Mechanism for cryptic splice site activation during pre-mRNA splicing.

The 5' splice site of a pre-mRNA is recognized by U1 small nuclear ribonucleoprotein particles (snRNP) through base pairing with the 5' end of U1 small nuclear RNA (snRNA). Single-base substitutions within a 9-nucleotide 5'-splice-site sequence can abolish or attenuate use of that site and, in higher eukaryotes, can also activate nearby "cryptic" 5' splice sites. Here we show that the effects of single-base substitutions within a 5' splice site can be completely or partially suppressed by cis mutations that improve the overall complementarity of the site to U1 snRNA. We further show that in the presence of the normal 5' splice site, a cryptic 5' splice site can be activated by increasing its complementarity to U1 snRNA. U1 snRNP binding experiments confirm that cryptic 5' splice sites are activated when their affinity for U1 snRNP approaches that of the authentic 5' splice site. Based upon these results, we propose a spliceosome competition model for 5'-splice-site selection and cryptic 5'-splice-site activation. We discuss our results with regard to the factors involved in 5'-splice-site recognition.     

Cellular immune response to cryptic epitopes during therapeutic gene transfer

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8⁺ T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8⁺ CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.     

Hilbert-Coding Metasurface for Diverse Electromagnetic Controls

Metamaterials, or metasurfaces, allow the flexible and efficient manipulation of electromagnetic (EM) wave. Although the passive coding metasurfaces have achieved a great deal of functionality, they also need a complex design process. In this paper, we propose Hilbert-coding metasurfaces for flexible and convenient EM regulation by arranging Hilbert-coding metamaterial units of different orders. To demonstrate this behavior, we designed 12 metasurfaces, then fabricated and measured 6 samples. Validation results on 6 Hilbert-coding metasurfaces show the deflection angles of the four single beam patterns obtained are about 21°, 13°, 12°, and 39°, with energy values of 7.75 dB, 7.3 dB, 7.2 dB, and 7.7 dB, respectively, and the deflection angles of the dual-beam patterns are 28.5° and 20° with energy values of 10.05 dB and 11.4 dB, respectively. The results are quite consistent with the simulation data, further confirming the feasibility of our idea. In addition, there are potential applications in Wireless Communications and Radar-imaging, like EM beam scanning and EM field energy distribution control in communication and imaging scenarios.     

Low atmospheric CO(2) levels during the Permo- Carboniferous glaciation inferred from fossil lycopsids

Earth history was punctuated during the Permo-Carboniferous [300-250 million years (Myr) ago] by the longest and most severe glaciation of the entire Phanerozoic Eon. But significant uncertainty surrounds the concentration of CO(2) in the atmosphere through this time interval and therefore its role in the evolution of this major prePleistocene glaciation. Here, I derive 24 Late Paleozoic CO(2) estimates from the fossil cuticle record of arborsecent lycopsids of the equatorial Carboniferous and Permian swamp communities. Quantitative calibration of Late Carboniferous (330-300 Myr ago) and Permian (270-260 Myr ago) lycopsid stomatal indices yield average atmospheric CO(2) concentrations of 344 ppm and 313 ppm, respectively. The reconstructions show a high degree of self-consistency and a degree of precision an order of magnitude greater than other approaches. Low CO(2) levels during the Permo-Carboniferous glaciation are in agreement with glaciological evidence for the presence of continental ice and coupled models of climate and ice-sheet growth on Pangea. Moreover, the Permian data indicate atmospheric CO(2) levels were low 260 Myr ago, by which time continental deglaciation was already underway. Positive biotic feedbacks on climate, and geotectonic events, therefore are implicated as mechanisms underlying deglaciation.     

Atrioventricular block during fetal life

Congenital complete atrioventricular (AV) block occurs in approximately 1 in 20,000 live births and is known to result in significant mortality and morbidity both during fetal life and postnatally. Complete AV block can occur as a result of an immune or a non-immune mediated process. Immune mediated AV block is a multifactorial disease, but is associated with the trans-placental passage of maternal autoantibodies (anti-Ro/SSA and/or anti-La/SSB). These autoantibodies attach to and subsequently damage the cardiomyocytes and conduction tissue in susceptible fetuses. In this report, we examine the evidence in reference to means of assessment, pathophysiology, and potential prenatal therapy of atrioventricular block.     

Diverse expression of ErbB receptor proteins during rat liver development and regeneration

BACKGROUND & AIMS: The protein expression and interactions of the ErbB receptors were examined in different liver proliferation models in vivo and in vitro, including ontogeny and regeneration following partial hepatectomy. METHODS: Expression and tyrosine phosphorylation status of specific ErbB proteins were studied by immunologic methods. RESULTS: The epidermal growth factor receptor, ErbB2, and ErbB3 were the only ErbB proteins detected in the liver parenchyma on embryonic day 19. ErbB2 disappeared by the third week after birth and could not be appreciably induced in the adult animal by partial hepatectomy. ErbB2 was also detected in multipotent stem (RLE) and hepatoma (H4IIe) cell lines as well as in fetal, but not adult, hepatocyte cultures. Only epidermal growth factor receptor and ErbB3 were detected in adult liver, and both showed circadian variation in protein expression. ErbB4 was not detected in any model. Patterns of ligand-induced ErbB phosphorylation differed between fetal and adult hepatocytes. CONCLUSIONS: Complex and independent programs regulate the ErbB receptors, with implications for differential cell signaling in hepatic development and regeneration.     

Plant Virology Delivers Diverse Toolsets for Biotechnology

Over a hundred years of research on plant viruses has led to a detailed understanding of viral replication, movement, and host–virus interactions. The functions of vast viral genes have also been annotated. With an increased understanding of plant viruses and plant–virus interactions, various viruses have been developed as vectors to modulate gene expressions for functional studies as well as for fulfilling the needs in biotechnology. These approaches are invaluable not only for molecular breeding and functional genomics studies related to pivotal agronomic traits, but also for the production of vaccines and health-promoting carotenoids. This review summarizes the latest progress in these forefronts as well as the available viral vectors for economically important crops and beyond.     

Dilated cardiomyopathy presenting during fetal life

OBJECTIVES: To describe the echocardiographic features, underlying causes, and outcome of fetuses with dilated cardiomyopathy. DESIGN: A retrospective observational study between 1983 and 2003 at a tertiary centre for fetal cardiology. PATIENTS: Affected fetuses were identified using a computerised database. We included fetuses with dilation and reduced systolic function of either the right ventricle, left ventricle, or both. We excluded fetuses with abnormal cardiac connections, arrhythmias, or stenosis of the aortic or pulmonary valves. In all, we identified 50 fetuses, born to 46 mothers. Of the fetuses, 24 had biventricular cardiomyopathy, 17 had isolated right ventricular cardiomyopathy, and 9 had isolated left ventricular cardiomyopathy. Two-thirds of the fetuses (32) were hydropic at some point during gestation. MAIN OUTCOMES: A cause of cardiomyopathy was identified in 37 cases (74 per cent). This was genetic or metabolic in 11 fetuses; infective in 11; fetal anaemia, without proven parvovirus infection, in 5; of cardiac origin in 5; and an association with renal disease in 5. In 10 cases (20 per cent), the pregnancy was terminated. Based on an intention to treat, the survival to delivery was 25 of 40 (62.5 per cent, 95 per cent confidence intervals from 46 to 77 per cent), at 28 days was 17 of 40 (42.5 per cent, 95 per cent confidence intervals from 27 to 59 per cent), and at 1 year was 15 of 40 (37.5 per cent, 95 per cent confidence intervals from 23 to 54 per cent). The overall survival of non-hydropic fetuses was 9 of 18 (50 per cent), compared to 6 of 32 (18 per cent) hydropic fetuses. CONCLUSIONS: Genetic, metabolic, infective, and cardiac diseases may present with dilated cardiomyopathy during fetal life. There is a high rate of spontaneous intra-uterine and early neonatal death. The prognosis is particularly poor for hydropic fetuses.     

Paradoxical embolism. Diagnosis during life

Of 128 reported cases of paradoxical embolism, only twelve have been diagnosed during life. However, five cases of paradoxical embolism have been detected during life at this hospital in two years. These five cases are presented to illustrate the pathophysiologic features that allow detection of paradoxical embolism during life.     

Health-related quality of life during natalizumab maintenance therapy for Crohn's disease

OBJECTIVES: We evaluated the effects of treatment on health-related quality of life (HRQoL) during a randomized controlled trial of natalizumab maintenance therapy (ENACT-2) using both disease-specific and generic measures. METHODS: Crohn's disease patients who received natalizumab as induction therapy in ENACT-1 (N=724) and responded (N=339) were re-randomized to ENACT-2 in which they received natalizumab 300 mg (N=168) or placebo (N=171) every 4 wk for 48 additional wk. Outcome measures were the change from baseline on the inflammatory bowel disease questionnaire (IBDQ), the short form-36 (SF-36), the EuroQol-5D (EQ-5D), and a subject global assessment. RESULTS: At entry into ENACT-1, scores indicated substantially impaired HRQoL for both the disease-specific and general measures. Natalizumab responders showed clinically meaningful improvement in HRQoL over the course of the ENACT-1 study. During maintenance therapy, IBDQ and SF-36 scale scores of patients who responded to natalizumab induction and were re-randomized to receive the drug in ENACT-2 (N=168) remained stable, while those re-randomized to placebo (N=171) worsened. At week 60, 48 wk after the initiation of maintenance therapy, the mean change from ENACT-1 baseline of all scales of the IBDQ and the SF-36 was significantly higher for those who continued to receive natalizumab (P<0.001 for all scales). The scores of patients who received maintenance natalizumab treatment were not statistically different from those of a cross-section of the U.S. population for 6 of 8 scales of the SF-36. CONCLUSIONS: The substantial improvement in HRQoL experienced by patients who responded to natalizumab induction therapy was maintained during an additional 48 wk of maintenance therapy.     

Diverse roles for the LDL receptor family.

The low-density lipoprotein (LDL) receptor family consists of several related scavenger receptors that not only function as important cargo transporters, but also inform the cell of changes in its environment by mediating signaling responses. The LDL receptor was the first family member to be characterized and its function seems to be restricted to lipoprotein metabolism. By contrast, lipoprotein metabolism does not appear to be the exclusive function of the other characterized LDL receptor family members. It is now apparent that cargo transport by members of the LDL receptor family is closely associated with regulation of cellular physiology and cellular signaling events. Here, we focus on the diverse biological activities of certain members of this family.     

Excretion of biliary compounds during intrauterine life

In adults, the hepatobiliary system, together with the kidney, constitute the main routes for the elimination of several endogenous and xenobiotic compounds into bile and urine, respectively. However, during intrauterine life the biliary route of excretion for cholephilic compounds, such as bile acids and biliary pigments, is very poor. Although very early in pregnancy the fetal liver produces bile acids, bilirubin and biliverdin, these compounds cannot be efficiently eliminated by the fetal hepatobiliary system, owing to the immaturity of the excretory machinery in the fetal liver. Therefore, the potentially harmful accumulation of cholephilic compounds in the fetus is prevented by their elimination across the placenta. Owing to the presence of detoxifying enzymes and specific transport systems at different locations of the placental barrier, such as the endothelial cells of chorionic vessels and trophoblast cells, this organ plays an important role in the hepatobiliary-like function during intrauterine life. The relevance of this excretory function in normal fetal physiology is evident in situations where high concentrations of biliary compounds are accumulated in the mother. This may result in oxidative stress and apoptosis, mainly in the placenta and fetal liver, which might affect normal fetal development and challenge the fate of the pregnancy. The present article reviews current knowledge of the mechanisms underlying the hepatobiliary function of the fetal-placental unit and the repercussions of several pathological conditions on this tandem.     

Estimating alveolar surface area during life.

Alveolar surface area (Sa) may be derived from measurements of total lung capacity (TLC) and mean linear intercept (Lm), an estimate of average airspace size. Because the index of pulmonary distensibility, K, is a function of Lm, we were able to derive Lm from ln K using their respective age regressions and estimated Sa in 147 healthy subjects. K was obtained from exponential analysis of static pressure-volume data. TLC was measured in a body plethysmograph. As an estimate of airspace size, the value used for Lm was shown to be appropriate for the air-inflated lung at TLC. In 95 men (mean age 40 +/- 16 SD years), Sa was 118 (SD 22) m2 and in 52 women (mean age 38 +/- 17 SD years) it was 91 (SD 18) m2 and Sa decreased with age (P less than 0.001). In a morphometric study, Thurlbeck (Am. Rev. Respir. Dis. 95: 765-773, 1967) obtained smaller values for Sa (owing to the use of a smaller lung volume) and a similar decrease in Sa with age as that found here. Providing a standardised methodology is used for measurement of K, the present method allows a reasonable estimate of Sa to be obtained during life.