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More than 20% of residents who have been in long-term care (LTC) facilities for 2 or more years will develop at least one pressure ulcer (PU). Residents suffer pain, disfigurement, and decreased quality of life, and their risk of illness and death increases. LTC facilities face censure from residents, their families, and surveyors and the threat of expensive lawsuits. Lawsuits are typically based on contentions of residents with a PU--or their advocates--that the LTC facility was negligent and failed to provide the care that, by industry standards, it must provide to prevent or manage such wounds (managing pressure, incontinence, and nutrition). In this article, data from 1999 and 2002 are presented, showing that lawsuits related to PUs are increasingly common and costly for LTC owners and care providers. Residents realized some type of recovery against the facility in 87% of the cases (verdicts for the resident plus settlements) and were awarded amounts as high as $312 million in damages. Even LTC administrators who believe that care in their facility equals or exceeds industry standards often settle lawsuits out of court to avoid jury verdicts. The data also show that jury awards were highest for PUs caused by multiple factors and that the highest awards for PUs caused by a single factor were seen when that factor was inadequate nutrition. LTC providers can help improve the health and quality of life of their residents, improve survey results, and minimize their risk of expensive lawsuits by developing, implementing, and documenting a plan of basic measures to prevent PUs. [ABSTRACT FROM AUTHOR]

The Goossen decarboxylative coupling reaction enables the union of thiazole-2-carboxylic acids with a 2-pyridyl triflate, leading to the formation of pyridine-thiazole clusters of the kind found in thiopeptide antibiotics. The method avoids problematic or technically challenging reaction sequences involving 2-thiazolyl organometallics, facilitating the investigation of the structure-activity relationship of the thiopeptides.

The nucleus, central to cellular activity, relies on both direct mechanical input as well as its molecular transducers to sense external stimuli and respond by regulating intra-nuclear chromatin organization that determines cell function and fate. In mesenchymal stem cells of musculoskeletal tissues, changes in nuclear structures are emerging as a key modulator of their differentiation and proliferation programs. In this review we will first introduce the structural elements of the nucleoskeleton and discuss the current literature on how nuclear structure and signaling are altered in relation to environmental and tissue level mechanical cues. We will focus on state-of-the-art techniques to apply mechanical force and methods to measure nuclear mechanics in conjunction with DNA, RNA, and protein visualization in living cells. Ultimately, combining real-time nuclear deformations and chromatin dynamics can be a powerful tool to study mechanisms of how forces affect the dynamics of genome function.

Genes are transcribed in a discontinuous pattern referred to as RNA bursting, but the mechanisms regulating this process are unclear. Although many physiological signals, including glucocorticoid hormones, are pulsatile, the effects of transient stimulation on bursting are unknown. Here we characterize RNA synthesis from single-copy glucocorticoid receptor (GR)-regulated transcription sites (TSs) under pulsed (ultradian) and constant hormone stimulation. In contrast to constant stimulation, pulsed stimulation induces restricted bursting centered around the hormonal pulse. Moreover, we demonstrate that transcription factor (TF) nuclear mobility determines burst duration, whereas its bound fraction determines burst frequency. Using 3D tracking of TSs, we directly correlate TF binding and RNA synthesis at a specific promoter. Finally, we uncover a striking co-bursting pattern between TSs located at proximal and distal positions in the nucleus. Together, our data reveal a dynamic interplay between TF mobility and RNA bursting that is responsive to stimuli strength, type, modality, and duration., (Published by Elsevier Inc.)

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation., (© 2019 The Authors.)

The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro., (© 2017 Panchapakesan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas., Methods: We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes., Results: Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma., Conclusions: The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.).

Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing, and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. In this study, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene. We find that kinetic competition results in multiple competing pathways for pre-mRNA splicing. Splicing of the terminal intron occurs stochastically both before and after transcript release, indicating there is not a strict quality control checkpoint. The majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. A single missense point mutation (S34F) in the essential splicing factor U2AF1 which occurs in human cancers perturbs this kinetic balance and defers splicing to occur entirely post-release.

Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes., (© 2014. Published by The Company of Biologists Ltd.)

Background: Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear., Methods: We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis., Results: AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories., Conclusions: We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

Single-cell studies of gene regulation suggest that transcription dynamics play a fundamental role in determining expression heterogeneity within a population. In addition, the three-dimensional organization of the nucleus seems to both reflect and influence expression patterns in the cell. Therefore, to gain a holistic understanding of transcriptional regulation, it is necessary to develop methods for studying transcription of single genes in living cells with high spatial and temporal resolution. In this chapter, we describe a recently developed approach for visualizing and quantifying pre-mRNA synthesis at a single active gene in the nucleus. The approach is based on the high-affinity interaction between MS2/PP7 bacteriophage coat proteins and RNA hairpins which are transcribed by the gene of interest. The MS2/PP7 coat protein is fused to a fluorescent protein and binds the nascent mRNA, allowing for detection of single transcription events in the fluorescence microscope. By time-lapse fluorescence imaging and quantitative image analysis, one can generate a time trace of fluorescence intensity at the site of transcription. By temporal autocorrelation analysis, one can determine enzymatic activities of RNAP such as initiation rate and elongation rate. In this protocol, we summarize the experimental concept, design, and execution for real-time observation of transcription in living cells.

Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell-to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.

Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 μM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Prehypertension is a major risk factor for hypertension. Blacks have more prevalent and severe hypertension than whites, but it is unknown whether progression from prehypertension is accelerated in blacks. We examined this question in a prospective cohort study of 18 865 nonhypertensive persons (5733 black [30.4%] and 13 132 white [69.6%]) aged 18 to 85 years. Electronic health record data were obtained from 197 community-based outpatient clinics in the Southeast United States. Days elapsing from study entry to hypertension diagnosis, mainly blood pressure ≥140 mm Hg systolic and/or ≥90 mm Hg diastolic on 2 consecutive visits established conversion time within a maximum observation period of 2550 days. Cox regression modeling was used to examine conversion to hypertension as a function of race, while controlling for age, sex, baseline systolic and diastolic blood pressures, body mass index, diabetes mellitus, and chronic kidney disease. The covariable adjusted median conversion time when 50% became hypertensive was 365 days earlier for blacks than whites (626 versus 991 days; P<0.001). Among covariables, baseline systolic blood pressure 130 to 139 mm Hg (hazard ratio: 1.77 [95% CI: 1.69 to 1.86]) and 120 to 129 mm Hg (hazard ratio: 1.52 [95% CI: 1.44 to 1.60]), as well as age ≥75 years (hazard ratio: 1.40 [95% CI: 1.29 to 1.51]) and 55 to 74 years (hazard ratio: 1.29 [95% CI: 1.23 to 1.35]) were the strongest predictors of hypertension. Additional predictors included age 35 to 54 years, diastolic blood pressure 80 to 89 mm Hg, overweight and obesity, and diabetes mellitus (all P<0.001). Conversion from prehypertension to hypertension is accelerated in blacks, which suggests that effective interventions in prehypertension could reduce racial disparities in prevalent hypertension.

Background: Cancer-associated weight loss may be mediated by an inflammatory response to cancer. Eicosapentaenoic acid (EPA) may suppress this response., Methods: Beginning no later than 2 weeks before surgery, patients with head and neck cancer and with weight loss, who were undergoing major resection with curative intent consumed a protein- and energy-dense nutritional supplement containing EPA from fish oil, in addition to usual diet or tube feed., Results: Thirty-one subjects consumed an average of 1.8 containers/day before surgery and 1.5/day during hospitalization (per container: 300 kilocalories, 16 grams (g) protein, 1.08 g EPA). Seventy percent of subjects maintained or gained weight before hospital admission. Mean weight gain was 0.71 kg at admission and 0.66 kg at discharge. At discharge lean body mass increased by 3.20 kg (p < .001) and fat decreased by 3.19 kg (p < .001)., Conclusions: An EPA-containing protein- and energy-dense nutritional supplement may help increase perioperative lean body mass in patients with head and neck cancer-related weight loss., (Copyright © 2010 Wiley Periodicals, Inc.)

Previous investigations showing that polydisperse biguanide (PDBG) molecules have activity against human immunodeficiency virus type 1 (HIV-1) also suggested a relationship between PDBG biologic activity and the lengths of hydrocarbon linkers surrounding the positively charged biguanide unit. To better define structure-activity relationships, PDBG molecules with select linker lengths were evaluated for cytotoxicity, anti-HIV-1 activity, and in vivo toxicity. Results of the in vitro experiments demonstrated that increases in linker length (and, therefore, increases in compound lipophilicity) were generally associated with increases in cytotoxicity and antiviral activity against HIV-1. However, a relationship between linker length asymmetry and in vitro therapeutic index (TI) suggested structural specificity in the mechanism of action against HIV-1. Polyethylene hexamethylene biguanide (PEHMB; biguanide units spaced between alternating ethylene and hexamethylene linkers) was found to have the highest in vitro TI (CC₅₀/IC₅₀) among the compounds examined. Recent improvements in PEHMB synthesis and purification have yielded preparations of PEHMB with in vitro TI values of 266 and 7000 against HIV-1 strains BaL and IIIB, respectively. The minimal toxicity of PEHMB relative to polyhexamethylene biguanide (PHMB; biguanide units alternating with hexamethylene linkers) in a murine model of cervicovaginal microbicide toxicity was consistent with considerable differences in cytotoxicity between PEHMB and PHMB observed during in vitro experiments. These structure-activity investigations increase our understanding of PDBG molecules as agents with activity against HIV-1 and provide the foundation for further preclinical studies of PEHMB and other biguanide-based compounds as antiviral and microbicidal agents., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, fluorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer-dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer-dimer contacts. We conclude that inducer-dependent dimer-dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members.

The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

Quantitative characterization of protein interactions in live cells remains one of the most important challenges in modern biology. In the present work we have used two-photon, two-color, fluorescence cross-correlation spectroscopy (FCCS) in transiently transfected COS-7 cells to measure the concentrations and interactions of estrogen receptor (ER) subtypes alpha and beta with one of their transcriptional coactivator proteins, TIF2, as well as heterodimerization between the two ER subtypes. Using this approach in a systematic fashion, we observed a strong ligand-dependent modulation of receptor-coactivator complexation, as well as strong protein concentration dependence for complex formation in the absence of ligand. These quantitative values for protein and complex concentrations provide the first estimates for the ER-TIF2 K(d) for the full-length proteins and in a cellular context (agonist, < approximately 6 nM; antagonist, > approximately 3 microM; unliganded, approximately 200 nM). Coexpression of the two ER subtypes revealed substantial receptor heterodimer formation. They also provide, for the first time, estimated homo- and heterodimerization constants found to be similar and in the low nanomolar range. These results underscore the importance of receptor and coregulator expression levels and stability in the tissue-dependent modulation of receptor function under normal and pathological conditions.

The clathrin triskelion, which is a three-legged pinwheel-shaped heteropolymer, is a major component in the protein coats of certain post-Golgi and endocytic vesicles. At low pH, or at physiological pH in the presence of assembly proteins, triskelia will self-assemble to form a closed clathrin cage, or "basket". Recent static light scattering and dynamic light scattering studies of triskelia in solution showed that an individual triskelion has an intrinsic pucker similar to, but differing from, that inferred from a high resolution cryoEM structure of a triskelion in a clathrin basket. We extend the earlier solution studies by performing small-angle neutron scattering (SANS) experiments on isolated triskelia, allowing us to examine a higher q range than that probed by static light scattering. Results of the SANS measurements are consistent with the light scattering measurements, but show a shoulder in the scattering function at intermediate q values (0.016 A(-1)), just beyond the Guinier regime. This feature can be accounted for by Brownian dynamics simulations based on flexible bead-spring models of a triskelion, which generate time-averaged scattering functions. Calculated scattering profiles are in good agreement with the experimental SANS profiles when the persistence length of the assumed semiflexible triskelion is close to that previously estimated from the analysis of electron micrographs.

Recent studies of cellulose-based polymers substituted with carboxylic acids like cellulose acetate phthalate (CAP) have demonstrated the utility of using carboxylic acid groups instead of the more common sulfate or sulfonate moieties. However, the pK(a) of the free carboxylic acid group is very important and needs careful selection. In a polymer like CAP the pK(a) is approximately 5.28. This means that under the low pH conditions found in the vaginal lumen, CAP would be only minimally soluble and the carboxylic acid would not be fully dissociated. These issues can be overcome by substitution of the cellulose backbone with a moiety whose free carboxylic acid group(s) has a lower pK(a). Hydroxypropyl methylcellulose trimellitate (HPMCT) is structurally similar to CAP; however, its free carboxylic acids have pK(a)s of 3.84 and 5.2. HPMCT, therefore, remains soluble and molecularly dispersed at a much lower pH than CAP. In this study, we measured the difference in solubility and dissociation between CAP and HPMCT and the effect these parameters might have on antiviral efficacy. Further experiments revealed that the degree of acid substitution of the cellulose backbone can significantly impact the overall efficacy of the polymer, thereby demonstrating the need to optimize any prospective polymer microbicide with respect to pH considerations and the degree of acid substitution. In addition, we have found HPMCT to be a potent inhibitor of CXCR4, CCR5, and dual tropic strains of human immunodeficiency virus in peripheral blood mononuclear cells. Therefore, the data presented herein strongly support further evaluation of an optimized HPMCT variant as a candidate microbicide.

A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral cages principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends upon the pH, with cages forming more readily at low pH and less readily at high pH. We have developed procedures, on the basis of static and dynamic light scattering, to determine the radius of gyration, R(g), and hydrodynamic radius, R(H), of isolated triskelia, under conditions where cage assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending the legs and a puckering at the vertex. We also show that the values of R(g) and R(H) measured for clathrin triskelia in solution are qualitatively consistent with the conformation of a triskelion in a "D6 barrel" cage assembly measured by cryoelectron microscopy.

Allylic phosphonium ylides are readily generated by the combination of an allylic alcohol, a carbene, and a chlorophosphite. Here we demonstrate that these intermediates undergo a thermal [3,3]-rearrangement to provide single isomers of homoallylic phosphonates in good to excellent yields. This new reaction manifold for phosphorus ylides is tolerant of a range of substitution patterns on the reactants and provides access to structurally complex intermediates for the synthesis of enzyme inhibitors, aminophosphonic acids, and natural products.

Objectives: To identify resident, wound, and treatment characteristics associated with pressure ulcer (PrU) healing in long-term care residents., Design: Retrospective cohort study with convenience sampling., Setting: Ninety-five long-term care facilities participating in the National Pressure Ulcer Long-Term Care Study throughout the United States., Participants: Eight hundred eighty-two residents, aged 18 and older, with length of stay of 14 days or longer, who had at least one Stage II to IV PrU., Measurements: Data collected for each resident over a 12-week period included resident characteristics, treatment characteristics, and change in PrU area. Data were obtained from medical records, Minimum Data Set, and other records., Results: Two multiple regression models, one for each stage grouping (Stage II, Stage III and IV), were completed. The area of Stage II PrU was reduced more with moist (F=21.91, P<.001) than with dry (F=13.41, P<.001) dressings. PrUs cleaned with saline or soap showed less decrease in area (F=12.34, P<.001) than PrUs cleaned with other cleansers such as antiseptic, antibiotic, or commercial cleansers. Change in area of Stage III and IV PrUs was related to sufficient enteral feeding (F=5.23, P=.02), enteral feeding without higher acuity levels (F=3.94, P=.048), size of PrU (very large (F=120.89, P=.001) and large (F=27.82, P=.001)), and type of dressing (moist (F=14.70, P<.001) and dry (F=5.88, P=.02)). Stage III and IV PrUs increased in area when debrided (F=5.97, P=.02). The overall models were significant (Stage III and IV, F=20.30, coefficient of determination (R2)=0.06, P<.001; Stage II, F=40.28, R2=0.13, P<.001) but explained little of the variation in change in PrU area., Conclusion: In this sample of nursing facility residents, use of moist dressings (Stage II, Stage III and IV) and adequate nutritional support (Stage III and IV) are strong predictors of PrU healing.

C31G, which has potent activity against the human immunodeficiency virus type 1 (HIV-1) and an established record of safety in animal studies and human trials, is a microbicidal agent comprised of a buffered equimolar mixture of two amphoteric, surface-active agents: an alkyl amine oxide (C14AO) and an alkyl betaine (C16B). Studies of long-term in vitro exposure to C31G and its constituents have suggested that the components of C31G may contribute differentially to its toxicity and efficacy. In the present studies, in vitro assays of cytotoxicity and anti-HIV-1 activity demonstrated that C16B was slightly less cytotoxic compared to either C31G or C14AO, whereas the anti-HIV-1 activities of C31G and its individual constituents were similar. In the murine model of cervicovaginal microbicide toxicity, in vivo exposure to C14AO resulted in severe cervical inflammation followed by a delayed disruption of the columnar epithelium. In contrast, exposure to C16B caused severe cervical epithelial disruption and a secondary, less intense inflammatory response. These results demonstrate that (i) there are both mechanistic and temporal differences in toxicity associated with the components of C31G not necessarily predicted by in vitro assessments of cytotoxicity and (ii) contributions of each component to the anti-HIV-1 activity of C31G appear to be equal. In addition, these findings indicate that direct and indirect mechanisms of in vivo toxicity can be observed as separate but interrelated events. These results provide further insight into the activity of C31G, as well as mechanisms potentially associated with microbicide toxicity.

Polyhexamethylene biguanide (PHMB) is a polybiguanide (PBG) oligomer with antimicrobial activity that is used extensively and safely as a disinfectant. The reported mechanism of PHMB antimicrobial activity, which involves interactions with cell membrane components, suggested that PHMB or other PBG-based compounds might also have antiviral or virucidal activity against the human immunodeficiency virus type 1 (HIV-1). PHMB had modest in vitro activity against both cell-free and cell-associated HIV-1, as well as the ability to interfere with viral binding and entry. However, PHMB was comparable in cytotoxicity to the spermicidal agent nonoxynol-9 (N-9), a compound that has been characterized in previous studies as generally cytotoxic and detrimental to cervicovaginal epithelial integrity. To identify structural variants of PHMB with greater anti-HIV-1 activity and/or less cytotoxicity, modified versions of PHMB incorporating length changes in the hydrocarbon linker units were synthesized and evaluated for in vitro cytotoxicity and inhibition of HIV-1 infectivity. These experiments demonstrated that the PHMB variant polyethylene hexamethylene biguanide (PEHMB) was just as active against HIV-1 as PHMB, yet was much less cytotoxic than either N-9 or PHMB, resulting in an in vitro therapeutic index (TI) approximately 114-fold greater than the TI of N-9. PEHMB, which has been identified in these studies as a promising microbicidal candidate in this family of compounds, will be the focus of further in vitro and in vivo evaluations of anti-HIV-1 activity, toxicity, and mechanisms of action.

Comparative assays of in vitro cytotoxicity using nonoxynol-9 (N-9) and the candidate microbicides C31G and sodium dodecyl sulfate (SDS) demonstrated that these agents, which are, respectively, characterized as nonionic, amphoteric, and anionic surfactants, differed in their concentration-dependent effects on cell viability, especially after prolonged exposure. We hypothesized that differences in cellular sensitivity may have been due, in part, to cellular changes induced by long-term exposure to each agent. To examine this possibility, HeLa cells were exposed to N-9, C31G, or SDS for extended periods of time and subsequently reassessed for sensitivity to each of these agents. Following 10 continuous days of C31G exposure, HeLa cells were less sensitive to a subsequent C31G exposure compared to cells that had not undergone long-term C31G treatment. Interestingly, long-term C31G exposure also changed subsequent sensitivity to N-9 but not SDS. In contrast, prolonged exposure to either N-9 or SDS did not reduce sensitivity to re-exposure. The effect of long-term C31G exposure was both concentration-dependent and transient, as treated cells reverted to pre-exposure sensitivity in a time-dependent manner following the cessation of C31G exposure. Lipid analyses of cells exposed to C31G for extended durations revealed altered phospholipid profiles relative to C31G-naïve cells. Experiments examining the individual components of C31G demonstrated the involvement of the amine oxide moiety in reductions in cellular sensitivity. These studies, which provide new information concerning the cytotoxicity of surfactant microbicides, suggest that cervicovaginal epithelial cells may have greater in vivo tolerance for products containing C31G through unique interactions between C31G and components of the cellular membranes.

C31G is currently the focus of clinical trials designed to evaluate this agent as a microbicidal and spermicidal agent. In the following studies, the in vivo safety of C31G was assessed with a Swiss Webster mouse model of cervicovaginal toxicity and correlated with results from in vitro cytotoxicity experiments and published clinical observations. A single exposure of unformulated 1% C31G resulted in mild-to-moderate epithelial disruption and inflammation at 2 and 4 h postapplication. The columnar epithelium of the cervix was the primary site of damage, while no perturbation of the vaginal mucosa was observed. In contrast, application of unformulated 1.7% C31G resulted in greater levels of inflammation in the cervical epithelium at 2 h postapplication and severe epithelial disruption that persisted to 8 h postapplication. Application of a nonionic aqueous gel formulation containing 1% C31G resulted in no apparent cervicovaginal toxicity at any time point evaluated. However, formulation of 1.7% C31G did not substantially reduce the toxicity associated with unformulated C31G at that concentration. These observations correlate with findings gathered during a recent clinical trial, in which once-daily applications resulted in no adverse events in women receiving the formulation containing 1% C31G, compared to moderate-to-severe adverse events in 30% of women receiving the 1.7% C31G formulation. The Swiss Webster mouse model was able to effectively discriminate between concentrations and formulations of C31G that produced distinct clinical effects in human trials. The Swiss Webster animal model may be a highly valuable tool for preclinical evaluation of candidate vaginal microbicides.

Objectives: To identify resident, treatment, and facility characteristics associated with pressure ulcer (PU) development in long-term care residents., Design: Retrospective cohort study with convenience sampling., Setting: Ninety-five long-term care facilities participating in the National Pressure Ulcer Long-Term Care Study throughout the United States., Participants: A total of 1,524 residents aged 18 and older, with length of stay of 14 days or longer, who did not have an existing PU but were at risk of developing a PU, as defined by a Braden Scale for Predicting Pressure Sore Risk score of 17 or less, on study entry., Measurements: Data collected for each resident over a 12-week period included resident characteristics (e.g., demographics, medical history, severity of illness using the Comprehensive Severity Index, Braden Scale scores, nutritional factors), treatment characteristics (nutritional interventions, pressure management strategies, incontinence treatments, medications), staffing ratios and other facility characteristics, and outcome (PU development during study period). Data were obtained from medical records, Minimum Data Set, and other written records (e.g., physician orders, medication logs)., Results: Seventy-one percent of subjects (n=1,081) did not develop a PU during the 12-week study period; the remaining 29% of residents (n=443) developed a new PU. Resident, treatment, and facility characteristics associated with greater likelihood of developing a Stage I to IV PU included higher initial severity of illness, history of recent PU, significant weight loss, oral eating problems, use of catheters, and use of positioning devices. Characteristics associated with decreased likelihood of developing a Stage I to IV PU included new resident, nutritional intervention (e.g., use of oral medical nutritional supplements and tube feeding for >21 days), antidepressant use, use of disposable briefs for more than 14 days, registered nurse hours of 0.25 hours per resident per day or more, nurses' aide hours of 2 hours per resident per day or more, and licensed practical nurse turnover rate of less than 25%. When Stage I PUs were excluded from the analyses, the same variables were significant, with the addition of fluid orders associated with decreased likelihood of developing a PU., Conclusion: A broad range of factors, including nutritional interventions, fluid orders, medications, and staffing patterns, are associated with prevention of PUs in long-term care residents. Research-based PU prevention protocols need to be developed that include these factors and target interventions for reducing risk factors.

Objectives: To describe and provide baseline data from The National Pressure Ulcer Long-Term Care Study (NPULS)., Design: Retrospective cohort study of detailed resident characteristics, treatments, and outcomes using convenience sampling., Setting: One hundred nine long-term care facilities throughout the United States., Participants: Two thousand four hundred twenty adult residents aged 18 and older, with a length of stay of 14 days or longer and who were at risk of developing a pressure ulcer, as defined by a Braden Scale for Predicting Pressure Sore Risk, Measurements: More than 500 characteristics were obtained for each resident over a 12-week period. This paper describes the NPULS database with respect to the resident (sex, age, diagnoses, severity of illness scores, Braden Scale score, activities of daily living, cognitive ability, mobility, bowel or bladder incontinence, laboratory values, nutritional assessment, and pressure ulcer assessment documentation), treatment (nutritional interventions, pressure relieving devices, incontinence interventions, protective devices, turning schedules, and pressure ulcer treatments), and outcome variables (pressure ulcer development and healing, pressure ulcer and systemic infection, changes in nutritional status, and discharge disposition) associated with pressure ulcers. Descriptive statistics and bivariate associations were used for preliminary analyses of resident, treatment, and outcome characteristics., Results: The average age +/- standard deviation was 79.7 +/- 14.2; 70% of the residents were female. Fifty-three percent of residents (n = 1,293) were at risk of developing a pressure ulcer but never developed one during the study (Group 1), 19% developed a new pressure ulcer during the study (n = 457) (Group 2), 22% had an existing pressure ulcer (n = 534) (Group 3), and 6% had an existing pressure ulcer and developed a new ulcer during the study (n = 136) (Group 4). Residents who developed a new pressure ulcer (Group 2) were more likely to be female, older, cognitively impaired, and immobile than residents who had an existing pressure ulcer (Group 3)., Conclusions: This baseline study describes the NPULS database with respect to the resident, treatment, and outcome variables associated with pressure ulcers. Future studies will focus on multivariate analyses for risk factor prediction of pressure ulcer development and pressure ulcer healing. Research-based pressure ulcer prevention and treatment protocols can then be developed.

The pathophysiologic significance of motor conduction slowing observed in diabetic distal symmetrical polyneuropathy (DSP) remains controversial. We have used multiple linear regression analysis of compound muscle action potential (CMAP) amplitude vs. motor conduction velocity (CV) and distal latency (DL) in 57 patients with diabetic DSP and 34 patients with amyotrophic lateral sclerosis (ALS) to determine whether motor conduction slowing in diabetic DSP is due mainly to loss of large axons as in ALS or whether there is an additional demyelinative component. We found amplitude-dependent slowing of CV and DL in both diabetic DSP and ALS in the upper and lower extremities, consistent with a loss of large myelinated fibers. However, in diabetic DSP, there was also significant amplitude-independent slowing in intermediate but not distal nerve segments, supportive of an additional demyelinative component. CMAP amplitude vs. CV and DL regression analyses using ALS as a control group for relatively pure axon loss may provide pathophysiologic information about motor nerves in other neuropathic disorders., (Copyright 2002 Wiley Periodicals, Inc.)

Gravitational billiards provide a simple method for the illustration of the dynamics of Hamiltonian systems. Here we examine a new billiard system with two parameters, which exhibits, in two limiting cases, the behaviors of two previously studied one-parameter systems, namely the wedge and parabolic billiard. The billiard consists of a point mass moving in two dimensions under the influence of a constant gravitational field with a hyperbolic lower boundary. An iterative mapping between successive collisions with the lower boundary is derived analytically. The behavior of the system during transformation from the wedge to the parabola is investigated for a few specific cases. It is surprising that the nature of the transformation depends strongly on the parameter values. (c) 1999 American Institute of Physics.

The yellow fever mosquito, Aedes aegypti, transmits pathogens that affect both humans and livestock, and has been the focus of extensive research to identify genetic loci that may be useful in control strategies. Fluorescence in situ hybridization (FISH) and digital imaging microscopy have provided a rapid mechanism to populate the physical map with probes derived from genetic markers, cDNAs and recombinant genomic libraries. When the physical and genetic linkage maps are aligned, map-based cloning will allow the rapid isolation of target genomic sequences. The strategy of FISH mapping and the results of initial hybridization studies are reviewed here by Martin Ferguson, Susan Brown and Dennis Knudson. An Ae. aegypti-specific genomic database, which collates data from mapping studies, sequences, references and other relevant information, is also discussed.

Few investigators have attempted to examine empirically the relationship between perception of autonomic arousal and emotional state. According to Jamesian theory, individuals who are more aware of their visceral activity should demonstrate greater affective response to emotion-laden stimuli than individuals who are not viscerally perceptive. Therefore, certain groups of 'unemotional' subjects, such as anhedonics, may report fewer or less intense emotional experiences due to an attenuation or lack of autonomic feedback. Results from studies by Katkin (Psychophysiology, 22, (1985) 125-137) suggest that there is a relationship between induced sympathetic arousal, the perception of that arousal, and the self-report of emotional experience. The present study sought to expand upon these findings by comparing visceral perception (i.e. heartbeat detection) performance and a multi-component assessment of emotion in normal and anhedonic subjects. No correlation was found between anhedonia and visceral perception; however, anhedonics were significantly different from normals across several measures of heart rate, self-report, and facial expressions in response to emotion-eliciting slides, with anhedonics consistently showing emotional hypo-responsiveness. Additionally, subjects who were able to perceive their own heartbeats were found to be more facially expressive than poor perceivers.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.

By using the computer in the ways mentioned, students learn important communication skills. The fact that they have fun in the process is an added bonus and a great motivator for changing behavior. The information given is by no means all-inclusive. Each speech-language pathologist can use his or her own ideas and creativity to develop treatment materials. The possibilities are endless.

This study evaluated the retest reliability of a number of inotropic and chronotropic indexes of cardiovascular reactivity to mental arithmetic stress over a 1-week interval. Differential habituation effects of the dependent measures were also assessed. 15 male and 15 female undergraduate Ss were administered a mental arithmetic task (serial subtractions by 13 from a 4-digit number) for 1-min on two successive weeks while their electrocardiogram and impedance cardiogram were recorded. Dependent measures included HR, PEP, HI, SV and CO. Results indicate that all dependent measures were reliable.

In Necturus gallbladders bathed in a NaCl Ringer solution buffered with 10 mM HCO3(-)-1% CO2, furosemide (added to the serosal solution) caused a concentration-dependent hyperpolarization of both cell membranes that was slow and reversible. At 10(-3) M furosemide, the basolateral membrane voltage (Vcs) increased significantly from -71 +/- 3 to -85 +/- 3 mV, the depolarization of Vcs elicited by a 10-fold rise in serosal [K+] increased from 34 +/- 4 to 50 +/- 1 mV, the depolarization elicited by lowering serosal [Cl-] from 98 to 8.1 mM was reduced from 15 +/- 1 to 1 +/- 1 mV, and the depolarization in response to lowering serosal [HCO3-] from 10 to 1 mM was reduced from 13 +/- 1 to 5 +/- 0.4 mV. Furosemide could in principle decrease the basolateral membrane Cl- conductance (Gcl), increase the basolateral membrane K+ conductance, or have a combined effect. To distinguish among these possibilities, we estimated the resistance of the basolateral membrane (Rb) by means of two-point intraepithelial cable analysis experiments. Furosemide increased Rb by 22%, which indicates that furosemide reduces basolateral membrane Gcl. The effect cannot be attributed to inhibition of apical membrane anion exchange by serosal addition of furosemide, because base secretion from cells to lumen is unchanged. We conclude that furosemide blocks reversibly basolateral membrane electrodiffusive Cl- permeability. A concomitant stimulation of basolateral membrane electrodiffusive K+ permeability is also possible.

Research on loneliness has been hindered by the lack of a simple and reliable assessment technique. The development of the UCLA Loneliness Scale, a short, 20-item general measure of loneliness is reported. The measure has high internal consistency (coefficient alpha = .96) and a test-retest correlation over a two-month period of .73. Concurrent and preliminary construct validity are indicated by correlations with self-reports of current loneliness and related emotional states, and by volunteering for a "loneliness clinic."