Using a flipped classroom in a veterinary systems physiology course increases student performance on basic knowledge and clinical applicability questions.

Flipped classrooms are being utilized more frequently in biomedical education to provide more active learning opportunities to students although there are mixed results on the benefits of the flipped classroom in biomedical education. In this study, the effects of using a flipped classroom with case-based learning in the endocrine section of a first-year veterinary-integrated histology and physiology course were investigated. Results demonstrated that the flipped classroom improved performance on the endocrine section exam by 15.9% (Cohen's d = 1.08; P < 0.001) with improvements on both clinically applicable and basic knowledge questions. Student satisfaction with the flipped classroom was also investigated. Students reported high satisfaction with the in-class case-based learning opportunities but lower satisfaction with the asynchronous content delivery and the time required outside of class. Student perceptions of the flipped classroom were measured again after being exposed to the results of the flipped classroom on student learning. After seeing the results, students were significantly more likely to value the time spent in the flipped classroom and to desire more opportunities for flipped classrooms in the future.NEW & NOTEWORTHY A flipped classroom using case-based learning can significantly improve student performance in a veterinary physiology course with the largest gains going to lower performing students. Student perception of the flipped classroom can be improved by showing students data on the improvement in performance on examinations.

System for delivering microwave ablation to subcutaneous tumors in small-animals under high-field MRI thermometry guidance.

PURPOSE: Bio-effects following thermal treatments are a function of the achieved temperature profile in tissue, which can be estimated across tumor volumes with real-time MRI thermometry (MRIT). Here, we report on expansion of a previously developed small-animal microwave hyperthermia system integrated with MRIT for delivering thermal ablation to subcutaneously implanted tumors in mice. METHODS: Computational models were employed to assess suitability of the 2.45 GHz microwave applicators for delivering ablation to subcutaneous tumor targets in mice. Phantoms and ex-vivo tissues were heated to temperatures in the range 47-67 °C with custom-made microwave applicators for validating MRIT with the proton resonance frequency shift method against fiberoptic thermometry. HAC15 tumors implanted in nude mice (n = 6) were ablated in vivo and monitored with MRIT in multiple planes. One day post ablation, animals were euthanized, and excised tumors were processed for viability assessment. RESULTS: Average absolute error between temperatures from fiberoptic sensors and MRIT was 0.6 °C across all ex-vivo ablations. During in-vivo experiments, tumors with volumes ranging between 5.4-35.9 mm3 (mean 14.2 mm3) were ablated (duration: 103-150 s) to achieve 55 °C at the tumor boundary. Thermal doses ≥240 CEM43 were achieved across 90.7-98.0% of tumor volumes for four cases. Ablations were incomplete for remaining cases, attributed to motion-affected thermometry. Thermal dose-based ablative tumor coverage agreed with viability assessment of excised tumors. CONCLUSIONS: We have developed a system for delivering microwave ablation to subcutaneous tumors in small animals under MRIT guidance and demonstrated its performance in-vivo.

Rationally designed peptide nanosponges for cell-based cancer therapy.

A novel type of supramolecular aggregate, named a "nanosponge" was synthesized through the interaction of novel supramolecular building blocks with trigonal geometry. The cholesterol-(K/D)nDEVDGC)3-trimaleimide unit consists of a trigonal maleimide linker to which homopeptides (either K or D) of variable lengths (n=5, 10, 15, 20) and a consensus sequence for executioner caspases (DEVDGC) are added via Michael addition. Upon mixing in aqueous buffer cholesterol-(K)nDEVDGC)3-trimaleimides and a 1:1 mixture of cholesterol-(K/D)nDEVDGC)3-trimaleimides form stable nanosponges, whereas cholesterol-(D)nDEVDGC)3-trimaleimide is unable to form supramolecular aggregates with itself. The structure of the novel nanosponges was investigated through explicit solvent and then coarse-grained molecular dynamics (MD) simulations. The nanosponges are between 80 nm and several micrometers in diameters and virtually non-toxic to monocyte/macrophage-like cells.

Nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases.

Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).

Thermosensitive Liposomes for Gemcitabine Delivery to Pancreatic Ductal Adenocarcinoma.

Treatment of pancreatic ductal adenocarcinoma with gemcitabine is limited by an increased desmoplasia, poor vascularization, and short plasma half-life. Heat-sensitive liposomes modified by polyethylene glycol (PEG; PEGylated liposomes) can increase plasma stability, reduce clearance, and decrease side effects. Nevertheless, translation of heat-sensitive liposomes to the clinic has been hindered by the low loading efficiency of gemcitabine and by the difficulty of inducing hyperthermia in vivo. This study was designed to investigate the effect of phospholipid content on the stability of liposomes at 37 °C and their release under hyperthermia conditions; this was accomplished by employing a two-stage heating approach. First the liposomes were heated at a fast rate, then they were transferred to a holding bath. Thermosensitive liposomes formulated with DPPC: DSPC: PEG2k (80:15:5, mole%) exhibited minimal release of carboxyfluorescein at 37 °C over 30 min, indicating stability under physiological conditions. However, upon exposure to hyperthermic conditions (43 °C and 45 °C), these liposomes demonstrated a rapid and significant release of their encapsulated content. The encapsulation efficiency for gemcitabine was calculated at 16.9%. Additionally, fluorescent analysis during the removal of unencapsulated gemcitabine revealed an increase in pH. In vitro tests with BxPC3 and KPC cell models showed that these thermosensitive liposomes induced a heat-dependent cytotoxic effect comparable to free gemcitabine at temperatures above 41 °C. This study highlights the effectiveness of the heating mechanism and cell models in understanding the current challenges in developing gemcitabine-loaded heat-sensitive liposomes.

T1-mapping characterization of two tumor types.

T1 mapping is a quantitative method to characterize tissues with magnetic resonance imaging in a quick and efficient manner. It utilizes the relaxation rate of protons to depict the underlying structures within the imaging frame. While T1-mapping techniques are used with some frequency in areas such as cardiac imaging, their application for understanding malignancies and identifying tumor structures has yet to be thoroughly investigated. Utilizing a saturation recovery method to acquire T1 maps for two different tumor models has revealed that longitudinal relaxation mapping is sensitive enough to distinguish between normal and malignant tissue. This is seen even with decreased signal/noise ratios using small voxel sizes to obtain high-resolution images. In both tumor models, it was revealed that relaxation mapping recorded significantly different relaxation values between regions encapsulating the tumor, muscle, kidney, or spleen, as well as between the cell lines themselves. This indicates a potential future application of relaxation mapping as a method to fingerprint various stages of tumor development and may prove a useful measure to identify micro-metastases.

Characterization of a Biochemical Mouse Model of Primary Aldosteronism for Thermal Therapies.

Introduction: Aldosterone-producing adenoma (APA) is the most common cause of endocrine-related hypertension but surgery is not always feasible. Current medical interventions are associated with significant side effects and poor patient compliance. New APA animal models that replicate basic characteristics of APA and give physical and biochemical feedback are needed to test new non-surgical treatment methods, such as image-guided thermal ablation. Methods: A model of APA was developed in nude mice using HAC15 cells, a human adrenal carcinoma cell line. Tumor growth, aldosterone production, and sensitivity to angiotensin II were characterized in the model. The utility of the model was validated via treatment with microwave ablation and characterization of the resulting physical and biochemical changes in the tumor. Results: The APA model showed rapid and relatively homogeneous growth. The tumors produced aldosterone and steroid precursors in response to angiotensin II challenge, and plasma aldosterone levels were significantly higher in tumor bearing mice two hours after challenge verses non-tumor bearing mice. The model was useful for testing microwave ablation therapy, reducing aldosterone production by 80% in treated mice. Conclusion: The HAC15 model is a useful tumor model to study and develop localized treatment methods for APA.

Channel blocking of MspA revisited.

Porin A from Mycobacterium smegmatis (MspA) is a highly stable, octameric channel protein, which acts as the main transporter of electrolytes across the cell membrane. MspA features a narrow, negatively charged constriction zone, allowing stable binding of various analytes thereby blocking the channel. Investigation of channel blocking of mycobacterial porins is of significance in developing alternate treatment methods for tuberculosis. The concept that ruthenium(II)quaterpyridinium complexes have the capability to act as efficient channel blockers for MspA and related porins, emerged after very high binding constants were measured by high-performance liquid chromatography and steady-state luminescence studies. Consequently, the interactions between the ruthenium(II) complex RuC2 molecules and MspA, leading to RuC2@MspA assemblies, have been studied utilizing time-resolved absorption/emission, atomic force microscopy, dynamic light scattering, ζ potential measurements, and isothermal titration calorimetry. The results obtained provide evidence for the formation of clusters/large aggregates of RuC2 and MspA. The results are of interest with respect to utilizing prospective channel blockers in porins. The combination of results from conceptually different techniques shed some light onto the chemical nature of MspA-channel blocker interactions thus contributing to the development of a paradigm for channel blocking.

In Vitro Measurement and Mathematical Modeling of Thermally-Induced Injury in Pancreatic Cancer Cells.

Thermal therapies are under investigation as part of multi-modality strategies for the treatment of pancreatic cancer. In the present study, we determined the kinetics of thermal injury to pancreatic cancer cells in vitro and evaluated predictive models for thermal injury. Cell viability was measured in two murine pancreatic cancer cell lines (KPC, Pan02) and a normal fibroblast (STO) cell line following in vitro heating in the range 42.5-50 °C for 3-60 min. Based on measured viability data, the kinetic parameters of thermal injury were used to predict the extent of heat-induced damage. Of the three thermal injury models considered in this study, the Arrhenius model with time delay provided the most accurate prediction (root mean square error = 8.48%) for all cell lines. Pan02 and STO cells were the most resistant and susceptible to hyperthermia treatments, respectively. The presented data may contribute to studies investigating the use of thermal therapies as part of pancreatic cancer treatment strategies and inform the design of treatment planning strategies.

A cell-delivered and cell-activated SN38-dextran prodrug increases survival in a murine disseminated pancreatic cancer model.

Enzyme-activated prodrugs have been investigated and sought after as highly specific, low-side-effect treatments, especially for cancer therapy. Unfortunately, excellent targets for enzyme-activated therapy are rare. Here a system based on cell delivery that can carry both a prodrug and an activating enzyme to the cancer site is demonstrated. Raw264.7 cells (mouse monocyte/macrophage-like cells, Mo/Ma) are engineered to express intracellular rabbit carboxylesterase (InCE), which is a potent activator of the prodrug irinotecan to SN38. InCE expression is regulated by the TetOn® system, which silences the gene unless a tetracycline, such as doxycycline, is present. Concurrently, an irinotecan-like prodrug, which is conjugated to dextran and can be loaded into the cytoplasm of Mo/Ma, is synthesized. To test the system, a murine pancreatic cancer model is generated by intraperitoneal (i.p.) injection of Pan02 cells. Engineered Mo/Ma are loaded with the prodrug and are injected i.p. Two days later, doxycycline was given i.p. to activate InCE, which activated the prodrug. A survival study demonstrates that this system significantly increased survival in a murine pancreatic cancer model. Thus, for the first time, a prodrug/activating enzyme system, which is self-contained within tumor-homing cells and can prolong the life of i.p. pancreatic tumor bearing mice, is demonstrated.

Stem cell-based photodynamic therapy.

We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.

A self-contained enzyme activating prodrug cytotherapy for preclinical melanoma.

Gene-directed enzyme prodrug therapy (GDEPT) has been investigated as a means of cancer treatment without affecting normal tissues. This system is based on the delivery of a suicide gene, a gene encoding an enzyme which is able to convert its substrate from non-toxic prodrug to cytotoxin. In this experiment, we have developed a targeted suicide gene therapeutic system that is completely contained within tumor-tropic cells and have tested this system for melanoma therapy in a preclinical model. First, we established double stable RAW264.7 monocyte/macrophage-like cells (Mo/Ma) containing a Tet-On® Advanced system for intracellular carboxylesterase (InCE) expression. Second, we loaded a prodrug into the delivery cells, double stable Mo/Ma. Third, we activated the enzyme system to convert the prodrug, irinotecan, to the cytotoxin, SN-38. Our double stable Mo/Ma homed to the lung melanomas after 1 day and successfully delivered the prodrug-activating enzyme/prodrug package to the tumors. We observed that our system significantly reduced tumor weights and numbers as targeted tumor therapy after activation of the InCE. Therefore, we propose that this system may be a useful targeted melanoma therapy system for pulmonary metastatic tumors with minimal side effects, particularly if it is combined with other treatments.

The Progress and Promise of RNA Medicine─An Arsenal of Targeted Treatments.

In the past decade, there has been a shift in research, clinical development, and commercial activity to exploit the many physiological roles of RNA for use in medicine. With the rapid success in the development of lipid-RNA nanoparticles for mRNA vaccines against COVID-19 and with several approved RNA-based drugs, RNA has catapulted to the forefront of drug research. With diverse functions beyond the role of mRNA in producing antigens or therapeutic proteins, many classes of RNA serve regulatory roles in cells and tissues. These RNAs have potential as new therapeutics, with RNA itself serving as either a drug or a target. Here, based on the CAS Content Collection, we provide a landscape view of the current state and outline trends in RNA research in medicine across time, geography, therapeutic pipelines, chemical modifications, and delivery mechanisms.

Lipophilic Near-Infrared Dyes for In Vivo Fluorescent Cell Tracking.

Cells can be easily and noninvasively tracked in the body by labeling them with a lipophilic, near-infrared dye and using a live fluorescence imaging system to image the position of the dye in the body. Near-infrared dyes provide several advantages, primarily that tissue is mostly highly transparent to near-infrared light, resulting in clearer and more accurate images. Briefly, cells are labeled with a near-infrared dye such as DiR and injected into a disease model. The model is then imaged using the live fluorescence imaging system on an hourly and/or daily basis to track cell migration and final location. The relative number of cells that migrate to the desired location can be measured by measuring the fluorescent intensity at the location versus elsewhere in the body. This paper describes a method for using DiR dye to label and track C17.2 neural progenitor cells to a murine model of mammary carcinoma.

Using Naturally Occurring Bioluminescent Enzymes to Track Specific Cell Populations.

Luminol-based bioluminescence imaging allows noninvasive tracking of oxidatively active cells such as neutrophils. Luminol is given intravenously or intraperitoneally, followed by bioluminescence imaging at 425 nm. Here we describe a method for tracking neutrophil extravasation into an inflammatory site, especially focusing on mammary carcinoma.

Direct treatment versus indirect: Thermo-ablative and mild hyperthermia effects.

Hyperthermia is a rapidly growing field in cancer therapy and many advances have been made in understanding and applying the mechanisms of hyperthermia. Secondary effects of hyperthermia have been increasingly recognized as important in therapeutic effects and multiple studies have started to elucidate their implications for treatment. Immune effects have especially been recognized as important in the efficacy of hyperthermia treatment of cancer. Both thermo-ablative and mild hyperthermia activate the immune system, but mild hyperthermia seems to be more effective at doing so. This may suggest that mild hyperthermia has some advantages over thermo-ablative hyperthermia and research into immune effects of mild hyperthermia should continue. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies Implantable Materials and Surgical Technologies > Nanoscale Tools and Techniques in Surgery.

Temperature estimation for MR-guided microwave hyperthermia using block-based compressed sensing.

Mild hyperthermia has been clinically employed as an adjuvant for radiation/chemotherapy and is under investigation for precise thermally-mediated delivery of cancer therapeutic agents. Magnetic Resonance Imaging (MRI) facilitates non-invasive, real-time spatial thermometry for monitoring and guiding hyperthermia procedures. Long image acquisition time during MR-guided hyperthermia may fail to capture rapid changes in temperature. This may lead to unwanted heating of healthy tissue and/or temperature rise above hyperthermic range. We have developed a block-based compressed sensing approach to reconstruct volumetric MR-derived microwave hyperthermia temperature profiles using a subset of measured data. This algorithm exploits the sparsity of MR images due to the presence of inter- and intra-slice correlation of hyperthermic MR-derived temperature profiles. We have evaluated the performance of our developed algorithm on a phantom and in vivo in mice using previously implemented microwave applicators. This algorithm reconstructs 3D temperature profiles with PSNR of 33 dB - 49 dB in comparison to the original profiles. In summary, this study suggests that microwave hyperthermia induced temperature profiles can be reconstructed using subsamples to reduce MR image acquisition time.

Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells.

Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n = 5-20) and a hydrophobic unit. In contrast to classical liposomes/vesicles, nanosponges feature interwoven hydrophilic and hydrophobic nanodomains and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by a poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC) because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and ζ potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum-containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.

Developing a xenograft human tumor model in immunocompetent mice.

Animal models are essential to cancer research, but current xenograft models are limited in their utility especially due to the lack of an immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and after birth mice were challenged with A375 cells to determine their ability to develop tumors. Intravenous injections of cells resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. These results were replicated with several other human tumor types: BxPC3 (human pancreatic adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma), M21 (human melanoma), and HeLa (human cervical adenocarcinoma). Development of an immunocompetent xenograft tumor model would allow the further elucidation of the interaction of the immune system with therapy in both preclinical research and patient derived xenografts.