Clinical Pilot of Bacterial Transcriptional Profiling as a Combined Genotypic and Phenotypic Antimicrobial Susceptibility Test.

Antimicrobial resistance is a growing health threat, but standard methods for determining antibiotic susceptibility are slow and can delay optimal treatment, which is especially consequential in severe infections such as bacteremia. Novel approaches for rapid susceptibility profiling have emerged that characterize either bacterial response to antibiotics (phenotype) or detect specific resistance genes (genotype). GoPhAST-R is a novel assay, performed directly on positive blood cultures, that integrates rapid transcriptional response profiling with detection of key resistance gene transcripts, thereby providing simultaneous data on both phenotype and genotype. Here, we performed the first clinical pilot of GoPhAST-R on 42 positive blood cultures: 26 growing Escherichia coli, 15 growing Klebsiella pneumoniae, and 1 with both. An aliquot of each positive blood culture was exposed to 9 different antibiotics, lysed, then underwent rapid transcriptional profiling on the NanoString® platform; results were analyzed using an in-house susceptibility classification algorithm. GoPhAST-R achieved 95% overall agreement with standard antimicrobial susceptibility testing methods, with the highest agreement for beta-lactams (98%) and the lowest for fluoroquinolones (88%). Epidemic resistance genes including the extended spectrum beta-lactamase bla CTX-M-15 and the carbapenemase bla KPC were also detected within the population. This study demonstrates the clinical feasibility of using transcriptional response profiling for rapid resistance determination, although further validation with larger and more diverse bacterial populations will be essential in future work. GoPhAST-R represents a promising new approach for rapid and comprehensive antibiotic susceptibility testing in clinical settings.

Environmental conditions during early life determine the consequences of inbreeding in Agrostemma githago (Caryophyllaceae).

In an inbred population, selection may reduce the frequency of deleterious recessive alleles through a process known as purging. Empirical studies suggest, however, that the efficacy of purging in natural populations is highly variable. This variation may be due, in part, to variation in the expression of inbreeding depression available for selection to act on. This experiment investigates the roles of life stage and early-life environment in determining the expression of inbreeding depression in Agrostemma githago. Four population-level crosses ('self', 'within', 'near' and 'far') were conducted on 20 maternal plants from a focal population. Siblings were planted into one of three early environmental treatments with varying stress levels. Within the focal population, evidence for purging of deleterious recessive alleles, as well as for variation in the expression of inbreeding depression across the life cycle was examined. In addition, the effect of early environment on the expression of inbreeding depression and the interaction with cross-type was measured. We find that deleterious recessive alleles have not been effectively purged from our focal population, the expression of inbreeding depression decreases over the course of the life cycle, and a stressful early environment reduces the variance in inbreeding depression expressed later in life, but does not consistently influence the relative fitness of inbred versus outcrossed individuals.

Secreted lymphotoxin-alpha is essential for the control of an intracellular bacterial infection.

Although the essential role of tumor necrosis factor (TNF) in the control of intracellular bacterial infection is well established, it is uncertain whether the related cytokines lymphotoxin-alpha (LTalpha3) and lymphotoxin-beta (LTbeta) have independent roles in this process. Using C57Bl/6 mice in which the genes for these cytokines have been disrupted, we have examined the relative contribution of secreted LTalpha3 and membrane-bound LTbeta in the host response to aerosol Mycobacterium tuberculosis infection. To overcome the lack of peripheral lymph nodes in LTalpha-/- and LTbeta-/- mice, bone marrow chimeric mice were constructed. LT-/- chimeras, which lack both secreted LTalpha3 and membrane-bound LTbeta (LT1beta2 and LT2beta1), were highly susceptible and succumbed 5 wk after infection. LTbeta-/- chimeras, which lack only the membrane-bound LTbeta, controlled the infection in a comparable manner to wild-type (WT) chimeric mice. T cell responses to mycobacterial antigens and macrophage responses in LTalpha-/- chimeras were equivalent to those of WT chimeras, but in LTalpha-/- chimeras, granuloma formation was abnormal. LTalpha-/- chimeras recruited normal numbers of T cells into their lungs, but the lymphocytes were restricted to perivascular and peribronchial areas and were not colocated with macrophages in granulomas. Therefore, LTalpha3is essential for the control of pulmonary tuberculosis, and its critical role lies not in the activation of T cells and macrophages per se but in the local organization of the granulomatous response.

Carotid Artery Calcification on Orthopantomograms (CACO Study) - is it indicative of carotid stenosis?

It is unclear whether incidental carotid artery calcification (CAC) on radiographs has a defined relationship to clinically significant carotid artery stenosis, and therefore risk of stroke. The primary objective of this study was to ascertain the relationship between dental radiograph detected carotid calcification and carotid artery stenoses ≥50% on carotid duplex ultrasound. We carried out an observational study of patients undergoing routine dental orthopantomogram (OPG) examinations. Consecutive patients with CAC on OPG were prospectively matched to those without CAC based on age and gender. Ultrasound of the carotid arteries was performed to determine the presence of stenosis (≥50%) in either vessel. Of 5780 consecutive OPG examinations with suitable images for analysis, CAC was detected in 10.8%. A total of 233 patients underwent carotid ultrasound (130 with and 103 without CAC on OPG). The prevalence of a clinically significant (≥50%) carotid stenosis on ultrasound was 15.4% (20/130) in those with CAC and 5.8% (6/103) for those without CAC on OPG. Incidental CAC detected on routine OPG requires both radiological reporting and clinical follow-up since 1 in 7 patients will have a clinically significant carotid artery stenosis as compared with 1 in 20 patients who do not have CAC. Trial Registration: Australian and New Zealand Clinical Trials Registry website (U1111-1148-1066). http://www.ANZCTR.org.au/ACTRN12613001038785.aspx.

Correlations between contouring similarity metrics and simulated treatment outcome for prostate radiotherapy.

Many similarity metrics exist for inter-observer contouring variation studies, however no correlation between metric choice and prostate cancer radiotherapy dosimetry has been explored. These correlations were investigated in this study. Two separate trials were undertaken, the first a thirty-five patient cohort with three observers, the second a five patient dataset with ten observers. Clinical and planning target volumes (CTV and PTV), rectum, and bladder were independently contoured by all observers in each trial. Structures were contoured on T2-weighted MRI and transferred onto CT following rigid registration for treatment planning in the first trial. Structures were contoured directly on CT in the second trial. STAPLE and majority voting volumes were generated as reference gold standard volumes for each structure for the two trials respectively. VMAT treatment plans (78 Gy to PTV) were simulated for observer and gold standard volumes, and dosimetry assessed using multiple radiobiological metrics. Correlations between contouring similarity metrics and dosimetry were calculated using Spearman's rank correlation coefficient. No correlations were observed between contouring similarity metrics and dosimetry for CTV within either trial. Volume similarity correlated most strongly with radiobiological metrics for PTV in both trials, including TCPPoisson (ρ = 0.57, 0.65), TCPLogit (ρ = 0.39, 0.62), and EUD (ρ = 0.43, 0.61) for each respective trial. Rectum and bladder metric correlations displayed no consistency for the two trials. PTV volume similarity was found to significantly correlate with rectum normal tissue complication probability (ρ = 0.33, 0.48). Minimal to no correlations with dosimetry were observed for overlap or boundary contouring metrics. Future inter-observer contouring variation studies for prostate cancer should incorporate volume similarity to provide additional insights into dosimetry during analysis.

Origins of heart rate variability. Inducibility and prevalence of a discrete, tachycardic event.

BACKGROUND: We propose that heart period sequences are linearly organized, like sentences, and that there is a lexicon of recurrent, similarly shaped transient structures like words. Each word (or lexon) has a characteristic physiological basis. One potential lexon is the transient, reversible tachycardia that is induced by exercise initiation under laboratory conditions. We hypothesized that this lexon was inducible and observable on ambulatory ECGs of most or all subjects, was morphologically similar in both induced and detected bursts, and shared a plausible origin in both circumstances. METHODS AND RESULTS: Ten healthy subjects (mean age, 36 years) underwent a protocol in which subjects rolled themselves from supine to lateral decubitus positions and back. Transient tachycardias ("bursts") were seen in 36 of 40 rollovers. Bursts were characterized by an initial monoexponential heart period decay (K=0.39+/-0.23 s-1), a maximum heart period decrease of 277+/-109 ms after 10.8+/-4.5 seconds, and a subsequent return to baseline 23.3+/-10.8 seconds after roll initiation. The roll-induced bursts were detected with 97% sensitivity and 99% specificity with a search algorithm that incorporated morphological parameters. In 24-hour ambulatory ECGs of 10 healthy subjects (mean age, 38 years; range, 17 to 69 years), 117+/-59 bursts were detected. Induced and detected bursts were similar in most morphological parameters. Finally, many bursts occurred at night, when rolling over also occurs. CONCLUSIONS: Bursts are inducible, transient tachycardias that occur clinically and constitute a lexon with an understandable physiology.

A novel tumor necrosis factor (TNF) mimetic peptide prevents recrudescence of Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in CD4+ T cell-depleted mice.

Tumor necrosis factor (TNF) is required to control mycobacterial infections, but its therapeutic value is limited by its in vivo instability and toxicity. The efficacy of a nontoxic TNF-mimetic peptide (TNF70-80) was tested in mice infected with Mycobacterium bovis bacillus Callette-Guerin (BCG). In vitro TNF70-80 and recombinant human TNF (hTNF) acted with interferon gamma (IFN-gamma) to reduce bacterial replication and to induce synthesis of bactericidal nitric oxide (NO) in BCG-infected, bone marrow-derived murine macrophages. The dose-dependent inhibitory effect on bacterial replication was blocked by neutralizing anti-IFN-gamma and anti-hTNF mAbs. Further, n-monomethyl-L-arginine (n-MMA) and a soluble TNF-receptor I (TNFRI-IgG) blocked bacterial growth and NO synthesis. Therefore, the peptide acted with IFN-gamma via induction of NO synthase and signaled through TNFRI receptors. Concomitant in vivo treatment with TNF70-80 or hTNF prevented reactivation of chronic BCG infection in mice depleted of CD4+ T cells by injecting anti-CD4 antibodies. Granuloma number and bacterial load were comparable in treated, T cell-depleted mice and in chronically infected, intact animals. Thus, TNF70-80 and hTNF can modulate recrudescent BCG infection in CD4+ T cell-deficient mice.

Environmental effects on age-dependent mortality: a test with a perennial plant species under natural and protected conditions.

Most experimental studies of senescence have been done with short-lived organisms under controlled laboratory conditions and it is not clear whether the insights gained from these studies can be broadly generalized. This study was designed to detect senescence in a natural population and to compare the patterns of mortality for a single species in natural and protected conditions. It was done with Plantago lanceolata, a perennial plant for which the demography of a large population of individuals in their natural environment is relatively straightforward. An initial cohort of 10,000 individuals was established in the natural field environment. In order to separate the effects of environment- and age-dependent factors on mortality, an additional cohort was planted in the field one year later. To study the demography of mortality under protected conditions, a population of 1000 individuals was established in the greenhouse. The results of the comparative analysis of two different-aged cohorts in the field and of the field and greenhouse populations show that senescence patterns can be very plastic. The results show that senescence in the natural environment is caused by an increased vulnerability of older individuals to environmental stress. Under the protected environmental conditions of the greenhouse senescence was negligible.

Tracheal and neck position influence upper airway airflow dynamics by altering airway length.

Upper airway obstruction during sleep is characterized by inspiratory airflow limitation and reductions in maximal inspiratory airflow (VImax). To determine how mechanical factors modulate VImax, we analyzed pressure-flow relationships obtained in the isolated upper airway of paralyzed cats. VImax and its determinants, the pharyngeal critical pressure (Pcrit) and the nasal resistance (Rn) upstream to the flow-limiting site (FLS), were measured as caudal tracheal displacement, neck position, and airway length were systematically varied. As the proximal tracheal stump was displaced caudally, graded increases in VImax from 145.3 +/- 90.8 (SD) to 285.9 +/- 117.5 ml/s (P < 0.02) and decreases in Pcrit from -3.0 +/- 3.0 to -9.5 +/- 3.4 cmH2O (P < 0.002) were seen without any significant change in Rn. During neck flexion, significant decreases in VImax from 192.1 +/- 68.5 to 87.2 +/- 48.4 ml/s (P = 0.001), increases in Pcrit from -5.3 +/- 2.03 cmH2O to -1.6 +/- 1.4 cmH2O (P < 0.001), and decreases in Rn from 29.7 +/- 12.2 cmH2O.l-1.s to 16.2 +/- 8.9 cmH2O.l-1.s (P < 0.001) were noted compared with the neutral or extended neck position. Relative to the neutral airway length, upper airway length was found to decrease by 1.15 +/- 0.14 cm during neck flexion and to lengthen by 0.45 +/- 0.12 cm during neck extension. When tracheal displacement and neck position were altered, VImax and Rn correlated directly and Pcrit correlated inversely with airway length (P < 0.001). We conclude that alterations in airflow mechanics with caudal tracheal displacement and changes in neck positions are primarily due to alterations in airway length.

Modulation of maximal inspiratory airflow by neuromuscular activity: effect of CO2.

To determine how maximal inspiratory airflow (VImax) is modulated by changes in airway neuromuscular activity, we analyzed pressure-flow relationships obtained during inspiration and expiration in isolated upper airways of anesthetized hyperoxic dogs at different levels of CO2. Inspiratory airflow (VI), hypopharyngeal pressure (Php), pharyngeal pressure at the flow-limiting site (FLS), and alae nasi (AN) and genioglossus (GG) electromyographic (EMG) activity were recorded while VI limitation was produced by rapidly lowering Php until VI plateaued at VImax. VImax and its mechanical determinants, pharyngeal critical pressure (Pcrit) and nasal resistance (Rn) upstream to the FLS, were measured. During hypercapnia (high CO2), VImax increased significantly during inspiration (217.3) and expiration (184.1%). These increases were associated with significant increases in phasic but not tonic AN and GG activity. They were also associated with decreases in Pcrit from -6.2 +/- 1.6 (SE) at hypocapnia to -9.3 +/- 3.0 and -11.8 +/- 3.4 cmH2O at high CO2 during expiration and inspiration, respectively. No significant changes in Rn occurred. When phasic neuromuscular activity was abolished by complete neuromuscular blockade in three dogs, these increases in VImax and decreases in Pcrit at high CO2 were eliminated. When phasic EMG activity was accentuated in four vagotomized dogs, significant increases in VImax and decreases in Pcrit were demonstrated during inspiration vs. expiration at high CO2. These findings indicate that upper airway neuromuscular activity increases VImax in the isolated upper airway by decreasing collapsibility (Pcrit) at the FLS site when neuromuscular activity is stimulated by hypercapnia.

Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats.

The aim of this study was to evaluate the acute lung toxicity of intratracheally instilled single-wall carbon nanotubes (SWCNT) in rats. The lungs of rats were instilled either with 1 or 5 mg/kg of the following control or particle types: (1) SWCNT, (2) quartz particles (positive control), (3) carbonyl iron particles (negative control), (4) phosphate-buffered saline (PBS) + 1% Tween 80, or (5) graphite particles (lung tissue studies only). Following exposures, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers and cell proliferation methods, and by histopathological evaluation of lung tissue at 24 h, 1 week, 1 month, and 3 months postinstillation. Exposures to high-dose (5 mg/kg) SWCNT produced mortality in ~15% of the SWCNT-instilled rats within 24 h postinstillation. This mortality resulted from mechanical blockage of the upper airways by the instillate and was not due to inherent pulmonary toxicity of the instilled SWCNT particulate. Exposures to quartz particles produced significant increases versus controls in pulmonary inflammation, cytotoxicity, and lung cell parenchymal cell proliferation indices. Exposures to SWCNT produced transient inflammatory and cell injury effects. Results from the lung histopathology component of the study indicated that pulmonary exposures to quartz particles (5 mg/kg) produced dose-dependent inflammatory responses, concomitant with foamy alveolar macrophage accumulation and lung tissue thickening at the sites of normal particle deposition. Pulmonary exposures to carbonyl iron or graphite particles produced no significant adverse effects. Pulmonary exposures to SWCNT in rats produced a non-dose-dependent series of multifocal granulomas, which were evidence of a foreign tissue body reaction and were nonuniform in distribution and not progressive beyond 1 month postexposure (pe). The observation of SWCNT-induced multifocal granulomas is inconsistent with the following: (1) lack of lung toxicity by assessing lavage parameters, (2) lack of lung toxicity by measuring cell proliferation parameters, (3) an apparent lack of a dose response relationship, (4) nonuniform distribution of lesions, (5) the paradigm of dust-related lung toxicity effects, (6) possible regression of effects over time. In addition, the results of two recent exposure assessment studies indicate very low aerosol SWCNT exposures at the workplace. Thus, the physiological relevance of these findings should ultimately be determined by conducting an inhalation toxicity study.

The detection of Hind III restriction-fragment length polymorphisms using a deoxyribonucleic acid probe for the beta subunit of follicle-stimulating hormone.

Molecular diagnosis of disorders of follicle-stimulating hormone (FSH) production may become possible now that the gene for FSH beta has been characterized. Restriction-fragment length polymorphism (RFLP) analysis provides a means of organized search for molecular variants of FSH. The purpose of this study was to screen controls for the presence of RFLPs using the deoxyribonucleic acid (DNA) probe pFSH beta -1.4. Genomic DNA was digested with 12 different restriction endonucleases; Southern blots were constructed and hybridized to pFSH beta -1.4. No polymorphisms were identified with 11 enzymes. Three of 24 (12.6%) Hind III digests demonstrated a polymorphic fragment of either 5.2, 4.7, or 4.3 kb. These are the first RFLPs identified for the FSH beta gene with pFSH beta -1.4. RFLPs for FSH beta constitute the first step in the molecular analysis of disorders of FSH production.

Identification of restriction-fragment length polymorphisms for the human chorionic gonadotropin-beta/luteinizing hormone-beta gene cluster.

OBJECTIVE: To determine whether restriction fragment length polymorphisms are present using a deoxyribonucleic acid (DNA) probe for human luteinizing hormone beta subunit (hLH-beta). If the gene for hLH-beta is polymorphic, genetic diagnosis of disorders of luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) production could become possible. DESIGN: Study of genomic DNA from controls with a variety of restriction enzymes to identify polymorphisms. SETTING: Laboratories of the Department of Obstetrics and Gynecology, Department of Oral Biology, Medical College of Georgia, Augusta, Georgia. PATIENTS: Unrelated control men and women seen in clinics at the Medical College of Georgia. INTERVENTIONS: Genomic DNA was extracted from patients and digested with eight different restriction enzymes for the study of the hLH-beta gene by Southern analysis. MAIN OUTCOME MEASURE: Fragment (band) sizes on radiographs from Southern blots were compared with those from molecular weight standards. CONCLUSIONS: Restriction fragment length polymorphisms were identified for four of the restriction enzymes, DraI, HincII, MboI, and KpnI. These polymorphisms may be useful in the diagnosis of disorders of hLH and hCG production.

Review of biotechnology applications to nuclear waste treatment.

This paper gives an overview of the feasibility of the application of biotechnology to nuclear waste treatment. The contents are based on a report which PA Technology carried out for the Department of the Environment (DoE Reference: DoE/RW/88.008 Sector No 2.3). Many living and dead organisms accumulate heavy metals and radionuclides. The controlled use of this phenomenon forms the basis for the application of biotechnology to the removal of radionuclides from nuclear waste streams. Indeed, biotechnology offers a series of new opportunities for removal of radionuclides from dilute aqueous process effluents. Such technology is already used for heavy metal removal on a commercial basis and could be optimised for radionuclide removal. An overview of biotechnology areas, namely the use of biopolymers and biosorption using biomass applicable to the removal of radionuclides from industrial nuclear effluents is given. The potential of biomagnetic separation technology, genetic engineering and monoclonal antibody technology is also to be examined. The most appropriate technologies to develop for radionuclide removal in the short term appear to be those based on biosorption of radionuclides by biomass and the use of modified and unmodified biopolymers in the medium term.

Data base management system for a radiation safety program.

A data base management system (DBMS) has been developed that simplifies the retrieval of data concerning radioisotope use at a university and hospital. The system customizes software that is commercially available to perform several functions. Reports can be developed concerning receipt of radioactive materials, radioactive waste disposal, and research proposals submitted by investigators. Reports can be prepared that utilize the software's ability to perform numerical calculations. The main advantage of the DBMS is that it allows the easy retrieval of information that is used in the day-to-day operation of a radiation safety office; it also provides easy access and manipulation of data for the preparation of reports, budget proposals, and justifications for purchases.

Implementation of a capillary array electrophoresis instrument.

A capillary array electrophoresis (CAE) apparatus capable of running and analyzing DNA samples in 48 capillaries simultaneously has been constructed. The capillaries are individually replaceable, and sieving buffer can be easily pumped in and out of the capillary array as necessary. Samples are injected electrokinetically from polymerase chain reaction (PCR, Hoffmann-LaRoche, Nutley, NJ, U.S.A.) tubes arranged in a 6 x 8 format and are detected by laser-induced fluorescence. Data analysis software has been developed for semiautomatic analysis, including peak finding and DNA fragment sizing. The system represents a robust apparatus for the rapid and convenient analysis of DNA fragments in a high-throughout environment.

High-throughput DNA sequencing on a capillary array electrophoresis system.

A capillary array electrophoresis apparatus capable of running and analyzing 48 DNA sequencing samples simultaneously has been constructed. The instrument uses a replaceable sieving buffer and incorporates a convenient method for introducing the buffer into the capillaries. Data from laser-induced fluorescence are collected as four separate images, one for each optical channel. The integrated data analysis software employs an open architecture that allows use of any DNA base-calling algorithm. DNA sequencing runs are completed in approx. 1 hr (approximately 500 bases), and instrument turnaround time between runs is less than 15 min. Overall, the instrument throughput is on the order of 720 templates/day, or 360,000 bases/day.

The compliance conundrum.

Complex regulations and statutes, murky guidance from government agencies, and overly zealous enforcement initiatives make the legal questions in the healthcare arena a veritable mine field. This Article examines some of the issues that arise in the healthcare legal setting, explains why compliance programs are difficult to establish, and analyzes the issues that arise when providers attempt to establish them. Finally, the authors suggest changes to the governing legal framework that would facilitate achievement of the government's goals in a more efficient and just manner.

Microleakage of composite resin restorations with various etching times.

Microleakage was assessed in Class V composite resin restorations, placed in extracted noncarious human premolar teeth after the enamel cavosurface margins of the preparations had been etched with phosphoric acid for 5, 15, or 30 seconds. The restored teeth were thermocycled, placed in methylene blue dye, invested, and sectioned. The extent of dye penetration along the tooth-restoration interface in the three etching groups was compared. The group etched for 5 seconds showed the greatest leakage; leakage was statistically significantly greater than that in the groups etched for 15 or 30 seconds. The tooth-restoration interface at the occlusal and gingival margins showed more leakage than did the mesial or distal margins.

Effects of some social variables on field dependence.

The effects of socio-economic status, father's absence, family size, and birth order on the field dependence of Grade 6 elementary school children in Jamaica, West Indies, were examined. To 206 boys and 212 girls from 5 urban schools the Hidden Figures Test and a questionnaire were administered. For girls, but not for boys, field independence correlated significantly and positively with socio-economic status, but significantly and negatively with father's absence. No significant relation existed for the boys. Field dependence had no significant relation with family size and birth order.