An intron-derived motif strongly increases gene expression from transcribed sequences through a splicing independent mechanism in Arabidopsis thaliana.

Certain introns significantly increase mRNA accumulation by a poorly understood mechanism. These introns have no effect when located upstream, or more than ~1 Kb downstream, of the start of transcription. We tested the ability of a formerly non-stimulating intron containing 11 copies of the sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, to affect expression from various positions. The activity profile of this intron at different locations was similar to that of a natural intron from the UBQ10 gene, suggesting that the motif increases mRNA accumulation by the same mechanism. A series of introns with different numbers of this motif revealed that the effect on expression is linearly dependent on motif copy number up to at least 20, with each copy adding another 1.5-fold increase in mRNA accumulation. Furthermore, 6 copies of the motif stimulated mRNA accumulation to a similar degree from within an intron or when introduced into the 5'-UTR and coding sequences of an intronless construct, demonstrating that splicing is not required for this sequence to boost expression. The ability of this motif to substantially elevate expression from several hundred nucleotides downstream of the transcription start site reveals a novel type of eukaryotic gene regulation.

Introns as Gene Regulators: A Brick on the Accelerator.

A picture is beginning to emerge from a variety of organisms that for a subset of genes, the most important sequences that regulate expression are situated not in the promoter but rather are located within introns in the first kilobase of transcribed sequences. The actual sequences involved are difficult to identify either by sequence comparisons or by deletion analysis because they are dispersed, additive, and poorly conserved. However, expression-controlling introns can be identified computationally in species with relatively small introns, based on genome-wide differences in oligomer composition between promoter-proximal and distal introns. The genes regulated by introns are often expressed in most tissues and are among the most highly expressed in the genome. The ability of some introns to strongly stimulate mRNA accumulation from several hundred nucleotides downstream of the transcription start site, even when the promoter has been deleted, reveals that our understanding of gene expression remains incomplete. It is unlikely that any diseases are caused by point mutations or small deletions that reduce the expression of an intron-regulated gene unless splicing is also affected. However, introns may be particularly useful in practical applications such as gene therapy because they strongly activate expression but only affect the transcription unit in which they are located.

Intron DNA Sequences Can Be More Important Than the Proximal Promoter in Determining the Site of Transcript Initiation.

To more precisely define the positions from which certain intronic regulatory sequences increase mRNA accumulation, the effect of a UBIQUITIN intron on gene expression was tested from six different positions surrounding the transcription start site (TSS) of a reporter gene fusion in Arabidopsis thaliana The intron increased expression from all transcribed positions but had no effect when upstream of the 5'-most TSS. While this implies that the intron must be transcribed to increase expression, the TSS changed when the intron was located in the 5'-untranslated region (UTR), suggesting that the intron affects transcription initiation. Remarkably, deleting 303 nucleotides of the promoter including all known TSSs and all but 18 nucleotides of the 5'-UTR had virtually no effect on the level of gene expression as long as an intron containing stimulatory sequences was included. Instead, transcription was initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. These results suggest that certain intronic DNA sequences play unexpectedly large roles in directing transcription initiation and constitute a previously unrecognized type of downstream regulatory element for genes transcribed by RNA polymerase II.

Intron sequences that stimulate gene expression in Arabidopsis.

KEY MESSAGE: Related motifs strongly increase gene expression when added to an intron located in coding sequences. Many introns greatly increase gene expression through a mechanism that remains elusive. An obstacle to understanding intron-mediated enhancement (IME) has been the difficulty of locating the specific intron sequences responsible for boosting expression because they are redundant, dispersed, and degenerate. Previously we used the IMEter algorithm in two independent ways to identify two motifs (CGATT and TTNGATYTG) that are candidates for involvement in IME in Arabidopsis. Here we show that both motifs are sufficient to increase expression. An intron that has little influence on expression was converted into one that increased mRNA accumulation 24-fold and reporter enzyme activity 40-fold relative to the intronless control by introducing 11 copies of the more active TTNGATYTG motif. This degree of stimulation is twice as large as that of the strongest of 15 natural introns previously tested in the same reporter gene. Even though the CGATT and TTNGATYTG motifs each increased expression, and CGATT matches the NGATY core of the longer motif, combining the motifs to make TTCGATTTG reduced the stimulating ability of the TTNGATYTG motif. Additional substitutions were used to test the contribution to IME of other residues in the TTNGATYTG motif. The verification that these motifs are active in IME will improve our ability to predict the stimulating ability of introns, to engineer any intron to increase expression to a desired level, and to explore the mechanism of IME by seeking factors that might interact with these sequences.

The enduring mystery of intron-mediated enhancement.

Within two years of their discovery in 1977, introns were found to have a positive effect on gene expression. Numerous examples of stimulatory introns have been described since then in very diverse organisms, including plants. In some cases, the mechanism through which the intron affects expression is readily understood. However, many introns that affect expression increase mRNA accumulation through an unknown mechanism, referred to as intron-mediated enhancement (IME). Despite several decades of research into IME, and the clear benefits of using introns to increase transgene expression, little progress has been made in understanding the mechanism of IME. Several fundamental questions regarding the role of transcription and splicing, the sequences responsible for IME, the involvement of other factors, and the relationship between introns and promoters remain unanswered. The more we learn about the properties of stimulating introns, the clearer it becomes that the effects of introns are unfamiliar and difficult to reconcile with conventional views of how transcription is controlled. We hypothesize that introns increase transcript initiation upstream of themselves by creating a localized region of accessible chromatin. Introns might represent a novel kind of downstream regulatory element for genes transcribed by RNA polymerase II.

The effects of a stimulating intron on the expression of heterologous genes in Arabidopsis thaliana.

Introns are often added to transgenes to increase expression, although the mechanism through which introns stimulate gene expression in plants and other eukaryotes remains mysterious. While introns vary in their effect on expression, it is unknown whether different genes respond similarly to the same stimulatory intron. Furthermore, the degree to which gene regulation is preserved when expression is increased by an intron has not been thoroughly investigated. To test the effects of the same intron on the expression of a range of genes, GUS translational fusions were constructed using the promoters of eight Arabidopsis genes whose expression was reported to be constitutive (GAE1, CNGC2 and ROP10), tissue specific (ADL1A, YAB3 and AtAMT2) or regulated by light (ULI3 and MSBP1). For each gene, a fusion containing the first intron from the UBQ10 gene was compared to fusions containing the gene's endogenous first intron (if the gene has one) or no intron. In every case, the UBQ10 intron increased expression relative to the intronless control, although the magnitude of the change and the level of expression varied. The UBQ10 intron also changed the expression patterns of the CNGC2 and YAB3 fusions to include strong activity in roots, indicating that tissue specificity was disrupted by this intron. In contrast, the regulation of the ULI3 and MSBP1 genes by light was preserved when their expression was stimulated by the intron. These findings have important implications for biotechnology applications in which a high level of transgene expression in only certain tissues is desired.

Rhizobial and mycorrhizal symbioses in Lotus japonicus require lectin nucleotide phosphohydrolase, which acts upstream of calcium signaling.

Nodulation in legumes requires the recognition of rhizobially made Nod factors. Genetic studies have revealed that the perception of Nod factors involves LysM domain receptor-like kinases, while biochemical approaches have identified LECTIN NUCLEOTIDE PHOSPHOHYDROLASE (LNP) as a Nod factor-binding protein. Here, we show that antisense inhibition of LNP blocks nodulation in Lotus japonicus. This absence of nodulation was due to a defect in Nod factor signaling based on the observations that the early nodulation gene NODULE INCEPTION was not induced and that both Nod factor-induced perinuclear calcium spiking and calcium influx at the root hair tip were blocked. However, Nod factor did induce root hair deformation in the LNP antisense lines. LNP is also required for infection by the mycorrhizal fungus Glomus intraradices, suggesting that LNP plays a role in the common signaling pathway shared by the rhizobial and mycorrhizal symbioses. Taken together, these observations indicate that LNP acts at a novel position in the early stages of symbiosis signaling. We propose that LNP functions at the earliest stage of the common nodulation and mycorrhization symbiosis signaling pathway downstream of the Nod factor receptors; it may act either by influencing signaling via changes in external nucleotides or in conjunction with the LysM receptor-like kinases for recognition of Nod factor.

Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants.

Introns in a wide range of organisms including plants, animals and fungi are able to increase the expression of the gene that they are contained in. This process of intron-mediated enhancement (IME) is most thoroughly studied in Arabidopsis thaliana, where it has been shown that enhancing introns are typically located near the promoter and are compositionally distinct from downstream introns. In this study, we perform a comprehensive comparative analysis of several sequenced plant genomes. We find that enhancing sequences are conserved in the multi-cellular plants but are either absent or unrecognizable in algae. IME signals are preferentially located towards the 5'-end of first introns but also appear to be enriched in 5'-UTRs and coding regions near the transcription start site. Enhancing introns are found most prominently in genes that are highly expressed in a wide range of tissues. Through site-directed mutagenesis in A. thaliana, we show that IME signals can be inserted or removed from introns to increase or decrease gene expression. Although we do not yet know the specific mechanism of IME, the predicted signals appear to be both functional and highly conserved.

Evidence for a DNA-Based Mechanism of Intron-Mediated Enhancement.

Many introns significantly increase gene expression through a process termed intron-mediated enhancement (IME). Introns exist in the transcribed DNA and the nascent RNA, and could affect expression from either location. To determine which is more relevant to IME, hybrid introns were constructed that contain sequences from stimulating Arabidopsis thaliana introns either in their normal orientation or as the reverse complement. Both ends of each intron are from the non-stimulatory COR15a intron in their normal orientation to allow splicing. The inversions create major alterations to the sequence of the transcribed RNA with relatively minor changes to the DNA structure. Introns containing portions of either the UBQ10 or ATPK1 intron increased expression to a similar degree regardless of orientation. Also, computational predictions of IME improve when both intron strands are considered. These findings are more consistent with models of IME that act at the level of DNA rather than RNA.

Aortic valve pathophysiology during left ventricular assist device support.

The increased applicability and excellent results with left ventricular assist devices (LVADs) have revolutionized the available treatment options for patients with advanced heart failure. Pre-existing valve abnormalities are common in this population, and subsequent development of valve abnormalities after LVAD placement is also often noted. Although native mitral and tricuspid valve disease is more common in heart failure patients before LVAD placement, aortic valves are much more likely to generate abnormal pathophysiology in the LVAD patient during as well as after LVAD placement. The aim of this comprehensive review is to review aortic valve function in LVAD patients and highlight the consideration of pre-existing valve disease on patient treatment at the time of LVAD implant. The basis for structural changes leading to valve pathophysiology during and after LVAD placement will be described, providing a basis for improved clinical understanding and new strategies to prevent these conditions.

Applying word-based algorithms: the IMEter.

Important patterns can be found in strings of characters such as nucleotides in a DNA sequence by examining the frequency of occurrence of specific character combinations or words. The abundance of words can reveal the presence of underlying trends governing the order of characters, even if the biological reasons for those trends remain mysterious. As an example of one way in which word frequencies have provided insight, we describe the IMEter, a word-based algorithm for analyzing introns and their effect on gene expression. The IMEter demonstrates that introns located near the beginning of genes are compositionally distinct from later introns and that these differences are closely related to the ability of some introns to increase gene expression. This word-based approach has proven more successful than deletion analysis at identifying the sequences responsible for elevating expression because they are dispersed throughout stimulatory introns.

Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression.

Introns that elevate mRNA accumulation have been found in a wide range of eukaryotes. However, not all introns affect gene expression, and direct testing is currently the only way to identify stimulatory introns. Our genome-wide analysis in Arabidopsis thaliana revealed that promoter-proximal introns as a group are compositionally distinct from distal introns and that the degree to which an individual intron matches the promoter-proximal intron profile is a strong predictor of its ability to increase expression. We found that the sequences responsible for elevating expression are dispersed throughout an enhancing intron, as is a candidate motif that is overrepresented in first introns and whose occurrence in tested introns is proportional to its effect on expression. The signals responsible for intron-mediated enhancement are apparently conserved between Arabidopsis and rice (Oryza sativa) despite the large evolutionary distance separating these plants.

The coronary artery disease quality dashboard: a chronic care disease management tool in an electronic health record.

Quality reporting tools, integrated with ambulatory electronic health records (EHRs), may help clinicians understand performance, manage populations, and improve quality. The Coronary Artery Disease Quality Dash board (CAD QD) is a secure web report for performance measurement of a chronic care condition delivered through a central data warehouse and custom-built reporting tool. Pilot evaluation of the CAD Quality Dash board indicates that clinicians prefer a quality report that combines not only structured data from EHRs but one that facilitates actions to be taken on individual patients or on a population, i.e., for case management.

Decision support for acute problems: the role of the standardized patient in usability testing.

For applications that require clinician use while interacting with patients, usability testing with standardized patients has the potential to approximate actual patient care in a controlled setting. We used hypothetical scenarios and a standardized patient to collect quantitative and qualitative results in testing an early prototype of a new application, the Acute Respiratory Infection (ARI) Smart Form. The standardized patient fit well into the usability testing sessions. Clinicians had a positive response to the standardized patients and behaved as they normally would during a clinical encounter. Positive findings of the ARI Smart Form included that users thought it had impressive functionality and the potential to save time. Criticism focused on the visual design, which could be streamlined, and navigation, which was difficult in some areas. Based on these results, we are modifying the ARI Smart Form in preparation for use in actual patient care. Standardized patients should be considered for usability testing, especially if an application is to be used during the patient interview.

Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection.

In 1990, an international grading system for cardiac allograft biopsies was adopted by the International Society for Heart Transplantation. This system has served the heart transplant community well, facilitating communication between transplant centers, especially with regard to patient management and research. In 2004, under the direction of the International Society for Heart and Lung Transplantation (ISHLT), a multidisciplinary review of the cardiac biopsy grading system was undertaken to address challenges and inconsistencies in its use and to address recent advances in the knowledge of antibody-mediated rejection. This article summarizes the revised consensus classification for cardiac allograft rejection. In brief, the revised (R) categories of cellular rejection are as follows: Grade 0 R--no rejection (no change from 1990); Grade 1 R--mild rejection (1990 Grades 1A, 1B and 2); Grade 2 R--moderate rejection (1990 Grade 3A); and Grade 3 R--severe rejection (1990 Grades 3B and 4). Because the histologic sub-types of Quilty A and Quilty B have never been shown to have clinical significance, the "A" and "B" designations have been eliminated. Recommendations are also made for the histologic recognition and immunohistologic investigation of acute antibody-mediated rejection (AMR) with the expectation that greater standardization of the assessment of this controversial entity will clarify its clinical significance. Technical considerations in biopsy processing are also addressed. This consensus revision of the Working Formulation was approved by the ISHLT Board of Directors in December 2004.

Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events.

The extensive mechanistic and regulatory interconnections between the various events of mRNA biogenesis are now recognized as a fundamental principle of eukaryotic gene expression, yet the specific details of the coupling between the various steps of mRNA biogenesis do differ, and sometimes dramatically, between the different kingdoms. In this review, we emphasize examples where plants must differ in this respect from other eukaryotes, and highlight a recurring trend of recruiting the conserved, versatile functional modules, which have evolved to support the general mRNA biogenesis reactions, for plant-specific functions. We also argue that elucidating the inner workings of the plant 'mRNA factory' is essential for accomplishing the ambitious goal of building the 'virtual plant'.

Pulmonary complications after bone marrow transplantation: an autopsy study from a large transplantation center.

CONTEXT: Bone marrow transplantation (BMT) is used to treat various malignant and nonmalignant disorders. Pulmonary complications are some of the most common causes of mortality in BMT recipients. Poor general health and bleeding tendency frequently preclude the use of definitive diagnostic tests, such as open lung biopsy, in these patients. OBJECTIVE: To identify pulmonary complications after BMT and their role as the cause of death (COD). DESIGN: The autopsy and bronchoalveolar lavage (BAL) slides and microbiology studies of BMT recipients from a 7-year period were reviewed. RESULTS: Pulmonary complications were identified in 40 (80%) of the 50 cases. The most common complications were diffuse alveolar damage (DAD) and diffuse alveolar hemorrhage (DAH). Pulmonary complications were the sole or 1 of multiple CODs in 37 cases (74%). All complications were more common in allogeneic BMT recipients. In 19 (51%) of the 37 cases in which pulmonary complications contributed to the death, cultures were negative. Both DAD and DAH, complications commonly reported in the early post-BMT period, were seen more than 100 days after BMT in 33% and 12% of cases, respectively. Five (83%) of 6 cases of invasive pulmonary aspergillosis diagnosed at autopsy were negative for fungi ante mortem (by BAL and cultures). CONCLUSIONS: Pulmonary complications are a significant COD in BMT recipients, many of which, especially the fungal infections, are difficult to diagnose ante mortem. The etiology of DAD and DAH is likely to be multifactorial, and these complications are not limited to the early posttransplantation period. Autopsy examination is important in determining the COD in BMT recipients.

Pathology in patients with ventricular assist devices: a study of 21 autopsies, 24 ventricular apical core biopsies and 24 explanted hearts.

BACKGROUND: Ventricular assist devices (VADs) are used as a bridge to cardiac transplantation or as a permanent or sometimes temporary treatment for end stage heart failure. METHODS: Our autopsy and surgical pathology experience with VADs prior to August 2002 was reviewed. Noted were patient's age, sex, underlying (UCOD) and proximate causes of death (PCOD), duration of VAD implantation, presence of native or prosthetic valvar disease and organ complications. Myocardium from biopsies and explanted hearts were blindly assessed for coagulative necrosis (CN), contraction bands (CB), myocytolysis (MC), increased eosinophilia (IE), myocyte waviness (MW) and fibrosis (F). Each was graded as either mild (score 1), moderate (score 2) or severe (score 3). RESULTS: Autopsy patients: Twenty-one patients, with mean age 55 years (range 10-73), comprised 10 women and 11 men. UCOD was ischemic disease in 16 patients, dilated cardiomyopathy in 4 and aortic valve disease in 1. The mean duration of VAD implantation was 125.7 days (range 1-1095 days, S.D.=253.6). Five patients had biventricular VADs, and 16 had LVAD only. Acquired aortic valve fusion was noted in three patients. PCOD was VAD related in six, donor heart problem in four, cerebrovascular accident in four, miscellaneous in three, pulmonary hypertension in two and aortic disease in two patients. Morbidity: local liver necrosis in seven, acquired aortic valve disease in four, gut infarction in three, abdominal aortic aneurysm in two and host cell assault against VAD porcine aortic valves in one case. Biopsies and explanted hearts: Twenty-four patients had a mean age of 53 years (range 38-68, S.D.=8.6). VADs were implanted for 177.8 days (range 7-593 days, S.D.=151.1). Comparison of histologic scores of biopsies with explanted hearts showed the following: CN 1.33 (S.D.=1.4)/0.21 (S.D.=0.66; P<.001); CB: 2.1 (S.D.=0.93)/0.83 (S.D.=0.28; NS); MC: 0.88 (S.D.=1.19)/0.13 (S.D.=0.34; P<.01); IE: 1.71 (S.D.=1.27)/0.38 (S.D.=0.65; NS); fibrosis: 1.08 (S.D.=1.35)/1.75 (S.D.=1.26; NS); and MW: 1.50 (S.D.=1.22)/0.59 (S.D.=0.73; P<.01). Acquired aortic stenosis developed in six hearts, and one heart showed thrombotic occlusion of the left ventricular outflow tract below an aortic bioprosthesis. CONCLUSIONS: VAD significantly reduced the amount of CN, MC and MW in the left ventricle but may lead to acquired aortic stenosis of native aortic valves or total occlusive thrombosis of aortic prosthetic valves. Proximate cause of death was, most often, VAD related.