Stabilization of the tau exon 10 stem loop alters pre-mRNA splicing

Neurofibrillary tangles containing filaments of the microtubule-associated protein tau are found in a variety of neurodegenerative diseases. Mutations in the tau gene itself cause frontotemporal dementia with parkinsonism, demonstrating the critical role of tau in pathogenesis. Many of these mutations in tau are silent, are found at the 5'-splice site of exon 10, and lead to increased inclusion of exon 10. These silent mutations are predicted to destabilize a stem loop structure at the exon 10 5'-splice site; however, the existence of this stem loop under physiological conditions and its role in splice regulation are controversial. Here we show that base changes that stabilize this stem loop in vitro substantially decrease exon 10 inclusion in a wild type tau minigene and rescue the increase in exon 10 splicing caused by a dementia-causing point mutation. Moreover, we probed the intracellular structure of the tau stem loop with antisense RNA and demonstrate that the stability of the stem loop dictates antisense effectiveness. Together these results validate the stem loop as a bona fide structure regulating tau exon 10 splicing.     

Modulation of connexin43 alters expression of osteoblastic differentiation markers

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43^–). hFOB/Cx43^– cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43^– cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43^– cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor 1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43^–. Osteopontin mRNA levels were increased in hFOB/Cx43^– relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43^– and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19     

Structure-activity relationships of triazolopyridine oxazole p38 inhibitors: identification of candidates for clinical development

The synthesis, structure-activity relationship, in vivo activity, and metabolic profile for a series of triazolopyridine-oxazole based p38 inhibitors are described. The deficiencies of the lead structure in the series, CP-808844, were overcome by changes to the C4 aryl group and the triazole side-chain culminating in the identification of several potential clinical candidates.     

Influence of food hoarding behavior on the over-winter survival of pikas in strongly seasonal environments

Food hoarding is a behavioral adaptation of some herbivores to manage food availability through time and space. In strongly seasonal environments, where summer growing seasons are short relative to winter, an earlier start to hoarding should increase the amount of vegetation stored for winter and improve subsequent survival. We examined hoarding behavior (‘haying’) and its impact on survival for a small alpine lagomorph, the collared pika (Ochotona collaris) in Yukon, Canada. We used a combination of video surveillance, haypile measurements, and survival data from marked individuals of known age and sex. Annual haypile initiation was strongly influenced by age and year. Adult pikas began haying an average of 16 days earlier in 2004 relative to 2005, whereas young of the year (juveniles) did not vary in the timing of haypile initiation. The mean haying rate per hour increased monthly from 3.7 ± 0.7 trips in June to 6.6 ± 0.8 trips in August. Simulation analysis estimated the median haypile mass (dry weight) by mid-September to be 5.5 kg. At least 75% of simulated haypiles had a minimum of 90 days (3 months) of food reserves, and 50% of simulated haypiles had a minimum of 177 days (5.9 months) of food reserves by mid-September, supporting the hypothesis that haypiles serve as the primary source of food during winter. Survival was greatest for pikas in 2005 when they began haying prior to 31 July, but the benefits of earlier accumulation of vegetation on survival also varied between years. The implications of earlier spring snowmelt are discussed with respect to pika foraging and overwinter survival.     

The podosomal-adaptor protein SH3PXD2B is essential for normal postnatal development

Podosome-type adhesions are actin-based membrane protrusions involved in cell-matrix adhesion and extracellular matrix degradation. Despite growing knowledge of many proteins associated with podosome-type adhesions, much remains unknown concerning the function of podosomal proteins at the level of the whole animal. In this study, the spontaneous mouse mutant nee was used to identify a component of podosome-type adhesions that is essential for normal postnatal growth and development. Mice homozygous for the nee allele exhibited runted growth, craniofacial and skeletal abnormalities, ocular anterior segment dysgenesis, and hearing impairment. Adults also exhibited infertility and a form of lipodystrophy. Using genetic mapping and DNA sequencing, the cause of nee phenotypes was identified as a 1-bp deletion within the Sh3pxd2b gene on mouse Chromosome 11. Whereas the wild-type Sh3pxd2b gene is predicted to encode a protein with one PX domain and four SH3 domains, the nee mutation is predicted to cause a frameshift and a protein truncation altering a portion of the third SH3 domain and deleting all of the fourth SH3 domain. The SH3PXD2B protein is believed to be an important component of podosomes likely to mediate protein-protein interactions with membrane-spanning metalloproteinases. Testing this directly, SH3PXD2B localized to podosomes in constitutively active Src-transfected fibroblasts and through its last SH3 domain associated with a transmembrane member of a disintegrin and metalloproteinase family of proteins, ADAM15. These results identify SH3PXD2B as a podosomal-adaptor protein required for postnatal growth and development, particularly within physiologic contexts involving extracellular matrix regulation.     

Growth-mortality trade-offs along a depth gradient in Cancer borealis

Intertidal and shallow subtidal ecosystems experience steep environmental gradients over short distances. Individual foraging rate, predation risk, and physiologic stress vary along these gradients, resulting in growth-mortality trade-offs with depth. In the summer, Cancer borealis commonly forage in the shallow subtidal in the Gulf of Maine. C. borealis are the favored invertebrate prey of the Herring Gull and the Great Black-backed Gull, which consume 25%-50% of available C. borealis (those in < 1 m water) during each daytime low tide. We investigated three possible explanations for the presence of C. borealis in the risky gull-predation zone. First, we tested whether predation risk in the gull-predation zone was matched at deeper depths by subtidal predators; we found predation risk decreases with depth. Second, we tested whether water temperatures were warmer in the gull-predation zone and whether these warmer temperatures resulted in increased growth rates. We found that, while waters were warmer in the gull-predation zone, crabs grew at similar rates above and below the thermocline when fed similar diets. Finally, we tested for differences in food availability with depth and whether these differences influenced C. borealis growth rates. Our results suggest a growth-mortality trade-off, where increased food availability provides sufficient growth benefit to outweigh the risk of foraging at shallower depths.     

High-Throughput,Sensitive Quantification of Repopulating Hematopoietic Stem Cell Clones

Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5′ and the 3′ vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.Integration of the retroviral DNA provirus into the host genome is an obligatory step in the retroviral life cycle. Because of this unique property, retroviruses have been adapted as vectors (24, 26) and used successfully to correct genetic diseases, such as X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA)-deficient SCID, and X-linked adrenoleukodystrophy, by stable genetic modification of hematopoietic progenitor/stem cells (HPSC) (1, 2, 5, 6, 13, 27, 29). However, the risk of insertional mutagenesis from therapeutic vectors has been demonstrated in several cases in which integration events near or within proto-oncogenes triggered leukemia (8, 12, 14, 16, 34). Therefore, it is important to understand the mechanisms for complex hematopoietic repopulation in humans and to study the behaviors of engineered HPSC clones following transplant.Since retrovirus vectors uniquely “mark” individual HPSC by vector integration sites (VIS), clonal repopulation by HPSC can be analyzed by tracking the VIS. Restriction enzyme-based assays are commonly used for the clonal tracking, where genomic DNA is digested with restriction enzymes to generate VIS DNA fragments of different lengths that can be detected by Southern blotting (9, 17, 18, 22) or nucleotide sequencing via linker-mediated PCR (LM-PCR) (32), inverse PCR (INV-PCR) (33), or linear amplification-mediated PCR (LAM-PCR) (30). These approaches have been widely used in biological and clinical research to study composition of the HPSC pool, stem cell engraftment, regulatory decisions of individual stem cells, and genotoxicity of retroviral vectors (17, 22, 23, 25, 29, 31, 35). While mouse HPSC repopulation is typically mono- or oligoclonal (17), the number of HPSC clones repopulating in humans or nonhuman primates is much larger, manifesting several hundreds to thousands of repopulating clones posttransplant (5, 31, 35). Recent advances in sequencing technology have enabled high-throughput and parallel clonality analysis through large-scale VIS sequencing and enumeration of VIS sequences (5, 15, 35, 36). However, these methods can detect only VIS that are proximal to restriction enzyme sites, and additional experimental limitations may exist (10, 15, 36). As a result, current assays can only roughly estimate clonal frequencies, so the current standard is to perform clone-specific real-time PCR for sensitive and accurate quantification. Recently, novel clonal tracking assays that do not require restriction enzyme usage have been described (10, 11). However, these methods involve experimental steps that are technically challenging, and they require further optimization to achieve reliable, high-throughput quantification.Here, we present a novel VIS detection and quantification system based on 454 pyrosequencing and accompanying guidelines for high-throughput quantification of multiple clonal populations. We used a novel bidirectional PCR method to concurrently analyze both the 5′ (left) and the 3′ (right) vector-host junctions in peripheral blood repopulating cells (PBC) in a rhesus macaque transplanted with autologous HPSC transduced with lentivirus vectors (3). The reproducibility and conditions for reliable quantification were tested by two independent experiments conducted on the same PBC collected at four posttransplant time points. The lengths of VIS PCR amplicons, the amount of genomic DNA for analysis, and the intensity of sequencing are important factors influencing the reliability and the sensitivity of the assay. Of 964 unique vector integrants analyzed, the relative quantities of a 398-member subset were determined, demonstrating heterogeneous and dynamic clonal frequency changes over time. Clonal frequencies were further confirmed by clone-specific real-time PCR. We show that this assay detects the majority of VIS that are present in a given clonal population and accurately measures their relative frequencies.     

Setting a standard for a "silent" disease: defining osteoporosis in the 1980s and 1990s

Osteoporosis, a disease of bone loss associated with aging and estrogen loss, can be crippling but is 'silent' (symptomless) prior to bone fracture. Despite its disastrous health effects, high prevalence, and enormous associated health care costs, osteoporosis lacked a universally accepted definition until 1992. In the 1980s, the development of more accurate medical imaging technologies to measure bone density spurred the medical community's need and demand for a common definition. The medical community tried, and failed, to resolve these differing definitions several times at consensus conferences and through published articles. These experts finally accepted a standard definition at an international consensus conference convened by the World Health Organization in 1992. The construction of osteoporosis as a disease of quantifiable risk diagnosed by medical imaging machines reflects contemporary trends in medicine, including the quantification of disease, the risk factor model, medical disciplinary boundaries, and global standardization of medical knowledge.     

Mouse Gestation Length Is Genetically Determined

Background

Preterm birth is an enormous public health problem, affecting over 12% of live births and costing over $26 billion in the United States alone. The causes are complex, but twin studies support the role of genetics in determining gestation length. Despite widespread use of the mouse in studies of the genetics of preterm birth, there have been few studies that actually address the precise natural gestation length of the mouse, and to what degree the timing of labor and birth is genetically determined.

Methodology/Principal Findings

To further develop the mouse as a genetic model of preterm birth, we developed a high-throughput monitoring system and measured the gestation length in 15 inbred strains. Our results show an unexpectedly wide variation in overall gestation length between strains that approaches two full days, while intra-strain variation is quite low. Although litter size shows a strong inverse correlation with gestation length, genetic difference alone accounts for a significant portion of the variation. In addition, ovarian transplant experiments support a primary role of maternal genetics in the determination of gestation length. Preliminary analysis of gestation length in the C57BL/6J-Chr#^A/J/NaJ chromosome substitution strain (B.A CSS) panel suggests complex genetic control of gestation length.

Conclusions/Significance

Together, these data support the role of genetics in regulating gestation length and present the mouse as an important tool for the discovery of genes governing preterm birth.     

Detection, analysis and clinical validation of chromosomal aberrations by multiplex ligation-dependent probe amplification in chronic leukemia

Current diagnostic screening strategies based on karyotyping or fluorescent in situ hybridization (FISH) for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are laborious, time-consuming, costly, and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA) can simultaneously detect copy number changes of multiple loci in one simple PCR reaction, making it an attractive alternative to FISH. To enhance the clinical robustness and further harness MLPA technology for routine laboratory operations, we have developed and validated a protocol for comprehensive, automatic data analysis and interpretation. A training set of 50 normal samples was used to establish reference ranges for each individual probe, for the calling of statistically significant copy number changes. The maximum normal ranges of 2 and 3 standard deviations (SD) are distributed between 0.82 and 1.18 (Mean ± 2SD, 95% CI, P = 0.05), and between 0.73 and 1.27 (Mean ± 3SD, 99% CI, P = 0.01), respectively. We found an excellent correlation between MLPA and FISH with 93.6% concordance (P<0.0001) from a testing cohort of 100 clinically suspected CLL cases. MLPA analyses done on 94/100 patients showed sensitivity and specificity of 94.2% and 92.9%, respectively. MLPA detected additional copy number gains on 18q21.1 and chromosome 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci in six samples. Three FISH-failed samples were tested positive by MLPA, while three 13q- cases with a low percentage of leukemia cells (7%, 12% and 19%) were not detected by MLPA. The improved CLL MLPA represents a high-throughput, accurate, cost-effective and user-friendly platform that can be used as a first-line screening test in a clinical laboratory.