Embryoid body morphology influences diffusive transport of inductive biochemicals: a strategy for stem cell differentiation

Differentiation of human embryonic stem (hES) cells into cells for regenerative medicine is often initiated by embryoid body (EB) formation. EBs may be treated with soluble biochemicals such as cytokines, growth factors and vitamins to induce differentiation. A scanning electron microscopy analysis, conducted over 14 days, revealed time-dependent changes in EB structure which led to the formation of a shell that significantly reduced the diffusive transport of a model molecule (374 Da) by >80%. We found that the shell consists of 1) an extracellular matrix (ECM) comprised of collagen type I; 2) a squamous cellular layer with tight cell-cell adhesions associated with E-cadherin; and 3) a collagen type IV lining indicative of a basement membrane. Disruption of the basement membrane, by either inhibiting its formation with noggin or permeabilizing it with collagenase, resulted in recovery of diffusive transport. Increasing the diffusive transport of retinoic acid (RA) and serum in EBs by a 15-min collagenase digestion on days 4, 5, 6 and 7 promoted neuronal differentiation. Flow cytometry and quantitative RT-PCR analysis of collagenase-treated EBs revealed 68% of cells expressing neural cell adhesion molecule (NCAM) relative to 28% for untreated EBs. Our results suggest that limitations in diffusive transport of biochemicals need to be considered when formulating EB differentiation strategies.     

The tandem bypass: subclavian artery-to-middle cerebral artery bypass with dacron and saphenous vein grafts. Technical case report

BACKGROUND: Fusiform or dolichoectatic intracranial aneurysms often cannot be managed with conventional surgical or endovascular techniques, and instead require trapping and revascularization techniques. On rare occasions in elderly patients, extracranial sites used for anastomosing the bypass have been previously repaired with synthetic vascular prostheses. This circumstance in an elderly subarachnoid hemorrhage patient led to a novel bypass procedure, the tandem bypass: a long extracranial-to-intracranial bypass with two grafts of different materials assembled in series. CASE DESCRIPTION: A 71-year-old man with carotid artery atherosclerotic disease and a previous vascular reconstruction (subclavian artery-to-internal carotid artery Dacron interposition graft) presented with a subarachnoid hemorrhage from a dolichoectatic supraclinoid ICA aneurysm. The aneurysm was treated with trapping and distal revascularization. The final construct was a subclavian artery-to-middle cerebral artery bypass, with the graft being the previous Dacron prosthesis and a long saphenous vein. The vein graft was anastomosed end-to-side to the Dacron graft proximally, and end-to side to the middle cerebral artery distally. Subsequently, inflow to the aneurysm was occluded with clips on the Dacron graft beyond the proximal anastomosis of the vein graft, and outflow from the aneurysm was occluded with clips on the supraclinoid ICA. CONCLUSIONS: The tandem bypass, which uses prosthetic graft material and saphenous vein in succession, is a technically straightforward technique in patients who need extracranial-to-intracranial bypasses and who also have pre-existing carotid reconstructions or lack sufficient saphenous vein to complete a long bypass.     

Assessing Advanced Communication Skills via Objective Structured Clinical Examination: A Comparison of Faculty Versus Self,Peer, and Standardized Patient Assessors

Abstract

Construct: The construct addressed in this study is assessment of advanced communication skills among senior medical students. Background: The question of who should assess participants during objective structured clinical examinations (OSCEs) has been debated, and options discussed in the literature have included peer, self, standardized patient, and faculty assessment models. What is not known is whether same-level peer assisted learning can be utilized for formative assessment of advanced communication skills when no faculty, standardized patients, or other trained assessors are involved in providing feedback. If successful, such an educational model would optimize resource utilization and broaden the scope of topics that could be covered in formative OSCEs. Approach: The investigators developed a 4-station formative OSCE focused on advanced communication skills for senior medical students, and evaluated the concordance of assessment done by same-level peers, self, standardized patients, and faculty for 45 students. After each station, examinees completed a self-assessment checklist and received checklist-based assessment and verbal feedback from same-level peers only. Standardized patients completed checklist-based assessments outside the room, and faculty did so after the OSCE via video review; neither group provided direct feedback to examinees. The investigators assessed inter-rater agreement and mean difference scores on the checklists using faculty score as the gold standard. Findings: There was fair to good overall agreement among self, same-level peer, standardized patient, and faculty-assessment of advanced communication skills. Relative to faculty, peer and standardized patient assessors overestimated advanced communication skills, while self-assessments underestimated skills. Conclusions: Self and same-level peer-assessment may be a viable alternative to faculty assessment for a formative OSCE on advanced communication skills for senior medical students.     

Effects of temporal lobectomy on consciousness-impairing and consciousness-sparing seizures in children

Purpose

Most children with medically refractory temporal lobe epilepsy (TLE) become seizure free after temporal lobectomy, but some individuals continue to seize. As studies of temporal lobectomy typically focus on seizure freedom, the effect of surgery on seizure type and frequency among children with persistent seizures is poorly understood. Seizures which impair consciousness are associated with increased morbidity compared to consciousness-sparing seizures.

Methods

A retrospective cohort study was performed to evaluate the effects of temporal lobectomy on seizure type and frequency in children with intractable TLE.

Results

Among 58 pediatric TLE patients with a mean (±SEM) age of 14.0?±?0.7 years who received temporal lobectomy, 46 (79.3 %) individuals achieved an Engel class I seizure outcome, including 38 (65.5 %) children who became completely seizure free (Engel IA). Mean follow-up was 2.7?±?0.4 years. While the number of patients experiencing simple partial seizures (SPSs) (consciousness sparing) decreased by only 23 % after surgery, the number of children having complex partial seizures and generalized tonic–clonic seizures (consciousness impairing) diminished by 87 and 83 %, respectively (p?<?0.01). SPS was the predominant seizure type in only 11.3 % of patients before resection, but in 42.1 % of patients with postoperative seizures (p?<?0.01). Children with postoperative seizures experienced a 70 % reduction in overall seizure frequency compared to baseline (p?<?0.05), having consciousness-impairing seizures 94 % less frequently (p?<?0.05), but having consciousness-sparing seizures 35 % more frequently (p?=?0.73).

Conclusions

Seizure type and frequency are important considerations in the medical and surgical treatment of children with epilepsy, although complete seizure freedom remains the ultimate goal.     

Computed axial tomography (CAT) contribution for dosimetry and treatment evaluation in lung cancer

The use of computed axial tomography (CAT) scans in postoperative patients with lung cancer was studied to evaluate its contribution in dosimetry and to study the late effects of irradiation. Comparisons were made between the treatment planning generated from CAT scan data and that obtained from two orthogonal radiographs. Both methods offered a good approximation but with orthogonal radiographs possible mediastinal and lung shift could not be seen and the dose delivered to the spinal cord was overestimated. A control CAT scan performed 6 to 18 months after treatment showed a lung fibrosis that was strictly correlated with the treatment planning and related with doses and volume treated. CAT scans allowed more accurate treatment planning.     

Effect of operative devascularization on estrogen and progesterone receptor levels in breast cancer specimens

To compare estrogen and progesterone receptor values between biopsy and mastectomy specimens, we prospectively studied 29 patients with breast cancer treated by incisional biopsy followed by mastectomy. The average tumor size was 5.4 +/- 0.5 cm and the mean age was 57.6 +/- 3.0 years. Nine patients were premenopausal and 20 were postmenopausal. Biopsies were performed without electrocautery, and tissue samples were promptly frozen and subsequently assayed for estrogen and progesterone receptor levels. After mastectomy, samples of residual tumor were excised from the biopsy site, promptly frozen, and assayed for estrogen and progesterone receptor levels. Operating time averaged 87.7 +/- 6.0 minutes. Estrogen receptor levels averaged 100.0 +/- 24.4 femtomole per mg on biopsy specimens and 29.5 +/- 8.2 fmol/mg on mastectomy specimens, representing a 70% decline (p less than 0.02). Of clinical significance is the fact that eight of 29 (27.6%) tumors changed from positive estrogen receptor values in the biopsy specimen to negative (four) and borderline (four) in the mastectomy specimens. Progesterone receptor levels were more variable, but their mean value decreased by 24.4% from 17.6 +/- 6.4 fmol/mg on biopsy samples to 13.3 +/- 4.3 fmol/mg on mastectomy samples (p, not significant). We conclude that the biopsy specimen is usually a more reliable indicator of hormonal receptor status than the mastectomy specimen and recommend that incisional or excisional biopsy specimens be taken for estrogen and progesterone receptor assays before mastectomy.     

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.     

Dot enzyme-linked immunosorbent assay for more reliable staging of patients with Human African trypanosomiasis

Human African trypanosomiasis (HAT) or sleeping sickness is a disease characterized by a hemolymphatic stage 1 followed by a meningoencephalitic stage 2 which is fatal without specific treatment. Furthermore, due to the toxicity of drugs used to treat stage 2 (mainly melarsoprol) accurate staging is required. Actual criteria employed during field surveys are not sensitive enough for precise staging. Antineurofilament (anti-NF) and antigalactocerebrosides (anti-GalC) antibodies have been identified in cerebrospinal fluid (CSF) as potential markers of central nervous system (CNS) involvement. We describe a dot enzyme-linked immunosorbent assay (dot-ELISA) to detect anti-GalC and anti-NF antibodies and its value in staging. NF- and GalC-dotted nitrocellulose strips were first developed in our laboratory. They were then evaluated in Angola and Central African Republic on 140 CSF samples. Compared to our staging criteria (i.e., CSF cell count > or = 20 cells/microl, CSF immunoglobulin M concentration > or = 100 mg/liter, and/or the presence of trypanosomes in the CSF), combined detection of both CSF anti-NF and CSF anti-GalC by dot-ELISA showed 83.2% sensitivity and 100.0% specificity. Dot-ELISA could be a useful test to diagnose CNS involvement in HAT in the less-equipped laboratories or in the field situation and to improve patient treatment.