Lighting the Path: Light Delivery Strategies to Activate Photoresponsive Biomaterials In Vivo

Photoresponsive biomaterials are experiencing a transition from in vitro models to in vivo demonstrations that point toward clinical translation. Dynamic hydrogels for cell encapsulation, light-responsive carriers for controlled drug delivery, and nanomaterials containing photosensitizers for photodynamic therapy are relevant examples. Nonetheless, the step to the clinic largely depends on their combination with technologies to bring light into the body. This review highlights the challenge of photoactivation in vivo, and presents strategies for light management that can be adopted for this purpose. The authors’ focus is on technologies that are materials-driven, particularly upconversion nanoparticles that assist in “direct path” light delivery through tissue, and optical waveguides that “clear the path” between external light source and in vivo target. The authors’ intention is to assist the photoresponsive biomaterials community transition toward medical technologies by presenting light delivery concepts that can be integrated with the photoresponsive targets. The authors also aim to stimulate further innovation in materials-based light delivery platforms by highlighting needs and opportunities for in vivo photoactivation of biomaterials.     

National survey of blindness and low vision in Lebanon

Here we report a case that presented with sudden onset of neurological symptoms and was treated with ganciclovir. Polymerase chain reaction analysis for human herpes virus-6 (HHV-6) from his cerebrospinal fluid was later found to be positive. He subsequently recovered with minimal residual neurological symptoms. Encephalitis secondary to this virus may be more common than previously thought in patients undergoing bone marrow transplantation. This condition should be considered in the differential diagnosis of sudden onset neurological symptoms after bone marrow transplantation.     

Particle-bombardment-mediated DNA vaccination with rotavirus VP4 or VP7 induces high levels of serum rotavirus IgG but fails to protect mice against challenge

We recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129-138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4. In vitro assays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus. In vitro expression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Delta1-162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge.     

Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores

BACKGROUND: Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo. RESULTS: A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here 'fluorettes', was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat pIII protein of the filamentous bacteriophage M13. The 'best' fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red. Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution. In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye. CONCLUSIONS: Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection. Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes. Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.     

Establishment and neurite outgrowth properties of neonatal and adult rat olfactory bulb glial cell lines

Two glial cell types surround olfactory axons and glomeruli in the olfactory bulb (OB) and may influence synapse development and regeneration. OB astrocytes resemble type-1 astrocytes, and OB ensheathing cells resemble non-myelinating Schwann cells. We have produced clonal OB astrocyte and ensheathing cell lines from rat neonatal and adult OB cultures by SV40 large T antigen transduction. These cell lines have been characterized by morphology, growth characteristics, immunophenotype, and ability to promote neurite outgrowth in vitro. Neonatal and adult ensheathing cell lines were found to support higher neurite outgrowth than OB astrocyte lines. Neonatal OB astrocyte lines were of two types, high and low outgrowth support. The low support astrocyte lines express J1 and a chondroitin sulfate-containing proteoglycan as do astrocytes encircling the neonatal glomeruli in vivo. The adult OB astrocyte cell lines supported lower levels of outgrowth than adult ensheathing cell lines. These results are consistent with a positive role for ensheathing cells in OB synapse regeneration, in vivo. Further, based on our results, we hypothesize that ensheathing cells and high-outgrowth astrocytes facilitate axon growth in vivo, while low outgrowth astrocytes inhibit axon growth and may facilitate glomerulus formation.     

Effects of fatty acid oxidation on glucose utilization by isolated hepatocytes

We have studied the inhibitory action of long- and short-chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2-3H] and [6-3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the 'cycling' between glucose and glucose-6-phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 mumol/min/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 mumol/min/g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.     

Ends,Means and Innovation Management

The management of innovation inevitably implies the management of uncertainty. Research and practise clearly indicate that judgements about the size and the nature of this are often inaccurate. This can result in poor performance against expectations and in some cases in complete failure. In this paper it is argued that prior analysis of the type of uncertainty can help to improve the success rate and/or reduce the time to completion. Early signs that discontinuation, or a significant change in direction, might be called for are also highlighted by this analysis.     

Parents and procedures: a randomized controlled trial

INTRODUCTION: Previous work has shown that parents prefer to be present when their children undergo common invasive procedures, although physicians are ambivalent about parental presence. PURPOSE: To determine the effect of a parent-focused intervention on the pain and performance of the procedure, anxiety of parents and clinicians, and parental satisfaction with care. POPULATION: Children younger than 3 years old undergoing venipuncture, intravenous cannulation, or uretheral catheterization. SETTING: Pediatric emergency department of Boston City Hospital. DESIGN: Randomized controlled trial with three groups; parents present and given instructions on how to help their children; parents present, but no instructions given; and parents not present. INTERVENTION: The parents were instructed to touch, talk to, and maintain eye contact during the procedure. RESULTS: A total of 431 parents was randomized to the intervention (N = 153), present (N = 147), and not present (N = 131) groups. The groups were equivalent with respect to measured sociodemographic variables and parents' previous experience in the pediatric emergency department. No differences emerged with respect to pain (3-point scale measured by parent and clinician, and analysis of cry); performance of the procedure (number of attempts, completion of procedure by first clinician, time); clinician anxiety; or parental satisfaction with care. Parents who were present were more likely to rate the pain of the children as extreme/severe (52%) in comparison to clinicians (15%, kappa .07, poor agreement) and were significantly less anxious than parents who were not present. CONCLUSION: Overall, the intervention was not effective in reducing the pain of routine procedures. Parental presence did not negatively affect performance of the procedure or increase clinician anxiety. Parents who were present were less anxious than those who were not present. CLINICAL IMPLICATION: In general, parents have indicated that they want to be present when their children undergo procedures. The results of this study challenge the traditional belief that parental presence negatively affects our ability to successfully complete procedures. We should encourage parents who want to be present to stay during procedures.     

Use of a subcutaneous glucose sensor to detect decreases in glucose concentration prior to observation in blood

The development of a hypoglycemic alarm system using a subcutaneous glucose sensor implies that a decrease in blood glucose is rapidly followed by a decrease in the signal generated by the sensor. In a first set of experiments the linearity and the kinetics of the response of sensors implanted in the subcutaneous tissue of normal rats were investigated during a progressive increase in plasma glucose concentration: the sensitivities determined between 5 and 10 mM and between 10 and 15 mM were not significantly different, and a 5-10 min delay in the sensor's response was observed. In a second set of experiments, performed in diabetic rats, the kinetics of the decrease in subcutaneous glucose concentration following insulin administration was monitored during a decrease in plasma glucose level, from 15 to 3 mmol/L. During the 20 first min following insulin administration, the sensor monitored glucose concentration in subcutaneous tissue with no lag time. Subsequently, the decrease in the estimation of subcutaneous glucose concentration preceded that of plasma glucose. This phenomenon was not observed when the same sensors were investigated in vitro during a similar decrease in glucose concentration and may be due to a mechanism occurring in vivo, such as the effect of insulin on glucose transfer from the interstitial space to the cells surrounding the sensor. It reinforces the interest of the use of implantable glucose sensors as a part of a hypoglycemic alarm.     

Rapid identification of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching. Characterization of a novel holoenzymic form of protein phosphatase 1

Microcystin-affinity chromatography was used to purify 15 protein phosphatase 1 (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle. To reduce the time and amount of material required to identify these proteins, proteome analysis by mixed peptide sequencing was developed. Proteins are resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to polyvinylidene fluoride membrane, and stained. Bands are sliced from the membrane, cleaved briefly with CnBr, and applied without further purification to an automated Edman sequencer. The mixed peptide sequences generated are sorted and matched against the GenBank using two new programs, FASTF and TFASTF. This technology offers a simple alternative to mass spectrometry for the subpicomolar identification of proteins in polyacrylamide gels. Using this technology, all 15 proteins recovered in PP-1C affinity chromatography were sequenced. One of the proteins, PP-1bp55, was homologous to human myosin phosphatase, MYPT2. A second, PP-1bp80, identified in the EST data bases, contained a putative PP-1C binding site and a nucleotide binding motif. Further affinity purification over ATP-Sepharose isolated PP-1bp80 in a quaternary complex with PP-1C and two other proteins, PP-1bp29 and human p20. Recombinant PP-1bp80 also bound PP-1C and suppressed its activity toward a variety of substrates, suggesting that the protein is a novel regulatory subunit of PP-1.