Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.

Evidence for multiple historical colonizations of an endoreic drainage basin by an Australian freshwater fish.

The contemporary and historical colonization capacity of an Australian freshwater fish, north-west glassfish Ambassis sp., was tested using mtDNA sequence data and six newly developed microsatellite loci in an endoreic basin in central Australia. Overall, Ambassis sp. exhibited weak genetic structure within catchments, suggesting some capacity to recolonize extirpated waterholes after disturbance. Genetic structure revealed that the historical pattern of connectivity among catchments in the Lake Eyre Basin was dramatically different from other species studied in this region. Two highly divergent clades were detected in separate catchments in the basin. mtDNA from individuals sampled in catchments north of the Lake Eyre Basin suggest that Ambassis sp. has colonized on two separate occasions from catchments in northern Australia, subsequently generating two highly divergent lineages.

Evaluation of PCDD/Fs patterns emitted from incinerator via direct ambient sampling and indirect serum levels assessment of Taiwanese.

The aim of this study was to evaluate the PCDD/Fs patterns in ambient air based on data information emitted from incinerator generated from ambient air measurements and those in serum. Four circular zones, namely A, B, C, and D, were identified based on simulated ambient annual average PCDD/Fs concentrations, from a selected municipal waste incinerator. Sixteen ambient samples were taken from the 4 circular zones across 4-seasons. Eighty-nine volunteers were recruited according to the demographic distribution within each zone. PCDD/Fs profiles were documented both for air and serum samples collected. Comparing to the congener patterns from ambient air and serum samples, we found that OCDD, OCDF, 1,2,3,4,6,7,8-HpCDD, and 1,2,3,4,6,7,8-HpCDF were the predominant groups among 17 congeners from both the ambient air and serum sample. And, factor analysis showed the distribution patterns of PCDD/Fs from ambient air and serum samples are almost identical across different zones, except for congener patterns of serum samples from residents in zone C. In addition, the average PCDD/Fs level significantly reduced for about 10 folds than those of the other three seasons when the incinerator was shut down in one of sampling periods. We might conclude that ambient air exposure was the most important contributor to PCDD/Fs levels in ambient air but not the single in serum. Therefore, another or more powerful source, such as occupational exposure, dietary intake or the consumption of local food, should be further investigated at the same time.

Multisite randomized controlled trials in health services research: scientific challenges and operational issues.

Although well-designed randomized controlled trials (RCT) provide the strongest evidence regarding causation, only relatively recently have they been used by health services researchers to study the organization, delivery, quality, and outcomes of care. More recent yet is the extension of multisite RCTs to health services research. Such studies offer numerous methodological advantages over single-site trials: (1) enhanced external validity; (2) greater statistical power when studying conditions with a low incidence or prevalence, small event rate in the outcome (eg, mortality), and/or large variance in the outcome (eg, health care costs); and (3) rapid recruitment to provide health care organizations and policy makers with timely results. This paper begins by outlining the advantages of multisite RCTs over single-site trials. It then discusses both scientific challenges (ie, standardizing eligibility criteria, defining and standardizing the intervention, defining usual care, standardizing the data collection protocol, blinded outcome assessment, data management and analysis, measuring health care costs) and operational issues (ie, site selection, randomization procedures, patient accrual, maintaining enthusiasm, oversight) posed by multisite RCTs in health services research. Recommendations are offered to health services researchers interested in conducting such studies.

Predicting non-elective hospital readmissions: a multi-site study. Department of Veterans Affairs Cooperative Study Group on Primary Care and Readmissions.

OBJECTIVE: To determine clinical and patient-centered factors predicting non-elective hospital readmissions. DESIGN: Secondary analysis from a randomized clinical trial. CLINICAL SETTING: Nine VA medical centers. PARTICIPANTS: Patients discharged from the medical service with diabetes mellitus, congestive heart failure, and/or chronic obstructive pulmonary disease (COPD). MAIN OUTCOME MEASUREMENT: Non-elective readmission within 90 days. RESULTS: Of 1378 patients discharged, 23.3% were readmitted. After controlling for hospital and intervention status, risk of readmission was increased if the patient had more hospitalizations and emergency room visits in the prior 6 months, higher blood urea nitrogen, lower mental health function, a diagnosis of COPD, and increased satisfaction with access to emergency care assessed on the index hospitalization. CONCLUSIONS: Both clinical and patient-centered factors identifiable at discharge are related to non-elective readmission. These factors identify high-risk patients and provide guidance for future interventions. The relationship of patient satisfaction measures to readmission deserves further study.

Complication after laparoscopic donor nephrectomy: a case report and review.

BACKGROUND: Laparoscopic procedures are gaining acceptance in the treatment of benign and some malignant urologic disorders. Recently, laparoscopic techniques have been applied to transplant surgery and touted as a safe alternative to traditional open techniques. METHODS: We present a patient who developed a complication from laparoscopic donor nephrectomy that required open corrective surgery. RESULTS: A 25-year-old man underwent laparoscopic donor nephrectomy at a large medical center familiar with the operation. There were no operative or early postoperative complications. Within 6 weeks of the operation, the patient developed signs and symptoms of partial small bowel obstruction. Further evaluation revealed an internal hernia in the retroperitoneum at the site of the nephrectomy. This required a second operation to reduce the hernia and close the defect. CONCLUSION: Laparoscopic donor nephrectomy remains an evolving technique that has not stood the test of time. Larger series will eventually reveal whether this is the procedure of choice as compared to traditional open donor nephrectomy.

Preparing obstetricians and gynecologists for capitation.

We are indeed facing a changing paradigm in the way medical services are delivered and reimbursed. Physicians are becoming entangled in the business world. Terms such as "medical loss ratio" must be integrated within the physician's existing language. According to Green and Barnett, "No group is so large or so small as to likely survive the next decade without being involved in a capitated contract." The change is not tomorrow, it is today. Do not be lost in the changing world of medicine without becoming involved, and see if it is for you and your practice. Progress of medicine may not be what you would like but do not be passed by without making an impact. Capitation offers an opportunity to regain control of the management of care, to develop innovative and cost-effective approaches to delivery, and to enhance income and revenue in the face of declining health care budgets.

Classifying general medicine readmissions. Are they preventable? Veterans Affairs Cooperative Studies in Health Services Group on Primary Care and Hospital Readmissions.

OBJECTIVES: To describe a new quality assessment method used to classify the preventability of hospitalization in terms of patient, clinician, or system factors. DESIGN: The instrument was developed in two phases. Phase 1 was a prospective comparison of admitting residents' and their attending physicians' classifications of the perceived preventability of consecutive admissions to one Veterans Affairs Medical Center (VAMC) excluding admissions to the intensive care unit (ICU). In phase 2, a panel of 10 physicians rated 811 abstracted records of readmissions from nine VAMCs. SETTING: Nine VAMCs across the United States with varying degrees of university hospital affiliation. PATIENTS: Phase 1, 156 patients admitted to the general medicine service at the Durham VAMC. Phase 2, 514 patients accounting for 811 readmissions within 6 months of a general medicine service discharge at nine VAMCs. MEASUREMENTS AND MAIN RESULTS: Physicians used a checklist to record the reason for hospitalization, the preventability of the hospitalization, and, if preventable, a reason defining preventability, which was classified in terms of system, clinician, and patient factors. In phase 2, two physician panelists assessed preventability for each chart. When two panelists disagreed on the preventability of hospitalization, a third panelist, blind to the original assessments, rated the chart. In phase 1, residents and attending physicians rated 33% and 34% of admissions as preventable (kappa = 0.41), respectively. In phase 2, 277 (34%) of 811 readmissions were deemed preventable. Intraobserver accuracy for the assessment of preventability was 96% (kappa = 0.89). interobserver accuracy was 73% (kappa = 0.43). Hospital system factors accounted for 37% of preventable readmissions, clinician factors for 38%, and patient factors for 21%. The nine hospitals differed markedly in their profile of reasons for preventable readmissions (p = .005). CONCLUSIONS: Using a new method of determining the preventability of hospitalizations, we identified several factors that might avert hospitalizations. Focusing efforts to identify preventable hospitalizations may yield better methods for managing patients' total health care needs; however, the content of those efforts will vary by institution.

[Common variable immunodeficiency (hypogammaglobulinemia of late onset or acquired hypogammaglobulinemia): initial follow-up of 11 cases]. / Imunodeficiência comum variável (hipogamaglobulinemia de início tardio ou hipogamaglobulinemia adquirida): seguimento inicial de 11 casos.

The present paper describes the clinical and laboratory follow-up of 11 patients with the diagnosis of common variable immunodeficiency. Their age varied from 8 to 45 years. The mean disease time was 12.6 years and mean diagnosis time 4.3 years. Infectious manifestations, mainly of the respiratory and digestive tracts, occurred in all patients. Polyadenomegaly was noted in seven, hepatomegaly in six, splenomegaly in five and arthralgia in four patients. All of them presented serum IgG less than 250 mg/dl. IgA less than 33 mg/dl and IgM less than 31 mg/dl, except one with IgM = 176 mg/dl. The isohaemagglutinin titers were less than 1/20 in all but one patient. The determination of the number of B lymphocytes in the peripheral blood revealed normal counts in three, elevated in one and decreased in five patients. The CD-4/CD-8 ratio was less than 1 in 8 and greater than 1 in three of them. Five patients had positive cutaneous late reactions to at least one of the following antigens: PPD, SK-SD (Varidase), Trichophytin and Levedurin (Candidin). A decrease of the proliferative activity of peripheral blood mononuclear cells stimulated by lectins (PHA, Con-A, PWM) was also noted. Natural killer function was decreased. The association a possible role of regulatory lymphocytes in the immunopathogenesis of this disease. The data presented here emphasize the diversity of clinical and immunological manifestations of this disease, which could be noted between diverse patients and in the follow-up of a single one. In our cases the disease had an evolutive character, with a primarily humoral dysfunction followed by cellular immunity disturbances that determined poorer prognosis and progressive difficulties in the therapeutics. We suggest a conceptual reevaluation of this condition and a new denomination, for instance "Late-Onset Combined Immunodeficiency". The long delay between the initial clinical manifestations of the disease and its diagnosis was a handicap for an adequate treatment. Early intervention could certainly decrease the morbidity and mortality of the disease.

Genital tuberculosis: case report and literature review.

A 64-year-old woman complained of abdominal pain and postmenopausal bleeding. A uterine curettage demonstrated acid fast bacilli and non caseating granulomas, indicating Mycobacterium tuberculosis. A chest roentgenogram revealed the presence of bilateral upper lobe calcific granulomas. The epidemiologic, diagnostic, and therapeutic implications of genital tuberculosis are discussed.

Clinical evaluation of intravenous labetalol for the treatment of hypertensive urgency.

Antihypertensive medications currently used in the treatment of hypertensive urgencies are limited due to deleterious side effects or requirements for sophisticated monitoring techniques. Labetalol HCl (Trandate) is a unique adrenergic blocking agent that can smoothly lower blood pressure following bolus injection without increasing heart rate or cardiac output. This study evaluates the efficacy and safety of intravenous boluses of labetalol HCl in the treatment of patients presenting to the hospital with a diagnosis of hypertensive urgency (diastolic blood pressure greater than or equal to 110 mm Hg). After baseline blood pressure and heart rate were recorded, 20 consecutive patients were treated with an initial 20-mg bolus of labetalol. Additional boluses of 40, 80, and 160 mg were administered at least 10 minutes apart in a step-wise fashion until control of blood pressure (diastolic blood pressure less than 100 mm Hg) was achieved or a total of 300 mg had been given. Blood pressures and heart rates were recorded at the time of response or following the last dose of labetalol. Mean (+/- SEM) supine systolic blood pressure decreased from 185 +/- 3 to 155 +/- 4 mm Hg (P less than 0.05) following labetalol therapy, and mean supine diastolic pressure decreased from 120 +/- 2 to 98 +/- 2 mm Hg (P less than 0.05). Mean heart rate did not change significantly. Eighteen of the 20 patients exhibited a therapeutic response; nine patients received a total of 20 mg, six required 60 mg, two required 140 mg, one received 300 mg. Of the two patients who did not respond, one received the maximum dose (300 mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Test Medium for the Growth of Nitrosomonas europaea.

A mineral medium for studying the growth of Nitrosomonas europaea was developed and examined. The medium was defined in terms of chemical speciation by using chemical equilibrium computer models. The medium significantly increased the metabolic activity of the organisms compared with previously developed media, yielding a specific growth rate as high as 3.0 day (generation time, 5.5 h). The specific growth rate was enhanced by increasing the inoculum and was linearly correlated with the inoculum-to-total-culture volume ratio on a semilog scale. A reproducible growth rate for N. europaea was obtained with this medium under controlled experimental conditions.

Effect of epidermal growth factor on the synthetic activity of human fibroblasts.

The influence of epidermal growth factor on DNA and protein synthesis by human gingival fibroblasts was studied. Synchronized cells treated with epidermal growth factor synthesized considerably greater amounts of DNA relative to 10% fetal calf serum and the peak of synthesis ocurred 6 h later than with serum. Epidermal growth factor caused a dose-dependent stimulation of protein synthesis (proline incorporation). Collagen synthesis remained unaffected and, as a result, the proportion of collagen synthesized decreased with increasing epidermal growth factor concentration. Aspirin and indomethacin did not abrogate these effects, indicating that prostaglandins may not be involved.

Intrapartum fetal heart rate: correlation with scalp pH in the preterm fetus.

The relationship of FHR patterns and fetal scalp blood pH is well documented in the term fetus. Although FHR monitoring has been increasingly used to guide intrapartum management of the preterm fetus, little documentation of efficacy and the interrelationship of FHR patterns with pH is available. This report of 62 preterm fetuses documents the relationship of FHR findings and their correlation with fetal pH. The ominous connotation of late deceleration is emphasized. The evaluation of the presence or absence of FHR variability and acceleration patterns are proved as significant factors which are helpful in making management decisions.

The evaluation of uterine activity: a comparative analysis.

In this study a comparison is made between the most widely accepted method of expressing uterine activity, Montevideo units, and an automated on-line method expressed as uterine activity units. Although the actual numerical expression differs, the percentage of variation when the two values are compared is small at various phases of labor. The variation between the methods for uterine activity required to progress from 4 to 6 cm cervical dilatation and from 6 to 10 cm cervical dilatation is less than 5%. Both methods closely agree on the actual expression of uterine activity or uterine work.

Monitoring of uterine activity.

The aim of modern obstetrics is to deliver a healthy, undamaged infant to a healthy, happy mother. The ability to classify and treat abnormalities of uterine activity safely with the plotting of cervical progress against time and to quantitate uterine activity as adequate or inadequate allows for more specific and rapid treatment. Treatment may be instituted more rapidly with monitoring and thus avoid prolonged labours with maternal exhaustion and dehydration. With normal fetal heart rate and variability Schifrin (1974) has stated that the differences in fetal outcome associated with various patterns of abnormal labour essentially disappear if mid-forceps procedures are abandoned and only spontaneous vaginal delivery or caesarean section is used.

Fetal heart rate variability: an approach to automated assessment.

Three hundred seventy-five hours of fetal heart rate (FHR) data derived from the direct fetal electrocardiogram (ECG) were studied. This data had been stored on magnetic tape from 83 intrapartum patients. By means of a computerized technique, the FHR variability was assessed quantitatively. The degree of variability was then related to: (1) state of labor, (2) fetal scalp pH values, and (3) the 1-minute Apgar score. FHR variability was computed from differences between consecutive R-R intervals measured from the R wave of each fetal ECG. A trend of increasing variability was seen with advancing labor, defined by either time prior to delivery or cervical dilatation, but values were not statistically significant. Significantly less FHR variability was encountered when fetal scalp pH values below 7.20 were compared to higher values. FHR variability assessed during the 20 minutes immediately preceding delivery was significantly lower in infants with 1-minute Apgar scores less than 7. Machine assessment of FHR variability thus could be correlated with fetal condition as determined by scalp pH and neonatal outcome determined by Apgar score.

Evaluation of tracheal incisions for tracheostomy.

Three standard tracheostomy incisions in dogs were compared to determine whether any were more likely associated with stomal stenosis. Each incision resulted in an average reduction in tracheal lumen of 25 per cent, with a 50 per cent maximum. The incision selected should depend on the conditions present.