An Alzheimer's Disease Mechanism Based on Early Pathology, Anatomy, Vascular-Induced Flow, and Migration of Maximum Flow Stress Energy Location with Increasing Vascular Disease.

This paper suggests a chemical mechanism for the earliest stages of Alzheimer's disease (AD). Cerebrospinal fluid (CSF) flow stresses provide the energy needed to induce molecular conformation changes leading to AD by initiating amyloid-ß (Aß) and tau aggregation. Shear and extensional flow stresses initiate aggregation in the laboratory and in natural biophysical processes. Energy-rich CSF flow regions are mainly found in lower brain regions. MRI studies reveal flow stress "hot spots" in basal cisterns and brain ventricles that have chaotic flow properties that can distort molecules such as Aß and tau trapped in these regions into unusual conformations. Such fluid disturbance is surrounded by tissue deformation. There is strong mapping overlap between the locations of these hot spots and of early-stage AD pathology. Our mechanism creates pure and mixed protein dimers, followed by tissue surface adsorption, and long-term tissue agitation ultimately inducing chemical reactions forming more stable, toxic oligomer seeds that initiate AD. It is proposed that different flow stress energies and flow types in different basal brain regions produce different neurotoxic aggregates. Proliferating artery hardening is responsible for enhanced heart systolic pulses that drive energetic CSF pulses, whose critical maximum systolic pulse energy location migrates further from the heart with increasing vascular disease. Two glymphatic systems, carotid and basilar, are suggested to contain the earliest Aß and tau AD disease pathologies. A key to the proposed AD mechanism is a comparison of early chronic traumatic encephalopathy and AD pathologies. Experiments that test the proposed mechanism are needed.

Vancomycin during delivery hospitalizations for women with group B streptococcus.

OBJECTIVE: Vancomycin use for intrapartum GBS prophylaxis is not well characterized. The objective of this study was to describe trends in the use of vancomycin among women undergoing vaginal delivery with group B Streptococcus (GBS) colonization. METHODS: An administrative inpatient database that includes medications was analyzed to evaluate antibiotic use in women undergoing vaginal delivery hospitalizations complicated by GBS colonization from January 2006 to March 2015. Patients with other obstetric or infectious indications for antibiotics were excluded. Frequency of use of individual antibiotic agents was determined. The Cochran-Armitage test was used to assess temporal trends. An adjusted log-linear regression model accounting for demographic and hospital factors with vancomycin receipt as the outcome was performed with adjusted risk ratios (aRR) and 95% confidence intervals (CI) as the measure of effect. Hospital level variation in administration of vancomycin was also evaluated. RESULTS: 469,717 deliveries met inclusion criteria and were included in this analysis. Use of vancomycin increased from 0.8% of patients in 2006 to 3.8% of patients in the first quarter of 2015. Comparing 2015 to 2006 both the unadjusted (relative risk 4.89 95% CI 4.26-5.60) and adjusted (aRR 4.52 95% 3.94-5.19) models demonstrated significantly increased likelihood of vancomycin administration. In evaluating hospital level vancomycin use, variation was noted with 8.0% of centers administering vancomycin to ≥6.0% of patients. CONCLUSIONS: Vancomycin is becoming increasingly commonly used for intrapartum GBS prophylaxis. Further research and quality improvements initiatives are indicated to optimize intrapartum GBS antibiotic prophylaxis.

Shear-Induced Amyloid Aggregation in the Brain: V. Are Alzheimer's and Other Amyloid Diseases Initiated in the Lower Brain and Brainstem by Cerebrospinal Fluid Flow Stresses?

Amyloid-ß (Aß) and tau oligomers have been identified as neurotoxic agents responsible for causing Alzheimer's disease (AD). Clinical trials using Aß and tau as targets have failed, giving rise to calls for new research approaches to combat AD. This paper provides such an approach. Most basic AD research has involved quiescent Aß and tau solutions. However, studies involving laminar and extensional flow of proteins have demonstrated that mechanical agitation of proteins induces or accelerates protein aggregation. Recent MRI brain studies have revealed high energy, chaotic motion of cerebrospinal fluid (CSF) in lower brain and brainstem regions. These and studies showing CSF flow within the brain have shown that there are two energetic hot spots. These are within the third and fourth brain ventricles and in the neighborhood of the circle of Willis blood vessel region. These two regions are also the same locations as those of the earliest Aß and tau AD pathology. In this paper, it is proposed that cardiac systolic pulse waves that emanate from the major brain arteries in the lower brain and brainstem regions and whose pulse waves drive CSF flows within the brain are responsible for initiating AD and possibly other amyloid diseases. It is further proposed that the triggering of these diseases comes about because of the strengthening of systolic pulses due to major artery hardening that generates intense CSF extensional flow stress. Such stress provides the activation energy needed to induce conformational changes of both Aß and tau within the lower brain and brainstem region, producing unique neurotoxic oligomer molecule conformations that induce AD.

Subfoveal Choroidal Thickness and Associated Changes of Angiogenic Factors in Women with Severe Preeclampsia.

OBJECTIVE: Severe preeclampsia complicates roughly 1% of all pregnancies. One defining feature of severe preeclampsia is new onset visual disturbance. The accessibility of the choroid to high-resolution, noninvasive imaging makes it a reasonable target of investigation for disease prediction, stratification, or monitoring in preeclampsia. This study aimed to compare subfoveal choroidal thickness between women with severe preeclampsia and those with normotensive pregnancies, and to investigate associations between such findings and other indicators of disease severity, including gestational age and serum angiogenic factors. STUDY DESIGN: We designed a case-control study comprised of 36 women diagnosed with severe preeclampsia (cases) matched to 37 normotensive women (controls) by race/ethnicity and parity, all diagnosed in the postpartum period. All patients underwent enhanced depth imaging spectral-domain optical coherence tomography and serum analysis. RESULTS: Cases showed no difference in subfoveal choroidal thickness compared with controls (p = 0.65). Amongst cases, subfoveal choroidal thickness and gestational age at delivery were inversely related (r = 0.86, p < .001). There was a positive association of placental growth factor with subfoveal choroidal thickness amongst cases (r = 0.54, p = 0.002). CONCLUSION: This study suggests a relationship between the degree of disease severity and the magnitude of choroidal thickening. We also show an association between this index and placental growth factor level in the postpartum period.

Clindamycin, Gentamicin, and Risk of Clostridium difficile Infection and Acute Kidney Injury During Delivery Hospitalizations.

OBJECTIVE: To describe risk of Clostridium difficile infection associated with clindamycin and acute kidney injury associated with gentamicin during delivery hospitalizations. METHODS: Women admitted for delivery from January 2006 to March 2015 were analyzed using an inpatient administrative database. Primary outcomes were C difficile infection and acute kidney injury. C difficile infection was compared between women receiving clindamycin (with or without other antibiotics) and women receiving antibiotics other than clindamycin. Acute kidney injury was compared between women receiving gentamicin (with or without other antibiotics), women receiving antibiotics other than gentamicin, and women receiving no antibiotics. Unadjusted and adjusted log linear models analyzing the role of patient demographics, mode of delivery, and hospital-level characteristics were created evaluating risk of C difficile infection and acute kidney injury with risk ratios (RRs) and adjusted risk ratios with 99% CIs as measures of association. A sensitivity analysis for gentamicin and acute kidney injury was performed restricted to women with preeclampsia. RESULTS: Of 5,657,523 women admitted for delivery hospitalization, 266,402 (4.7%) received clindamycin and 165,726 (2.9%) received gentamicin. C difficile infection was diagnosed in 0.04% of women receiving clindamycin. Compared with women receiving other antibiotics, clindamycin was associated with a nearly threefold increased risk of C difficile infection (RR 2.93, 99% CI 2.21-3.90). Acute kidney injury was diagnosed in 0.24% of women receiving gentamicin. Gentamicin was associated with a threefold increased risk of acute kidney injury (RR 3.01, 99% CI 2.62-3.45) compared with women receiving other antibiotics, whereas receipt of no antibiotics was associated with significantly lower risk (RR 0.18, 99% CI 0.15-0.20). In adjusted analyses, these associations retained significance. Significantly increased risk of acute kidney injury was noted for women with preeclampsia receiving gentamicin (RR 2.04, 99% CI 1.64-2.53). CONCLUSION: Receipt of clindamycin was associated with significantly increased likelihood for C difficile infection and receipt of gentamicin with significantly increased likelihood of acute kidney injury, although the absolute risk of these complications was low.

Antibiotics for 3rd and 4th Degree Vaginal Lacerations, Uterine Tamponade, and Manual Placental Extraction.

OBJECTIVE: Trends in use of antibiotics during delivery hospitalizations complicated by (1) 3rd/4th degree vaginal lacerations, (2) manual placenta extraction, and (3) uterine tamponade are not well characterized. The objective of this study was to analyze trends in antibiotic use during vaginal delivery hospitalizations complicated by these three clinical scenarios. STUDY DESIGN: An administrative inpatient database was used to perform a serial cross-sectional analysis of antibiotic administration during delivery hospitalizations in the United States from January 2006 to March 2015. The primary outcome was receipt of antibiotics during vaginal delivery hospitalizations complicated by (1) 3rd and 4th degree vaginal lacerations, (2) manual placenta extraction, and (3) uterine tamponade. Patients with other indications for antibiotics were excluded. The Cochran-Armitage test was used to assess trends. Adjusted log linear regression analyses including demographic, hospital, and obstetric factors were performed to analyze factors associated with antibiotic receipt for each of these three clinical scenarios in both primary and sensitivity analyses. RESULTS: From 2006 to 2015 the rate of antibiotic administration during delivery hospitalizations decreased from 43.1% in 2006 to 25.5% for 3rd and 4th degree lacerations and from 59.6% to 49.2% for manual extraction (p < 0.01). Administration of antibiotics in the setting of uterine tamponade decreased from 48.6% in 2006 to 27.6% in 2009 before rising to 62.5% in the first quarter of 2015. In adjusted analyses, comparing the first quarter of 2015 to 2006 adjusted risk ratios for antibiotic administration were 0.61 (95% confidence interval [CI] 0.56-0.66) for 3rd and 4th degree vaginal lacerations, 0.76 (95% CI 0.53-1.09) for manual placental extraction, and 0.83 (95% CI 0.76-0.92) for uterine tamponade. CONCLUSION: Antibiotics are not used consistently during vaginal deliveries complicated by 3rd/4th degree lacerations, manual placenta extraction, and uterine tamponade. These findings support that a significant opportunity exists for comparative effectiveness research to assist in characterizing best practices.

Preliminary Capillary Flow Experiments with Amyloid-ß, Possible Needle and Capillary Aß Adsorption, and a Proposal for Drug Evaluation Under Shear Conditions.

Amyloid-ß (Aß) solution injections into an aqueous mobile phase moving through narrow bore stainless-steel capillary tubing results in adsorption of at least 99% Aß within the tubing or injection valve. However, if flow is stopped for a period of 5-10 minutes, then started, wall desorption yields Aß-containing molecules in the new effluent. The amount of desorbed Aß-containing effluent depends on flow rate, period of flow cessation, and number of successive Aß injections into the same tube without cleaning between injections. Unexpected multiple chromatographic peaks in these experiments seem to imply "separation" of released, previously adsorbed Aß-containing products in the empty capillary tubing. These preliminary experiments raise questions about possible errors in Alzheimer's disease (AD) spinal tap analyses, which use stainless-steel needles of approximately the same inner diameter and encounter similar flow rates as those in our capillary experiments. Microliter syringes and HPLC connectors also contain stainless-steel tubing that have similar inner diameter dimensions and similar flow rates. The capillary system involved in these experiments has previously been proposed as a model system for studying the effects of shear on Aß within the brain because it offers a research environment that provides highly restrictive flow through very small dimension channels. A suggestion is made for the use of this system in exploratory anti-amyloid drug studies in which both the drug and Aß are injected in the same solution so that both drug and Aß are subjected to the same shear environment. Reduction in adsorbed Aß is suggested as an indicator of effective anti-Aß drugs.

Shear-induced amyloid formation of IDPs in the brain.

The IDP amyloid ß-protein (Aß) has been both the prime causative suspect and drug development target in the fight against Alzheimer's disease (AD). Unfortunately, all clinical trials against Aß based on this assumption have failed. This review proposes that the Aß IDP conformation ensembles in the research laboratory may be somewhat different than those of Aß found within the human brain. It is argued that the multiple quiescent Aß molecular conformations may be the incorrect drug targets. Instead there may be much more complex ensembles found in the flowing liquids within the brain. The highly flexible Aß is quite sensitive to shear-induced aggregation. Different shear types and shear energies must be generated by Aß-containing fluids flowing through different constricted brain spaces at different velocities. A model is proposed in which Aß subjected to shear-induced aggregation produces toxic Aß oligomers that are different from those produced in the laboratory. It is proposed that amyloid researchers seeking Alzheimer drug candidates should perform experiments under shear conditions that attempt to mimic those found in the brain. Because Aß experiments in devices with narrow bore capillaries are rather limited, it is imperative that further experiments of this type be carried out using spinal tap needles and microliter syringes. It is believed that analytical errors may be generated by plating out of amyloids on the inner wall surfaces of these capillary devices. It is suggested that shear-induced aggregation due to flow in confined brain fluid flow pathways may be responsible for many related amyloid diseases and brain trauma events.

Antibiotic Use Without Indication During Delivery Hospitalizations in the United States.

OBJECTIVE: To analyze trends in unindicated antibiotic use during vaginal delivery hospitalization. METHODS: This study used an administrative database to analyze antibiotic use during delivery hospitalizations from January 2006 to March 2015. Women were classified by mode of delivery and whether they had an evidence-based indication for antibiotics. Indications for antibiotics included preterm prelabor rupture of membranes (PROM), cesarean delivery, group B streptococcus (GBS) colonization, chorioamnionitis, endometritis, urinary tract infections, and other infections. The Cochran-Armitage test was used to assess trends of antibiotic administration. Unadjusted and adjusted analyses for antibiotic receipt including demographic, hospital, and obstetric and medical factors were performed with unadjusted and adjusted risk ratios (RRs) with 95% CIs as measures of association. RESULTS: A total of 5,536,756 delivery hospitalizations, including 2,872,286 vaginal deliveries without an indication for antibiotics, were analyzed. The most common indication for antibiotics was cesarean delivery (33.6% of the entire cohort), followed by GBS colonization (15.8%), chorioamnionitis (1.7%), preterm PROM (1.6%), endometritis (1.2%), urinary tract infections (0.6%), and other infections (total less than 0.5%). The proportion of women receiving unindicated antibiotics decreased 44.4%, from 38.1% in 2006 to 21.2% in 2015. Adjusted risk for receipt of unindicated antibiotics was lower in 2015 vs 2006 (adjusted RR 0.56, 95% CI 0.55-0.57). CONCLUSION: Use of antibiotics during vaginal delivery hospitalizations without an indication for antibiotic use declined significantly based on an analysis of a large administrative data set.

Shear-Induced Amyloid Formation in the Brain: IV. Effects on Synapses Surrounding Senile Plaque and in Plaque-Free Regions.

Amyloid-ß oligomers (AßO) have been proposed as neurotoxins in the synaptic dysfunction that precedes Alzheimer's disease symptoms. Human and animal model studies report that senile plaques contain a halo of AßO molecules surrounding these plaques. A far smaller number of oligomers are distributed widely in plaque-free regions. It has been suggested that oligomers migrate from halos to nearby synapses and are incorporated into both pre- and postsynaptic terminals. These two types of oligomers have two different toxicities when extracted and injected in animal models. This paper proposes a shear-energy based explanation for the data in these studies. Shear hypotheses in the preceding three papers in this series are applied to suggest how the hydrodynamics and resulting shear patterns explain the spatial distribution of both AßO types, the apparent synapse loss in the vicinity of plaque particles, and possible reasons for the differing toxicities. A shear-based mechanism is proposed for the preferential migration of locally shear-excited Aß molecules into the synaptic cleft. It is proposed that high energy laminar shear generated by the forced diversion of interstitial fluid around the flow-impeding plaque particle is responsible for the formation of AßOs around the plaque. It is suggested that in plaque-free regions, a different type of AßO with different toxicity is generated by lower energy shear flow around synapses, depositing AßO within the synapse from either the neuron membrane surface or by prion-like seeding within the synaptic cleft by locally-sheared Aß molecules near the synapse entry.

Shear-Induced Amyloid Formation in the Brain: III. The Roles of Shear Energy and Seeding in a Proposed Shear Model.

If cerebrospinal and interstitial fluids move through very narrow brain flow channels, these restrictive surroundings generate varying levels of fluid shear and different shear rates, and dissolved amyloid monomers absorb different shear energies. It is proposed that dissolved amyloid-ß protein (Aß) and other amyloid monomers undergo shear-induced conformational changes that ultimately lead to amyloid monomer aggregation even at very low brain flow and shear rates. Soluble Aß oligomers taken from diseased brains initiate in vivo amyloid formation in non-diseased brains. The brain environment is apparently responsible for this result. A mechanism involving extensional shear is proposed for the formation of a seed Aß monomer molecule that ultimately promotes templated conformational change of other Aß molecules. Under non-quiescent, non-equilibrium conditions, gentle extensional shear within the brain parenchyma, and perhaps even during laboratory preparation of Aß samples, may be sufficient to cause subtle conformational changes in these monomers. These result from brain processes that significantly lower the high activation energy predicted for the quiescent Aß dimerization process. It is further suggested that changes in brain location and changes brought about by aging expose Aß molecules to different shear rates, total shear, and types of shear, resulting in different conformational changes in these molecules. The consequences of such changes caused by variable shear energy are proposed to underlie formation of amyloid strains causing different amyloid diseases. Amyloid researchers are urged to undertake studies with amyloids, anti-amyloid drugs, and antibodies while all of these are under shear conditions similar to those in the brain.

Shear-Induced Amyloid Formation in the Brain: II. An Experimental System for Monitoring Amyloid Shear Processes and Investigating Potential Spinal Tap Problems.

Liquid sheared amyloid-ß (Aß) initiates amyloid cascade reactions, producing unstable, potentially toxic oligomers. There is a need for new analytical tools with which to study these oligomers. A very small bore capillary flow system is proposed as a tool for studying the effects of liquid shear in amyloid research. This simple system consists of injecting a short cylindrical liquid sample plug containing dissolved amyloid into a liquid mobile phase flowing through an empty, very small internal diameter capillary tube. For liquid samples containing a single protein sample, under conditions in which there is laminar flow and limited sample protein molecular diffusion, chromatograms monitoring the optical protein absorbance of capillary effluent contain either one or two peaks, depending on the mobile phase flow rate. By controlling the sample diffusion times through changes in flow rate and/or capillary diameter, this tool can be used to generate aliquot samples with precise, reproducible amounts of shear for exploring the effects of variable shear on amyloid systems. The tool can be used for producing in-capillary stopped flow spectra of shear-stressed Aß monomers as well as for kinetic studies of Aß dimer- and oligomer-forming reactions between shear stressed Aß monomers. Many other experiments are suggested using this experimental tool for studying the effects of shear on different Aß and other amyloid systems, including testing for potentially serious amyloid sampling errors in spinal tap quantitative analysis. The technique has potential as both a laboratory research and a clinical tool.

The BMP2 nuclear variant, nBMP2, is expressed in mouse hippocampus and impacts memory.

The novel nuclear protein nBMP2 is synthesized from the BMP2 gene by translational initiation at an alternative start codon. We generated a targeted mutant mouse, nBmp2NLStm, in which the nuclear localization signal (NLS) was inactivated to prevent nuclear translocation of nBMP2 while still allowing the normal synthesis and secretion of the BMP2 growth factor. These mice exhibit abnormal muscle function due to defective Ca2+ transport in skeletal muscle. We hypothesized that neurological function, which also depends on intracellular Ca2+ transport, could be affected by the loss of nBMP2. Age-matched nBmp2NLStm and wild type mice were analyzed by immunohistochemistry, behavioral tests, and electrophysiology to assess nBMP2 expression and neurological function. Immunohistochemical staining of the hippocampus detected nBMP2 in the nuclei of CA1 neurons in wild type but not mutant mice, consistent with nBMP2 playing a role in the hippocampus. Mutant mice showed deficits in the novel object recognition task, suggesting hippocampal dysfunction. Electrophysiology experiments showed that long-term potentiation (LTP) in the hippocampus, which is dependent on intracellular Ca2+ transport and is thought to be the cellular equivalent of learning and memory, was impaired. Together, these results suggest that nBMP2 in the hippocampus impacts memory formation.

Shear-Induced Amyloid Formation in the Brain: I. Potential Vascular and Parenchymal Processes.

Shear distortion of amyloid-beta (Aß) solutions accelerates amyloid cascade reactions that may yield different toxic oligomers than those formed in quiescent solutions. Recent experiments indicate that cerebrospinal fluid (CSF) and interstitial fluid (ISF) containing Aß flow through narrow brain perivascular pathways and brain parenchyma. This paper suggests that such flow causes shear distortion of Aß molecules involving conformation changes that may be one of the initiating events in the etiology of Alzheimer's disease. Aß shearing can occur in or around brain arteries and arterioles and is suggested as the origin of cerebral amyloid angiopathy deposits in cerebrovascular walls. Comparatively low flow rates of ISF within the narrow extracellular spaces (ECS) of the brain parenchyma are suggested as a possible initiating factor in both the formation of neurotoxic Aß42 oligomers and amyloid fibrils. Aß42 in slow-flowing ISF can gain significant shear energy at or near the walls of tortuous brain ECS flow paths, promoting the formation of a shear-distorted, excited state hydrophobic Aß42* conformation. This Aß42* molecule could possibly be involved in one of two paths, one involving rapid adsorption to a brain membrane surface, ultimately forming neurotoxic oligomers on membranes, and the other ultimately forming plaque within the ECS flow pathways. Rising Aß concentrations combined with shear at or near critical brain membranes are proposed as contributing factors to Alzheimer's disease neurotoxicity. These hypotheses may be applicable in other neurodegenerative diseases, including tauopathies and alpha-synucleinopathies, in which shear-distorted proteins also may form in the brain ECS.

Epitope mapping of mAbs to denatured human testicular ACE (CD143).

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

Frontal electroencephalogram asymmetry during affective processing in children with Down syndrome: a pilot study.

BACKGROUND: Although the pattern of frontal electroencephalogram (EEG) asymmetry during the processing of emotion has been examined in many studies of healthy adults and typically developing infants and children, no published work has used these theoretical and methodological approaches to study emotion processing in children with Down syndrome. The purpose of this pilot study was to examine the feasibility of using brain-based measures of emotion (i.e. regional EEG asymmetry measures) with children with Down syndrome, and whether children with Down syndrome exhibit patterns of frontal brain activity during the processing of affective stimuli that are not different from typically developing children, but of lesser magnitude. METHODS: Regional brain electrical activity (EEG) was measured in response to the presentation of popular children's video clips that varied in affective content in three children with Down syndrome and three typically developing children who were matched on reading level. RESULTS: The children with Down syndrome appeared to show a similar pattern of frontal EEG asymmetry as the typically developing children for the video clips depicting happiness, sadness and fear. However, the magnitude of the frontal asymmetry scores for the children with Down syndrome was large across the affective stimuli, and they appeared to process the video clip depicting anger differently from the typically developing children. CONCLUSION: This preliminary evidence suggests that brain-based measures of affective processing can be used to study the differentiation of emotion on an electrocortical level among children with Down syndrome.

The challenge of viral snRNPs.

Some gammaherpesviruses encode nuclear noncoding RNAs (ncRNAs) that assemble with host proteins. Their conservation and abundance implies that they serve important functions for the virus. This paper focuses on our studies of three classes of nuclear noncoding herpesvirus RNAs. (1) EBERs 1 and 2 are expressed by Epstein-Barr virus in latent infection of human B lymphocytes. Recent studies revealed three sites on EBER1 that associate with ribosomal protein L22. In addition, heterokaryon assays have definitively shown that both EBERs are confined to the nucleus, arguing that their contribution to viral latency is purely nuclear. (2) HSURs 1-7 are U RNAs encoded by Herpesvirus saimiri, which causes aggressive T-cell leukemias and lymphomas. Comparison of monkey T cells transformed with wild-type or mutant virus lacking HSURs 1 and 2 revealed significant changes in host mRNAs implicated in T-cell signaling. (3) PAN is a 1-kb polyadenylated RNA that accumulates in the nucleus of Kaposi's sarcoma-associated herpesvirus lytically infected cells. A novel element, the ENE, is essential for its high accumulation. Recent results indicate that the ENE functions to counteract poly(A)-dependent RNA degradation, which we propose contributes to nuclear surveillance of mRNA transcripts in mammalian cells. Continuing studies of these viral RNAs will provide insights into both cellular and viral gene expression.

Expression of endomyocardial nitric oxide synthase and coronary endothelial function in human cardiac allografts.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is expressed and is functionally active in the presence of transplant arteriosclerosis. However, the early involvement of iNOS in alterations of microvascular endothelial function in the absence of preexisting lesions remains unclear; this information would be of prognostic value. We studied the course of iNOS mRNA expression, transcardiac nitric oxide production, and their potential association with microvascular coronary endothelial dysfunction in human cardiac allografts. METHODS AND RESULTS: A total of 42 patients were studied at 1, 6, and 12 months after heart transplantation. Microvascular coronary flow velocity reserve (CFVR) was tested in an endothelium-dependent (acetylcholine) and -independent manner (adenosine) using a Doppler flow wire. Endomyocardial iNOS expression was determined by reverse transcription polymerase chain reaction. iNOS protein and nitrotyrosine levels were detected by immunohistochemistry. Transcardiac plasma nitrite/nitrate (NOx) levels were measured by the Griess reaction. CFVR was impaired in 26.1% of patients (n=11) at 1 month and in 31% of patients (n=13) at 12 months after heart transplantation. Patients who developed impaired CFVR in the first year showed a significant increase in iNOS gene expression. Patients with impairment of CFVR 1 month after heart transplantation had higher levels of iNOS mRNA than patients with a normal CFVR. Patients with an initial impairment of CFVR who did not improve over time presented with significantly higher iNOS mRNA levels. iNOS protein and nitrotyrosine were expressed in the endomyocardial vessels of patients with impaired CFVR. Transcardiac NOx release was higher in patients with impaired CFVR. CONCLUSIONS: In human cardiac allografts, microvascular endothelial dysfunction is associated with an enhanced endomyocardial iNOS mRNA expression and higher transcardiac NOx production and is accompanied by the expression of nitrotyrosine protein, suggesting peroxynitrite plays a role in the disease process. The data from the present study suggest an important role for the iNOS/nitric oxide pathway in the regulation of microvascular function in the absence of preexisting atherosclerotic lesions.

RNA-binding protein Nrd1 directs poly(A)-independent 3'-end formation of RNA polymerase II transcripts.

A eukaryotic chromosome contains many genes, each transcribed separately by RNA polymerase (pol) I, II or III. Transcription termination between genes prevents the formation of polycistronic RNAs and anti-sense RNAs, which are generally detrimental to the correct expression of genes. Terminating the transcription of protein-coding genes by pol II requires a group of proteins that also direct cleavage and polyadenylation of the messenger RNA in response to a specific sequence element, and are associated with the carboxyl-terminal domain of the largest subunit of pol II (refs 1, 2, 3, 4, 5, 6). By contrast, the cis-acting elements and trans-acting factors that direct termination of non-polyadenylated transcripts made by pol II, including small nucleolar and small nuclear RNAs, are not known. Here we show that read-through transcription from yeast small nucleolar RNA and small nuclear RNA genes into adjacent genes is prevented by a cis-acting element that is recognized, in part, by the essential RNA-binding protein Nrd1. The RNA-binding protein Nab3, the putative RNA helicase Sen1, and the intact C-terminal domain of pol II are also required for efficient response to the element. The same proteins are required for maintaining normal levels of Nrd1 mRNA, indicating that these proteins may control elongation of a subset of mRNA transcripts.