Enzyme aggregation and fragmentation induced by catalysis relevant species.

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.

Preferential uptake of antibody targeted calcium phosphosilicate nanoparticles by metastatic triple negative breast cancer cells in co-cultures of human metastatic breast cancer cells plus bone osteoblasts.

Calcium phosphosilicate nanoparticles (CPSNPs) are bioresorbable nanoparticles that can be bioconjugated with targeting molecules and encapsulate active agents and deliver them to tumor cells without causing damage to adjacent healthy tissue. Data obtained in this study demonstrated that an anti-CD71 antibody on CPSNPs targets these nanoparticles and enhances their internalization by triple negative breast cancer cells in-vitro. Caspase 3,7 activation, DNA damage, and fluorescent microscopy confirmed the apoptotic breast cancer response caused by targeted anti-CD71-CPSNPs encapsulated with gemcitabine monophosphate, the active metabolite of the chemotherapeutic gemcitabine used to treat cancers including breast and ovarian. Targeted anti-CD71-CPSNPs encapsulated with the fluorophore, Rhodamine WT, were preferentially internalized by breast cancer cells in co-cultures with osteoblasts. While osteoblasts partially internalized anti-CD71-GemMP-CPSNPs, their cell growth was not affected. These results suggest that CPSNPs may be used as imaging tools and selective drug delivery systems for breast cancer that has metastasized to bone.

Effective encapsulation and biological activity of phosphorylated chemotherapeutics in calcium phosphosilicate nanoparticles for the treatment of pancreatic cancer.

Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive, phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. These findings demonstrate a novel methodology to efficiently encapsulate chemotherapeutic agents into the CPSNPs and to effectively deliver them to pancreatic tumor cells.

VCAM-1-targeted nanoparticles to diagnose, monitor and treat atherosclerosis.

Vascular cell adhesion molecule-1 (VCAM-1) was identified over 2 decades ago as an endothelial adhesion receptor involved in leukocyte recruitment and cell-based immune responses. In atherosclerosis, a chronic inflammatory disease of the blood vessels that is the leading cause of death in the USA, endothelial VCAM-1 is robustly expressed beginning in the early stages of the disease. The interactions of circulating immune cells with VCAM-1 on the activated endothelial cell surface promote the uptake of monocytes and the progression of atherosclerotic lesions in susceptible vessels. Herein, we review the role of VCAM-1 in atherosclerosis and the use of VCAM-1 binding peptides, antibodies and aptamers as targeting agents for nanoplatforms for early detection and treatment of atherosclerotic disease.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Addition of in situ clay catalysts at different process points in a cascaded hydrothermal carbonization-pyrolysis process for agro-industrial waste valorization.

Agro-industrial wastes can be thermochemically converted to sustainable fuels and upcycled carbon products. However, processing such feedstocks through pyrolysis or hydrothermal carbonization (HTC) alone yields fuels that require significant downstream upgrading. In this work, apple pomace was treated via a cascaded HTC-pyrolysis process using inexpensive and abundant clay catalysts, montmorillonite and attapulgite. Clays were added pre-HTC to raw biomass or to hydrochar pre-pyrolysis to examine the effect of addition as a function of process insertion point. Both clays produce similar bio-oils when they are added at the same process point. However, bio-oil was affected by the point in which clay was added to the process (before or after HTC). When clay was added pre-HTC, the bio-oil had an average hydrocarbon content twice that when clay was added to the hydrochar after HTC, prior to pyrolysis.

Reactive Oxygen Species-Triggered Hydrogen Sulfide Release and Cancer-Selective Antiproliferative Effect of Anethole Dithiolethione-Containing Polymeric Micelles.

Hydrogen sulfide (H2 S) is a gaseous signaling molecule in the human body and has attracted attention in cancer therapy due to its regulatory roles in cancer cell proliferation and migration. Accumulating evidence suggests that continuous delivery of H2 S to cancer cells for extended periods of time suppresses cancer progression. However, one major challenge in therapeutic applications of H2 S is its controlled delivery. To solve this problem, polymeric micelles are developed containing H2 S donating-anethole dithiolethione (ADT) groups, with H2 S release profiles optimal for suppressing cancer cell proliferation. The micelles release H2 S upon oxidation by reactive oxygens species (ROS) that are present inside the cells. The H2 S release profiles can be controlled by changing the polymer design. Furthermore, the micelles that show a moderate H2 S release rate exert the strongest anti-proliferative effect in human colon cancer cells in in vitro assays as well as the chick chorioallantoic membrane cancer model, while the micelles do not affect proliferation of human umbilical vein endothelial cells. This study shows the importance of fine-tuning H2 S release profiles using a micelle approach for realizing the full therapeutic potential of H2 S in cancer treatment.

Validation of a novel laparoscopic adjustable gastric band simulator.

BACKGROUND: Morbid obesity accounts for more than 90,000 deaths per year in the United States. Laparoscopic adjustable gastric banding (LAGB) is the second most common weight loss procedure performed in the US and the most common in Europe and Australia. Simulation in surgical training is a rapidly advancing field that has been adopted by many to prepare surgeons for surgical techniques and procedures. The aim of our study was to determine face, construct, and content validity for a novel virtual reality laparoscopic adjustable gastric band simulator. METHODS: Twenty-eight subjects were categorized into two groups (expert and novice), determined by their skill level in laparoscopic surgery. Experts consisted of subjects who had at least 4 years of laparoscopic training and operative experience. Novices consisted of subjects with medical training but with less than 4 years of laparoscopic training. The subjects used the virtual reality laparoscopic adjustable band surgery simulator. They were automatically scored according to various tasks. The subjects then completed a questionnaire to evaluate face and content validity. RESULTS: On a 5-point Likert scale (1 = lowest score, 5 = highest score), the mean score for visual realism was 4.00 ± 0.67 and the mean score for realism of the interface and tool movements was 4.07 ± 0.77 (face validity). There were significant differences in the performances of the two subject groups (expert and novice) based on total scores (p < 0.001) (construct validity). Mean score for utility of the simulator, as addressed by the expert group, was 4.50 ± 0.71 (content validity). CONCLUSION: We created a virtual reality laparoscopic adjustable gastric band simulator. Our initial results demonstrate excellent face, construct, and content validity findings. To our knowledge, this is the first virtual reality simulator with haptic feedback for training residents and surgeons in the laparoscopic adjustable gastric banding procedure.

Aptamer-Targeted Calcium Phosphosilicate Nanoparticles for Effective Imaging of Pancreatic and Prostate Cancer.

PURPOSE: Accurate tumor identification and staging can be difficult. Aptamer-targeted indocyanine green (ICG)-nanoparticles can enhance near-infrared fluorescent imaging of pancreatic and prostate tumors and could improve early cancer detection. This project explored whether calcium-phosphosilicate nanoparticles, also known as NanoJackets (NJs), that were bioconjugated with a tumor-specific targeting DNA aptamer could improve the non-invasive detection of pancreatic and prostate tumors. METHODS: Using in vivo near-infrared optical imaging and ex vivo fluorescence analysis, DNA aptamer-targeted ICG-loaded NJs were compared to untargeted NJs for detection of tumors. RESULTS: Nanoparticles were bioconjugated with the DNA aptamer AP1153, which binds to the CCK-B receptor (CCKBR). Aptamer bioconjugated NJs were not significantly increased in size compared with unconjugated nanoparticles. AP1153-ICG-NJ accumulation in orthotopic pancreatic tumors peaked at 18 h post-injection and the ICG signal was cleared by 36 h with no evidence on uptake by non-tumor tissues. Ex vivo tumor imaging confirmed the aptamer-targeted NJs accumulated to higher levels than untargeted NJs, were not taken up by normal pancreas, exited from the tumor vasculature, and were well-dispersed throughout pancreatic and prostate tumors despite extensive fibrosis. Specificity for AP1153-NJ binding to the CCK-B receptor on pancreatic tumor cells was confirmed by pre-treating tumor-bearing mice with the CCK receptor antagonist proglumide. Proglumide pre-treatment reduced the in vivo tumoral accumulation of AP1153-NJs to levels comparable to that of untargeted NJs. CONCLUSION: Through specific interactions with CCK-B receptors, tumor-targeted nanoparticles containing either ICG or rhodamine WT were well distributed throughout the matrix of both pancreatic and prostate tumors. Tumor-targeted NJs carrying various imaging agents can enhance tumor detection.

Single-incision laparoscopic right colectomy: experience with 17 consecutive cases and comparison with multiport laparoscopic right colectomy.

BACKGROUND: Recently, single-incision laparoscopic surgery has begun to develop as an extension of standard laparoscopic minimally invasive procedures. However, there have been a limited number of reports of single-incision procedures in colorectal disease. PURPOSE: The aim of this study is to describe our initial experience with single-incision laparoscopic right colectomy and to make comparisons with the current standard of care, multiport laparoscopic right colectomy. METHODS: Data from consecutive patients undergoing single-incision laparoscopic right colectomy were analyzed and compared with case-matched multiport laparoscopic right colectomies. Indications for surgery, type of port used, operative time, number of nodes harvested, length of hospital stay, and complications were the outcomes measured. RESULTS: During the study period, 17 patients underwent single-incision laparoscopic colectomy. Of the planned single-incision laparoscopic cases, 15 (88%) were completed with a single incision, whereas 2 required an additional port placement. There were no conversions to open surgery during any of the cases. Indications for surgery were similar between the 2 groups. Operative time was not significantly different in single-incision laparoscopic right colectomy compared with multiport laparoscopic right colectomy (139 min vs 134 min, respectively; P = .61). Length of stay and number of nodes harvested also had no significant differences between the 2 groups. There was one death after discharge to home secondary to pulmonary embolism and one delayed thermal injury in the single-incision laparoscopic group. CONCLUSION: Single-incision laparoscopic right colectomy is feasible, and appears to have results similar to standard multiport right colectomy in our initial comparisons. Ongoing development in instrumentation may help to further shorten operative time and minimize complications, and may make this an equivalent or preferred method for minimally invasive colorectal surgery. Large, prospective, randomized, controlled trials should be conducted to further compare the safety and efficacy of this approach.

Photophysics of Cy3-encapsulated calcium phosphate nanoparticles.

Progress toward clinical application of biodegradable fluorescent calcium phosphate (CP) nanoparticles as a bioimaging agent requires detailed knowledge of chromophore interaction with CP. As readouts of this cargo-matrix interaction, we determined the principle photophysical properties of Cy3 encapsulated in CP nanparticles (CPNPs) using steady-state and time-resolved fluorescence spectroscopy. Fluorescence correlation spectroscopy (FCS)-determined diffusion coefficients and associated hydrodynamic radii confirmed the presence of highly monodisperse CPNPs with radii ranging from 7 to 10 nm. Single CP nanoparticles were 20 times brighter than free dye molecules because of a CP-induced 5-fold increase in quantum efficiency and encapsulation of four dye molecules per particle. Solvatochromic shifts resulting from hydrogen bonding between free dye and solvent or restricted intramolecular mobility by solvent viscosity were absent when Cy3 was encapsulated in CP. Encapsulation-mediated increases in radiative decay rates and decreases in nonradiative decay rates resulting in longer fluorescence lifetimes of Cy3 were attributed to solvent and CP-related local refractive indices and restricted flexibility of dye by rigid CP. Enhanced brightness of CPNPs enabled imaging of single nanoparticles under epifluorescence using both standard and total internal reflection fluorescence (TIRF) modes with camera exposure times on the order of tens of milliseconds. These enhanced photophysical properties together with excellent biocompatibility make CPNPs ideal for bioimaging applications ranging from single-molecule tracking to in vivo tumor detection and offer the possibility of timed codelivery of drugs to control cell function.

Lost Mold-Rapid Infiltration Forming of Mesoscale Ceramics: Part 2, Geometry and Strength Improvements.

Iterative process improvements have been used to eliminate strength-limiting geometric flaws in mesoscale bend bars composed of yttria-tetragonal zirconia polycrystals (Y-TZP). These improvements led to large quantities of high bend strength material. The metrology of Y-TZP mesoscale bend bars produced using a novel lost mold-rapid infiltration-forming process (LM-RIF) is characterized over several process improvements. These improvements eliminate trapezoidal cross sections in the parts, reduce concave upper surfaces in cross section, and minimize warping along the long axis of 332 x 26 x 17 mum mesoscale bend bars. The trapezoidal cross sections of earlier, first-generation parts were due to the absorption of high-energy ultraviolet (UV) light during the photolithographic mold-forming process, which produced nonvertical mold walls that the parts mirrored. The concave upper surfaces in cross section were eliminated by implementing a RIF-buffing process. Warping during sintering was attributed to impurities in the substrate, which creates localized grain growth and warping as the tetragonal phase becomes destabilized. Precision in the part dimensions is demonstrated using optical profilometry on bend bars and a triangular test component. The bend bar dimensions have a 95% confidence interval of < +/-1 mum, and the tip radius of the triangular test component is 3 mum, consistent with the UV-photolithographic process used to form the mold cavities. The average bend strength of the mesoscale Y-TZP bend exceeds 2 GPa with a Weibull modulus equal to 6.3.

Lost Mold Rapid Infiltration Forming of Mesoscale Ceramics: Part 1, Fabrication.

Free-standing mesoscale (340 mum x 30 mum x 20 mum) bend bars with an aspect ratio over 15:1 and an edge resolution as fine as a single grain diameter ( approximately 400 nm) have been fabricated in large numbers on refractory ceramic substrates by combining a novel powder processing approach with photoresist molds and an innovative lost-mold thermal process. The colloid and interfacial chemistry of the nanoscale zirconia particulates has been modeled and used to prepare highly concentrated suspensions. Engineering solutions to challenges in mold fabrication and casting have yielded free-standing, crack-free parts. Molds are fabricated using high-aspect-ratio photoresist on ceramic substrates. Green parts are formed using a rapid infiltration method that exploits the shear thinning behavior of the highly concentrated ceramic suspension in combination with gelcasting. The mold is thermally decomposed and the parts are sintered in place on the ceramic substrate. Chemically aided attrition milling disperses and concentrates the as-received 3Y-TZP powder to produce a dense, fine-grained sintered microstructure. Initial three-point bend strength data are comparable to that of conventional zirconia; however, geometric irregularities (e.g., trapezoidal cross sections) are present in this first generation and are discussed with respect to the distribution of bend strength.

Silica encapsulated CdS tabular nanocomposites via a template directed agglomeration mechanism.

Silica coated CdS tabular nanocomposites were synthesized through precipitation of CdS nanoparticles in octylamine/water bilayer system followed by in situ hydrolysis of tetraethoxylsilicate (TEOS) precursor. Face diameter of the nanoplatelets was in the range of 50-250 nm with a variable thickness (3 to 25 nm) dictated by octylamine content or R ratio ([water]/[octylamine]). A uniform SiO2 outer shell of about 15 nm was observed regardless of the size of the high aspect ratio CdS nanoplatelets, which appeared to be agglomerated primarily owing to the confined bilayer template. Morphology and microstructure of the CdS/SiO2 tabular nanocomposites were characterized using atomic force microscope (AFM) and high resolution transmission electron microscope (HRTEM). A noticeable enhancement in absorbance for the UV-vis spectra was observed due to the SiO2 coating layer. Growth mechanism of nanocomposite platelets and potential applications associated with this anisotropic nanocomposite are discussed.

Inferior vena cava filter migration to right ventricle with destruction of tricuspid valve: a case report.

Inferior vena cava filter migration is an uncommon event. Temporary inferior vena cava filters offer protection against pulmonary embolism in the trauma patient in whom anticoagulation is contraindicated. We present the case of a 53-year-old man who suffered a lower extremity injury, which left him unable to walk for an extended period of time. The patient developed a deep venous thrombosis in the early postoperative course and decision was made to place a retrievable inferior vena cava (IVC) filter. One week later the IVC filter had migrated to the right ventricle and destroyed the tricuspid valve. Although there are a limited number of cases describing the migration of IVC filters to the heart, there have been no cases in the literature, to our knowledge, where an IVC filter has destroyed the tricuspid valve and required valve replacement.

Seat belt trauma: pectoralis muscle rupture and delayed mesh repair.

To our knowledge, this is the first reported case of a seat belt-related rupture of the pectoralis major muscle and its successful delayed repair using mesh. We report a case of a 34-year-old white man who sustained a right pectoralis major muscle rupture from a seat belt during a motor vehicle crash. The patient presented to us 2 years after the injury. We introduce a technique using mesh that results in a successful repair of a cosmetically disfiguring chest wall defect.

The use of nanoparticulates to treat breast cancer.

Breast cancer is a major ongoing public health issue among women in both developing and developed countries. Significant progress has been made to improve the breast cancer treatment in the past decades. However, the current clinical approaches are invasive, of low specificity and can generate severe side effects. As a rapidly developing field, nanotechnology brings promising opportunities to human cancer diagnosis and treatment. The use of nanoparticulate-based platforms overcomes biological barriers and allows prolonged blood circulation time, simultaneous tumor targeting and enhanced accumulation of drugs in tumors. Currently available and clinically applicable innovative nanoparticulate-based systems for breast cancer nanotherapies are discussed in this review.

A Cholecystokinin B Receptor-Specific DNA Aptamer for Targeting Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against "exposed" CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bioconjugation of AP1153 to the surface of fluorescent NPs greatly facilitated delivery of NPs to PDAC tumors in vivo. The selectivity of this AP-targeted NP delivery system holds promise for enhanced early detection of PDAC lesions as well as improved chemotherapeutic treatments for PDAC patients.

Dispersion of SiO2-based nanocomposites with high performance liquid chromatography.

Core-shell structured Ag/SiO2 nanocomposite has been synthesized by a cyclohexane/Igepal/water reverse micelle system. The spherical nanocomposite particles were washed and concentrated with high performance liquid chromatography (HPLC) to remove the surfactant added during synthesis. Spherical SiO2 micrometer-scale particles were packed in the HPLC column as a stationary phase for the washing and dispersing of Ag/SiO2 nanocomposite particles. Surface modification of Ag/SiO2 nanocomposite particles and SiO2 microspheres with silane coupling agent enhanced the surface charge of the particles and improved the efficiency of washing with HPLC. Well-dispersed Ag/SiO2 stable suspensions were successfully attained in ethanol/water mixed solvents after HPLC washing. The state of dispersion for the Ag/SiO2 nanocomposite suspension was systematically assessed using dynamic light scattering (DLS) and transmission electron microscope (TEM) and spin coat/atomic force microscope (AFM) analyses. The mechanism of the enabling HPLC washing protocol for SiO2-based nanoparticles is discussed.

3D Printing of Personalized Artificial Bone Scaffolds.

Additive manufacturing technologies, including three-dimensional printing (3DP), have unlocked new possibilities for bone tissue engineering. Long-term regeneration of normal anatomic structure, shape, and function is clinically important subsequent to bone trauma, tumor, infection, nonunion after fracture, or congenital abnormality. Due to the great complexity in structure and properties of bone across the population, along with variation in the type of injury or defect, currently available treatments for larger bone defects that support load often fail in replicating the anatomic shape and structure of the lost bone tissue. 3DP could provide the ability to print bone substitute materials with a controlled chemistry, shape, porosity, and topography, thus allowing printing of personalized bone grafts customized to the patient and the specific clinical condition. 3DP and related fabrication approaches of bone grafts may one day revolutionize the way clinicians currently treat bone defects. This article gives a brief overview of the current advances in 3DP and existing materials with an emphasis on ceramics used for 3DP of bone scaffolds. Furthermore, it addresses some of the current limitations of this technique and discusses potential future directions and strategies for improving fabrication of personalized artificial bone constructs.