Gentamicin Eluting 3D-Printed Implants for Preventing Post-Surgical Infections in Bone Fractures.

A surgically implantable device is an inevitable treatment option for millions of people worldwide suffering from diseases arising from orthopedic injuries. A global paradigm shift is currently underway to tailor and personalize replacement or reconstructive joints. Additive manufacturing (AM) has provided dynamic outflow to the customized fabrication of orthopedic implants by enabling need-based design and surface modification possibilities. Surgical grade 316L Stainless Steel (316L SS) is promising with its cost, strength, composition, and corrosion resistance to fabricate 3D implants. This work investigates the possibilities of application of the laser powder bed fusion (L-PBF) technique to fabricate 3D-printed (3DP) implants, which are functionalized with a multilayered antimicrobial coating to treat potential complications arising due to postsurgical infections (PSIs). Postsurgical implant-associated infection is a primary reason for implantation failure and is complicated mainly by bacterial colonization and biofilm formation at the installation site. PLGA (poly-d,l-lactide-co-glycolide), a biodegradable polymer, was utilized to impart multiple layers of coating using the airbrush spray technique on 3DP implant surfaces loaded with gentamicin (GEN). Various PLGA-based polymers were tested to optimize the ideal lactic acid: glycolic acid ratio and molecular weight suited for our investigation. 3D-Printed PLGA-GEN substrates sustained the release of gentamicin from the surface for approximately 6 weeks. The 3DP surface modification with PLGA-GEN facilitated cell adhesion and proliferation compared to control surfaces. The cell viability studies showed that the implants were safe for application. The 3DP PLGA-GEN substrates showed good concentration-dependent antibacterial efficacy against the common PSI pathogen Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). The GEN-loaded substrates demonstrated antimicrobial longevity and showed significant biofilm growth inhibition compared to control. The substrates offered great versatility regarding the in vitro release rates, antimicrobial properties, and biocompatibility studies. These results radiate great potential in future human and veterinary clinical applications pertinent to complications arising from PSIs, focusing on personalized sustained antibiotic delivery.

Online geometry calibration for retrofit computed tomography from a mouse rotation system and a small-animal imager.

BACKGROUND: Computed tomography (CT) generates a three-dimensional rendering that can be used to interrogate a given region or desired structure from any orientation. However, in preclinical research, its deployment remains limited due to relatively high upfront costs. Existing integrated imaging systems that provide merged planar X-ray also dwarfs CT popularity in small laboratories due to their added versatility. PURPOSE: In this paper, we sought to generate CT-like data using an existing small-animal X-ray imager with a specialized specimen rotation system, or MiSpinner. This setup conforms to the cone-beam CT (CBCT) geometry, which demands high spatial calibration accuracy. Therefore, a simple but robust geometry calibration algorithm is necessary to ensure that the entire imaging system works properly and accurately. METHODS: Because the rotation system is not permanently affixed, we propose a structure tensor-based two-step online (ST-TSO) geometry calibration algorithm. Specifically, two datasets are needed, namely, calibration and actual measurements. A calibration measurement detects the background of the system forward X-ray projections. A study on the background image reveals the characteristics of the X-ray photon distribution, and thus, provides a reliable estimate of the imaging geometry origin. Actual measurements consisted of an X-ray of the intended object, including possible geometry errors. A comprehensive image processing technique helps to detect spatial misalignment information. Accordingly, the first processing step employs a modified projection matrix-based calibration algorithm to estimate the relevant geometric parameters. Predicted parameters are then fine-tuned in a second processing step by an iterative strategy based on the symmetry property of the sum of projections. Virtual projections calculated from the parameters after two-step processing compensate for the scanning errors and are used for CT reconstruction. Experiments on phantom and mouse imaging data were performed to validate the calibration algorithm. RESULTS: Once system correction was conducted, CBCT of a CT bar phantom and a cohort of euthanized mice were analyzed. No obvious structure error or spatial artifacts were observed, validating the accuracy of the proposed geometry calibration method. Digital phantom simulation indicated that compared with the preset spatial values, errors in the final estimated parameters could be reduced to 0.05° difference in dominant angle and 0.5-pixel difference in dominant axis bias. The in-plane resolution view of the CT-bar phantom revealed that the resolution approaches 150 µ $\umu$ m. CONCLUSIONS: A constrained two-step online geometry calibration algorithm has been developed to calibrate an integrated X-ray imaging system, defined by a first-step analytical estimation and a second-step iterative fine-tuning. Test results have validated its accuracy in system correction, thus demonstrating the potential of the described system to be modified and adapted for preclinical research.

Evaluation of transfection efficacy, biodistribution, and toxicity of branched amphiphilic peptide capsules (BAPCs) associated with mRNA.

Nanoparticles (NPs) have been shown to be a suitable mRNA delivery platform by conferring protection against ribonucleases and facilitating cellular uptake. Several NPs have succeeded in delivering mRNA intranasally, intratracheally, and intramuscularly in preclinical settings. However, intravenous mRNA delivery has been less explored. Only a few NPs have been tested for systemic delivery of mRNA, many of which are formulated with polyethylene glycol (PEG). The incorporation of PEG presents some tradeoffs that must be carefully considered when designing a systemic delivery model. For example, while the addition of PEG may prolong circulation time by preventing early clearance by the mononuclear phagocytic system (MPS), it has also been reported that treating patients with PEGylated drugs can result in hypersensitivity reactions due to anti-PEG antibodies. Thus, it is desirable to have alternative PEG-free delivery methods for mRNA to avoid these adverse effects while preserving the beneficial effects. Our research group developed BAPCs (branched amphiphilic peptide capsules), a peptide-based nanoparticle that resists disruption by chaotropes, proteases, and elevated temperature, thus displaying significant stability and shelf-life. In this study, we demonstrated that similarly to PEG, mRNA shields the BAPC cationic surface to avoid early clearance by the MPS. Multispectral optoacoustic tomography (MSOT) and fluorescence reflectance imaging were imaging techniques used to analyze biodistribution within major MPS organs. Analysis of pro-inflammatory cytokine expression showed that BAPC-mRNA complexes do not cause chronic inflammation. Additionally, BAPCs enhance intracellular delivery of mRNA with negligible cytotoxicity or oxidative stress. These results might pave the way for future therapeutic applications of BAPCs as a delivery platform for systemic mRNA delivery.

Sugar Shock: Probing Streptococcus pyogenes Metabolism Through Bioluminescence Imaging.

Streptococcus pyogenes (S. pyogenes) can thrive in its host during an infection, and, as a result, it must be able to respond to external stimuli and available carbon sources. The preclinical use of engineered pathogens capable of constitutive light production may provide real-time information on microbial-specific metabolic processes. In this study, we mapped the central metabolism of a luxABCDE-modified S. pyogenes Xen20 (Strep. Xen20) to its de novo synthesis of luciferase substrates as assessed by the rate of light production in response to different environmental triggers. Previous characterization predicted that the lux operon was under the myo-inositol iolE promotor. In this study, we revealed that supplementation with myo-inositol generated increased Strep. Xen20 luminescence. Surprisingly, when supplemented with infection-relevant carbon sources, such as glucose or glycine, light production was diminished. This was presumably due to the scavenging of pyruvate by L-lactate dehydrogenase (LDH). Inhibition of LDH by its inhibitor, oxamate, partially restored luminescent signal in the presence of glucose, presumably by allowing the resulting pyruvate to proceed to acetyl-coenzyme A (CoA). This phenomenon appeared specific to the lactic acid bacterial metabolism as glucose or glycine did not reduce signal in an analogous luxABCDE-modified Gram-positive pathogen, Staph. Xen29. The Strep. Xen20 cells produced light in a concentration-dependent manner, inversely related to the amount of glucose present. Taken together, our measures of microbial response could provide new information regarding the responsiveness of S. pyogenes metabolism to acute changes in its local environments and cellular health.

Tunable three-dimensional engineered prostate cancer tissues for in vitro recapitulation of heterogeneous in vivo prostate tumor stiffness.

In this manuscript we report the establishment and characterization of a three-dimensional in vitro, coculture engineered prostate cancer tissue (EPCaT) disease model based upon and informed by our characterization of in vivo prostate cancer (PCa) xenograft tumor stiffness. In prostate cancer, tissue stiffness is known to impact changes in gene and protein expression, alter therapeutic response, and be positively correlated with an aggressive clinical presentation. To inform an appropriate stiffness range for our in vitro model, PC-3 prostate tumor xenografts were established. Tissue stiffness ranged from 95 to 6,750 Pa. Notably, xenograft cell seeding density significantly impacted tumor stiffness; a two-fold increase in the number of seeded cells not only widened the tissue stiffness range throughout the tumor but also resulted in significant spatial heterogeneity. To fabricate our in vitro EPCaT model, PC-3 castration-resistant prostate cancer cells were co-encapsulated with BJ-5ta fibroblasts within a poly(ethylene glycol)-fibrinogen matrix augmented with excess poly(ethylene glycol)-diacrylate to modulate the matrix mechanical properties. Encapsulated cells temporally remodeled their in vitro microenvironment and enrichment of gene sets associated with tumorigenic progression was observed in response to increased matrix stiffness. Through variation of matrix composition and culture duration, EPCaTs were tuned to mimic the wide range of biomechanical cues provided to PCa cells in vivo; collectively, a range of 50 to 10,000 Pa was achievable. Markedly, this also encompasses published clinical PCa stiffness data. Overall, this study serves to introduce our bioinspired, tunable EPCaT model and provide the foundation for future PCa progression and drug development studies. STATEMENT OF SIGNIFICANCE: The development of cancer models that mimic the native tumor microenvironment (TME) complexities is critical to not only develop effective drugs but also enhance our understanding of disease progression. Here we establish and characterize our 3D in vitro engineered prostate cancer tissue model with tunable matrix stiffness, that is inspired by this study's spatial characterization of in vivo prostate tumor xenograft stiffness. Notably, our model's mimicry of the TME is further augmented by the inclusion of matrix remodeling fibroblasts to introduce cancer-stromal cell-cell interactions. This study addresses a critical unmet need in the field by elucidating the prostate tumor xenograft stiffness range and establishing a foundation for recapitulating the biomechanics of site-of-origin and soft tissue metastatic prostate tumors in vitro.

Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis.

Interactions between the immune and central nervous systems strongly influence brain health. Although the blood-brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain's interstitial and perivascular spaces, facilitates outward signaling beyond the blood-brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull's hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders.

Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region.

Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the prototypical SC contains one pseudorepeat (PR) and seven repeats (R1 → R7) that bind fibrinogen/Fbn fragment D (frag D) by a mechanism that is unclear. Here, we define affinities and stoichiometries of frag D binding to C-terminal SC constructs, using fluorescence equilibrium binding, NMR titration, alanine scanning, and native PAGE. We found that constructs containing the PR and single repeats bound frag D with KD ∼50 to 130 nM and a 1:1 stoichiometry, indicating a conserved binding site bridging the PR and each repeat. NMR titration of PR-R7 with frag D revealed that residues 22 to 49, bridging PR and R7, constituted the minimal peptide (MP) for binding, corroborated by alanine scanning, and binding of labeled MP to frag D. MP alignment with the PR-R and inter-repeat junctions identified critical conserved residues. Full-length PR-(R1 → R7) bound frag D with KD ∼20 nM and a stoichiometry of 1:5, whereas constructs containing the PR and various three repeats competed with PR-(R1 → R7) for frag D binding, with a 1:3 stoichiometry. These findings are consistent with binding at PR-R and R-R junctions with modest inter-repeat sequence variability. CD of PR-R7 and PR-(R1 → R7) suggested a disordered flexible structure, allowing binding of multiple fibrin(ogen) molecules. Taken together, these results provide insights into pathogen localization on host fibrin networks.

Estimating the Center of Rotation of Tomographic Imaging Systems with a Limited Number of Projections.

For a tomographic imaging system, image reconstruction quality is dependent on the accurate determination of coordinates for the true center of rotation (COR). A significant COR offset error may introduce ringing, streaking, or other artifacts, while smaller error in determining COR may blur the reconstructed image. Well known COR correction techniques including image registration, center of mass calculation, or reconstruction evaluation work well under certain conditions. However, many of these methods do not consider various real-world cases such as a tilted sensor or non-parallel projections. Furthermore, a limited number of projections introduces stripe artifacts into the image reconstruction that interfere with many of these classic COR correction techniques. In this paper, we propose a revised variance-based algorithm to find the correct COR position automatically prior to tomographic reconstruction. The algorithm was tested on both simulated phantoms and acquired datasets, and our results show improved reconstruction accuracy.

Bacillus velezensis AP183 Inhibits Staphylococcus aureus Biofilm Formation and Proliferation in Murine and Bovine Disease Models.

The increasing frequency of S. aureus antimicrobial resistance has spurred interest in identifying alternative therapeutants. We investigated the S. aureus-inhibitory capacity of B. velezensis strains in mouse and bovine models. Among multiple B. velezensis strains that inhibited S. aureus growth in vitro, B. velezensis AP183 provided the most potent inhibition of S. aureus proliferation and bioluminescence in a mouse cutaneous wound (P = 0.02). Histology revealed abundant Gram-positive cocci in control wounds that were reduced in B. velezensis AP183-treated tissues. Experiments were then conducted to evaluate the ability of B. velezensis AP183 to prevent S. aureus biofilm formation on a tracheostomy tube substrate. B. velezensis AP183 could form a biofilm on a tracheostomy tube inner cannula substrate, and that this biofilm was antagonistic to S. aureus colonization. B. velezensis AP183 was also observed to inhibit the growth of S. aureus isolates originated from bovine mastitis cases. To evaluate the inflammatory response of mammary tissue to intramammary inoculation with B. velezensis AP183, we used high dose and low dose inocula in dairy cows. At the high dose, a significant increase in somatic cell count (SCC) and clinical mastitis was observed at all post-inoculation time points (P < 0.01), which resolved quickly compared to S. aureus-induced mastitis; in contrast, the lower dose of B. velezensis AP183 resulted in a slight increase of SCC and no clinical mastitis. In a subsequent experiment, all mammary quarters in four cows were induced to have grade 1 clinical mastitis by intramammary inoculation of a S. aureus mastitis isolate; following mastitis induction, eight quarters were treated with B. velezensis AP183 and milk samples were collected from pretreatment and post-treatment samples for 9 days. In groups treated with B. velezensis AP183, SCC and abundance of S. aureus decreased with significant reductions in S. aureus after 3 days post-inoculation with AP183 (P = 0.04). A milk microbiome analysis revealed significant reductions in S. aureus relative abundance in the AP183-treated group by 8 days post-inoculation (P = 0.02). These data indicate that B. velezensis AP183 can inhibit S. aureus biofilm formation and its proliferation in murine and bovine disease models.

Recent Advances in Lipid-Based Nanovesicular Delivery Systems for Melanoma Therapy.

Melanoma is one of the most aggressive forms of cancer with limited treatment options available. Successful treatment involves a combination of surgical resection of the tumor; chemotherapy and immunotherapy. Given their complex nature, the rapid development of drug resistance and metastatic spread, nanotechnology-based therapeutics are an attractive option for effective melanoma treatment. Nano-vesicular-based delivery systems hold the promise of aiding in the diagnosis and treatment of melanoma. These formulations can improve targeted delivery, deliver insoluble drugs belonging to class II, biopharmaceutical classification system, and alter drug pharmacokinetics and exposure profiles. These nanometer-sized carriers predominantly bypass the reticuloendothelial system and, thereby, improve blood circulation time and enhance tumor cell uptake with reduced toxicity. In this review, various lipid-based nano-formulations used in the diagnosis, treatment, or both for melanoma are discussed. Utilization of these na-no-formulations with a single drug or a combination of drugs, nucleic acid-based compounds (small interfering RNA, DNA) and targeting antibodies as other possibilities for melanoma are reviewed. We also present a state-of-the-art overview of alternative therapeutic approaches for the treatment of melanoma, such as photodynamic, immune, and gene therapies.

Spontaneous In Vitro and In Vivo Interaction of (-)-Oleocanthal with Glycine in Biological Fluids: Novel Pharmacokinetic Markers.

Since the first discovery of its ibuprofen-like anti-inflammatory activity in 2005, the olive phenolic (-)-oleocanthal gained great scientific interest and popularity due to its reported health benefits. (-)-Oleocanthal is a monophenolic secoiridoid exclusively occurring in extra-virgin olive oil (EVOO). While several groups have investigated oleocanthal pharmacokinetics (PK) and disposition, none was able to detect oleocanthal in biological fluids or identify its PK profile that is essential for translational research studies. Besides, oleocanthal could not be detected following its addition to any fluid containing amino acids or proteins such as plasma or culture media, which could be attributed to its unique structure with two highly reactive aldehyde groups. Here, we demonstrate that oleocanthal spontaneously reacts with amino acids, with high preferential reactivity to glycine compared to other amino acids or proteins, affording two products: an unusual glycine derivative with a tetrahydropyridinium skeleton that is named oleoglycine, and our collective data supported the plausible formation of tyrosol acetate as the second product. Extensive studies were performed to validate and confirm oleocanthal reactivity, which were followed by PK disposition studies in mice, as well as cell culture transport studies to determine the ability of the formed derivatives to cross physiological barriers such as the blood-brain barrier. To the best of our knowledge, we are showing for the first time that (-)-oleocanthal is biochemically transformed to novel products in amino acids/glycine-containing fluids, which were successfully monitored in vitro and in vivo, creating a completely new perspective to understand the well-documented bioactivities of oleocanthal in humans.

Multimodal imaging of bacterial-host interface in mice and piglets with Staphylococcus aureus endocarditis.

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart's valves. S. aureus relies on virulence factors to evade therapeutics and the host's immune response, usurping the host's clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor-binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor-binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases.

Co-Delivery of Hispolon and Doxorubicin Liposomes Improves Efficacy Against Melanoma Cells.

Hispolon is a small molecular weight polyphenol that has antioxidant, anti-inflammatory, and anti-proliferative activities. Our recent study has demonstrated hispolon as a potent apoptosis inducer in melanoma cell lines. Doxorubicin is a broad spectrum first-line treatment for various kinds of cancers. In this study, co-delivery of doxorubicin and hispolon using a liposomal system in B16BL6 melanoma cell lines for synergistic cytotoxic effects was investigated. Liposomes were prepared using a lipid film hydration method and loaded with doxorubicin or hispolon. The formulations were characterized for particle size distribution, release profile, and encapsulation efficiency (EE). In addition, in vitro cytotoxicity, in vitro cell apoptosis, and cellular uptake were evaluated. Liposomes exhibited small particle size (mean diameter ~ 100 nm) and narrow size distribution (polydispersity index (< 0.2) and high drug EE% (> 90%). The release from liposomes showed slower release compared to free drug solution as an additional time required for the release of drug from the liposome lipid bilayer. Liposome loaded with doxorubicin or hispolon exhibited significantly higher cytotoxicity against B16BL6 melanoma cells as compared to doxorubicin solution or hispolon solution. Likewise, co-delivery of hispolon and doxorubicin liposomes showed two-fold and three-fold higher cytotoxicity, as compared to hispolon liposomes or doxorubicin liposomes, respectively. In addition, co-delivery of doxorubicin and hispolon in liposomes enhanced apoptosis more than the individual drugs in the liposome formulation. In conclusion, the co-delivery of hispolon and doxorubicin could be a promising therapeutic approach to improve clinical outcomes against melanoma.

Design and implementation of a molecular imaging elective for third-year pharmacy students.

OBJECTIVE: To design, implement, and evaluate a molecular imaging elective course that would expose Doctor of Pharmacy (PharmD) students to fundamentals of various imaging modalities and their pre-clinical and clinical applications. METHODS: The "Surveys of Multi-Modality Imaging" course is a two-credit hour elective course offered to third-year PharmD and doctoral students. Experiential learning methods including active learning application-based exercises were used to supplement didactic lectures in the form of field trips (with follow-up debriefings), small group team-based tasks, hands-on demonstrations, visual modelling, gamification with problem sets, concept maps regarding given modalities, and concluding with written summary reports and formal in-class group presentations. In addition to standard course evaluations, a pre- and post-course survey were conducted to assess the students' confidence regarding course content. RESULTS: Since its implementation in 2013, 101 students have completed the course with 72% being PharmD students (n = 73) and the remainder being doctoral students in pharmaceutical science (n = 28). Pre- and post-assessments completed by the students the last two offerings (n = 40 of a possible 43) indicated a shift in students' self-reported confidence in discussing imaging modalities from a total of 2.4% confidence (pre-course) to 97.4% confidence (post-course). Also, post-course survey indicated that 77.5% (n = 31 of 40 participants) students strongly agreed that our immersive and experiential learning activities were beneficial to overall learning for this elective. CONCLUSION: Students who participated in this innovative experiential learning-grounded course gained an appreciation for molecular imaging and its value and role in modern drug therapy.

Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.

In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

The Cardioprotective Mechanism of Phenylaminoethyl Selenides (PAESe) Against Doxorubicin-Induced Cardiotoxicity Involves Frataxin.

Doxorubicin (DOX) is an anthracycline cancer chemotherapeutic that exhibits cumulative dose-limiting cardiotoxicity and limits its clinical utility. DOX treatment results in the development of morbid cardiac hypertrophy that progresses to congestive heart failure and death. Recent evidence suggests that during the development of DOX mediated cardiac hypertrophy, mitochondrial energetics are severely compromised, thus priming the cardiomyocyte for failure. To mitigate cumulative dose (5 mg/kg, QIW x 4 weeks with 2 weeks recovery) dependent DOX, mediated cardiac hypertrophy, we applied an orally active selenium based compound termed phenylaminoethyl selenides (PAESe) (QIW 10 mg/kg x 5) to our animal model and observed that PAESe attenuates DOX-mediated cardiac hypertrophy in athymic mice, as observed by MRI analysis. Mechanistically, we demonstrated that DOX impedes the stability of the iron-sulfur cluster biogenesis protein Frataxin (FXN) (0.5 fold), resulting in enhanced mitochondrial free iron accumulation (2.5 fold) and reduced aconitase activity (0.4 fold). Our findings further indicate that PAESe prevented the reduction of FXN levels and the ensuing elevation of mitochondrial free iron levels. PAESe has been shown to have anti-oxidative properties in part, by regeneration of glutathione levels. Therefore, we observed that PAESe can mitigate DOX mediated cardiac hypertrophy by enhancing glutathione activity (0.4 fold) and inhibiting ROS formation (1.8 fold). Lastly, we observed that DOX significantly reduced cellular respiration (basal (5%) and uncoupled (10%)) in H9C2 cardiomyoblasts and that PAESe protects against the DOX-mediated attenuation of cellular respiration. In conclusion, the current study determined the protective mechanism of PAESe against DOX mediated myocardial damage and that FXN is implicitly involved in DOX-mediated cardiotoxicity.

Quantitative, real-time in vivo tracking of magnetic nanoparticles using multispectral optoacoustic tomography (MSOT) imaging.

The goal of this work was to demonstrate real-time tracking of in vivo nanoparticle concentrations utilizing multispectral optoacoustic tomography (MSOT). Combining the high contrast of optical imaging with the high resolution of ultrasound imaging, MSOT was utilized for non-invasive, real-time tomographic imaging of particles in mice and the results calibrated against analysis of tissue samples with electron paramagnetic resonance (EPR) spectroscopy. In a longitudinal study, the pharmacokinetics (pK) and biodistribution of Cyanine-7 (Cy7) conjugated superparamagnetic iron oxide nanoparticles (Cy7-SPIONs) were monitored after intravenous administration into the tail vein of healthy B6-albino mice. Concentrations of Cy7-SPIONs determined by MSOT image analysis of the liver, spleen, and kidneys showed excellent agreement with EPR data obtained on tissue samples ‒ validating MSOT's ability to quantify SPION concentrations with high spatial resolution. Both methods of analysis indicated highest accumulation of Cy7-SPIONs in the liver followed by the spleen, and negligible accumulation in the kidneys; SPION accumulation in organs with high concentrations of mononuclear phagocytic system macrophages is typical. Additionally, our study observed that particles modified with a 2 kDa polyethylene glycol (PEG) demonstrated significantly prolonged half-life in circulation compared to particles with 5 kDa PEG. The study demonstrates the potential of Cy7-SPIONs and MSOT for quantitative localization of magnetic nanoparticles in vivo, which can potentially be used to study their toxicity, quantify the efficacy of targeted drug delivery (e.g. within tumors), and their use as a multi-modal diagnostic agent to monitor disease progression.

Correlation of 360-degree Surface Mapping In Vivo Bioluminescence with Multi-Spectral Optoacoustic Tomography in Human Xenograft Tumor Models.

Pre-clinical monitoring of tumor growth and identification of distal metastasis requires a balance between accuracy and expediency. Bioluminescence imaging (BLI) is often used to track tumor growth but is primarily limited to planar 2-dimensional (2D) imaging. Consistent subject placement within a standard top-mounted, single-detector small animal imager is vital to reducing variability in repeated same-animal measures over time. Here, we describe a method for tracking tumor development using a multi-angle BLI and photo-acoustic workflow. We correlate serial caliper measurements and 2D BLI to 360° BLI and photo-acoustic datasets for the same animals. Full 360° BLI showed improved correlations with both volumes obtained from caliper measurements and photo-acoustic segmentation, as compared to planar BLI. We also determined segmented tumor volumes from photo-acoustic datasets more accurately reflects true excised tumors' volumes compared to caliper measurements. Our results demonstrate the distinct advantages of both 360° surface mapping by BLI and photo-acoustic methodologies for non-invasive tracking of tumor growth in pre-clinical academic settings. Furthermore, our design is fully implementable in all top-mounted, single-detector imagers, thereby providing the opportunity to shift the paradigm away from planar BLI into rapid BLI tomography applications.

Nanoparticle-based probes to enable noninvasive imaging of proteolytic activity for cancer diagnosis.

Proteases play a key role in tumor biology, with high expression levels often correlating with poor prognosis for cancer patients - making them excellent disease markers for tumor diagnosis. Despite their significance, quantifying proteolytic activity in vivo remains a challenge. Nanoparticles, with their ability to serve as scaffolds having unique chemical, optical and magnetic properties, offer the promise of merging diagnostic medicine with material engineering. Such nanoparticles can interact preferentially with proteases enriched in tumors, providing the ability to assess disease state in a noninvasive and spatiotemporal manner. We review recent advances in the development of nanoparticles for imaging and quantification of proteolytic activity in tumor models, and prognosticate future advancements.

Complete genome of Staphylococcus aureus Tager 104 provides evidence of its relation to modern systemic hospital-acquired strains.

BACKGROUND: Staphylococcus aureus (S. aureus) infections range in severity due to expression of certain virulence factors encoded on mobile genetic elements (MGE). As such, characterization of these MGE, as well as single nucleotide polymorphisms, is of high clinical and microbiological importance. To understand the evolution of these dangerous pathogens, it is paramount to define reference strains that may predate MGE acquisition. One such candidate is S. aureus Tager 104, a previously uncharacterized strain isolated from a patient with impetigo in 1947. RESULTS: We show here that S. aureus Tager 104 can survive in the bloodstream and infect naïve organs. We also demonstrate a procedure to construct and validate the assembly of S. aureus genomes, using Tager 104 as a proof-of-concept. In so doing, we bridged confounding gap regions that limited our initial attempts to close this 2.82 Mb genome, through integration of data from Illumina Nextera paired-end, PacBio RS, and Lucigen NxSeq mate-pair libraries. Furthermore, we provide independent confirmation of our segmental arrangement of the Tager 104 genome by the sole use of Lucigen NxSeq libraries filled by paired-end MiSeq reads and alignment with SPAdes software. Genomic analysis of Tager 104 revealed limited MGE, and a νSaß island configuration that is reminiscent of other hospital acquired S. aureus genomes. CONCLUSIONS: Tager 104 represents an early-branching ancestor of certain hospital-acquired strains. Combined with its earlier isolation date and limited content of MGE, Tager 104 can serve as a viable reference for future comparative genome studies.