Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein.

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host's cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2-6.4 ID50 g-1 was reduced to a remaining infectivity of 104.6-5.7 ID50 g-1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein.

Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.

Surface micromachined electrostatically actuated micro peristaltic pump.

An electrostatically actuated micro peristaltic pump is reported. The micro pump is entirely surface micromachined using a multilayer parylene technology. Taking advantage of the multilayer technology, the micro pump design enables the pumped fluid to be isolated from the electric field. Electrostatic actuation of the parylene membrane using both DC and AC voltages was demonstrated and applied to fluid pumping based on a 3-phase peristaltic sequence. A maximum flow rate of 1.7 nL min(-1) and an estimated pumping pressure of 1.6 kPa were achieved at 20 Hz phase frequency. A dynamic analysis was also performed with a lumped-parameter model for the peristaltic pump. The analysis results allow a quantitative understanding of the peristaltic pumping operation, and correctly predict the trends exhibited by the experimental data. The small footprint of the micro pump is well suited for large-scale integration of microfluidics. Moreover, because the same platform technology has also been used to fabricate other devices (e.g. valves, electrospray ionization nozzles, filters and flow sensors), the integration of these different devices can potentially lead to versatile and functional micro total analysis systems (microTAS).

Extracellular production of a functional soy cystatin by Bacillus subtilis.

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.

Implantable MicroPump for Drug Delivery in Patients with Diabetic Macular Edema.

PURPOSE: To demonstrate the safety and surgical feasibility of the first-in-man ocular implant of a novel Posterior MicroPump Drug Delivery System (PMP) in patients with diabetic macular edema (DME) and to report on the device capabilities for delivering a programmable microdose. METHODS: This was a single center, single arm, open-label, prospective study. Eleven patients with DME and visual acuity equal to or worse than 20/40 were included. The PMP prefilled with ranibizumab was implanted into the subconjunctival space. After implantation, the PMP was wirelessly controlled to deliver a programmed microdose. Comprehensive ophthalmic exams and optical coherence tomography were performed biweekly for 90 days. At the end of the study, the PMP was explanted and the subjects thereafter received standard of care for DME (i.e., laser or intravitreal injections). RESULTS: All 11 surgical implantations were without complications and within the skill sets of a retinal surgeon. No serious adverse events occurred during the follow-up period. At no point were visual acuity and central foveal thickness worse than baseline in the implanted eye. The PMP delivered the programmed ranibizumab dosage in seven subjects. The remaining four patients received a lower than target dose, and the treatment was complemented with standard intravitreal injection. CONCLUSIONS: This study demonstrates the first-in-man safety of the Replenish MicroPump implant for a period of 90 days and its capability to deliver a microdose into the vitreous cavity. Further studies to enable longer-term safety and to demonstrate the feasibility of multiple programmable drug delivery are necessary.

One-Year Feasibility Study of Replenish MicroPump for Intravitreal Drug Delivery: A Pilot Study.

PURPOSE: To determine the feasibility of the surgical procedure and to collect some safety data regarding the bioelectronics of a novel micro drug pump for intravitreal drug delivery in a Beagle dog model for up to 1 year. METHODS: Thirteen Beagle dogs were assigned to two groups. The experimental group (n = 11) underwent pars plana implantation of MicroPump; the body of which was sutured episclerally, while its catheter was secured at a pars plana sclerotomy. The control group (n = 2) underwent sham surgeries in the form of a temporary suturing of the MicroPump, including placement of the pars plana tube. Baseline and follow-up exams included ophthalmic examination and imaging. The experimental animals were euthanized and explanted at predetermined time points after surgery (1, 3, and 12 months), while the control animals were euthanized at 3 months. All operated eyes were submitted for histopathology. RESULTS: Eyes were scored according to a modified McDonald-Shadduck system and ophthalmic imaging. Neither the implanted eyes nor the control eyes showed clinically significant pathological changes beyond the expected surgical changes. The operated eyes showed neither significant inflammatory reaction nor tissue ingrowth through the sclerotomy site compared with the fellow eyes. CONCLUSION: This study shows that the Replenish Posterior MicroPump could be successfully implanted with good safety profile in this animal model. TRANSLATIONAL RELEVANCE: The results of this study in a Beagle dog model are supportive of the biocompatibility of Replenish MicroPump and pave the way to the use of these devices for ocular automated drug delivery after further testing in larger animal models.

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures.

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.

Increased production of Bacillus keratinase by chromosomal integration of multiple copies of the kerA gene.

To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3-5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.

Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems.

Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems.

An electrochemical pumping system for on-chip gradient generation.

Within the context of microfluidic systems, it has been difficult to devise pumping systems that can deliver adequate flow rates at high pressure for applications such as HPLC. An on-chip electrochemical pumping system based on electrolysis that offers certain advantages over designs that utilize electroosmotic driven flow has been fabricated and tested. The pump was fabricated on both silicon and glass substrates using photolithography. The electrolysis electrodes were formed from either platinum or gold, and SU8, an epoxy-based photoresist, was used to form the pump chambers. A glass cover plate and a poly(dimethylsiloxane) (PDMS) gasket were used to seal the chambers. Filling of the chambers was accomplished by using a syringe to inject liquid via filling ports, which were later sealed using a glass cover plate. The current supplied to the electrodes controlled the rate of gas formation and, thus, the resulting fluid flow rate. At low backpressures, flow rates >1 microL/min have been demonstrated using <1 mW of power. Pumping at backpressures as high as 200 psi have been demonstrated, with 20 nL/min having been observed using <4 mW. By integrating two electrochemical pumps with a polymer electrospray nozzle, we have confirmed the successful generation of a solvent gradient via a mass spectrometer.

Enzymatic degradation of prion protein in brain stem from infected cattle and sheep.

Prions-infectious agents involved in transmissible spongiform encephalopathies-normally survive proteolytic and mild protein-destructive processes. Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, we tested conditions to accomplish the full degradation of prion protein (PrP) in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie. The detection of PrPSc, the disease-associated isoform of PrP, in homogenates was done by Western blotting and various antibodies. The results indicated that only in the presence of detergents did heat pretreatment at >100 degrees C allow the extensive enzymatic breakdown of PrPSc to a state where it is immunochemically undetectable. Proteinase K and 2 other subtilisin proteases, but not trypsin and pepsin, were also effective. This enzymatic process could lead to the development of a method for the decontamination of medical and laboratory equipment. The ultimate effectiveness of this method of prion inactivation has to be tested in mouse bioassays.