Strong intermolecular antiferromagnetic verdazyl-verdazyl coupling in the solid state.

Strong magnetic couplings are generally observed intramolecularly in organic diradicals or in systems in which they are promoted by crystal engineering strategies involving, for example, transition metal ligation. We herein present a strong intermolecularly coupling verdazyl radical in the solid state without the use of such design strategies. The crystal structure of an acetylene-substituted verdazyl radical shows a unique antiparallel face-to-face orientation of the neighboring verdazyl molecules along with verdazyl-acetylene interactions giving rise to an alternating antiferromagnetic Heisenberg chain. Single crystal structural data at 80, 100, 173, and 223 K show that one of the π-stacking distances depends on temperature, while heat capacity data indicate the absence of a phase transition. Based on this structural input, broken symmetry DFT calculations predict a change from an alternating linear Heisenberg chain with two comparable coupling constants J1 and J2 at higher temperatures towards dominant pair interactions at lower temperatures. The predicted antiferromagnetic coupling is confirmed experimentally by magnetic susceptibility, solid-state EPR and NMR spectroscopic results.

The ethical problem of false positives: a prospective evaluation of physician reporting in the medical record.

OBJECTIVE: To determine if the medical record might overestimate the quality of care through false, and potentially unethical, documentation by physicians. DESIGN: Prospective trial comparing two methods for measuring the quality of care for four common outpatient conditions: (1) structured reports by standardised patients (SPs) who presented unannounced to the physicians' clinics, and (2) abstraction of the medical records generated during these visits. SETTING: The general medicine clinics of two veterans affairs medical centres. PARTICIPANTS: Twenty randomly selected physicians (10 at each site) from among eligible second and third year internal medicine residents and attending physicians. MAIN MEASUREMENTS: Explicit criteria were used to score the medical records of physicians and the reports of SPs generated during 160 visits (8 cases x 20 physicians). Individual scoring items were categorised into four domains of clinical performance: history, physical examination, treatment, and diagnosis. To determine the false positive rate, physician entries were classified as false positive (documented in the record but not reported by the SP), false negative, true positive, and true negative. RESULTS: False positives were identified in the medical record for 6.4% of measured items. The false positive rate was higher for physical examination (0.330) and diagnosis (0.304) than for history (0.166) and treatment (0.082). For individual physician subjects, the false positive rate ranged from 0.098 to 0.397. CONCLUSIONS: These data indicate that the medical record falsely overestimates the quality of important dimensions of care such as the physical examination. Though it is doubtful that most subjects in our study participated in regular, intentional falsification, we cannot exclude the possibility that false positives were in some instances intentional, and therefore fraudulent, misrepresentations. Further research is needed to explore the questions raised but incompletely answered by this research.

The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development, in egg cells, and early embryogenesis.

MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.

Effect of pravastatin-to-simvastatin conversion on low-density-lipoprotein cholesterol.

The effects of a pravastatin-to-simvastatin conversion program on low-density-lipoprotein (LDL) cholesterol levels were studied. Patients receiving pravastatin at a Veterans Affairs medical center were switched to simvastatin beginning in 1997. The dosage of simvastatin was based on the additional percent reduction in LDL cholesterol needed to achieve the goal specified by the National Cholesterol Education Program. The primary endpoint was the change in the percentage of patients meeting their LDL cholesterol goal at baseline and follow-up. Changes in lipid indices, the relative risk (RR) of coronary heart disease (CHD), and program costs were also evaluated. A total of 1032 patients completed the program. The mean +/- S.D. daily doses of pravastatin and simvastatin were 25.2 +/- 11.3 and 22.7 +/- 13.3 mg, respectively. Median baseline and follow-up LDL cholesterol concentrations were 116 and 99 mg/dL, respectively (p < 0.001). Overall, 44% of the patients met their LDL cholesterol goal while taking pravastatin, compared with 69% after conversion to simvastatin (p < 0.001). The predicted mean RR of a future CHD event (based on changes in serum lipids) was 0.87 (95% confidence interval, 0.83-0.91) four years after conversion. The total cost of the program was $40,644 in the first year, and there was a net saving thereafter. Therapeutic interchange between pravastatin and simvastatin increased the number of patients meeting their LDL cholesterol goal.

Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family.

Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (somatic embryogenesis receptor-like kinase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

ZmES genes encode peptides with structural homology to defensins and are specifically expressed in the female gametophyte of maize.

All four members of a gene family, which are highly expressed in the cells of the female gametophyte (ZmES1--4: Zea mays embryo sac), were isolated from a cDNA library of maize egg cells. High expression of ZmES genes in the synergids around the micropylar region was detected in thin sections of maize ovaries. Single-cell RT--PCR analyses with the various cells of the female gametophyte confirmed the expression in synergids and also showed expression in the egg cell and central cell, and low expression in the antipodals. The expression of the whole gene family is suppressed after fertilization of the embryo sac, and expression in two-cell or later embryo stages or other tissues of maize could not be detected. In order to investigate ZmES mRNA gradients in the highly polarized and vacuolized cells of the maize embryo sac, a whole-mount in situ protocol with isolated single cells was developed: as for total RNA, ZmES transcripts are uniformly distributed in the cytoplasm of egg cell, synergids and central cell. ZmES genes encode small, cysteine-rich proteins with an N-terminal signal peptide, probably for translocation into the embryo sac cell wall. The four ZmES proteins display high sequence identity with each other, and the proposed tertiary structure of the mature peptides is similar to that of plant and animal defensins. The function of ZmES1-4 during the fertilization process is discussed.

Tripsacum dactyloides (Poaceae): a natural model system to study parthenogenesis.

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.35× larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (BIII hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin:: ß -glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.

Measuring compliance with preventive care guidelines: standardized patients, clinical vignettes, and the medical record.

OBJECTIVE: To determine how accurately preventive care reported in the medical record reflects actual physician practice or competence. DESIGN: Scoring criteria based on national guidelines were developed for 7 separate items of preventive care. The preventive care provided by randomly selected physicians was measured prospectively for each of the 7 items. Three measurement methods were used for comparison: (1) the abstracted medical record from a standardized patient (SP) visit; (2) explicit reports of physician practice during those visits from the SPs, who were actors trained to present undetected as patients; and (3) physician responses to written case scenarios (vignettes) identical to the SP presentations. SETTING: The general medicine primary care clinics of two university-afflliated VA medical centers. PARTICIPANTS: Twenty randomly selected physicians (10 at each site) from among eligible second- and third-year general internal medicine residents and attending physicians. MEASUREMENTS AND MAIN RESULTS: Physicians saw 160 SPs (8 cases x 20 physicians). We calculated the percentage of visits in which each prevention item was recorded in the chart, determined the marginal percentage improvement of SP checklists and vignettes over chart abstraction alone, and compared the three methods using an analysis-of-variance model. We found that chart abstraction underestimated overall prevention compliance by 16% (P < .01) compared with SP checklists. Chart abstraction scores were lower than SP checklists for all seven items and lower than vignettes for four items. The marginal percentage improvement of SP checklists and vignettes to performance as measured by chart abstraction was significant for all seven prevention items and raised the overall prevention scores from 46% to 72% (P < .0001). CONCLUSIONS: These data indicate that physicians perform more preventive care than they report in the medical record. Thus, benchmarks of preventive care by individual physicians and institutions that rely solely on the medical record may be misleading, at best.

How well does chart abstraction measure quality? A prospective comparison of standardized patients with the medical record.

PURPOSE: Despite widespread reliance on chart abstraction for quality measurement, concerns persist about its reliability and validity. We prospectively evaluated the validity of chart abstraction by directly comparing it with the gold standard of reports by standardized patients. SUBJECTS AND METHODS: Twenty randomly selected general internal medicine residents and attending faculty physicians at the primary care clinics of two Veterans Affairs Medical Centers blindly evaluated and treated actor-patients (standardized patients) who had one of four common diseases: diabetes, chronic obstructive pulmonary disease, coronary artery disease, or low back pain. Charts from the visits were abstracted using explicit quality criteria; standardized patients completed a checklist containing the same criteria. For each physician, quality was measured for two different cases of the four conditions (a total of 160 physician-patient encounters). We compared chart abstraction with standardized-patient reports for four aspects of the encounter: taking the history, examining the patient, making the diagnosis, and prescribing appropriate treatment. The sensitivity and specificity of chart abstraction were calculated. RESULTS: The mean (+/- SD) chart abstraction score was 54% +/- 9%, substantially less than the mean score on the standardized-patient checklist of 68% +/- 9% (P <0.001). This finding was similar for all four conditions and at both sites. "False positives"-chart-recorded necessary care actions not reported by the standardized patients-resulted in a specificity of only 81%. The overall sensitivity of chart abstraction for necessary care was only 70%. CONCLUSIONS: Chart abstraction underestimates the quality of care for common outpatient general medical conditions when compared with standardized-patient reports. The medical record is neither sensitive nor specific. Quality measurements derived from chart abstraction may have important shortcomings, particularly as the basis for drawing policy conclusions or making management decisions.

Comparison of vignettes, standardized patients, and chart abstraction: a prospective validation study of 3 methods for measuring quality.

CONTEXT: Better health care quality is a universal goal, yet measuring quality has proven to be difficult and problematic. A central problem has been isolating physician practices from other effects of the health care system. OBJECTIVE: To validate clinical vignettes as a method for measuring the competence of physicians and the quality of their actual practice. DESIGN: Prospective trial conducted in 1997 comparing 3 methods for measuring the quality of care for 4 common outpatient conditions: (1) structured reports by standardized patients (SPs), trained actors who presented unannounced to physicians' clinics (the gold standard); (2) abstraction of medical records for those same visits; and (3) physicians' responses to clinical vignettes that exactly corresponded to the SPs' presentations. Setting Outpatient primary care clinics at 2 Veterans Affairs medical centers. PARTICIPANTS: Ninety-eight (97%) of 101 general internal medicine staff physicians, faculty, and second- and third-year residents consented to be randomized for the study. From this group, 10 physicians at each site were randomly selected for inclusion. MAIN OUTCOME MEASURES: A total of 160 quality scores (8 cases x 20 physicians) were generated for each method using identical explicit criteria based on national guidelines and local expert panels. Scores were defined as the percentage of process criteria correctly met and were compared among the 3 methods. RESULTS: The quality of care, as measured by all 3 methods, ranged from 76.2% (SPs) to 71.0% (vignettes) to 65.6% (chart abstraction). Measuring quality using vignettes consistently produced scores closer to the gold standard of SP scores than using chart abstraction. This pattern was robust when the scores were disaggregated by the 4 conditions (P<.001 to <.05), by case complexity (P<.001), by site (P<.001), and by level of physician training (P values from <.001 to <.05). The pattern persisted, although less dominantly, when we assessed the component domains of the clinical encounter--history, physical examination, diagnosis, and treatment. Vignettes were responsive to expected directions of variation in quality between sites and levels of training. The vignette responses did not appear to be sensitive to physicians' having seen an SP presenting with the same case. CONCLUSIONS: Our data indicate that quality of health care can be measured in an outpatient setting by using clinical vignettes. Vignettes appear to be a valid and comprehensive method that directly focuses on the process of care provided in actual clinical practice. Vignettes show promise as an inexpensive case-mix adjusted method for measuring the quality of care provided by a group of physicians.

A transcript encoding translation initiation factor eIF-5A is stored in unfertilized egg cells of maize.

Differential screening of cDNA libraries of unfertilized egg cells and in vitro zygotes of maize resulted in the isolation of more than 50 different genes whose expression is up- or down-regulated after in vitro fertilization (IVF). Among these genes, we identified a cDNA encoding the eukaryotic translation initiation factor eIF-5A. This highly conserved factor is thought to be necessary for selective mRNA stabilization and translation. It is also the only known protein that contains the unusual amino acid hypusine which is required for biological activity. High transcript amounts are stored in the egg cell, which is, in terms of metabolism, relatively inactive. Upon fertilization transcript amounts decrease, in contrast to metabolically inactive embryos in which the transcript cannot be detected and transcript levels increase upon germination. The expression pattern during the first embryonic cell cycle is also different from that observed during the somatic cell cycle: egg cells in the G0 phase contain high transcript levels, while arrested suspension cells contain few transcripts. In the somatic cell cycle, eif-5A is strongly induced during the G1 phase and transcripts are continuously degraded during the S, G2 and M phases until new induction during the G1 phase of the next cycle. eif-5A, a member of a small gene family in maize, is expressed in most maize tissues investigated. Based on our results, we suggest that the unfertilized egg cell of maize, although relatively inactive regarding its metabolism, is prepared for selective mRNA translation that is quickly triggered after fertilization. We also suggest that the regulation of eif-5A in the first embryonic cell cycle is different from the somatic cell cycle.

Novel ribosomal genes from maize are differentially expressed in the zygotic and somatic cell cycles.

We have isolated cDNAs representing more than 50 different genes from libraries of unfertilised egg cells and zygotes of maize, expression of which is up- or downregulated after in vitro fertilisation (IVF). Among the cDNAs isolated are seven which encode proteins that are probably involved in translation and two encoding proteins probably involved in DNA replication. The latter genes are strongly induced on fertilisation. This indicates that zygotic gene activation (ZGA) - the switch from maternal to embryonic control of development - occurs in the zygote shortly after fertilisation in higher plants - earlier than in animal systems so far investigated. Four novel transcripts for ribosomal proteins (S21A, S21B, L39, P0) involved in ribosome biosynthesis and translation were analysed in more detail. The expression of all four genes correlates with cell division activity and is strongly induced during the G1 phase of the somatic cell cycle. A different mode of regulation operates in the first embryonic cell cycle: relatively large amounts of transcript are stored in the unfertilised egg cells, and by 18 h after IVF, two ribosomal genes are induced while a third is downregulated. These results indicate that using the combination of single-cell culture techniques with novel molecular methods, it is possible to isolate and study numerous genes expressed in female gametes and zygotes of higher plants. The detailed analysis of the four ribosomal protein genes demonstrates that the zygotic and somatic cell cycles are differentially regulated.

Analyzing the time and value of housestaff inpatient work.

OBJECTIVE: To determine time allocation and the perceived value to education and patient care of the weekday activities of internal medicine housestaff on inpatient rotations and to compare the work activities of interns and residents. DESIGN: An observational study. We classified activities along five dimensions (association, location, activity, time, and value), developed a computer-assisted self-interview survey, and demonstrated its face and content validity, internal consistency, and interrater reliability. Subjects were assigned survey computers for 5 consecutive weekdays over a 24-week period, into which they entered data when prompted several times a day. SETTING: The medical service of a university-affiliated Veterans Administration Medical Center. PARTICIPANTS: Sixty housestaff (36 interns, 24 residents) rotating on the inpatient wards. MEASUREMENTS AND MAIN RESULTS: We analyzed activities according to content (direct patient care, indirect patient care, education), association, and location. Likert-scale ratings of perceived value to education and patient care were also obtained. Housestaff provided complete responses to 3,812 (95%) of 3,992 prompts by a median of 11 seconds; 93% of responses were logically consistent across the measured dimensions. Housestaff spent more time in indirect patient care (56%) than in direct patient care (14%) or educational activities (45%). Formal educational activities had the highest educational value (66 on 0-100 scale), and direct care had the highest value to patient care (81). Over 30% of time was spent in administrative activities, which had low educational value(40). Compared with residents, interns allocated significantly less time to educational activities (38% vs 57%) and more time to lower-value activities such as documentation (19% vs 12%). CONCLUSIONS: Improved data collection methods demonstrate that housestaff in our program, particularly interns, spend much of their workday in activities that are low in educational and patient care value. Selective elimination or delegation of such activities would preserve higher-value experiences during reductions in overall inpatient training time. Planners can use automated random sampling to guide the rational redesign of housestaff work.

Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize.

A full-size cDNA clone (1614 bp) encoding calreticulin was isolated from a PCR-based cDNA library of maize in vitro zygotes. Calreticulin is a major Ca2+ storage protein located mainly in the lumen of the endoplasmic reticulum but also in the nucleus and/or cytoplasm of some cells. A differential screening between cDNA libraries originating from 104 in vitro zygotes (18 h after in vitro fertilization) and 128 unfertilized egg cells was performed to isolate newly expressed genes or genes expressed more abundantly after fertilization. The expression of the isolated cDNA clone is enhanced after fertilization and strongly correlated to cell division. Sequence comparison to a shorter maize calreticulin cDNA isolated from a PCR based cDNA library construction from a few plant cells [12]. It is further shown that calreticulins in maize are probably transcribed from a small gene family differentially expressed in abundance in diverse tissues. The deduced amino acid sequence encodes an acidic protein (pI 4.17) of 48 kDa sharing 77-92% and 50-54% homology to other plant and animal calreticulins, respectively. The described calreticulin gene represents to our knowledge the first cDNA clone isolated from a RT/PCR cDNA library originating from only a few plant cells and is the first gene isolated from zygotes of higher plants.

Isolation and characterization of a Tritordeum cDNA encoding S-adenosylmethionine decarboxylase that is circadian-clock-regulated.

Sequence analysis of the two cDNA clones 47/11 and 50A which were isolated by differential screening of an explant cDNA library obtained from the monocot Tritordeum (hexaploid hybrid of diploid wild barley and tetraploid wheat lines) reveals that both clones include the same open reading frame (ORF). The sequence of this ORF shows a high degree of similarity with dicot S-adenosylmethionine decarboxylase (SAMDC) gene sequences and contains regions highly conserved in all known SAMDC sequences. It is further shown that the sequence represented by the cDNA clones 47/11 and 50A is derived from the wild barley (Hordeum chilense) genome, where it is present as a single-copy gene. Northern analyses indicate the corresponding transcript to accumulate in response to wounding and the transcript level changes with a circadian rhythm, having a beak in the middle of the light period. The periodicity continues in constant light, but is changed in constant darkness.

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase.

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3'-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5' end of the cDNA was amplified by a 5'-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.

Representative cDNA libraries from few plant cells.

A reverse transcriptase/polymerase chain reaction (RT/PCR) method for the construction of representative cDNA libraries originating from few isolated cells is described. Poly(A)+ RNA was extracted from an average of 100 maize cells and reverse transcribed into sscDNA. The sscDNA was dG-tailed at its 3' end and amplified during a two-step PCR reaction. The generated PCR products were analysed and the majority < or = 2 kbp were full-size cDNAs. A fraction of the amplified cDNA from 128 isolated maize egg cells was cloned into the lambda Uni-ZAP XR vector and a primary library of 6.8 x 10(6) p.f.u. was obtained. The average insert size is 860 bp. It was further determined, that 0.31% of the clones hybridized to a cytosolic GAPDH probe. It is thought that, with this method, the first cDNA library of egg cells in higher plants was generated.

Bronchial asymmetry and Fibonacci scaling.

The irregular branching pattern of the bronchial tree in multiple mammalian species is consistent with a process of morphogenetic self-similarity described by Fibonacci scaling.