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PubMed2001

Anticipating incoming events: an impaired cognitive process in schizophrenia.

Intentions are central to guiding actions to their completion because they generate expectations which precede the realization of a task. This ability to manage time was investigated by using a cognitive task which involves several highly integrated processes: sequential learning, explicit processing, and working memory. In this task, participants are required to explicitly learn a repeating color sequence before receiving an instruction to give an anticipatory motor response concerning the next element. Two types of sequences (temporal and spatial) and three experimental conditions were tested in both a group of normal participants and a group of schizophrenic patients. Schizophrenics were included because their condition is known to alter conscious executive function. Our results showed that schizophrenic patients have a strong deficit in performing anticipation tasks. Although they learned the sequences almost normally, their anticipatory ability was reduced in comparison to normal participants in all the tested conditions. These results expand the notion of a working memory deficit in schizophrenia and bear strong implications for understanding executive disorders observed in such patients.

Adult↗

PubMed2002

Interactive computer simulations of knee-replacement surgery.

OBJECTIVE: Current surgical training programs in the United States are based on an apprenticeship model. This model is outdated because it does not provide conceptual scaffolding, promote collaborative learning, or offer constructive reinforcement. Our objective was to create a more useful approach by preparing students and residents for operative cases using interactive computer simulations of surgery. Total-knee-replacement surgery (TKR) is an ideal procedure to model on the computer because there is a systematic protocol for the procedure. Also, this protocol is difficult to learn by the apprenticeship model because of the multiple instruments that must be used in a specific order. We designed an interactive computer tutorial to teach medical students and residents how to perform knee-replacement surgery. We also aimed to reinforce the specific protocol of the operative procedure. Our final goal was to provide immediate, constructive feedback. DESCRIPTION: We created a computer tutorial by generating three-dimensional wire-frame models of the surgical instruments. Next, we applied a surface to the wire-frame models using three-dimensional modeling. Finally, the three-dimensional models were animated to simulate the motions of an actual TKR. The tutorial is a step-by-step tutorial that teaches and tests the correct sequence of steps in a TKR. The student or resident must select the correct instruments in the correct order. The learner is encouraged to learn the stepwise surgical protocol through repetitive use of the computer simulation. Constructive feedback is acquired through a grading system, which rates the student's or resident's ability to perform the task in the correct order. The grading system also accounts for the time required to perform the simulated procedure. We evaluated the efficacy of this teaching technique by testing medical students who learned by the computer simulation and those who learned by reading the surgical protocol manual. Both groups then performed TKR on manufactured bone models using real instruments. Their technique was graded with the standard protocol. The students who learned on the computer simulation performed the task in a shorter time and with fewer errors than the control group. They were also more engaged in the learning process. DISCUSSION: Surgical training programs generally lack a consistent approach to preoperative education related to surgical procedures. This interactive computer tutorial has allowed us to make a quantum leap in medical student and resident teaching in our orthopedic department because the students actually participate in the entire process. Our technique provides a linear, sequential method of skill acquisition and direct feedback, which is ideally suited for learning stepwise surgical protocols. Since our initial evaluation has shown the efficacy of this program, we have implemented this teaching tool into our orthopedic curriculum. Our plans for future work with this simulator include modeling procedures involving other anatomic areas of interest, such as the hip and shoulder.

Arthroplasty, Replacement, Knee↗

PubMedDate not supplied

Structure-activity analysis of taxane-based broad-spectrum multidrug resistance modulators.

BACKGROUND: Clinical drug resistance is frequently associated with overexpression of the multidrug resistance (MDR) proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP). Taxanes are substrates for Pgp and MRP-1, but not BCRP. Taxane-based reversal agents (tRAs) are non-cytotoxic MDR modulators previously examined for broad-spectrum modulation of Pgp, MRP-1 and BCRP. MATERIALS AND METHODS: Modulation by tRAs was studied by flow cytometry and resistance to taxanes was studied in cytotoxicity assays in the parental HL60/wt, 8226/wt and MCF7/S, and the resistant HL60/ADR, 8226/Dox6, 8226/MR20 and MCF7 AdVp3000 cell lines. Amino acid sequence (BLAST) alignments were performed using ClustalW. RESULTS: Structure-activity analysis demonstrated greatest alignment of BCRP with the transmembrane 7-12 region of Pgp and identified tRA side groups that contributed or were detrimental to modulation. CONCLUSION: Identification of tRA side groups contributing to modulation of Pgp, MRP-1 and BCRP will allow the design of a next generation of tRAs and will optimize their potential clinical applicability.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Intronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity.

A rat testicular luteinizing hormone (LH) receptor cDNA containing a 266-base pair deletion resulting in the omission of the 1st transmembrane region and truncation of the open reading frame was isolated using a rat ovarian LH receptor cDNA probe. Comparison of this clone with a restriction fragment from the LH receptor genomic DNA revealed potential alternative splice sites following the consensus sequence TTXCAG that is present at an intron acceptor splice site and also within the next exon, accounting for the specific deletion mutation observed in this cDNA. Expression of the testicular cDNA in COS1 cells resulted in synthesis and secretion of a soluble binding protein with high affinity and specificity for LH and human chorionic gonadotropin. These studies have demonstrated that the LH receptor gene contains intron(s) within the region coding for the extracellular domain of the molecule, which determine the nature and generation of LH receptor isoforms. Expression of the soluble form of the LH receptor has indicated that the amino-terminal extracellular region plays a major role in gonadotropin binding. These features of the LH receptor are distinct from those of most other G protein-coupled receptors, which are intronless and contain their binding sites within the transmembrane region rather than the extracellular domain.

Amino Acid Sequence↗

PubMed1996

Non-Mendelian, heritable blocks to DNA rearrangement are induced by loading the somatic nucleus of Tetrahymena thermophila with germ line-limited DNA.

Site-specific DNA deletion occurs at thousands of sites within the genome during macronuclear development of Tetrahymena thermophila. These deletion elements are usually not detected in macronuclear chromosomes. We have interfered with the normal deletion of two of these elements, the adjacent M and R elements, by loading vegetative macronuclei with these elements prior to sexual conjugation. Transformed cell lines containing the exogenous M or R element, carried on high-copy-number vectors containing genes encoding rRNA within parental (old) macronuclei, consistently failed to excise chromosomal copies of the M or R element during formation of new macronuclei. Little or no interference with the deletions of adjacent elements or of unlinked elements was observed. The micronucleus (germ line)-limited region of each element was sufficient to inhibit specific DNA deletion. This interference with DNA deletion usually is manifested as a cytoplasmic dominant trait: deletion elements present in the old macronucleus of one partner of a mating pair were sufficient to inhibit deletion occurring in the other partner. Remarkably, the failure to excise these elements became a non-Mendelian, inheritable trait in the next generation and did not require the high copy number of exogenously introduced elements. The introduction of exogenous deletion elements into parental macronuclei provides us with an epigenetic means to establish a heritable pattern of DNA rearrangement.

Animals↗

PubMed2006

Fifty years of genetic epidemiology, with special reference to Japan.

Genetic epidemiology deals with etiology, distribution, and control of disease in groups of relatives and with inherited causes of disease in populations. It took its first steps before its recognition as a discipline, and did not reach its present scope until the Human Genome Project succeeded. The intimate relationship between genetics and epidemiology was discussed by Neel and Schull (1954), just a year after Watson and Crick reported the DNA double helix, and 2 years before human cytogenetics and the Japan Society of Human Genetics were founded. It is convenient to divide the next half-century into three phases. The first of these (1956-1979) was before DNA polymorphisms were typed, and so the focus was on segregation and linkage of major genes, cytogenetics, population studies, and biochemical genetics. The next phase (1980-2001) progressively identified DNA polymorphisms and their application to complex inheritance. The last phase began with a reliable sequence of the human genome (2002), followed by exploration of genomic diversity. Linkage continues to be useful to study recombination and to map major genes, but association mapping gives much greater resolution and enables studies of complex inheritance. The generation now entering human genetics will have collaborative opportunities undreamed of a few years ago, without the independence that led to great advances during the past half-century.

Epidemiology↗

PubMed1986

Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter.

The Xenopus hsp70 promoter contains three copies of the consensus heat shock element (HSE) between positions -260 and -100. When the gene is transfected into mammalian cells, maximal heat-induced expression requires two HSEs in addition to a CCAAT box located next to the TATA box. The HSE-containing region can be separated from the CCAAT/TATA region without affecting expression of the gene, and it can enhance transcription of a linked beta-globin gene upon heat shock. It thus has the properties of a heat-inducible enhancer. Such an enhancer can also be generated by duplication of HSE sequences from the Drosophila hsp70 promoter, which were previously identified as an upstream promoter element and are known to bind a purified heat shock transcription factor in vitro.

Animals↗

PubMed2002

T-DNA insertional mutagenesis for activation tagging in rice.

We have developed a new T-DNA vector, pGA2715, which can be used for promoter trapping and activation tagging of rice (Oryza sativa) genes. The binary vector contains the promoterless beta-glucuronidase (GUS) reporter gene next to the right border. In addition, the multimerized transcriptional enhancers from the cauliflower mosaic virus 35S promoter are located next to the left border. A total of 13,450 T-DNA insertional lines have been generated using pGA2715. Histochemical GUS assays have revealed that the GUS-staining frequency from those lines is about twice as high as that from lines transformed with the binary vector pGA2707, which lacks the enhancer element. This result suggests that the enhancer sequence present in the T-DNA improves the GUS-tagging efficiency. Reverse transcriptase-PCR analysis of a subset of randomly selected pGA2715 lines shows that expression of the genes immediately adjacent to the inserted enhancer is increased significantly. Therefore, the large population of T-DNA-tagged lines transformed with pGA2715 could be used to screen for promoter activity using the gus reporter, as well as for creating gain-of-function mutants.

Caulimovirus↗

PubMed1979

Development, ultrastructure, and mode of transmission of Amblyospora sp. (Microspora) in the mosquito.

Amblyospora sp. in Culex salinarius (Coquillett) is transovarially transmitted and has 2 developmental sequences, one in each host sex. In females, the entire life cycle is restricted to oenocytes which become greatly hypertrophied due to the multiplication of diplokaryotic cells during merogony and come to lie next to ovaries. Sporulation occurs only after a blood meal is taken and is shortly followed by infection of the oocytes and subsequent transmission to the next host generation. In the male host, infections spread from oenocytes to adipose tissue where diplokaryotic cells undergo a 2nd merogony. During this merogonic cycle, the number of diplokaryotic cells greatly increases and the infection is spread throughout the body of the larval host. Sporulation is initiated with the physical separation of the 2 members of the diplokaryon and the simultaneous secretion of a pansporoblastic membrane. Subsequent meiotic division and morphogenesis result in the formation of 8 haploid spores enclosed with a pansporoblastic membrane. Buildup of spores and subsequent destruction of host adipose tissue prove fatal to the male host during the 4th larval stage.

Animals↗

PubMed1990

Equivalence classes generated by sequence training.

In Experiment 1, 3, adult females were taught with verbal instructions and contingencies to select, in sequence, three arbitrary visual stimuli from an array of five stimuli. After four different sequences were taught, match-to-sample tests assessed emergent conditional relations among all stimuli that had been selected in the same order in the sequences. Subjects' performances indicated development of four stimulus classes, three based on ordinal position and one based on nonselection. Next, match-to-sample training established conditional relations between each of four novel figures and one member of each of the ordinal stimulus classes. Tests confirmed that the classes were equivalence classes, each expanded by one new member. In subsequent sequence tests, the new stimuli were selected in a sequence that was consistent with ordinal class membership. Experiment 2 replicated Experiment 1 with 2 different adult females, but the verbal instructions were omitted. Results were similar to Experiment 1, except that extensive review and retesting were required before expansion of the ordinal classes with the novel figures was observed.

Adult↗

PubMed2025

Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

Gene Editing↗

PubMed2001

Trap a gene and find out its function: toward functional genomics in Drosophila.

Many declared aims of the genome projects have been achieved. The total genomic sequences of several relatively noncomplex/complex organisms (such as E. coli, yeast, Caenorhabditis, Drosophila) are being determined, and the nucleotide sequencing of the entire human genome will be complete in the near future. However, this achievement is not the end of the road but rather the first step toward the functional understanding of the genome of humans and other organisms. The determined linear nucleotide sequences remain only lists of A, C, G and T, unless they are given functional significance. The coding sequences of genes can be identified in a relatively reliable manner by computational methods, but the exact function of their protein products can rarely be determined without obtaining much additional information, e.g., by biochemical or cell biological methods. Thus, following sequencing, the next step must be to assign functions to the identified genes. The final goal of genome research today may look futuristic, but the knowledge of the function of every single gene and the interactions between them will finally allow us to understand the development and functioning of an organism as a whole. Gene-trapping methodology is a powerful strategy for cloning and identifying functional genes, as it marks a gene with a tag and simultaneously generates a corresponding genetic variation for that particular locus. Therefore, gene trapping is an extremely useful tool for functional genomics, establishing a correlation between the physical and genetic maps of the genome. The relative simplicity of its genome and the availability of huge bodies of genetic and molecular information make Drosophila melanogaster one of the most important model organisms. Its genome will serve as a "reference" for the in-depth analysis of the organization of more complex eukaryotic genomes. Multifaceted approaches to Drosophila functional genomics and the dual-tagging gene trap system newly developed for functional analysis of Drosophila genes are discussed in this review.

Animals↗

PubMed2004

Natal homing in juvenile loggerhead turtles (Caretta caretta).

Juvenile loggerhead turtles (Caretta caretta) from West Atlantic nesting beaches occupy oceanic (pelagic) habitats in the eastern Atlantic and Mediterranean, whereas larger juvenile turtles occupy shallow (neritic) habitats along the continental coastline of North America. Hence the switch from oceanic to neritic stage can involve a trans-oceanic migration. Several researchers have suggested that at the end of the oceanic phase, juveniles are homing to feeding habitats in the vicinity of their natal rookery. To test the hypothesis of juvenile homing behaviour, we surveyed 10 juvenile feeding zones across the eastern USA with mitochondrial DNA control region sequences (N = 1437) and compared these samples to potential source (nesting) populations in the Atlantic Ocean and Mediterranean Sea (N = 465). The results indicated a shallow, but significant, population structure of neritic juveniles (PhiST = 0.0088, P = 0.016), and haplotype frequency differences were significantly correlated between coastal feeding populations and adjacent nesting populations (Mantel test R2 = 0.52, P = 0.001). Mixed stock analyses (using a Bayesian algorithm) indicated that juveniles occurred at elevated frequency in the vicinity of their natal rookery. Hence, all lines of evidence supported the hypothesis of juvenile homing in loggerhead turtles. While not as precise as the homing of breeding adults, this behaviour nonetheless places juvenile turtles in the vicinity of their natal nesting colonies. Some of the coastal hazards that affect declining nesting populations may also affect the next generation of turtles feeding in nearby habitats.

Analysis of Variance↗

PubMed2007

Influence of Ki-ras-driven oncogenic transformation on the protein network of murine fibroblasts.

Ki-ras gene mutations that specifically occur in codons 12, 13 and 61 are involved in the carcinogenesis of acute myeloid leukemia, melanoma and different carcinomas. In order to define potential mutation-specific therapeutic targets, stable transfectants of NIH3T3 cells carrying different Ki-ras4B gene mutations were generated. Wild type Ki-ras transformants, mock transfectants and parental cells served as controls. These in vitro model systems were systematically analyzed for their protein expression pattern using two-dimensional gel electrophoresis followed by mass spectrometry and/or protein sequencing. Using this approach, a number of target molecules that are differentially but coordinately expressed in the ras transfectants were identified next to other proteins that exhibit a distinct regulation pattern in the different cell lines analyzed. The differentially expressed proteins predominantly belong to the families of cytoskeletal proteins, heat shock proteins, annexins, metabolic enzymes and oxidoreductases. Their validation was assessed by real-time quantitative RT-PCR and/or Western blot analysis. Our results suggest that the Ki-ras-transformed cells represent a powerful tool to study Ki-ras gene mutation-driven protein expression profiles. In addition, this approach allows the discovery of ras-associated cellular mechanisms, which might lead to the identification of physiological targets for pharmacological interventions of the treatment of Ki-ras-associated human tumors.

Animals↗

PubMed1999

An infectious molecular clone of a Japanese genotype 1b hepatitis C virus.

We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a third-generation enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera.

3' Untranslated Regions↗

PubMed2006

Reproducing bursting interspike interval statistics of the gustatory cortex.

Cortical neurons in vivo generate highly irregular spike sequences. Recently, it was experimentally found that the local variation of interspike intervals, LV, is nearly constant for every spike sequence for the same neurons. On the contrary, the coefficient of variation, CV, varies over different spike sequences. Here, we first show that these characteristic features are also applicable in bursting spike sequences that are obtained from the rat gustatory cortex. Next, we show that the conventional leaky integrate-and-fire model does not fully account for reproducing these statistical features in data of real bursting spike sequences. We resolve this difficulty by proposing an alternative neuron model which is a reduction of the bursting neuron model involving the persistent sodium current. Our study implies that (1) the characteristic features of CV and LV are the results of the endogenous bursting and (2) the bursting behavior in the gustatory cortex is caused mainly by the persistent sodium current.

Animals↗

PubMed2006

Identifying the interacting positions of a protein using Boolean learning and support vector machines.

It is known that in the three-dimensional structure of a protein, certain amino acids can interact with each other in order to provide structural integrity or aid in its catalytic function. If these positions are mutated the loss of this interaction usually leads to a non-functional protein. Directed evolution experiments, which probe the sequence space of a protein through mutations in search for an improved variant, frequently result in such inactive sequences. In this work, we address the use of machine learning algorithms, Boolean learning and support vector machines (SVMs), to find such pairs of amino acid positions. The recombination method of imparting mutations was simulated to create in silico sequences that were used as training data for the algorithms. The two algorithms were combined together to develop an approach that weighs the structural risk as well as the empirical risk to solve the problem. This strategy was adapted to a multi-round framework of experiments where the data generated in the present round is used to design experiments for the next round to improve the generated library, as well as the estimation of the interacting positions. It is observed that this strategy can greatly improve the number of functional variants that are generated as well as the average number of mutations that can be made in the library.

Algorithms↗

PubMed1988

Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.

Animals↗