Packed bed compression visualisation and flow simulation using an erosion-dilation approach.

X-ray computed tomography has been demonstrated to be capable of imaging 1 mL (5 mm diameter, 50 mm height) chromatography packed beds under compression, visualising the 3D structure and measuring changes to geometry of the packing. 1 mL pre-packed columns did not exhibit any structural changes at vendor specified flow rate limits, however cellulose beds did compress at higher flow rates that were imaged before, during and after flow. This was used to visualise and quantitate changes to porosity, tortuosity and permeability based on simulation of flow through the packed bed structure using the imaging data. When using a high flow rate it was found that a decrease in porosity could be measured during compression before reverting after flow had ceased, with corresponding changes to tortuosity and permeability also occurring. X-ray CT imaging of packed beds and individual beads exposed to foulant-rich process streams resulted in considerable image quality loss, associated with residual biological material. In order to address this, digital processing using an erosion-dilation method was applied at bead and bed scales to computationally alter the porosity by adding or removing material from the existing surface to calculate the impact upon tortuosity factor. The eroded and dilated bead volumes of agarose, cellulose and ceramic materials were used to simulate diffusivity whilst mimicking internal bead pore constriction and blocking mechanisms.

Three dimensional characterisation of chromatography bead internal structure using X-ray computed tomography and focused ion beam microscopy.

X-ray computed tomography (CT) and focused ion beam (FIB) microscopy were used to generate three dimensional representations of chromatography beads for quantitative analysis of important physical characteristics including tortuosity factor. Critical-point dried agarose, cellulose and ceramic beads were examined using both methods before digital reconstruction and geometry based analysis for comparison between techniques and materials examined. X-ray 'nano' CT attained a pixel size of 63 nm and 32 nm for respective large field of view and high resolution modes. FIB improved upon this to a 15 nm pixel size for the more rigid ceramic beads but required compromises for the softer agarose and cellulose materials, especially during physical sectioning that was not required for X-ray CT. Digital processing of raw slices was performed using software to produce 3D representations of bead geometry. Porosity, tortuosity factor, surface area to volume ratio and pore diameter were evaluated for each technique and material, with overall averaged simulated tortuosity factors of 1.36, 1.37 and 1.51 for agarose, cellulose and ceramic volumes respectively. Results were compared to existing literature values acquired using established imaging and non-imaging techniques to demonstrate the capability of tomographic approaches used here.

CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

Granuloma faciale in a child successfully treated with the pulsed dye laser.

Granuloma faciale, a rare, chronic, cutaneous disorder, is uncommon in children. Numerous treatment modalities have been used to treat granuloma faciale but none are consistently effective. In addition, many of the previously used therapies have unwanted side effects or are not well tolerated by children. We report a case of granuloma faciale in an 11-year-old child successfully treated with the pulsed dye laser. To our knowledge, this is the first report of this treatment modality for granuloma faciale.

Tracheobronchial mucoepidermoid carcinoma in childhood and adolescence: case report and review of the literature.

In children and adolescents, primary neoplasms of the tracheobronchial tree and lungs are rare, with most tumors involving the respiratory system being metastatic, small, blue cell tumors of childhood. Of the primary pulmonary neoplasms, most are malignant with mucoepidermoid carcinoma representing about 10% of these malignant tumors. We present an 8-year-old Hispanic male with hemoptysis and several episodes of pneumonia which initially was thought to be infectious upon biopsy during bronchoscopy, but proved to be mucoepidermoid carcinoma of the tracheobronchial tree by microscopic examination during an open lung biopsy. This rare tumor is more common in adults than in children, and infrequently presents with hemoptysis. Mucoepidermoid tumors of the tracheobronchial tree carry a more favorable prognosis in children than adults. In the adult population, the overall mortality is slightly less than 30%. In contrast, of the 31 reported cases of tracheobronchial mucoepidermoid carcinoma in pediatrics, all children are free of tumor involvement with a mean follow-up period of 5.8 years (range, 0.7-21 years). Based upon the available clinical outcome and survival data, it would appear that tracheobronchial mucoepidermoid carcinoma may be successfully managed by surgical intervention alone in children and adolescents.

Subacute care: evolution in search of value.

The postacute continuum of nonhospital-based services has experienced rapid growth over the past decade because of a variety of economic factors. Medicare's prospective payment system for inpatient hospital stays combined with the diagnostic requirements of patients in acute medical rehabilitation and the growth of managed care have all reinforced the development of alternative levels and venues of care. Market forces are leading to consolidation and integrated health delivery systems offering both acute and postacute service options. The cost-effectiveness of these new models indicates promise for increased value measured by comparing resource utilization with outcomes. Reimbursement of the developing continuum, however, offers little incentive for efficiency. This article discusses the present dynamics of postacute growth and examines the issues that should be evaluated to determine its relevance in the spectrum of care.

Beta-turns induced in bradykinin by (S)-alpha-methylproline.

The ability of (S)-alpha-methylproline (alpha-MePro) to stabilise reverse-turn conformations in the peptide hormone bradykinin (BK = Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) has been investigated. Two BK analogues containing alpha-MePro at position 3 or position 7 were synthesised and their conformations in aqueous solution investigated by NMR spectroscopy. Whereas BK is largely disordered on the NMR time scale both analogues showed ROE connectivities in 2D-ROESY spectra indicative of reverse-turn conformations at both Pro2-Phe5 and Ser6-Arg9, whose formation appears to be cooperative. Some potential applications of alpha-MePro as a reverse-turn mimetic in the construction of synthetic peptide libraries is discussed.

Synthesis, conformational properties, and antibody recognition of peptides containing beta-turn mimetics based on alpha-alkylproline derivatives.

Peptide recognition by monoclonal antibodies may provide a useful model for drug development, in particular to test the effects of conformational restriction on ligand binding. We have tested the influence of novel peptide mimetics upon conformation and binding affinity for the case of monoclonal antibodies raised to a peptide antigen which displays a preference for a beta-turn conformation in aqueous solution. Two monoclonals were isolated that recognized the peptide Ac-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala specifically at the beta-turn formed by Tyr-Pro-Tyr-Asp. Peptide analogues were then synthesized containing mimetics designed to stabilize this conformation. One, analogue (3), contained a spirocyclic gamma-lactam bridge between the alpha-position of proline-2 and the N atom of tyrosine-3, while another (2) contained (S)-alpha-methylproline at position 2. NMR spectroscopy and molecular modeling suggest that both analogues adopt reverse-turn conformations stabilized relative to that in the native sequence. For the (S)-alpha-methylproline analogue binding to both monoclonal antibodies was substantially improved, compared with the native antigen, whereas the gamma-lactam analogue (3) was not recognized by either antibody. Quantitative equilibrium ultrafiltration binding assays showed that the affinities of the (S)-alpha-methylproline analogue (2) for the two antibodies were improved over those measured with the native antigen by -2.3 and -0.65 kcal/mol. The origins of these free energy differences cannot be explained wholly on the basis of presumed extra hydrophobic contacts between the new methyl substituent and the antigen binding sites. We propose that the increased conformational stability of the analogue plays a decisive role, implying that the reverse turn detected in the native antigen, possibly a type-I turn, is important for recognition by the two antibodies.