Advancing brain barriers RNA sequencing: guidelines from experimental design to publication.

BACKGROUND: RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process. MAIN BODY: In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN ( https://www.btrain-2020.eu/ ) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood-brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community. CONCLUSION: Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community.

Screening Spirometry in Military Personnel Correlates Poorly with Exercise Tolerance and Asthma History.

BACKGROUND: Spirometry is an easy-to-perform test for evaluating pulmonary symptoms but has several limitations to include adequate test performance for valid results. Spirometry is not recommended to screen a general population for evidence of pulmonary disease unless symptoms are present or longitudinal screening is done for potential occupational exposures. METHODS: A single-spirometry examination was performed on 900 active duty Army soldiers, ages 18-35 yr, without documented respiratory disease. Abnormal studies were identified (obstructive, restrictive, mixed, and flow volume loop abnormalities) and compared with reported respiratory symptoms, smoking history, prior diagnosis of asthma, and failure of the Army physical fitness test 2-mile run using generalized linear modeling techniques. RESULTS: The cohort population had spirometry values comparable with published NHANES III reference values. Ninety-eight subjects (10.9%) were identified with abnormal spirometry included 33 obstructive, 44 restrictive, 3 mixed, and 18 isolated flow volume loop abnormalities. Historical features (smoking, exertional dyspnea, cough, asthma, or APFT failure) had no effect on the probability of an abnormal spirometry result (p = 0.56). Although APFT failure probability is strongly affected by exertional dyspnea (p = 0.00) and current smoking (p = 0.01), abnormal spirometry results did not have a statistically significant effect (p = 0.38). DISCUSSION: For potential screening of military personnel with spirometry to detect pulmonary disease, study findings identified a significant percentage with non-specific abnormalities requiring further evaluation. Spirometry may be indicated in those individuals with a history of asthma or active dyspnea symptoms. Spirometry as a screening tool is poorly (and often incorrectly) predictive for respiratory symptoms or decreased exercise tolerance in a military cohort. CONCLUSION: Spirometry should not be used to screen the military force for the presence of respiratory disorders. In those individuals with a history of asthma, in-depth testing should be performed to fully evaluate any non-specific findings identified during spirometry.

Open versus Endovascular Repair of Abdominal Aortic Aneurysm in the Elective and Emergent Setting in a Pooled Population of 37,781 Patients: A Systematic Review and Meta-Analysis.

Background. We evaluated the incidence of mortality and myocardial infarction (MI) in endovascular repair (EVAR) as compared to open aneurysm repair (OAR) in both elective and ruptured abdominal aortic aneurysm (AAA ) setting. Methods. We analyzed the rates of 30-day mortality, 30-day MI, and hospital length of stay (LOS) based on comparative observation and randomized control trials involving EVAR and OAR. Results. 41 trials compared EVAR to OAR with a total pooled population of 37,781 patients. Analysis of elective and ruptured AAA repair favored EVAR with respect to 30-day mortality with a pooled odds ratio of 0.19 (95% CI 0.17-0.20; I (2) = 88.9%; P < 0.001). There were a total of 1,835 30-day MI events reported in the EVAR group as compared to 2,483 events in the OAR group. The pooled odds ratio for elective AAA was 0.74 (95% CI 0.58-0.96; P = 0.02) in favor of EVAR. The average LOS was reduced by 296.75 hrs (95% CI 156.68-436.82 hrs; P < 0.001) in the EVAR population. Conclusions. EVAR has lower rates of 30-day mortality, 30-day MI, and LOS in both elective and ruptured AAA repair.

Studies on the toxicity of an aqueous suspension of C60 nanoparticles using a bacterium (gen. Bacillus) and an aquatic plant (Lemna gibba) as in vitro model systems.

The increasing use of C60 nanoparticles and the diversity of their applications in industry and medicine has led to their production in a large scale. C60 release into wastewaters and the possible accumulation in the environment has raised concerns about their ecotoxicological impact. In the present study, an aqueous suspension of C60 nanoparticles was prepared and its potential toxicity studied in laboratory, using a bacterium (Bacillus stearothermophilus) and an aquatic plant (Lemna gibba) as model systems. C60 nanoparticles inhibited the growth of L. gibba, in contrast to that of the bacterium. Consistently, the ultrastructure and respiratory activity of bacterial cells were not affected by C60, but the contents of chlorophylls a and b and chloroplast oxygen production decreased considerably in L. gibba. Altogether, our results suggest that C60 aqueous dispersions must be viewed as an environmental pollutant, potentially endangering the equilibrium of aquatic ecosystems.

The tones of the kalimba (African thumb piano).

The acoustic spectrum of the kalimba (African thumb piano) is measured and analyzed for tonal structure. The frequency f(1) of the fundamental tone of each tine (key) is investigated in relation to the frequencies of its two dominant overtones, f(2) and f(3). These frequencies are identified as the first three modes of transverse vibration of a beam of rectangular cross section. As is typical for vibrating-beam instruments, the overtone sequence is inharmonic, that is, the sequence f(1), f(2), f(3),[ellipsis (horizontal)] is unevenly spaced and the frequency ratios f(2)/f(1) and f(3)/f(1) are not integers. The kalimba tines are modeled by applying the Euler-Bernoulli beam equation with one end clamped, the other end free, and an intermediate point (the bridge) simply supported. Unlike the cases of free-free and clamped-free beams, it is found that the clamped-supported-free frequency ratios f(2)/f(1) and f(3)/f(1) are not fixed values, but depend uniquely upon where the bridge supports and subdivides the tine. The model solution is more thoroughly investigated analytically for the special case in which the beam segment ratio is unity, which has some analytic solutions. Numerically computed mode frequencies agree well with acoustic measurements, validating the model. Mode shapes are computed for the first three modes of a typical tine.

Using streamlines to visualize acoustic energy flow across boundaries.

For spherical waves that radiate from a point source in a homogeneous fluid and propagate across a plane boundary into a dissimilar homogeneous fluid, the acoustic field may differ significantly from the geometric acoustic approximation if either the source or receiver is near the interface (in acoustic wavelengths) or if the stationary phase path is near the critical angle. In such cases, the entire acoustic field must be considered, including inhomogeneous waves associated with diffraction (i.e., those components that vanish with increasing frequency). The energy flow from a continuous-wave monopole point source across the boundary is visualized by tracing acoustic streamlines: those curves whose tangent at every point is parallel to the local acoustic intensity vector, averaged over a wave cycle. It is seen that the acoustic energy flow is not always in line with the "Snell's law" or stationary phase path. Also, plots of acoustic energy streamlines do not display unusual behavior in the vicinity of the critical angle. Finally, it is shown that there exists a law of refraction of acoustic energy streamlines at boundaries with density discontinuities analogous to Snell's law of refraction of ray paths across sound speed discontinuities. Examples include water-to-seabed transmission and water-to-air transmission.

Medical qualification of a commercial spaceflight participant: not your average astronaut.

BACKGROUND: Candidates for commercial spaceflight may be older than the typical astronaut and more likely to have medical problems that place them at risk during flight. Since the effects of microgravity on many medical conditions are unknown, physicians have little guidance when evaluating and certifying commercial spaceflight participants. This dynamic new era in space exploration may provide important data for evaluating medical conditions, creating appropriate medical standards, and optimizing treatment alternatives for long-duration spaceflight. CASE: A 57-yr-old spaceflight participant for an ISS mission presented with medical conditions that included moderately severe bullous emphysema, previous spontaneous pneumothorax with talc pleurodesis, a lung parenchymal mass, and ventricular and atrial ectopy. The medical evaluation required for certification was extensive and included medical studies and monitoring conducted in analogue spaceflight environments including altitude chambers, high altitude mixed-gas simulation, zero-G aircraft, and high-G centrifuge. To prevent recurrence of pneumothorax, we performed video-assisted thoracoscopic pleurodesis, and to assess lung masses, several percutaneous or direct biopsies. The candidate's 10-d mission was without incident. CONCLUSION: Non-career astronauts applying for commercial suborbital and orbital spaceflight will, at least in the near future, challenge aerospace physicians with unknowns regarding safety during training and flight, and highlight important ethical and risk-assessment problems. The information obtained from this new group of space travelers will provide important data for the evaluation and in-flight treatment of medical problems that space programs have not yet addressed systematically, and may improve the medical preparedness of exploration-class missions.

The effects of ignored seabed variability in geoacoustic inversion.

Geoacoustic inversion using a matched-field inversion algorithm is a well-established technique for estimating the geoacoustic parameters of the seabed. This paper demonstrates how parameter estimation can be affected by unknown or wishfully ignored random range dependence of the true environment when the inversion model is--for practicality--assumed to be range independent. Simulations with controlled statistics were carried out using a simple shallow water model: an isospeed water column over a homogeneous elastic halfspace. The inversion parameters included water depth, compressional speed in the seabed, seabed density, and compressional wave attenuation. On average the environment is range independent: some parameters are constant while other parameters are random with range-independent means and variances. A Parabolic Equation underwater acoustic propagation model is used to calculate the simulated data fields for the range-dependent environment as well as to calculate the model fields for the range-independent inversion model. The Adaptive Simplex Simulated Annealing inversion algorithm is used to estimate the best-fit solution. It is found that ignoring the variability of even a single geoacoustic parameter leads to significant and correlated uncertainty (bias and variance) in the estimation of all inverted parameters. Results are presented for range variation of compressional sound speed and water depth.

Thirty-day mortality of nosocomial systemic bacterial infections according to antibiotic susceptibility in an 800-bed teaching hospital in France.

Overall 30-day mortality among patients with nosocomial bacterial infections was analysed according to antibiotic susceptibility in an 800-bed hospital. Survival analysis identified three factors associated with an increased mortality rate: (1) a longer interval between admission and onset of infection; (2) bacterial resistance; and (3) the severity of the initial illness in the intensive care unit. The increased mortality rate associated with antibiotic resistance was observed for all bacterial species with the exception of Gram-positive cocci, and remained significant or near-significant regardless of the hospital ward.

The role of tropomyosin in the regulation of myocardial contraction and relaxation.

Studies over the last 30 years have demonstrated the essential nature of the evolutionarily highly conserved tropomyosin (TM) protein. TM-deficient cells neither function properly nor survive, and mutations within this protein impair severely its function within the sarcomere. The ability to manipulate TM isoform expression genetically within functioning cardiomyocytes and the whole heart has proven essential in deciphering the physiological significance of the different TM isoforms. It is now apparent that alpha-TM and actin serve as the requisite backbone of the thin filament, with varying levels of beta- and gamma-TM, together with the troponin complex serving to modulate sarcomere function. Defining the mechanisms whereby specific TM isoform amino acid differences alter thin filament dynamics will enhance greatly the understanding of muscle contraction during both normal and pathological states.

Underwater measurements and modeling of a sonic boom.

During a sea trial on the Scotian Shelf, acoustic signals from a sonic boom were recorded on 11 hydrophones of a vertical array. The array spanned the lower 50 m of the water column above a sand bank at 76 m water depth. The source of the sonic boom was deduced to be a Concorde supersonic airliner traveling at about Mach 2. The waterborne waveform was observed to decay as an evanescent wave below the sea surface, as expected. The calm weather (sea state 1) resulted in low ambient noise and low self-noise at the hydrophones, and good signal-to-noise ratio on the upper hydrophones; however, the decreased signal amplitude is more difficult to detect towards the lower part of the water column. The period of the observed waveform is of the order 0.23 s, corresponding to a peak frequency of about 3 Hz. The shape of the measured waveform differs noticeably from the theoretical N-shape waveform predicted with Sawyers' theory [J. Acoust. Soc. Am. 44, 523-524 (1968)]. A simple shallow-ocean geoacoustic model suggests that this effect may be caused in part by seismo-acoustic interaction of the infrasonic waves with the elastic sediments that form the seabed.

Educational benefits of student self-marking of short-answer questions.

A pilot study was undertaken to evaluate the feasibility and reliability of undergraduate medical student selfmarking of degree written examinations, and to survey student opinion regarding the process. The correlation between student and faculty staff scores for individual questions and the total examination was high (correlation coefficient ranged from 0.77 to 0.91: p < 0.001). There were no significant differences between the mean student and mean faculty staff scores for individual questions or the total examination: 98% (97199) of student scores fell within ± 15% of the faculty staff score, with 92% (91199) of students falling within ± 10%. Although the approach was demonstrated to be reliable, students generally failed to acknowledge the potential value of self-marking in terms of feedback and as a learning opponunity, and found the process stressful.

The role of assessment in expanding professional horizons.

This paper explores assessment innovations which have a system-wide effect on medical education and the medical profession. Important assessment approaches such as the objective structured clinical examination (OSCE), the portfolio, and hi-tech simulations are examples of reform-driven developments. A detailed account is provided on assessment areas that require further developments. The identified areas reflect current thinking in the Centre for Medical Education, University of Dundee Medical School.The assessment innovations are being developed alongside the implementation of the outcome-based curriculum. Areas that require extensive work are: assessment of progression towards defined outcomes, assessment of integrated abilities, assessment of different forms of medical knowledge, assessment of on-the-job learning, learning through assessment, assessment of error management and assessment of portfolio evidence. The identified areas for further assessment development are discussed and where appropriate a theoretical framework is provided.

Long-term ganciclovir therapy for hepatitis B virus infection after liver transplantation.

BACKGROUND/AIMS: Hepatitis B virus (HBV) disease on a liver graft is associated with florid viral replication and graft failure. The aim of this study performed between 1992 and 1995 was to investigate the safety and efficacy of long-term intravenous ganciclovir for HBV infection in liver transplant recipients. METHODS: Twelve patients with HBV re-infection and four with de novo HBV infection were studied. HBV DNA was positive in all (median titer: 437.5 pg/ml) and HBeAg was positive in seven. Intravenous ganciclovir was started after a median of 14.5 months from HBsAg positivation and continued for a median of 10 months. RESULTS: A complete response with HBV DNA negativation was seen in ten cases, a partial response with a decrease of more than 50% of initial HBV DNA levels in four and no response in two. Overall tolerance was good. Among the ten complete responders, two seroconverted for both HBsAg and HBeAg and one for HBsAg alone. Among these ten patients, three were re-transplanted for liver failure: two of them are alive; three had a viral breakthrough during treatment; and four remained HBV DNA negative: two are alive and two died. Partial responders and nonresponders were treated with other antiviral agents and three were re-transplanted, two of them are alive. Overall 12 out of 16 patients (75%) survived with a median follow up of 46 months. CONCLUSIONS: Long-term intravenous ganciclovir can persistently inhibit HBV DNA replication in liver transplant recipients and is well tolerated. Further evaluation should be encouraged, especially for HBV recurrence after first-line treatments.

The performance of foreign medical graduates on the National Board of Medical Examiners (NBME) standardized patient examination prototype: a collaborative study of the NBME and the Educational Commission for Foreign Medical Graduates (ECFMG).

OBJECTIVES: The purpose of the study was to explore foreign medical graduates' (FMGs) performance on a clinical skills (SPX) examination. The National Board of Medical Examiners (NBME) is in the process of developing an SPX for potential use in the United States Medical Licensing Examination (USMLE). The Educational Commission for Foreign Medical Graduates (ECFMG) is developing the Clinical Skills Assessment (CSA) as an additional requirement for FMGs who wish to be certified by ECFMG. DESIGN: Thirty-three FMGs and 151 United States medical students (USMSs) took the SPX during the winter of 1996 as part of the ongoing pilot studies conducted by the NBME. Four clinical skill areas were assessed: history-taking, physical examination, communication and interpersonal skills. The examination used in this research consisted of 12 cases. The examination utilizes standardized patients (SPs) who are trained to document examinee behaviours and evaluate the communication component of the test. The SPs were also trained to evaluate the English proficiency of the candidates. Candidates were also administered the Test of Spoken English developed by the Educational Testing Services (ETS). SETTING: The examination was conducted in one medical school which served as an SPX centre for NBME pilot studies. SUBJECTS: Thirty-three foreign medical students and 151 US medical students. RESULTS: The indications were that the majority of candidates in both groups felt the examination was moderately fair but 78% of FMGs felt moderately pressed for time, vs. 80% of the USMSs who did not feel pressed for time. Reliabilities obtained for the various SPX components were somewhat higher for the FMGs reflecting the heterogeneity of this group. CONCLUSIONS: The NBME-ECFMG collaborative study yielded important information regarding the NBME SPX prototype as a performance measure for FMGs.

[Assessment of clinical competence of medical school graduates in Italy with standardized patients. The opinion of the examinees]. / Sperimentazione in Italia del metodo del paziente standardizzato per la valutazione della competenza clinica. L'opinione dei neolaureati.

An international collaborative project for the evaluation of clinical competence at the end of the Medical School curriculum using the ECFMG-CSA (Educational Commission for Foreign Medical Graduates--Clinical Skills Assessment) prototype was started in Italy in April 1996. Faculty representatives from Italian Medical Schools and experts from the ECFMG in Philadelphia participated in the Project. The CSA consists of integrated clinical encounters with 10 standardized patients during which the examinee is asked to obtain a focused history, perform a relevant physical examination and communicate initial diagnoses and management plan to the Standardized Patient (SP). The SP then completes checklists that are scored by Faculty members. The project was concluded in Spring 1998 and a total of 173 new graduates were examined. The data elaborated by the primary site in Chieti University will be available in the Fall 1998 by the ECFMG in Philadelphia. This preliminary communication reports the opinions of the examinees on the ECFMG-CSA, contained in the questionnaires administered after the test. Most of the examinees considered this new methodology as a valid tool for the assessment of clinical competence, especially history-taking and interpersonal skills and stated that the SP simulations were realistic. The 72% of examinees indicated that the Medical School curriculum does not adequately prepare for the CSA examination. Lastly, 68% was in favour of including the SP in the Medical Licensing Examination.

Lessons learned from six years of international administrations of the ECFMG's SP-based clinical skills assessment.

PURPOSE: The Educational Commission on Foreign Medical Graduates (ECFMG) conducted international clinical skills assessments (CSAs) to evaluate the readiness of foreign medical graduates to enter U.S. residency programs, to validate national medical examinations in other countries, and to introduce other countries to new methods of evaluating medical students. METHOD: The ECFMG conducted CSA studies in the United States, Israel, Spain (Madrid and Barcelona), Ukraine, and Brazil between 1989 and 1995. ECFMG staff worked with local teams in following a seven-phase implementation process. The CSAs were conducted in each country's native language, and clinical cases were translated from English and culturally adapted. A total of 636 examinees took a ten-station standardized patient-based examination. RESULTS: A comparison of test results indicated stable psychometric properties and similar patterns of relationships among test components across all six countries. In every country, physical-examination and patient-note mean scores were lower than were history-taking scores, indicating the possibility of common skill deficiencies. CONCLUSION: The successful completion of international CSA projects in six countries suggests that high-quality standardized CSA projects are feasible and can be implemented from long distances.

De novo and apparent de novo hepatitis B virus infection after liver transplantation.

BACKGROUND/AIMS: The aim of this study was to clarify the aetiology of apparent de novo HBV infection after liver transplantation. METHODS: Twenty out of 570 HBsAg negative patients (3.5%) became HBsAg positive after transplantation and were studied. Donor and recipient sera were retrospectively tested for HBsAg, anti-HBs, anti-HBc, and HBV DNA by PCR. Donor and recipient livers were tested for HBV DNA by PCR on paraffin-embedded tissue. RESULTS: Group 1: HBV infection of donor origin (eight patients): one donor serum was HBsAg positive, three were serum HBV DNA positive, four were liver HBV DNA positive. Group 2: reactivation of latent HBV infection (eight patients) with detection of HBV DNA in pretransplant serum (seven patients) or in native liver (one patient): three were anti-HBs positive, two anti-HBc positive, and three with fulminant hepatitis had no serological HBV markers. Group 3: undetermined origin (four patients) defined by absence of HBV DNA in pretransplant donor and/or recipient sera and liver; however, acquired infection was suspected from two anti-HBs and anti-HBc positive donors. Two patients became HBsAg negative, and five HBV DNA negative. One died from HBV-cirrhosis and two were retransplanted. In the others, the last histology showed cirrhosis (three), chronic hepatitis (nine), acute hepatitis (one), and non-specific change (four patients). CONCLUSIONS: The prevalence of de novo HBV infection in liver transplant patients was 3.5%; the aetiology was determined in 16/20 patients: from the donor in eight, and from the recipient in eight. One should be cautious when donors or recipients are anti-HBc or both anti-HBs and anti-HBc positive.