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Purpose: Automated pupillometry (AP) facilitates objective pupillary assessment. In this study, we aimed at assessing nursing perspective about the utility of AP in neurocritically ill children to understand acceptance and usage barriers to guide development of a standardized use protocol., Methods: We conducted a web-based, cross-sectional, anonymous, Google™ survey of nurses at two independent pediatric ICUs which have been using AP over last four years. The survey included questions related to user-friendliness, barriers, acceptance, frequency of use, and method of documenting AP findings., Results: A total of 31 nurses responded to the survey. A total of 25 nurses (80.6%) used the automated pupillometer and 19 (61.3%) nurses preferred to use the automated pupillometer on critically ill intubated patients. Respondents rated the pupillometer a median [IQR] frequency of use of 7/10 [4-9] and a mean user-friendliness of 8/10 [7-10]. Barriers to pupillometer use included pupillometer unavailability, technical issues, lack of perceived clinical significance, and infection control., Conclusion: Nurses have widely adopted the use of automated pupillometer in the PICU especially for critically ill intubated patients and rate it favorably for user-friendliness. Barriers against its use include limited resources, infection concerns, technical issues, and a lack of perceived clinical significance and training. Implementation of standardized PICU protocol for AP usage in critically ill children, may enhance the acceptance, increase usage and aid in objective assessments., Practice Implications: These findings can be used to create a standardized protocol on implementing automated pupillometry in the PICU for critically ill children., Competing Interests: Declaration of competing interest None of the authors have any conflicts of interest to disclose., (Published by Elsevier Inc.)

Aim/objective Neurological Pupil Index (NPi), measured by automated pupillometry (AP), allows the objective assessment of pupillary light reflex (PLR). NPi ranges from 0 (non-reactive) to 5 (normal). In this study, we aimed to compare neurologic and functional outcomes in children admitted for neurologic injury with normal (≥3) versus abnormal (<3) NPi measured during their pediatric intensive care unit (PICU) stay. Materials and methods We conducted a retrospective chart review of children between one month and 18 years admitted to our PICU with a diagnosis of neurologic injury between January 2019 and June 2022. We collected demographic, clinical, pupillometer, and outcome data, including mortality, Pediatric Cerebral Performance Category (PCPC), Pediatric Overall Performance Category (POPC), and Functional Status Score (FSS) at admission, at discharge, and at the three to six-month follow-up. We defined abnormal pupil response as any NPi <3 at any point during the PICU stay. Using the student's t-test and chi-square test, we compared the short-term and long-term outcomes of children with abnormal NPi (<3) versus those with normal NPi (≥3). Results There were 49 children who met the inclusion criteria and who had pupillometry data available for analysis. The mean (SD) Glasgow Coma Scale (GCS) in the study cohort was 5.6 (4.3), and 61% had low (<3) NPi during ICU stay. Mortality was significantly higher among patients with an abnormal NPi as compared to those with normal NPi. Children with abnormal NPi exhibited significant worsening of neurologic and functional status (ΔPCPC, ΔPOPC, and ΔFSS) from admission to discharge (mean (SD): 3.55(1.5), 3.45(1.43), 16.75(7.85), p<0.001) as compared to those with normal NPi (mean (SD): 1.45(0.93), 1.73(0.90), 3.55(2.07), p>0.05). The significant difference in neurologic and functional status persisted at the three to six-month follow-up between the two groups - children with abnormal NPi (mean (SD): 2.0(1.41), 2.08(1.38), 6.92(6.83), p<0.01) and children with normal NPi (mean (SD): 0.82(1.01), 0.94(1.03), 1.53(1.70), p>0.05). Conclusion In our retrospective cohort study, children admitted to the PICU for a neuro injury and with abnormal NPi (< 3) have higher mortality, and worse short-term and long-term neurologic and functional outcomes as compared to those with normal NPi (≥ 3) measured during the PICU course. AP provides an objective assessment of PLR and has potential applications for neuro-prognostication. More research needs to be done to elucidate the prognostic value of NPi in pediatrics., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Jiang et al.)

The occurrence of antibiotic-resistant bacteria (ARB) in wastewater treatment plants (WWTPs) has become an occupational and environmental concern. WWTPs are engineered systems that treat wastewater to meet public health standards before release into the environment. The residuals, as either effluent or solids, are then discharged or beneficially recycled into the environment. Since these wastes contain a diverse array of microorganisms, some of which are resistant to commonly used antibiotics, there is a potential for these organisms to spread in the environment via residual recycling and effluent discharge. Human infections with ARB are increasing, and it is not well known how the interaction between humans and the environment plays a role in this process. WWTP workers, who are on the front lines, may come into direct contact with materials containing these microbes. This study aimed to determine the number of ARB present in both air and sewage sludges in a WWTP using nonselective media supplemented with two antibiotics (ciprofloxacin and azithromycin). The densities of total heterotrophic bacteria, ciprofloxacin-resistant bacteria, and azithromycin-resistant bacteria were 7.82 × 10 5 - 4.7 × 10 9 , 7.87 × 10 3 - 1.05 × 10 8 , and 2.27 × 10 5 - 1.16 × 10 9 CFU/g, respectively. The prevalence [(concentration on medium with antibiotics/concentration on medium without antibiotics) × 100] of ciprofloxacin-resistant bacteria in treated sludge was twice as low as in digested sludge and approximately three times lower than in raw sludge. For azithromycin, the prevalence of resistant bacteria in treated sludge was about the same in digested and nearly twice lower than in raw sludge. Despite a marked reduction in the mean prevalence of resistant bacteria in dewatered treated sludge for both antibiotics, these differences were not significant. The highest prevalence of antibiotic resistance was observed for azithromycin. Similarly, the prevalence of airborne azithromycin-resistant bacteria inside the belt filter press room (BFPR) was nearly seven times higher than the prevalence of airborne ciprofloxacin-resistant bacteria. These concentrations of ARB were not negligible and may represent an exposure pathway for some workers in WWTPs.

Background and Objectives: Antibiotic overuse is associated with adverse neonatal outcomes. Many medically underserved centers lack pediatric antibiotic stewardship program (ASP) support. Telestewardship may mitigate this disparity. Authors of this study aimed to determine the effectiveness and safety of a nursery-specific ASP delivered remotely., Methods: Remote ASP was implemented in 8 medically underserved newborn nurseries using a stepped-wedge, cluster-randomized design over 3 years. This included a 15-month baseline period, a 9-month "step-in" period using random nursery order, and a 12-month postintervention period. The program consisted of education, audit, and feedback; and 24/7 infectious diseases provider-to-provider phone consultation availability. Outcomes included each center's volume of antibiotic use and the proportion of infants exposed to any antibiotics. Safety measures included length of stay, transfer to another facility, sepsis, and mortality., Results: During the study period, there were 9277 infants born (4586 preintervention, 4691 postintervention). Infants exposed to antibiotics declined from 6.2% pre-ASP to 4.2% post-ASP (relative risk 0.68 [95% confidence interval, 0.63% to 0.75%]). Total antibiotic use declined from 117 to 84.1 days of therapy per 1000 patient-days (-28% [95% confidence interval -22% to -34%]. No safety signals were observed. Most provider-to-provider consultations were <5 minutes in duration and occurred during normal business hours., Conclusions: The number of infants exposed to antibiotics and total antibiotic use declined in medically underserved nurseries after implementing a remote ASP. No adverse safety events were seen, and the remote ASP time demands were manageable. Remote stewardship may be a safe and effective strategy for optimizing antibiotic use in medically underserved newborn nurseries., (Copyright © 2022 by the American Academy of Pediatrics.)

Background: Surgical repair of primary mitral regurgitation (MR) is considered an indicator of quality performance. Therefore, accurate data reporting is critical for quality assessment. During an institutional quality review, MR etiology could not be determined in 40% of operations in The Society of Thoracic Surgeons database entries, and therefore our true repair rate could not be reliably ascertained. Therefore, we reviewed all source documents and echocardiograms to assess our true disease etiology and repair rate., Methods: Source records and echocardiograms of all operations performed in a single health care system for a 1-year period were reviewed by an experienced mitral valve surgeon, an echocardiographic core laboratory, and a data manager. Disease etiology and operation were compared with data previously entered in the database by post hoc chart abstraction., Results: In all, 314 isolated mitral valve operations were performed. The MR was originally classified as primary, 163 (52%); secondary, 22 (7%); rheumatic, 37 (12%); endocarditis, 24 (8%); other, 33 (10%); and unknown, 35 (11%). Reported repair rate for primary MR was 142 of 163 (87.1%). After review, etiology was determined to be primary, 177 (56%); secondary, 33 (11%); rheumatic, 61 (20%); endocarditis, 25 (8%); and others, 18 (5%)-resulting in a change of classification in 99 of 314 patients (31.5%) and a true repair rate for primary MR of 165 of 177 (93.2%)., Conclusions: Source document and imaging review of mitral valve surgery revealed significant discordance with post hoc chart abstraction methods. A more detailed data entry methodology is necessary to accurately report the true disease etiology and repair rates for primary MR., (Copyright © 2020 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Functional cilia and flagella are crucial to the propulsion of physiological fluids, motile cells, and microorganisms. Motility assessment of individual cells allows discrimination of normal from dysfunctional behavior, but cell-scale analysis of individual trajectories to represent a population is laborious and impractical for clinical, industrial, and even research applications. We introduce an assay that quantifies swimming capability as a function of the variation in polar moment of inertia of cells released from an acoustic trap. Acoustic confinement eliminates the need to trace discrete trajectories and enables automated analysis of hundreds of cells in minutes. The approach closely approximates the average speed estimated from the mean squared displacement of individual cells for wild-type Chlamydomonas reinhardtii and two mutants (ida3 and oda5) that display aberrant swimming behaviors. Large-population acoustic trap-and-release rapidly differentiates these cell types based on intrinsic motility, which provides a highly sensitive and efficient alternative to conventional particle tracing.

To test the hypothesis that drugs of abuse and their conditioned stimuli (CSs) enhance memory consolidation, the effects of post-training exposure to cocaine and nicotine were compared to the effects of post-training exposure to contextual stimuli that were paired with the effects of these drugs. Using the object recognition (OR) task, it was first demonstrated that both 10 and 20 mg/kg cocaine, and 0.2 and 0.4 mg/kg nicotine, enhanced recognition memory when administered immediately after, but not 6 h after the sample phase. To establish the drug CSs, rats were confined for 2 h in a chamber (the CS+) after injections of 20 mg/kg cocaine, or 0.4 mg/kg nicotine, and in another chamber (the CS-) after injections of vehicle. This was repeated over 10 d (5 drug/CS+ and 5 vehicle/CS- pairings in total). At the end of this conditioning period, when tested in a drug-free state, rats displayed conditioned hyperactivity in the CS+ relative to the CS-. More important, immediate, but not delayed, post-sample exposure to the cocaine CS+, or nicotine CS+, enhanced OR memory. Therefore, this study reports for the first time that contextual stimuli paired with cocaine and nicotine, like the drugs themselves, have the ability to enhance memory consolidation., (© 2019 Wolter et al.; Published by Cold Spring Harbor Laboratory Press.)

The Centers for Medicare and Medicaid Services National Coverage Determination requires centers performing transcatheter aortic valve implantation (TAVI) to report clinical outcomes up to 1 year. Many sites encounter challenges in obtaining complete 1-year follow-up. We report our process to address this challenge. A multidisciplinary process involving clinical personnel, data and quality managers, and research coordinators was initiated to collect TAVI data at baseline, 30 days, and 1 year. This process included (1) planned clinical follow-up of all patients at 30 days and 1 year; (2) query of health-care system-wide integrated data warehouse (IDW) to ascertain last date of clinical contact within the system for all patients; (3) online obituary search, cross-referencing for unique patient identifiers to determine if mortality occurred in remaining unknown patients; and (4) phone calls to remaining unknown patients or patients' families. Between January 2012 and December 2016, 744 patients underwent TAVI. All 744 patients were eligible for 30-day follow-up and 546 were eligible for 1-year follow-up. At routine clinical follow-up of 22 of 744 (3%) patients at 30 days and 180 of 546 (33%) patients at 1 year had unknown survival status. The integrated data warehouse query confirmed status-alive for an additional 1 of 22 patients at 30 days (55%) and 91 of 180 patients at 1 year (51%). Obituaries were identified for 23 of 180 additional patients at 1 year (13%). Phone contact identified the remaining unknown patients at 30 days and 1 year, resulting in 100% known survival status for patients at 30 days (744 of 744) and at 1 year (546 of 546). In conclusion, using a comprehensive approach, we were able to determine survival status in 100% of patients who underwent TAVI., (Copyright © 2017 Elsevier Inc. All rights reserved.)

New microtubule depolymerizing agents with potent cytotoxic activities have been prepared with a 5-cyano or 5-oximino group attached to a pyrrole core. The utilization of ortho activation of a bromopyrrole ester to facilitate successful Suzuki-Miyaura cross-coupling reactions was a key aspect of the synthetic methodology. This strategy allows for control of regiochemistry with the attachment of four completely different groups at the 2, 3, 4 and 5 positions of the pyrrole scaffold. Biological evaluations and molecular modeling studies are reported for these examples., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cell culture based assays used to detect waterborne viruses typically call for incubating the sample for at least two weeks in order to ensure that all the culturable virus present is detected. Historically, this estimate was based, at least in part, on the length of time used for detecting poliovirus. In this study, we have examined A549 cells infected with human adenovirus type 2, and have found that a three week incubation of virus infected cells results in a higher number of detected viruses by quantal assay than what is seen after two weeks of incubation, with an average 955% increase in Most Probable Number (MPN) from 2 weeks to 3 weeks. This increase suggests that the extended incubation time is essential for accurately estimating viral titer, particularly for slow-growing viruses, UV treated samples, or samples with low titers of virus. In addition, we found that for some UV-treated samples, there was no detectable MPN at 2 weeks, but after 3 weeks, MPN values were obtained. For UV-treated samples, the average increase in MPN from 2 weeks to 3 weeks was 1401%, while untreated samples averaged a change in MPN of 674%, leading us to believe that the UV-damaged viral DNA may be able to be repaired such that viral replication then occurs., (Published by Elsevier B.V.)

Background: Many lower limb amputees experience thermal discomfort as a result of wearing a prosthesis. The development of new prosthetic technology to address thermal discomfort requires an understanding of how activity (or inactivity) affects residual limb skin temperatures and how skin temperatures are mapped across the skin-prosthesis interface., Questions/purposes: We studied skin temperatures inside the socket and suspension system of unilateral transtibial amputees to determine the following: (1) Does residual limb skin temperature change as a function of activity and its cessation? (2) If changes occur, are there regional differences (circumferential or proximal-distal) in temperature?, Methods: Nine unilateral transtibial amputees provided informed consent to participate in this institutional review board-approved study. Residual limb skin temperatures inside their prosthesis were measured at 16 distributed sites using thermistor sensors and a portable data acquisition system. The 150-minute protocol began with a 60-minute seated rest, continued with a 30-minute treadmill walk at a self-selected speed, and concluded after a second 60-minute seated rest. Data from the last minute of each of the three periods were used for analysis., Results: The skin temperature was 31.0° ± 1.5° C (mean ± SD) at the end of the initial rest period. After 30 minutes of treadmill walking, skin temperature increased to 34.1° ± 1.3° C, an increase of 3.1° C (95% confidence interval [CI], 2.4-3.8; p < 0.001). After the final 60 minutes of rest, the skin temperature was 33.2° ± 1.2° C, 0.9° C lower (95% CI, 0.5-1.2; p < 0.001) than at the end of treadmill walking but 2.2° C higher (95% CI, 1.4-2.9; p < 0.001) than the temperature observed at the end of the initial rest period. Skin temperatures were warmest over the tibialis anterior region (p < 0.006) and decreased from the most proximal to the most distal locations on the residual limb (p = 0.001)., Conclusions: Walking causes a dramatic increase in skin temperatures inside the prosthesis and subsequent rest of twice the walking duration fails to return temperatures to their initial condition. Rest alone is likely to be insufficient to provide thermal relief without doffing the prosthesis. New prosthetic technology is needed to address this problem. Skin temperatures also varied by residual limb location, suggesting that the development of location-specific technology would be advantageous.

Background: Patients started on long term hemodialysis have typically had low rates of reported renal recovery with recent estimates ranging from 0.9-2.4% while higher rates of recovery have been reported in cohorts with higher percentages of patients with acute renal failure requiring dialysis., Study Design: Our analysis followed approximately 194,000 patients who were initiated on hemodialysis during a 2-year period (2008 & 2009) with CMS-2728 forms submitted to CMS by dialysis facilities, cross-referenced with patient record updates through the end of 2010, and tracked through December 2010 in the CMS SIMS registry., Results: We report a sustained renal recovery (i.e no return to ESRD during the available follow up period) rate among Medicare ESRD patients of > 5% - much higher than previously reported. Recovery occurred primarily in the first 2 months post incident dialysis, and was more likely in cases with renal failure secondary to etiologies associated with acute kidney injury. Patients experiencing sustained recovery were markedly less likely than true long-term ESRD patients to have permanent vascular accesses in place at incident hemodialysis, while non-White patients, and patients with any prior nephrology care appeared to have significantly lower rates of renal recovery. We also found widespread geographic variation in the rates of renal recovery across the United States., Conclusions: Renal recovery rates in the US Medicare ESRD program are higher than previously reported and appear to have significant geographic variation. Patients with diagnoses associated with acute kidney injury who are initiated on long-term hemodialysis have significantly higher rates of renal recovery than the general ESRD population and lower rates of permanent access placement.

To facilitate isolation and characterization of disease and insect resistance genes important to potato, two bacterial artificial chromosome (BAC) libraries were constructed from genomic DNA of the Mexican wild diploid species, Solanum pinnatisectum, which carries high levels of resistance to the most important potato pathogen and pest, the late blight and the Colorado potato beetle (CPB). One of the libraries was constructed from the DNA, partially digested with BamHI, and it consists of 40328 clones with an average insert size of 125 kb. The other library was constructed from the DNA partially digested with EcoRI, and it consists of 17280 clones with an average insert size of 135 kb. The two libraries, together, represent approximately six equivalents of the wild potato haploid genome. Both libraries were evaluated for contamination with organellar DNA sequences and were shown to have a very low percentage (0.65-0.91%) of clones derived from the chloroplast genome. High-density filters, prepared from the two libraries, were screened with ten restriction fragment length polymorphism (RFLP) markers linked to the resistance genes for late blight, CPB, Verticillium wilt and potato cyst nematodes, and the gene Sr1 for the self-incompatibility S-locus. Thirty nine positive clones were identified and at least two positive BAC clones were detected for each RFLP marker. Four markers that are linked to the late blight resistance gene Rpi1 hybridized to 14 BAC clones. Fifteen BAC clones were shown to harbor the PPO (polyphenol oxidase) locus for the CPB resistance by three RFLP probes. Two RFLP markers detected five BAC clones that were linked to the Sr1 gene for self-incompatibility. These results agree with the library's predicted extent of coverage of the potato genome, and indicated that the libraries are useful resources for the molecular isolation of disease and insect resistance genes, as well as other economically important genes in the wild potato species. The development of the two potato BAC libraries provides a starting point, and landmarks for BAC contig construction and chromosome walking towards the map-based cloning of agronomically important target genes in the species.

25 undergraduate listeners judged that a male speaker with normal speech, who dentalized (lisped) or devoiced the /z/ phoneme in the context of a sentence, sounded significantly more "gay" in terms of judged sexual orientation than did the same speaker producing /z/ without phonological processes. Speech-language pathologists should be aware of possible social consequences of speech production disorders.

A Bacterial Artificial Chromosome (BAC) genomic DNA library of Anopheles gambiae, the major human malaria vector in sub-Saharan Africa, was constructed and characterized. This library (ND-TAM) is composed of 30,720 BAC clones in eighty 384-well plates. The estimated average insert size of the library is 133 kb, with an overall genome coverage of approximately 14-fold. The ends of approximately two-thirds of the clones in the library were sequenced, yielding 32,340 pair-mate ends. A statistical analysis (G-test) of the results of PCR screening of the library indicated a random distribution of BACs in the genome, although one gap encompassing the white locus on the X-chromosome was identified. Furthermore, combined with another previously constructed BAC library (ND-1), ~2,000 BACs have been physically mapped by polytene chromosomal in situ hybridization. These BAC end pair mates and physically mapped BACs have been useful for both the assembly of a fully sequenced A. gambiae genome and for linking the assembled sequence to the three polytene chromosomes. This ND-TAM library is now publicly available at both http://www.malaria.mr4.org/mr4pages/index.html/ and http://hbz.tamu.edu/, providing a valuable resource to the mosquito research community.

The relation of reading rate and rapid automatic naming (RAN) for pictures was investigated for 31 third graders. Reading rate was measured as the number of seconds required for reading aloud a third-grade passage. Rapid automatic naming was assessed with the RAN portion of the Woodcock Johnson III Tests of Cognitive Abilities. A moderate correlation of -.61 between the sets of scores suggests RAN assessment may be useful in screening for reading deficits in third-grade children.

Heterozygous familial hypercholesterolemia (FH) is a relatively common autosomal dominant disorder, which is characterized by elevated plasma concentrations of low density lipoprotein (LDL) cholesterol and early coronary heart disease. FH results from mutations in the gene encoding the LDL receptor (LDLR). In Canada, there is a founder effect for LDLR mutations in FH among individuals of French descent, most of whom reside in the province of Quebec. However, the spectrum of mutations in other regions, specifically in the populous and predominantly English-speaking province of Ontario, has not been studied. We sequenced the coding regions, promoter and intron-exon boundaries of the LDLR gene in 60 Ontario FH subjects from a variety of ethnic backgrounds other than French Canadian. We found 25 LDLR mutations in 34 subjects. Eleven LDLR mutations were novel, including two in-frame deletions of a single amino acid (one each in exons 2 and 4), two larger deletions that shifted the reading frame (one each in exons 4 and 10), five missense mutations (C42R, A370T, T413M, L561P and E760D) and two splice acceptor mutations (one each in introns 3 and 8). The results indicate that FH is more genetically diverse in Ontario than in Quebec. The results are also consistent with findings from investigations of the LDLR in FH conducted in other countries, in which PCR-based, exon-by-exon sequencing uncovers small mutations in about half of the subjects screened. The gap in molecular diagnosis suggests that lesions not found by this sequencing strategy, such as larger scale LDLR mutations that cannot be amplified, may underlie a substantial number of cases of FH. Alternatively, there might be genetic heterogeneity underlying the FH phenotype, with contributions from other single or multiple genes. Hum Mutat 18:359, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Access to outpatient services within the first 30 days after an inpatient mental health episode may influence relapse risk. A retrospective cohort of 3,755 adult Medicaid mental health inpatients discharged from their first managed care acute episode of care from July 1, 1996, through May 20, 1998, were studied. Results showed patients' utilization of any psychotherapy (OR = .43), medication management (OR = .41), or diagnostic evaluation services (OR = .61), relative to no utilization, was associated with significantly lower 30-day readmission rates, and longer times in remission. However, patients receiving above the median total number of ambulatory services, or having contact with more providers showed significantly greater likelihood of 30-day readmission, and shorter time in remission. Findings heighten the need for the availability of timely risk-reducing mental health outpatient services, the continuity and risk of fragmentation of therapeutic relationships, as well as crisis planning before an inpatient discharge.

The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7-2.5 million deaths each year. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).

The dynamics of individual DNA molecules in a thin gel were studied with fluorescence microscopy. Driven by an electric field, molecules hooked around isolated obstacles and became extended. By analyzing molecular images, we identified the reptation tube and primitive chain. When the field was turned off, the molecules relaxed. The relaxation time tau1 and primitive chain length at equilibrium depend on N, the size of the molecule in base pairs, consistently with reptation theory. Using five yeast chromosomal DNAs ranging in size from 245 kb to 980 kb, we found that: These results constitute a way of sizing individual DNA molecules by imaging rather than by gel electrophoresis., (Copyright 1999 Academic Press.)

New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis. The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I. We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (i) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high-resolution restriction maps from images of individual DNA molecules. The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.

To test whether conventional data reliability assessment overestimates reliability, an assessment and a comparison of the reliability of complex quality indicators and their simpler components were conducted. Medical records of 1078 Medicare cases with principal diagnoses of initial episodes of acute myocardial infarction (AMI) were independently reabstracted at two national Clinical Data Abstraction Centers (CDACs). The inter-rater agreement beyond chance (kappa) of reabstracted and original quality indicators and key components were computed and compared. Results showed excellent agreement (kappas ranging from 0.88 to 0.95) for simple determinations of whether standard medical therapies were provided. Repeatability of eligibility status and the more complex determinations of whether "ideal" candidates were not treated showed moderate to excellent kappa values ranging from 0.41 to 0.79. A planned comparison of five similar quality indicators and their key components showed that the simpler treatment components, as a group, had significantly higher kappas than the more complexly derived eligibility components and composite indicators (Fisher's exact, p < 0.02). Reliability assessment of quality indicators should be based upon the repeatability of the whole indicator, accounting for both data and logic, and not just one simple element.

Objective: To demonstrate the use of quality indicators developed through claims data augmentation., Method: A retrospective examination of field-tested acute care quality indicators and methods used in developing them to identify opportunities for quality improvement was used. The settings were 46 acute care hospitals and physician staffs serving Medicare beneficiaries in Vermont and New Hampshire, with more than 60,000 Medicare inpatient admissions per year. The objective was to measure hospital and provider initiatives in quality improvement projects., Result: More than 100 projects have been developed from hospital-specific comparative analyses and presentations about peer group profiles on indicator and outcome variables. Successfully completed projects have resulted in significant improvements in patient out comes, including reduced acute myocardial infarction mortality, reduced rates of discharges against medical advice, and increased use of pressure sore prevention and prophylactic antibiotics guidelines., Conclusion: Certain essential enabling data elements permit construction of a credible claims-based acute care quality indicator dataset. Unique individual patient identifiers and validated deaths are essential for conducting readmission and mortality analyses, respectively. Risk adjustment is necessary for making provider comparisons on indicators influenced by patient severity of illness. Cross-validation of outcome patterns with multiple indicators, and project successes have built provider interest and confidence in the use of such a data base.

Optical mapping is an emerging single molecule approach for the rapid generation of ordered restriction maps, using fluorescence microscopy. We have improved the size resolution of optical mapping by imaging individual DNA molecules elongated and fixed onto derivatized glass surfaces. Averaged fluorescence intensity and apparent length measurements accurately determined the mass of restriction fragments 800 basepairs long. We have used optical mapping to create ordered restriction maps for lambda clones derived from the mouse pygmy locus.

Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.

Case counts below 50 can compromise the careful use of disease- or procedure-specific standardized mortality ratio comparisons across small hospitals in predominantly rural areas. Linking data series where data are of the same quality and are continuous across the series is one way of handling the problem of small case sizes. Examination of three different annual reports of Health Care Financing Administration (HCFA) mortality findings for 1987 of one state's providers showed significantly lower actual and predicted mortality rates reported in 1989, compared with the same mortality rates reported about 1987 published in 1988 and 1987. One hypothesis for the downward trend is the change in case selection method used for constructing the mortality model from last discharge (used in 1987 and 1988 reports) to random selection of cases with multiple admissions (initiated in 1989). A test of both case selection methods is presented and shows the consequences of mortality model changes that create discontinuities with previously reported findings, changes that limit or at least need to be taken into account when linking current HCFA mortality data series with historic series.

A light microscope-based technique for rapidly constructing ordered physical maps of chromosomes has been developed. Restriction enzyme digestion of elongated individual DNA molecules (about 0.2 to 1.0 megabases in size) was imaged by fluorescence microscopy after fixation in agarose gel. The size of the resulting individual restriction fragments was determined by relative fluorescence intensity and apparent molecular contour length. Ordered restriction maps were then created from genomic DNA without reliance on cloned or amplified sequences for hybridization or analytical gel electrophoresis. Initial application of optical mapping is described for Saccharomyces cerevisiae chromosomes.

We present details on how to implement a newly developed methodology, Optical Contour Maximization (OCM), for accurately sizing large DNA molecules. Agarose gel containing stained DNA is cast between a slide and a coverslip, and molecules moved by an applied electrical field are observed using fluorescence microscopy. The molecules of interest lie in the interface formed between the coverslip and gel. DNA movement in this region is largely confined to lateral motions. When a DNA molecule snags an obstacle, it elongates, forming a metastable hook that can persist for several seconds. We found that the longest observed hook contour length can be determined from rapidly collected images. This maximized length shows a linear correlation with reported size [X.H. Guo, E.J. Huff & D.C. Schwartz, Nature 359, 783 (1992)] Successful measurements require a critical balance between the voltage needed for full elongation, and the unwanted effects of too large a voltage: fewer metastable hooks form, they dissipate faster, molecule breakage becomes a problem, and faster image collection becomes necessary. We measured apparent contour length as a function of applied voltage and determined an optimal voltage for our apparatus, using a singly anchored 114kb molecule. The measurement precision is estimated from the distribution of results. We expect that OCM will find utility in physical mapping and molecular karyotyping of lower eucaryotes of medical importance.

A 7-year-old boy had pure red cell aplasia and clinically significant hepatitis during isoniazid therapy. The former complication had been reported only in adults, and the latter is rare in children.

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).

Commonly used procedures effected the extraction of only 10% of the lipoteichoic acid of stationary-phase cells of Staphylococcus aureus and Streptococcus faecium, unless the cells were first disrupted.

Mesosomal vesicles and plasma membranes were isolated from Staphylococcus aureus ATCC 6538P by protoplasting and differential centrifugation. The lipids of each of the two membrane fractions were extracted with pyridine-acetic acid-N-butanol, and the nonlipid contaminants were removed by Sephadex treatment. The lipids were then separated by passage through diethylaminoethyl-cellulose columns and characterized by thin-layer chromatographic, chemical, and spectral analyses. The lipids were separated into four discrete diethylaminoethyl fractions: (i) vitamin K2, carotenoids, C55 isoprenoid alcohol, and monoglucosyl diglyceride; (ii) cardiolipin, carotenoids, phosphatidyl glycerol, diglucosyl diglyceride, and an unidentified ninhydrin-positive component; (iii) cardiolipid and phosphatidyl glyderol; (iv) cardiolipin, phosphatidyl glycerol, and phosphatidyl glucose. Qualitatively, no difference in lipid composition between mesosomal vesicles and plasma membranes was found. However, based on equal dry weights of membrane materials, a relative quantitative difference in the amount of specific lipids in mesosomal vesicles and plasma membranes was observed. There are 4 times more monoglucosyl diglyceride, 2.6 times more diglucosyl diglyceride, 3.8 times more phosphatidyl glucose, 2 times more carotenoids, and 2 times more vitamin K2 found in mesosomal vesicles than in plasma membranes. The concentration of cardiolipin and phosphatidyl glycerol is 3.6 and 6 times greater, respectively, in mesosomal vesicles.

Staphylococus aureus, ATCC 6538P, was fractionated into protoplast membranes, mesosomal vesicles, periplasm, and cytoplasm. These fractions and the culture fluid were then assayed for various degradative enzyme activities. They were not restricted to a single fraction nor dispersed homogeneously, but were distributed predominantly (on the basis of specific activity) as follows: nuclease in the culture fluid; alkaline phosphatase, 5'-nucleotidase, and acid phosphatase in the periplasm; adenosine triphosphatase in the protoplast membrane; and protease (low levels) in mesosomal vesicles. No significant esterase nor cell wall hydrolytic activity was found in any fraction. S. aureus 80/81 was studied for penicillinase activity after induction with benzyl penicillin; this enzyme was localized in the mesosomal vesicles. Electron microscopy did not reveal any ultrastructural changes associated with secretion of the extracellular fraction. Overall, these studies demonstrate that degradative enzymes are located in several surface compartments and that, therefore, the mesosome does not function as a prototype lysosome in S. aureus.

Mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P have been prepared and examined for the presence of lipoteichoic acid. Lipids were first removed by treatment with pyridine-acetic acid-butanol (22:31:100, vol/vol/vol) and chloroform-methanol (2:1, vol/vol). Subsequently, lipoteichoic acid was removed with 40% phenol in water. The lipoteichoic acid from mesosomal vesicles was characterized by (i) equimolar glycerol and phosphate, (ii) alanine upon hydrolysis (2 N NH(4)OH, 18 h, 22 C), and (iii) fatty acids, diglycerol triphosphate, glycerol monophosphate, and glycerol diphosphate upon alkaline hydrolysis (1 N NaOH, 3h, 100 C). The plasma membranes contained no lipoteichoic acid. The presence in mesosomal vesicles of 18% of the dry weight as lipoteichoic acid and its absence from plasma membranes provide the first major chemical differences between these organelles. A study of the lipoteichoic acid content in various fractions of the cell showed that the mesosomal vesicles were the major and probably the sole site for the localization of the lipoteichoic acid in these organisms. A new method for the preparation of mesosomes in increased yields is reported. A theory for the control of cell division involving lipoteichoic acid and the mesosome is proposed.