Translating Nanomedicine to Comparative Oncology-the Case for Combining Zinc Oxide Nanomaterials with Nucleic Acid Therapeutic and Protein Delivery for Treating Metastatic Cancer.

The unique anticancer, biochemical, and immunologic properties of nanomaterials are becoming a new tool in biomedical research. Their translation into the clinic promises a new wave of targeted therapies. One nanomaterial of particular interest are zinc oxide (ZnO) nanoparticles (NPs), which has distinct mechanisms of anticancer activity including unique surface, induction of reactive oxygen species, lipid oxidation, pH, and also ionic gradients within cancer cells and the tumor microenvironment. It is recognized that ZnO NPs can serve as a direct enzyme inhibitor. Significantly, ZnO NPs inhibit extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) associated with melanoma progression, drug resistance, and metastasis. Indeed, direct intratumoral injection of ZnO NPs or a complex of ZnO with RNA significantly suppresses ERK and AKT phosphorylation. These data suggest ZnO NPs and their complexes or conjugates with nucleic acid therapeutic or anticancer protein may represent a potential new strategy for the treatment of metastatic melanoma, and potentially other cancers. This review focuses on the anticancer mechanisms of ZnO NPs and what is currently known about its biochemical effects on melanoma, biologic activity, and pharmacokinetics in rodents and its potential for translation into large animal, spontaneously developing models of melanoma and other cancers, which represent models of comparative oncology.

Probing the Interaction at the Nano-Bio Interface Using Raman Spectroscopy: ZnO Nanoparticles and Adenosine Triphosphate Biomolecules.

With the advent of nanobiotechnology, there will be an increase in the interaction between engineered nanomaterials and biomolecules. Nanoconjugates with cells, organelles, and intracellular structures containing DNA, RNA, and proteins establish sequences of nano-bio boundaries that depend on several intricate complex biophysicochemical reactions. Given the complexity of these interactions, and their import in governing life at the molecular level, it is extremely important to begin to understand such nanoparticle-biomaterial association. Here we report a unique method of probing the kinematics between an energy biomolecule, adenosine triphosphate (ATP), and hydrothermally synthesized ZnO nanostructures using micro Raman spectroscopy, X-ray diffraction, and electron microscopy experiments. For the first time we have shown by Raman spectroscopy analysis that the ZnO nanostructures interact strongly with the nitrogen (N7) atom in the adenine ring of the ATP biomolecule. Raman spectroscopy also confirms the importance of nucleotide base NH2 group hydrogen bonding with water molecules and phosphate group ionization and their pH dependence. Calculation of molecular bond force constants from Raman spectroscopy reinforces our experimental data. These data present convincing evidence of pH-dependent interactions between ATP and zinc oxide nanomaterials. Significantly, Raman spectroscopy is able to probe such difficult to study and subtle nano-bio interactions and may be applied to elegantly elucidate the nano-bio interface more generally.

Investigations of peritoneal and intestinal infections of adult hookworms (Uncinaria spp.) in northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups on San Miguel Island, California (2003).

The peritoneal cavity (PNC) and intestine of northern fur seal (Callorhinus ursinus) pups and California sea lion (Zalophus californianus) pups that died in late July and early August, 2003, on San Miguel Island, California, were examined for hookworms. Prevalence and morphometric studies were done with the hookworms in addition to molecular characterization. Based on this and previous molecular studies, hookworms from fur seals are designated as Uncinaria lucasi and the species from sea lions as Uncinaria species A. Adult hookworms were found in the PNC of 35 of 57 (61.4%) fur seal pups and of 13 of 104 (12.5%) sea lion pups. The number of hookworms located in the PNC ranged from 1 to 33 (median = 3) for the infected fur seal pups and 1 to 16 (median = 2) for the infected sea lion pups. In addition to the PNC, intestines of 43 fur seal and 32 sea lion pups were examined. All of these pups were positive for adult hookworms. The worms were counted from all but one of the sea lion pups. Numbers of these parasites in the intestine varied from 3 to 2,344 (median = 931) for the fur seal pups and 39 to 2,766 (median = 643) for the sea lion pups. Sea lion pups with peritoneal infections had higher intensity infections in the intestines than did pups without peritoneal infections, lending some support for the hypothesis that peritoneal infections result from high-intensity infections of adult worms. There was no difference in intestinal infection intensities between fur seal pups with and without peritoneal infections. Female adult hookworms in the intestines of both host species were significantly larger than males, and sea lion hookworms were larger than those in fur seals. Worms in the intestine also were larger than worms found in the PNC. Gene sequencing and (RFLP) analysis of (PCR) amplified (ITS) ribosomal DNA were used to diagnose the species of 172 hookworms recovered from the PNC and intestine of 18 C. ursinus and seven Z. californianus hosts. These molecular data revealed that U. lucasi (hookworm of C. ursinus) and Uncinaria species A (of Z. californianus) infrequently mature in the intestine of the opposite host species in California rookeries. However, there is no support from molecular data for the hypothesis that cross-infection with "the wrong" Uncinaria species is a contributing factor in these cases of host peritonitis. The major significance of this research is the unusual finding of adult hookworms in the PNC of so many dead pups. No obvious explanation for this occurrence could be determined. Further research, like in the present study, should help understand and monitor the apparent ever changing role of hookworm disease in the health of northern fur seal and California sea lion pups on SMI.

Effects of nanomaterials on luciferase with significant protection and increased enzyme activity observed for zinc oxide nanomaterials.

This principle goal of this research was to examine the effects of various nanomaterials on the activity and behavior of the firefly enzyme luciferase. Nanomaterials have been found to stabilize, and in some instances, shown to increase the activity of enzymes. In this study gold, manganese oxide (MnO), and zinc oxide (ZnO) nanomaterials were utilized in order to test their effects on enzyme activity. Luciferase was used because its activity is easy to analyze, as it typically produces a large amount of bioluminescence easily detected by a Microtiter plate reader. Following incubation with the various nanomaterials, luciferase was subjected to degradation by several protein denaturing agents, such as heat, SDS, urea, ethanol, protease, hydrogen peroxide, and pH changes. Results indicated that luciferase activity is indeed affected when combined with nanomaterials, accompanied by both increases and decreases in enzyme activity depending on the type of nanomaterial and denaturing agent used. In most of the experiments, when incubated with ZnO nanomaterials, luciferase depicted significant increases in activity and bioluminescence. Additional experiments, in which human A375 cells were treated with luciferase-nanomaterial mixtures, also depicted increased enzyme activity and bioluminescence for luciferase incubated with ZnO nanomaterials. Ultimately, our findings indicated that when luciferase was subjected to multiple types of denaturation, zinc oxide nanomaterials dramatically preserved and increased enzyme activity and bioluminescence.

Interaction of MnO and ZnO nanomaterials with biomedically important proteins and cells.

Zinc and manganese nanomaterials may have potential for biomedical nanotechnology. Here first generation Zn and Mn oxide nanomaterials were prepared as determined by XRD. Transmission electron microscopy confirmed their nanoscale in two dimensions and revealed a rod or belt-like morphology for MnO or ZnO respectively. Association of MnO and ZnO to three model biomedically important proteins (albumin, protamine and thrombin) has been characterized by ultra-violet and dynamic laser light spectroscopy, UVS and DLLS respectively. UVS demonstrated a concentration-dependent loss of protein from the supernatant upon sedimentation of MnO or ZnO. Shifts in the surface charge of the MnO or ZnO by DLLS confirmed the protein's adsorption to the surface. MnO and ZnO were incubated with live human cells in culture (HeLa, A375 or 1321N1). A marked difference was observed for the two nanomaterials behavior in cell culture where the MnO could be discerned associating at the cell surface whereas the ZnO caused the cells to exhibit a rounded up morphology. Trypan blue dye exclusion studies demonstrated cytotoxicity of the ZnO at high concentrations 62.5-31.5 microg/mL whereas surprisingly the MnO demonstrated no cytotoxicity at any of the concentrations tested.

A novel statistical model for mandibular helical axis analysis.

The purpose of this study was to establish a new statistical method for the analysis of noisy mandibular helical axis parameters, especially the position vector of the finite helical axis (FHA). The subjects were children with anterior cross-bite who had received orthodontic treatment. Maximum mouth-opening was measured by means of an opto-electronic motion analysis system. These movements were compared with similar movement in the same group after treatment of their anterior cross-bite. Each curve of FHA position vectors was modelled as a spline function with random coefficients. To determine the optimal number of knots, two criteria were used: deviance information criteria (DIC) and mean squared prediction error (MSE). We were interested in estimating a typical curve for a population. Self-modelling regression (SEMOR) was extended to three dimensions to model groups of three-dimensional curves. Each curve was modelled as a spline function using nine knots. Population average curves were created using SEMOR. This study provided detailed information about jaw movement for comparing cross-bite to normal occlusion by calculating the population mean curves of the position vector of the FHA. Our results suggested that the two population mean curves for the position vector of the FHA were significantly different in the closing phase. The combination of a spline function with random coefficients and SEMOR extended to three dimensions can be used not only for FHA analysis but also for the analysis of other jaw movements.

Challenges and potential for RNA nanoparticles (RNPs).

The current research of our group focuses on delivery of oligonucleotides and gene vaccines targeted against infectious disease and cancer. Recently we reported a strategy for associating DNA to gold via protamine delivery enhancing material which here we suggest might be applicable to RNA and to other nanoparticles. An important new modality for such RNA based nanoparticles (RNPs) is the myriad of genes now known to undergo alternative splicing. Here we will review some important issues for the binding, stabilization and delivery of RNA, particularly splice-site switching oligomers (SSOs) via these RNPs in order to achieve selective molecular therapeutic effects and unlock their potential as chemotherapeutic agents.

Prevalence and characterization of Salmonella spp. among marine animals in the Channel Islands, California.

Salmonella enterica is a zoonotic pathogen that has been isolated from free-ranging marine mammals throughout the world, with animals in the Channel Islands of California (USA) showing the highest prevalence. The goal of this study was to determine prevalence, antimicrobial sensitivity and genetic similarity using pulsed-field gel electrophoresis (PFGE) of Salmonella in several non-domestic animal species on San Miguel and San Nicolas Islands. Fecal samples were collected from 90 California sea lion Zalophus californianus pups, 30 northern elephant seal Mirounga angustirostris pups and 87 western gulls Larus occidentalis in the Channel Islands and 59 adult male sea lions in Puget Sound, WA (USA). Salmonella were isolated, identified and serotyped, followed by antimicrobial susceptibility testing and PFGE. Of the California sea lion pups that were sampled on the islands, 21% (n = 19) were positive for Salmonella, whereas no adults males in Puget Sound were positive. Of the northern elephant seal pups sampled, 87% (n = 26) were harboring Salmonella. Only 9% (n = 8) of western gulls were shedding Salmonella, with one of these gulls harboring the only antimicrobial resistant isolate. The serotypes found in these animals were Enteritidis, Montevideo, Newport, Reading, and Saint Paul. The only serotype that showed variation on PFGE was Newport. The pinnipeds of the Channel Islands harbor Salmonella at a higher prevalence than pinnipeds from other geographic areas observed in previous studies. Researchers and veterinarians should exercise increased caution when working with these animals due to the zoonotic potential of Salmonella.

Accuracy of contacts calculated from 3D images of occlusal surfaces.

OBJECTIVES: Compare occlusal contacts calculated from 3D virtual models created from clinical records to contacts identified clinically using shimstock and transillumination. METHODS: Upper and lower full arch alginate impressions and vinyl polysiloxane centric interocclusal records were made of 12 subjects. Stone casts made from the alginate impressions and the interocclusal records were optically scanned. Three-dimensional virtual models of the dental arches and interocclusal records were constructed using the Virtual Dental Patient Software. Contacts calculated from the virtual interocclusal records and from the aligned upper and lower virtual arch models were compared to those identified clinically using 0.01mm shimstock and transillumination of the interocclusal record. Virtual contacts and transillumination contacts were compared by anatomical region and by contacting tooth pairs to shimstock contacts. Because there is no accepted standard for identifying occlusal contacts, methods were compared in pairs with one labeled "standard" and the second labeled "test". Accuracy was defined as the number of contacts and non-contacts of the "test" that were in agreement with the "standard" divided by the total number of contacts and non-contacts of the "standard". RESULTS: Accuracy of occlusal contacts calculated from virtual interocclusal records and aligned virtual casts compared to transillumination were: 0.87+/-0.05 and 0.84+/-0.06 by region and 0.95+/-0.07 and 0.95+/-0.05 by tooth, respectively. Comparisons with shimstock were: 0.85+/-0.15 (record), 0.84+/-0.14 (casts), and 81+/-17 (transillumination). CONCLUSIONS: The virtual record, aligned virtual arches, and transillumination methods of identifying contacts are equivalent, and show better agreement with each other than with the shimstock method.

R4 peptide-pDNA nanoparticle coated HepB vaccine microparticles: sedimentation, partitioning, and spray freeze dry bioprocesses.

Broad therapeutic application of nucleic acid micro- and nanoparticles will require bioprocesses capable of achieving high loads of structurally intact and functionality active DNA. Here we report condensation of pDNA into nanoparticles by sedimentation through R4 peptide and partitioning at a hydrophobic interface. > or = 90% coating efficiency onto microparticles is achieved via this combined bioprocess with the pDNA retaining 85-90% intact supercoil after bioprocessing. SEM analyses of the microparticles produced therefrom reveals bound pDNA and R4 peptide nanoparticles. HPLC and chemical analyses afford quantification of the particle-associated pDNA and R4 peptide along with lactose, raffinose, or trehalose carbohydrate stabilizer, surface coatings uniformly applied by spray freeze-drying. Administration of these particles by gene gun demonstrates delivery to the nucleus of expressive nanoparticles and into rodents and pigs pronounced immunogenicity even after bioprocessing and accelerated degradation. These data support the discovery of a robust bioprocess platform for preparing macromolecule bound bioparticles with potential relevance beyond simple preparation of bioactive DNA vaccine.

MHC gene configuration variation in geographically disparate populations of California sea lions (Zalophus californianus).

Major histocompatibility complex (MHC) class II DRB genotypes were examined in two geographically isolated populations of California sea lions (Zalophus californianus) (Gulf of California and California coastal Pacific Ocean). Genomic DNA from 227 California sea lions was examined using eight sequence-specific primer (SSP) pairs flanking the putative peptide-binding site. A total of 40 different Zaca-DRB genotype configurations were identified among the 227 individuals. Using SSP-PCR, significant differences were found between coastal California and Gulf of California Zalophus populations in numbers of DRB sequences per individual and configuration of sequences within individuals. Additionally, unique local patterns of MHC diversity were identified among the Midriff Island animals. These population differences are consistent with either ecologically distinct patterns of selection pressures and/or geographical isolation. The consequences of these partitioned MHC configurations at the population level are as yet unknown; however, the worldwide increase in emerging marine diseases lends urgency to their examination.

Seasonal prevalence and intensity of hookworms (Uncinaria spp.) in California sea lion (Zalophus californianus) pups born in 2002 on San Miguel Island, California.

Intestines of California sea lion (Zalophus californianus) pups (n= 204), born in 2002 on San Miguel Island, California, were examined for hookworms (Uncinaria spp.) as part of a seasonal mortality study from June through December 2002 and January 2003. The investigation was planned to coincide with most of the previously established hookworm infection period of the pups. Prevalence of hookworms in dead pups was 100% for each month of the study. The geometric mean intensity of infections per month was: 94.03 (n=30) for June, 629.09 (n=50) for July, 319.90 (n=31) for August, 159.90 (n=30) for October, 109.03 (n=30) for November, 37.84 (n=24) for December 2002 and 11.05 (n=9) for January 2003. In addition to the temporal pattern, the infection intensity was higher for pups in good condition and for male pups. An inter-year comparison of hookworm counts from dead pups collected in July of 1996, 2000, and 2002 also demonstrated higher intensity in pups in better condition but sex-differences in intensity were inconsistent across years. The inter-year comparison also demonstrated higher intensities in dead pups collected from portions of the rookery with sandy substrate versus rocky substrate. No annual differences in intensity were found after adjusting for substrate and condition.

Photomicrographic images of some features of Uncinaria spp (Nematoda: Ancylostomatidae) from otariid pinnipeds.

Photomicrographs of several morphologic features of hookworms (Uncinaria spp) from northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups are presented. The main purpose is to show and describe some physical characteristics of hookworms from the two hosts; it is not to decide from these attributes whether the Uncinaria spp are the same species. The number of species of Uncinaria in pinnipeds is uncertain and specimens need to be examined from the various infected seals and sea lions before the taxonomy of these parasites can be clarified. Information in the present paper should aid in this determination.

pDNA bioparticles: comparative heterogeneity, surface, binding, and activity analyses.

New applications for nucleic acid-bound micro/nanoparticles are emerging in drug delivery, biocatalysis, diagnostics, and toxicology. Bioactivity of viral or liposomal based technologies is limited by heterogeneity, partitioning, aggregation, and protein binding in physiological fluids, underlying immunotoxicity, and poor in vitro and cell-culture corollaries. Here we have systematically investigated novel pDNA bioparticles formed through complexation to model non-viral/non-lipid materials, peptides, aminoglycans, and small molecules (polybrene, chitosan, butirosin, protamine, Lys10, RGDS, bupivacaine, and chlorpromazine). On the basis of characterization by heterogeneity, kinetics, partitioning in physiological fluid and serum protein-binding, surface, size and electrophoretic behavior, transfection, and immunotoxicity, notably protamine, and chitosan DNA particles gave a long lifetime (12-18h), low protein-binding (<10microg/ml), good transfection activity (10(2)-10(4)RLU/mg cell protein), and low immunotoxicity. Our results support further evaluation of these materials as potential alternatives to viral or liposomal approaches, in combination with pDNA as binding, expression or therapeutic agents.

Penetration of the small intestine of a California sea lion (Zalophus californianus) pup by adult hookworms (Uncinaria spp).

During a study on the mortality of California sea lion (Zalophus californianus) pups born on San Miguel Island, California in 2002, two adult female hookworms (Uncinaria spp) were found penetrating the serosal surface of the intestinal wall and protruding into the peritoneal cavity of one pup. Documentation and a description of this unexpected finding and associated lesions are presented here. Also, adult hookworms were found in the peritoneal fluid of two other dead Z. californianus pups.

Evaluation of dental arch reproduction using three-dimensional optical digitization.

STATEMENT OF PROBLEM: Numerous investigations have been done to determine the most accurate method to reproduce a dental arch. Investigations have used different definitive cast configurations, materials, and measuring techniques to evaluate dimensional change, leaving clinicians undecided as to the most accurate method to reproduce a dental arch. PURPOSE OF STUDY: The purpose of this investigation was to evaluate the effect of impression tray selection on accuracy of reproductions of a dental arch using a 3-dimensional optical digitizer. MATERIALS AND METHODS: Impressions were made of a stone cast of a dental arch using vinyl polysiloxane impression material in custom and stock impression trays (n=12). Custom trays, designed with full palatal coverage, were fabricated on a separate standard stone cast using 2 layers of pink baseplate wax as the spacer and tissue stops on 1 anterior tooth and both first molar teeth. Impressions were poured with type IV gypsum and allowed to set with the tray inverted (n=5) or not inverted (n=5). The standard cast and resulting casts were digitized using an optical digitizer. Custom software was used to align and process the 3-dimensional images created by the digitizer. Each image was superimposed onto the standard cast image, curves were created of the percent of points less than a defined distance between the 2 superimposed images, and the area under the curve was calculated. A weighted average area was calculated for each cast, and these values were analyzed using a 2-way ANOVA (alpha=.05). RESULTS: The main effect of tray type, laboratory setting technique, and the interaction between these 2 characteristics was analyzed. None of these effects was significant. CONCLUSION: The differences in accuracy of reproductions of a dental arch, regardless of the impression and laboratory techniques, were not significant when evaluated using a 3-dimensional optical digitizer.

Elasticity of alveolar bone near dental implant-bone interfaces after one month's healing.

Information is scarce about Young's modulus of healing bone surrounding an implant. The purpose of this preliminary study is to quantify elastic properties of pig alveolar bone that has healed for 1 month around titanium threaded dental implants, using the nanoindentation method. Two 2-year-old Sinclair miniswine were used for the study. Nanoindentation tests perpendicular to the bucco-lingual cross section were performed on harvested implant-bone blocks using the Hysitron TriboScope III. Nomarski differential interference contrast microscopy was used to identify pyramidal indentation measurements that were from bone. Reduced moduli, averaged for all anatomical regions, were found to start low (6.17 GPa) at the interface and gradually increase (slope=0.014) to a distance of 150 microm (7.89 GPa) from the implant surface, and then flatten to a slope of 0.001 from 150 to 1500 microm (10.13 GPa). Mean reduced modulus and its relationship to distance did not differ significantly by anatomic location (e.g., coronal, middle, and apical third; P>/=0.28 for all relevant tests) at 1 month after implantation.

Effects of implant healing time on crestal bone loss of a controlled-load dental implant.

The universally accepted concept of delay-loaded dental implants has recently been challenged. This study hypothesizes that early loading (decreased implant healing time) leads to increased bone formation and decreased crestal bone loss. We used 17 minipigs to study implants under a controlled load, with non-loaded implants for comparison. Radiographic and histological assessments were made of the osseointegrated bone changes for 3 healing times (between implant insertion and loading), following 5 months of loading. The effect of loading on crestal bone loss depended on the healing time. Early loading preserved the most crestal bone. Delayed loading had significantly more crestal bone loss compared with the non-loaded controls (2.4 mm vs. 0.64 mm; P < 0.05). The histological assessment and biomechanical analyses of the healing bone suggested that loading and bioactivities of osteoblasts exert a synergistic effect on osseointegration that is likely to support the hypothesis that early loading produces more favorable osseointegration.

Accuracy of a system for creating 3D computer models of dental arches.

Three-dimensional imaging of dental tissues will have a major impact in dentistry if the images are accurate. The purpose of this study was to measure the accuracy and precision of a system for creating three-dimensional images of dental arches. Using vinyl polysiloxane impression materials and improved dental stone, we made 10 stone casts of a "dental" standard with known dimensions. The impressions and casts were scanned by means of a Comet 100 optical scanner. Custom software created three-dimensional images (computer models) from the scanned data. Accuracy was defined as the average of the absolute differences between the computer models and the standard. Precision was the standard deviation of accuracy over 10 repeated measures. Software processing improved the accuracy of the scanner data. Accuracy +/- precision for the casts and impressions was 0.024 +/- 0.002 mm and 0.013 +/- 0.003 mm, respectively. The system produced computer models with sufficient accuracy for clinical application.

Observations in 2001 on hookworms ( Uncinaria spp.) in otariid pinnipeds.

Uncinaria spp. were recovered from the milk of California sea lions ( Zalophus californianus) collected from the: (1) teats of a cow just after parturition (one parasitic third-stage larva, L(3)), (2) stomach of her nursing pup (two L(3)), and (3) stomach of a dead pup about 2 days old (one L(3), one headless, probably L(3), and four L(4)) on San Miguel Island, California in May 2001. This, in addition to earlier research, indicates transmammary transmission of hookworms in this host. Uncinaria spp. were found in dead northern fur seals ( Callorhinus ursinus) in the: (1) intestines of 2 of 75 pups (either one or two adult specimens in each infected pup) and (2) ventral abdominal blubber of 3 of 78 subadult males (one to seven L(3) in each infected seal) on St. Paul Island (SPI), Alaska in July and August 2001. These findings verify the low current prevalence of Uncinariaspp. in fur seals on SPI. Rectal fecal samples taken from 50 live Steller sea lion ( Eumetopias jubatus) pups, about 1 month old, on Rogue Reef in Curry County, Oregon in July 2001, were all negative for the eggs of Uncinaria spp. The apparent zero infection rate in these pups is possibly because the rocky terrain of this rookery is not suitable for hookworm transmission.