisms, single gene mutations can dramatically influence aging. Systems that allow researchers to control a gene's temporal and spatial expression pattern, known as inducible gene‐expression systems, are a valuable asset for the study of the influence of single genes on aging. One inducible gene‐expression system reported to allow temporal and tissue‐specific control of gene expression in Drosophila is the Gene‐Switch system. However, this system has not been extensively characterized in the context of aging research. This report uses six Gene‐Switch strains to examine the tissue localization and amount of expression achievable in the major tissue types of the fly. The quantitative analysis of adult flies fed with inducer through life reveals that the levels of expression are influenced by both the inducer concentration and the age of the animal in a strain‐specific manner. Furthermore, the relationship between inducer concentration and expression level is unique to each strain and, in some cases, to each gender. The analysis of the spatial expression patterns in several strains revealed expression in more tissue types than previously assumed. Finally, most Gene‐Switch strains display expression in the absence of inducer during development and/or during adulthood. These findings have important implications that may reconcile contradictions reported in studies investigating the effects of dFOXO on longevity. This study is an important guide to the design and interpretation of aging studies based on the Gene‐Switch system.
p16INK4a‐induced senescence is disabled by melanoma‐associated mutations
Authors: Haferkamp Sebastian , Becker Therese M. , Scurr Lyndee L. , Kefford Richard F. , Rizos Helen 
Published: August 2008

SummaryThe p16INK4a‐Rb tumour suppressor pathway is required for the initiation and maintenance of cellular senescence, a state of permanent growth arrest that acts as a natural barrier against cancer progression. Senescence can be overcome if the pathway is not fully engaged, and this may occur when p16INK4a is inactivated. p16INK4a is frequently altered in human cancer and germline mutations affecting p16INK4a have been linked to melanoma susceptibility. To characterize the functions of melanoma‐associated p16INK4a mutations, in terms of promoting proliferative arrest and initiating senescence, we utilized an inducible expression system in a melanoma cell model. We show that wild‐type p16INK4a promotes rapid cell cycle arrest that leads to a senescence programme characterized by the appearance of chromatin foci, activation of acidic β‐galactosidase activity, p53 independence and Rb dependence. Accumulation of wild‐type p16INK4a also promoted cell enlargement and extensive vacuolization independent of Rb status. In contrast, the highly penetrant p16INK4a variants, R24P and A36P failed to arrest cell proliferation and did not initiate senescence. We also show that overexpression of CDK4, or its homologue CDK6, but not the downstream kinase, CDK2, inhibited the ability of wild‐type p16INK4a to promote cell cycle arrest and senescence. Our data provide the first evidence that p16INK4a can initiate a CDK4/6‐dependent autonomous senescence programme that is disabled by inherited melanoma‐associated mutations.
TNF, IFNG, IL6, IL10 and TGFB1 gene polymorphisms in South and Southeast Brazil
Authors: Visentainer Jeane Eliete Laguila , Sell Ana Maria , Da Silva Gisele Cristina , Cavichioli Ariza Danussa Gonçalves , Franceschi Danilo Santana Alessio , Lieber Sofia Rocha , De Souza Cármino Antonio 
Published: August 2008

SummaryThis study attempted to establish single nucleotide polymorphism frequencies of TNF, IL6, IFNG, IL10 and TGFB1 genes among healthy individuals from South and Southeast Brazil. The sample included 108 healthy individuals from South and 106 from Southeast Brazil. Polymerase chain reaction using sequence‐specific primers genotyping was performed for these gene cytokines with Cytokine Genotyping Primers (One Lambda, Canoga Park, CA, USA). Differences in genotypic and allelic frequencies between the populations were assessed by chi‐square with either Yates’ correction or Fisher's exact test. Our investigations showed that there were not any significant differences between these two Brazilian populations for these polymorphisms. A statistically significant difference in the distribution of alleles and genotypes for both IL6 and IL10 genes was observed between the Brazilian population and the African‐derived populations. IL6‐174GG genotype and allele G and IL10‐819CT/‐592CA genotypes are more frequent in African‐derived populations than in this mixed Brazilian population, while IL10‐1082GG genotype is more frequent in our population. This mixed Brazilian population is closer to those of Joinville's, Santa Catarina, and Rio de Janeiro's, Rio de Janeiro (RJ), Euro‐Brazilian populations than to those of Salvador's, Bahia, and Rio de Janeiro's, RJ, African‐Brazilian populations. These findings have an enormous importance for experimental design and empowering future linkage and association mapping studies of the role of cytokines in human diseases and allotransplantation outcome in Brazil.
Localization and Dynamic Change of Saikosaponin in Root of Bupleurum chinense
Authors: Tan Ling‐Ling , Cai Xia , Hu Zheng‐Hai , Ni Xi‐Lu 
Published: August 2008

Abstract Anatomical, histochemical and phytochemical methods were used to investigate the structure, the localization and content changes of total saikosaponin and saikosaponin‐a of the roots of Bupleurum chinense DC. at different developmental stages. Results showed that saikosaponin was mainly distributed in pericycle and primary phloem in the young root; but in the mature root, it was mainly distributed in vascular cambium and secondary phloem. During the whole growth period from the pre‐blossom, blossom, fruit, and fruit mature periods until the pre‐withering period, it was in the fruit mature period that both the total saikosaponin content and the saikosaponin‐a content reached the highest level. So the last 20 d of October was considered as the right collecting season for the drug of B. chinense. In addition, the quality of 1‐year‐old drug was better than that of 2‐year‐old drug due to its higher saikosaponin content. On the other hand, judging from the external characteristics of the drug, the one with an acerose taproot and more lateral roots was of better quality. The results offered theoretical bases for selecting medicinal material of high quality and determining the most appropriate harvesting stage and part of B. chinense.